modulators of protein–protein interactions

Modulators of Protein−Protein InteractionsLech-Gustav Milroy,† Tom N. Grossmann,‡,§ Sven Hennig,‡ Luc Brunsveld,† and Christian Ottmann*,†

†Laboratory of Chemical Biology and Institute of Complex Molecular Systems, Department of Biomedical Engineering, TechnischeUniversiteit Eindhoven, Den Dolech 2, 5612 AZ Eindhoven, The Netherlands‡Chemical Genomics Centre of the Max Planck Society, Otto-Hahn Straße 15, 44227 Dortmund, Germany§Department of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany

CONTENTS

1. Introduction 46952. Biochemical Techniques for Ligand Identification 4696

2.1. Surface-Based Binding Assays, ELISA 46972.2. Surface Plasmon Resonance, SPR 46972.3. Fluorescence Polarization 46982.4. Proximity-Based Methods 4699

2.4.1. Fluorescence Resonance Energy Trans-fer (FRET) 4699

2.4.2. Time-Resolved FRET (TR-FRET) 46992.4.3. Bioluminescence Resonance Energy

Transfer (BRET) 47002.4.4. Amplified Luminescent Proximity Ho-

mogeneous Assay Screen (ALPHAScreen) 4700

3. Approaches for Hit Identification of PPI Modu-lators 47013.1. Structure-Based Design 4701

3.1.1. Hotspot Residue Theory 47013.1.2. Conformationally Constrained Peptides

and Miniproteins 47033.1.3. Peptidomimetics 47093.1.4. Mimetics of Protein Secondary Structure 4712

3.2. Natural-Product Inspired PPI Modulation 47143.2.1. Natural Products 47143.2.2. Approaches to Diversifying Natural

Products 47153.3. Supramolecular-Induced PPI Modulation 4719

3.3.1. Supramolecular-Induced Protein Dime-rization and Oligomerization 4719

3.3.2. Supramolecular-Modulation of the Pro-tein Surface 4720

3.4. Compound Library Generation 47213.4.1. Biological Techniques 4721

3.4.2. Microarrays 47233.4.3. One-Bead-Two-Compound Approach

(OBTC) 47233.4.4. Fragment-Based Drug Discovery (FBDD) 47233.4.5. In Silico Screening 47253.4.6. Multicomponent Reactions (MCRs) 4726

4. Stabilization of PPIs 47274.1. Cyclosporin A, FK506 and Rapamycin 47274.2. Forskolin and Brefeldin A 47284.3. Inositol Tetraphosphate 47284.4. Phytohormones Auxin, Jasmonate, and

Brassinolide 47284.4.1. Auxin 47284.4.2. Jasmonate 47294.4.3. Brassinolide 4729

4.5. Small-Molecule Stabilizers of Oligomeric-State Homocomplexes 4730

4.5.1. Tafamidis 47304.5.2. Phenothiazines 47304.5.3. Influenza Nucleoprotein (NP) 47314.5.4. MDMX Homodimer Stabilizers 47314.5.5. Topoisomerase II/ICRF-187 47324.5.6. 1-EBIO Class Stabilizers of SK-Channel-

CaM Interaction 47334.5.7. RAR-NCoR 4733

4.6. PPI Stabilizers of 14-3-3 Protein−ProteinInteractions 4734

4.6.1. Fusicoccin A 47344.6.2. Cotylenin A 47354.6.3. Pyrrolidone1 and Epibestatin 4735

5. Concluding Remarks 4736Author Information 4737

Corresponding Author 4737Notes 4737Biographies 4737

Acknowledgments 4738References 4738

1. INTRODUCTION

Since Hedin’s characterization of trypsin and antitrypsin in1906,1 arguably the first account of a regulatory protein−protein interaction (PPI), contemporary understanding ofproteins and PPIs has been progressively transformed by

Special Issue: Chemical Biology of Protein-Protein Interactions

Received: December 10, 2013Published: April 15, 2014

Review

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© 2014 American Chemical Society 4695 dx.doi.org/10.1021/cr400698c | Chem. Rev. 2014, 114, 4695−4748

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landmark conceptual and technological advances in molecularcell biology, biochemistry and biophysics,2 not least, thesequencing of the human genome and the ensuing genomictechnologies. Today, proteins can be viewed as the molecularsmart phones of the cell, genetically programmed to enactspecific cellular functions in response to external stimuli.Individually, proteins perform essential functions such ascatalysis and the transport of molecules and ions. However,their effectiveness in the crowded cellular environment is onlyshort-range and insufficient to sustain life without theinvolvement of other biomolecules such as other proteins ormetabolites. Proteins manage long-range effectiveness throughwider interactomes, highly organized and responsive proteinnetworks, which relay protein function cell-wide via interactionsbetween protein nodes: so-called protein−protein interactions(PPIs).3−6 This heightened awareness of a significant andubiquitous role for PPIs and PPI networks in cellularphysiology has created numerous new opportunities for drugdiscovery, as in principle every pathologically significant PPIbecomes a potential drug target.7,8 The challenge though now isfinding the small molecule modulators capable of performingthe task.9,10

The major motivation behind campaigns in PPI drugdiscovery today is the urgent need for safer and more effectivemedicines for the benefit of patients. The targeting of smallmolecule modulators at conventional protein targets, forinstance enzymes11 and ligand-activated transcription factors,12

has historically been a highly effective (and profitable) way totreat many types of diseases. However, a perceived crisis inresearch and development (R&D) productivity in thepharmaceutical industry today, which is connected with anover-reliance on the same drugs targeting the same proteintargets, has called for innovative approaches to discover newsmall molecule modulators as a basis for new drugs.13,14

Furthermore, diseases common to old-age, such as cancer andAlzheimer’s are expected to become more prevalent in thefuture as average life expectancies in countries across the worldcontinue to nudge upward, and yet, disturbingly, for many ofthese diseases there is currently no effective treatment, orcurrent drugs suffer from issues of drug resistance, high toxicityor low efficacy. As this review seeks to clarify, PPI drugdiscovery has the potential to deliver on both fronts, given theubiquitous importance of PPIs to cellular pathology, and theirorthogonality to conventional drug targets.With reference to a recent review article on PPI drug

discovery,15 we define a PPI as, “an interaction of two identicalor dissimilar proteins at their domain interfaces that regulatesthe function of the protein complex (interactions involvingenzyme active sites are not termed PPIs in drug discovery)”,15

and a small molecule modulator as, “a low-molecular weightnatural product or synthetic agent with a significant degree ofstructural complexity, thus allowing target selectivity and goodbinding affinity, which can regulate a PPI through either director allosteric inhibition, or stabilization”.15

The review’s focus on drug-like small molecule modulatorsunfortunately means that no-less-relevant, larger biomolecules,for example nucleic acid as well as peptide aptamers16 andantibodies,17 have been excluded from consideration this time.The generality of the review’s title, “Modulators of Protein−Protein Interactions”, means that rational, structure-basedapproaches as well as random approaches based on high-throughput screening to identifying small molecule PPImodulators are equally considered. Furthermore, the modulator

compounds have been descended from a naturally occurringagent such as a protein, peptide or secondary metabolite. Thisreview will emphasize actual and emerging approaches todesigned molecules targeting binary PPIs with a well-definedinterface. Work published from 2008 onward receives thehighest priority as does work reporting high resolution proteinX-ray crystallographic data as evidence of the interaction of asmall molecule with its target protein−protein interface.Interfacial18 as well as allosteric PPI modulators are bothcovered in this review, whereas higher-order multiproteincomplexes are not.19,20 Given the stringent selection criteriaand despite our best attempts to comprehensively cover asmany different aspects of PPI modulation as possible, we regretthat some important pieces of work and topics may have beenomitted, and that some of the topics covered may not havereceived as much focus as others. In this case, where possiblewe cite references to relevant and comprehensive reviewarticles.This review begins with a up-to-date account of the principle

biochemical techniques used to identify PPI modulators(section 2), e.g., ELISA and FRET based techniques, many ofwhich are referred to in later sections. In the next section, acomprehensive account is provided of the different chemicalmethods currently used to access designed small moleculeinhibitors of PPIs, including such details as the synthetic routesto the target molecules, as well as their molecular structures andPPI targeting properties (section 3). Finally, a highly detailedatomistic-level account of different established and new smallmolecule stabilizers of PPIs is given (section 4), to highlight theenormous potential of PPI stabilization as a drug discoverystrategy, and yet it remains a conceptually underexplored modeof PPI modulation.21−24

2. BIOCHEMICAL TECHNIQUES FOR LIGANDIDENTIFICATION

To interfere with a PPI of interest, compounds fit for thepurpose must first be identified. Nowadays, various interdisci-plinary techniques appear in the literature ranging from cellbased high content screening and biochemical in vitro assays tobioinformatical methods. Cell based assays often address acertain cellular phenotype (proliferation, apoptosis, andmorphology) rather than targeting a defined PPI. Therefore,these formats are not within the scope of this review.Bioinformatical methods usually require a high degree ofinsight into the PPI of interest including for example structuraldata. In silico screenings appear increasingly in the literature,and we describe some examples in section 3.4.5. However, thevast majority of compounds are still identified and validatedwith biochemical assays. Depending on the knowledge of thePPI, different sized collections of these molecules ranging fromsmall arrays to large library collections of several hundredthousand compounds need to be tested in a reliable and fastmanner (high-throughput screening, HTS). Accordingly,depending on the general principle of these screening methodsand the biophysical characteristics that the proteins of interesthave to possess, a differentiation can be made between surfacebased and proximity based assays. Hereafter, we will describethe general idea and the advantageous as well as disadvanta-geous features of these different assay types. New technologiesand assay principles, claiming to be beneficial for theidentification of small molecules in drug discovery, areconstantly appearing in the literature. We have decided to

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focus on successful and well-established assay formats, withsome examples in the context of PPI drug discovery.

2.1. Surface-Based Binding Assays, ELISA

Enzyme-linked immunosorbent assays (ELISAs) were firstdescribed in 1971 as a nonradioactive technology investigatingantibody−antigen interactions.25 Decades later, these are stillvalid tools for the investigation of PPIs and their modulation. Inbrief: one binding partner is bound to a surface by nonspecificadsorption, covalent linkage, or affinity tags (streptavidin−

biotin, GST-GSH, and GFP-trap). Subsequently, residualbinding spots on the surface are blocked and an incubationwith the second binding partner is performed. Quantification ofbinding events is achieved by a specific antibody for the bindingpartner that is in turn recognized by an enzyme-coupledsecondary antibody (Figure 1).Commonly used conjugated enzymes are horseradish

peroxidase (HRP) and alkaline phosphatase.26,27 Miniaturiza-tion into microtiter plates of decreasing size as well astechnological advances, such as automated liquid handlingtechnologies, and increased sample throughput has enabled thismethod for HTS. The introduction of lanthanide basedfluorophores instead of cross-linked enzymes enhanced thesystem, as it enabled time-resolved measurements.28,29

Dissociation-enhanced lanthanide fluorescence immunoassay(DELFIA, Perkin-Elmer) makes use of these fluorophores in anadvanced manner. Here, the secondary antibody carrieslanthanide ions (e.g., europium, samarium, or terbium). Afterextensive washing steps the ion is liberated. Once free insolution, the ions are incorporated into chelating complexesthat enhance the fluorescent signal. These caged lanthanidefluorophores feature sharp, red-shifted emission peaks whichallow detection in a spectral range where usual biologicalmaterial or compounds do not interfere with the read out.Additionally, these chromophores feature long half-lifetimesthat enable time-resolved measurements.Nowadays, the term ELISA is invariantly used to describe all

sorts of surface binding based assays. Detection strategies varyfrom chromogenic, fluorescent to chemoluminescent read outs,

whereas the types of ELISAs vary from indirect, sandwich, tocompetitive setups. A general advantage of this method is thesimplicity of instrumentation, as probes as well as plate readersare offered by numerous vendors. ELISAs show a high degreeof flexibility and a rather high sensitivity. They are easy to setup using standard laboratory equipment and therefore suitablefor small to medium sized libraries. On the other hand, assaydevelopment still requires specific antibodies and detectionreagents. Moreover, the rather harsh washing conditions couldaffect weak and transient interaction, as they are common forPPIs, resulting in false hit detection (false positives or falsenegatives depending on the assay layout). Additionally,extensive blocking and washing steps as well as antibodyincubations make these assay types a bigger effort forautomated liquid handling systems during HTS. However,the ELISA technology is a very versatile tool in PPI modulationstudies. This is illustrated by the fact that the inhibitors ofprotein−protein database (iPPI-DB) reports usage of ELISAsin about 20% of their collection.30

ELISAs performed during SAR studies are, for example, theLFA-1−ICAM interaction that plays a role in cell−cell adhesionof autoimmune diseases,31,32 acylpyrogallols as potent Bcl-2inhibitors33 as well as the derivatives of an isoindolinonescaffold as MDM2−p53 inhibitors.34 Also, many examples frompeptide ensembles of different size can be found for instance asantibiotics against the E. coli RNA polymerase σ(70)−coreinteraction35 or the modulation of the glycoprotein Ib−vonWillebrand factor complex important during platelet-dependentthrombus formation.36 Another example is the PED/PEA15−D4a complex, which plays a crucial role in insulin resistance intype 2 diabetes and was successfully addressed in a screening of∼20 000 peptides.37

2.2. Surface Plasmon Resonance, SPR

When a gold coated glass chip is illuminated with polarizedlight under conditions of total reflection, a proportion of thelight enters the coated surface as a so-called evanescent wave.This is the underlying principle of surface plasmon resonance(SPR) experiments. The energy of this evanescent wave ismissing in the reflected light beam and can be detected at acertain angle (SPR angle). Upon binding of macromolecules onthe surface of the sensor, the SPR angle changes (Figure 2).These changes can be measured over time. SPR measurementshave become popular in many fields over the years. Thetechnique is also applicable for sensing changes in proteincomplexes on the surface of the chip which makes it a valuabletool for PPI investigations.38,39

SPR measurements are performed in a label-free manner; yet,one partner must be surface immobilized requiring themodification of involved proteins. Initial shortcomings of thismethod such as the limited number of flow chambers weresubstantially improved by more advanced microfluidic systemsand the introduction of more advanced automated samplehandling systems (e.g., 96- and 384-well systems). Additionally,the sensitivity of the machines increased, allowing the precisemeasurement of the direct binding of small molecule fragmentlibraries with weak affinities to an immobilized protein.40,41 Upto now, SPR systems are not applicable to initial compoundscreening in large formats such as HTS. This is due to thelimited sample number these systems can handle in parallel.Also, the financial cost of purchasing such a machine issignificant. Nevertheless, SPR offers an extensive pool ofinformation as a secondary or downstream in depth validation

Figure 1. Assay principal for the ELISA. A surface bound PPI complexis exposed to either an inhibitory molecule (upper case, light redcompound) or a noninhibitory molecule (lower case, light bluecompound). Specific antibodies recognize the PPI complex and resultin a signal derived from the activity of a conjugated enzyme (e.g., HRP,yellow circle). Differences in enzymatic activity are a measure ofdifferent efficacies of inhibitors.



tool. Since kinetic measurements are performed, quantitativebinding characteristics such as on- and off-rates can be obtainedin addition to merely qualitative affinities.41 The iPPI-DBcurrently lists SPR in about 5% of their biochemical tests as themethod of choice. These involve the inhibition of BETrecruitment to chromatin,42 the inhibition of the cytokine

interleukin-2 to its receptor43 and the in vivo activation by

MDM2 antagonists.44

2.3. Fluorescence Polarization

The principle of fluorescence polarization (FP) is thatfluorescent molecules emit light of a different plane as beforewhen excited with linear polarized light. This effect is caused bythe movement of the molecules in solution between excitationand emission. Since large molecules rotate slower than smallerentities, a difference in polarization can be observed forfluorescently labeled species of different size (Figure 3). Thebasic mechanism of this effect was initially described in 1926 byPerrin.45 FP can be quantified by measuring the contributionsof the light intensity parallel and perpendicular to the plane ofthe excitation light beam. In the following decades, FP was usedintensively in analyses of biological molecules in general and indrug discovery approaches including PPI investigations.46,47

Various ways of fluorescently labeling proteins are known.Some of these are not suitable for FP based assays as theyincrease the mass of the protein under study significantly (likefluorescent protein fusions e.g. GFP or fluorescently labeledantibodies). Chemical modifications with commercially avail-able rhodamine, fluorescein or BODIPY dyes using thiol,amines, or acid reactive groups of proteins are currently acommon choice. Once a protein of interest is labeled potentialbinding partners can be added and differences in polarizationinduced by compound binding can be observed (Figure 3). Theassay essentially relies on a mass difference (preferably ∼10×)of the molecule carrying the fluorophore unbound vs bound toits protein partner. Therefore, the assay is often used in acompetition format where the fluorescent part is represented bya peptide derived from the original binding partner. Thispeptide is usually dispatched from its unlabeled partner uponcompetitive compound binding, thus leading to a strongdifference in FP.As the assay is not relying on surface decoration of

macromolecules it is relatively straightforward to miniaturizeinto simple uncoated microtiter plates of all sizes (96-, 384-,and 1536-well plates), saving biological reagents as well ascompound by simply reducing the assay volume to a few μL.Also, all components of the assay can be mixed prior tocompound administration, dispensed into plates, and read outdirectly (“mix-and-read-assay”). In contrast to ELISA assays,

Figure 2. Principle of surface plasmon resonance (SPR). Undercondition of total reflection, polarized light is penetrating the goldcoated surface of a glass chip as an evanescent wave. The energy of thelatter is missing in the detector in a certain angle (SPR angle). TheSPR angle is measured over time. Molecules binding on the surfaceshift the angle and can therefore be detected. (a) Shows ligand 1immobilized on the chip surface. (b) Upon application of a bindingpartner, the signal increases. (c) Application of an inhibitor lets thesignal decreases.

Figure 3. Assay principle for a fluorescence polarization assay (FP). Afluorescently labeled molecule rotates slower when complexed to itsprotein binding partner (light gray body) compared to its rotationwhen free in solution upon disruption of the complex, e.g., by acompetitor (light red compound). Therefore the FP signals differ, aprinciple which can be used to identify PPI modulating compounds.



blocking, extended antibody incubation periods, and platewashing are not required during the screening process.Fluorescent probes are used in rather low concentrations(depending on the PPI complex) so that the total costs per wellare comparably low. For larger libraries, automated liquidhandling is beneficial, but as FP is a mix-and-read-assay, asimple liquid dispenser is sufficient.One disadvantage of FP assays is that the assay cannot be

performed in a time-resolved manner, as the signal is to bemeasured instantly after excitation. Nonetheless, FP is the mostfrequently used assay format for the identification of PPImodulators. It is one of the most common primary assays onHTS platforms as its throughput can easily be scaled up. FPassays used for identification of PPI modulators can be found invarious biological fields. The iPPI-DB reports FP assays in topranking 51% of their cases.30 Additionally, there are severalrecent examples using FP assays targeting MDM2-MDMXinteraction with the tumor suppressor p53,48 the hypoxiainducible factor-1α (Hif1α) in complex with p30049 that plays arole in activating VEGF and its pro-angiogenic function, or theoncogene Casitas B-lineage Lymphoma (Cbl).50 The modu-lation of pro-apoptotic regulators Bcl-x(L) and Mcl-151 as wellas inhibiting the negative regulator Keap1 from its interactionwith the anti-inflammatory and anticancerous transcriptionfactor Nrf252 utilized successfully FP assays to identifyfunctional peptides. Also, the 14-3-3 adaptor protein involvedin many cellular functions was addressed several times using FPto find different classes of inhibitors like covalent binders,natural products from beetle extracts or phosphonates.53−55 FPplayed and will play a major role in drug discovery targetingPPIs. Nonetheless, FP is limited to PPI systems composed ofpartners whose size difference is large enough for a significantdifference in FP signal. In practice, this often means that onebinding partner has to be minimized to the essential interactingpatches of peptide size to serve as a model system. Toovercome these limitations, proximity based assay formatsrepresent suitable alternative solutions (section 2.4).

2.4. Proximity-Based Methods

2.4.1. Fluorescence Resonance Energy Transfer(FRET). Forster (or fluorescence) resonance energy transfer(FRET) is a phenomenon that occurs if the emission energy ofa given excited donor molecule matches the excitation energyof an acceptor fluorophore which is in close proximity and canbe excited (Figure 4). This acceptor molecule then emits theaccording energy as fluorescence. As each of these steps can becoupled with radiation-less relaxation of each state, energy isremoved from the system so that the wavelength is red-shiftedduring FRET. This process was described by Theodor Forsterin 1948.56 Since then this principle was used for many kinds ofphysical and biological analyses especially in imagingapplications.57

FRET signals are strictly dependent on the distance (d) ofthe two fluorophores as FRET efficiency is proportional to d−6.Thus, energy transfer occurs within a range of 10−100 Å.58 ForPPI measurements, two proteins need to be labeled withFRET-pairing fluorophores. Upon interaction the proteinsbring the two fluorophores in proximity, within the suitablerange, and FRET occurs.59 As this system is not size dependent,fluorescent labeling of the two proteins can be achieved eitherby chemical modification as described before in section 2.2(using, e.g., dansyl-fluorescin, Cy3-Cy5, or Alexa488-Alexa594as suitable FRET pairs) or by using specific fluorescentlyconjugated antibodies or fluorescent fusion proteins (e.g., CFP-YFP and GFP-mCherry are suitable FRET pairs) or mixtures ofboth systems.60 Also, quenching molecules like dabsyl anddabcyl can be used in FRET assays. In these cases thefluorescence of the donor conjugated molecule can only beobserved upon displacement of the quencher conjugatedbinding partner.61 Due to relatively low costs per well(depending on the complexity of the protein labeling strategy),FRET is highly applicable to compound screening. Also, itovercomes the size limitation problem of FP assays, its signal isrobust, and these types of assays are straightforward to handlein terms of instrumentation. A typical FRET assay requiresdispensing of protein mixtures and compound addition withsubsequent fluorescent read out, which is supported by anyplate reader.However, a downside of classical FRET assays in HTS is that

compounds excited with the rather high energetic excitationbeam often lead to false positive read outs. Several approacheshave been taken to improve the assay performance of FRET forinstance by substituting the initial excitation of the system withan enzymatic reaction, thereby utilizing bioluminescence as theenergy source (BRET, section 2.4.3). Alternatively, donorfluorophores with long half-lifetimes where successfullyintroduced, shifting the measurement window in order todistinguish rapidly decaying signals of false positives from truehits (TR-FRET, section 2.4.2). As these improved formats havebeen used widely in PPI modulations, the iPPI-database onlyreports 0.3% of their cases as classical FRET based assays.62−64

The FRET technology has constantly been developed furtherand is still an ongoing field of research. Lately, miniaturizationattempts were made for instance in a Lab-on-a-Chip mannerwhere Benz and colleagues were able to measure PPIs indroplets of pL size.33 However, these technical novelties haveyet to result in successful drug discoveries.

2.4.2. Time-Resolved FRET (TR-FRET). In order toeliminate the excitation and emission signals of biological aswell as small chemical molecule reagents in the homogeneousassay mixture of FRET based assays, a modulation of the

Figure 4. Theoretical constraints for FRET. (a) Jablonski diagramshowing the principal of FRET. A donor molecule becomes excitedand instead of directly emitting fluorescence, the energy is transferredto an acceptor molecule that is excited and emits light. Radiation-lessrelaxation of excited states shifts the energy downhill and causes a red-shift in the final fluorescence. (b) Schematic spectra of a FRET-pairingdonor and acceptor molecule. Emission spectrum of the donormolecule (EmD) overlaps (gray area) with the exitation spectrum ofthe acceptor molecule (ExA) for FRET. Excitation wavelength λExDand emission wavelength λEmA of the FRET-pairing system areindicated (arrows).



system was required. In TR-FRET lanthanide ions are used asfluorophores, exploiting their extended half-lifetimes in theright environment and in combination with suitable shortlifetime acceptor molecules.28,65,66 Different vendors offerdifferent solutions for coordination of the lanthanide ions(e.g., chelating methods: lanthanide chelate excite ‘LANCE’,Perkin-Elmer, or cryptates: homogeneous time-resolved fluo-rescence ‘HTRF’, CisBio).67 The general principle for time-

resolved measurements is the same: long-lived donorfluorescence is used in combination with a delay time periodof some μs in which unspecific excited molecules will rapidlydecay their fluorescence. Subsequent read out of the acceptorsignal is possible and enhances the specificity of the read out(Figure 5). Ratiometric correction (read out signal/donorfluorescence intensity) for differences in donor fluorescencefrom well to well are possible for a better signal comparison.Common TR-FRET-pairs are commercially available.

Remarkably, Tb2+-cryptates (CisBio) can also be paired withgreen acceptors such as fluorescein or GFP enabling multiplexassays.68 Unlike FP assays TR-FRET assays are mass differenceindependent allowing different ways of labeling. Thus, vendorsusually provide probes for classic chemical labeling of proteinsas well as conjugated antibodies against common protein-tags(e.g., GST, His6, Myc, HA, Flag, etc.). TR-FRET assays findbroad application in various fields of biology like receptorstudies (GPCRs), kinase studies, protein−DNA interactionstudies, cell surface protein interaction as well as intracellularPPIs.68 Compared to other assays like FP or ELISA, TR-FRETusually is more cost intensive per well. Additionally, the readout instrument of choice requires the implementation of a time-resolved method. Collectively, despite somewhat higher costs,the minimized false positive rate and the good statistical

parameters of TR-FRET assays are accountable for the rise inusage of these types of assays.In the field of PPI modulation TR-FRET systems play a role

at all stages of drug development, ranging from primary assaysfor HTS, secondary assays and IC50 determinations. The iPPI-DB quotes 20% of their content as derived from TR-FRETbased assays.30 An example of successfully utilized TR-FRET isthe inhibition of eph-kinase,69 where the authors used LANCEto validate and quantify their hits from an ELISA. TheTWEAK-Fn14 complex plays a role in inflammation, auto-immune diseases, and cancer and was successfully evaluated in a60 000 compound HTS using HTRF.70 Chung and Bam-borough used time-resolved measurements in a fragment basedsetup identifying bromodomain inhibitors.71,72 Despite itsslightly advanced instrumentation and costs, TR-FRET assaysdeliver many advantageous features and represent a valuabletool for PPI identification.

2.4.3. Bioluminescence Resonance Energy Transfer(BRET). In another derivative of FRET assays, the donormolecule is conjugated with an enzyme (e.g., Renilla luciferase,Rluc) that causes bioluminescence upon substrate addition(e.g., coelenterazine, coelenterazine 400a or DeepBlueC). Thehigh energetic light pulse which leads to undesired excitation ofbiomolecules and small chemical compounds at the beginningof the measurement is lacking and the acceptor molecule isexcited by the internal bioluminescence as light source.73,74

Suitable BRET-pairing systems are: Rluc-eYFP, Rluc-GFP2,Rluc8-GFP2, Firefly-DsRed, and Rluc/Rluc8-QDOT.75 BRETassays are most broadly applied to the analyses of membranesignaling such as GPCRs,76,77 whereas only few examples existcurrently for the successful usage of BRET in PPI modulation:Hamdan and colleagues showed a β-arrestin recruitment to thechemokine receptor CCR5 using a cellular BRET screening of26 000 compounds.78 Further examples are the CDK5−p25interaction which is of crucial relevance to neurodegenerativediseases as well as the Bax-Bcl-x(L) interaction which isimportant for the regulation of apoptosis.79,80

2.4.4. Amplified Luminescent Proximity Homogene-ous Assay Screen (ALPHA Screen). ALPHAScreen(amplified luminescent proximity homogeneous assay Screen,Perkin-Elmer) represents a remarkable advancement in the fieldof proximity based assays. It is a bead based proximity assay thathas its origins in diagnostic assay technology termed LOCI(luminescent oxygen channeling immunoassay). The proximityprinciple is similar to FRET based systems, as the interaction oftwo labeled proteins result in the transfer of a signal from adonor to an acceptor, which generates a read out signal (Figure6). The molecular details, however, are strikingly different:ALPHAScreen is a bead based assay, binding partners need tobe tagged (e.g., via epitope recognition, GST, His6, HA, Flag,etc.).In ALPHAScreen, upon excitation of the donor bead with

low energy red-shifted light (680 nm), the photosensitizingphthalocyanines release electronically excited singlet oxygenmolecules that can diffuse in solution within a radius of 200 nm(compared to 1−10 nm with FRET) limited by their halflifetime. The acceptor beads contain three dyes, namelythioxene, anthracene, and rubrene. Once the excited oxygenmolecule interacts with the thioxenes energy is released, whichis subsequently transferred to the cascade of anthracene andrubrene and finally results in the emission of light at 520−620nm.81 As one donor bead is able to release up to 60 000 singletoxygen molecules the signal of one biological molecule bound

Figure 5. Assay principle of TR-FRET. (a) After donor excitation(blue arrow, λExD), PPI-dependent FRET occurs and red-shiftedfluorescent light is emitted (red arrow, λEmA). The maximum distancefor the resonance energy transfer is indicated (10−100 Å). Upontreatment with an appropriate inhibitor the FRET signal decreases. (b)Diagram indicates the long lifetime of caged lanthanide-ionfluorophores such as Terbium or Europium after excitation overtime. These fluorophores are used in time-resolved measurements(gray area) in which the plate reader starts collecting photons after ashort delay time to let the intrinsic fluorescence of matrix andcompounds decay.68



to this bead is highly amplified, resulting in advantageoussensitivity with a remarkable dynamic range for this assay.82

Additionally, the energy of the photosensitizing reagentphthalocyanine results in a wavelength shift from low, duringexcitation, to higher energy during read out, resulting in verylow background signal.The chemical probes needed for ALPHAScreen (Perkin-

Elmer) are rather cost intensive and the plate reader of choicehas to be ALPHAScreen enabled. Additionally, as the chemicalreaction on the donor beads is excitable in the low energyrange, undesired irradiation such as sunlight has to beminimized while preparing the measurement, complicatingthe handling. However, the assay statistics are outstanding, thebeads do not interfere with automated liquid handling and theassay is titratable. Therefore, the assay is used for a broadspectrum of biological questions especially in PPI modulationinvolving primary screening, secondary validation as well asIC50 determination.81,83 Up to now just 1.6% of the iPPI-DBdepict ALPHAScreen as their utilized assay, but the numbersare rising and remarkable examples have utilized ALPHAScreenso far: Examples published very recently involve the inhibitionof Checkpoint Kinase 2 in a fragment based setup,84 Skp2−Cks1 anticancer target,85 the AF4−AF9 interaction as a noveltarget for MLL-R leukemia86 or the HIV-1−LEDGF inter-action87,88 were studied in HTS or in combination withprevious computational bioinformatics filters. Also, the PDE δinhibitor impairing oncogenic KRAS signaling was foundduring a HTS using the ALPHAScreen technology.89

3. APPROACHES FOR HIT IDENTIFICATION OF PPIMODULATORS

PPIs are difficult, unconventional drug targets due to theirunique molecular topologies.90 They operate via shallowextended solvent-exposed surfaces, compared to the drugpockets of conventional protein targets, such as enzyme activesites and transcription factors, which tend to be deeper and lessaccessible to bulk solvent. Consequently, the traditional

medicinal chemistry methods which have been successful ataddressing classical drug targets, have so far been less effectivefor PPIs. There is thus an urgent demand for innovativeapproaches to small molecule modulators designed specificallywith the topologies of PPIs in mind. For this purpose, the fieldof synthetic organic chemistry is an unending source of newsynthetic methodology and complex molecular architectures,which is proving highly beneficial for innovation in chemicalbiology91 and PPI drug discovery.92,93

This section has been subdivided into four inter-relatedtopics − namely, structure-based design (section 3.1), naturalproduct-inspired PPI modulation (section 3.2), supramolecular-induced PPI modulation (section 3.3) and compound librarygeneration (section 3.4). Section 3.1 describes methods whichuse the peptide binding epitope of PPIs as a lead structure,including a discussion of hotspot residue theory, conforma-tionally constrained peptides and miniproteins, peptidomi-metics, and mimetics of protein secondary structure. In section3.2, the discussion moves to chemical methods inspired by theguiding principles of biologically active natural products andways to diversify natural products, including semisynthesis,diverted total synthesis, mutagenesis, and biology-orientedsynthesis (BIOS). Section 3.3 introduces the exciting field ofsupramolecular-induced PPI modulation, in which smallsynthetic host molecules are designed to bind amino acidresidues site-selectively at the target protein. Finally, section 3.4discusses some of the powerful biological and chemicalapproaches to screening large compound libraries, includingbiological display techniques, microarrays, on-bead approaches,fragment-based drug discovery (FBDD), in silico screening andmulticomponent reactions (MCRs). In line with the rest of thereview, section 3 will emphasize recent work towards thedevelopment of PPI inhibitors, with priority given to workpublished from 2008 onwards. For an exclusive discussion ofsmall molecule PPI stabilization, please refer to section 4.

3.1. Structure-Based Design

The binding epitope of either one of the two protein partnershas been a widely exploited starting point for the developmentof small molecule PPI inhibitors, in which a considerableemphasis has been placed on structure-based design and theguiding principle of hotspot residue theory.

3.1.1. Hotspot Residue Theory. Alanine scanning muta-genesis studies have concluded that PPI formation ispredominantly driven by the need to maximize hydrophobicinteractions between amino acid residues at the PPI interface(although electrostatic interactions do sometimes dominate, forinstance in the case of phosphorylated peptide motifs), and thatthe free energy of binding is often focused at only a few keyamino acid residues, termed hotspot residues.94 Hotspotresidue theory has been ground-breaking for PPI drugdiscovery, as it implies that targeting hotspot residues withsmall molecules is an energetically feasible way to inhibitPPIs.15,95,96 Recent developments in this direction include newcomputational tools to better predict and characterize hotspotresidues, in particular for PPI targets lacking structuralinformation and for which alanine scanning mutagenesis isnot an option. For example, cosolvent molecular dynamics(MD) simulations have recently been used to investigateprotein−protein interacting surfaces.97,98 An alignment-freecomputational method termed “iPred” has recently beenreported, which predicts PPIs and the location of hotspotresidues.99 Large-scale screening and meta-analysis of all

Figure 6. Schematic overview of the ALPHAScreen principle. PPI thatbrings donor and acceptor beads in close proximity (minimally 200nm) results in an ALPHAScreen signal (upper panel). Low energy redlight (680 nm) excites photosensitizing phthalocyanines on the donorbead which release singlet oxygen. This results in an orchestratedexcitation−emission cascade of three dyes on the acceptor bead if thisis in close proximity. Rubrene finally emits higher energy light at 520−620 nm. Upon inhibitor administration the signal is decreased, becausethe distance of the two beads has increased beyond the diffusionthreshold of singlet oxygen.



protein−protein complexes in the PDB and MolecularModeling Database (MMDB) has recently been performed toidentify potential small-molecule “multibinding sites”, whichoverlap with the protein−protein interface.100 The existence of“stability patches”, which sit in the vicinity of hotspot residueshas also been postulated. Stability patches are well-definedclusters of highly immobile amino acids on the surface of theinteracting proteins, which are believed to contribute to thehigh binding energy at protein interfaces. Evidence for theexistence of stability patches has been put forward in studies onthree different classes of PPIInterleukin 2 (IL2)-IL2-receptor(R) complex, mouse double minute protein 2 (MDM2)-p53,proliferating cell nuclear antigen (PNCA)-(Flap endonuclease

1) Fen-1 PPIsusing steered molecular dynamics (SMD)simulations.101

A computational fragment-based mapping approach, com-bined with alanine scanning mutagenesis studies, was used toidentify the hotspot residues at the binding interface of nuclearfactor kappa B (NF-κB) essential modulator (NEMO)-IKKβPII − an important PPI for NF-κB signaling pathway (Figure7).102 The energetic contribution of individual amino acids wasquantified and a distinction made between “pocket-forming”

and “pocket-occupying” hotspot residues. The authors not onlyconfirm that the most energetic hotspot residues areconcentrated in a region of IKKβ previously known to bindat the NEMO surfaceW739, W741, and L742but that thisdruggable region extends to include residues L737 and F734(Figure 7). Significantly, two previously unidentified hotspotregions on the IKK β surface, specifically residues L708/V709and L719/I723, were also identified as potentially druggablepockets (Figure 7).

In addition to the amino acid side-chain residues, the amidebackbone also contributes significant energy to the binding ofprotein partners. Recently, the role of hydrogen bondingbetween the amide backbone of the PPI binding epitopes ofpostsynaptic density 95 (PSD-95)/discs large/zonula occludens1 (PDZ) domains was investigated using systematic syntheticamide-to-ester mutagenesis (Figure 8).103 For this work, fourPDZ-peptide interactions were studies: the PDZ2 of SAP97,protein tyrosine phosphate-BL and the PDZ3 of PSD-95 andPTP-BL (Figure 8a). The synthesis of a focused library ofdepsideptides was performed (Figure 8b), corresponding toamide-to-ester mutants of short PDZ binding peptides derived

Figure 7. Mapping of the hotspot residues at the NEMO-IKKβinterface using FTMap. (a) An overlay of individual amino acid sidechain groups with consensus cluster sites, which indicate bindinghotspots on the NEMO protein surface, with a zoomed-in view of theidentified hot-spot regions (NEMO protein removed for clarity). (b)Results from focused mapping of the same three hot-spot regions(colored blue, magenta, and yellow) within the search volumes(transparent gray) in the presence of the NEMO protein.102 Reprintedwith permission from ref 102. Copyright 2013 American ChemicalSociety.

Figure 8. Synthetic amide-to-ester mutation of PDZ domain-interacting peptides derived from naturally occurring proteininteracting partners. (a) X-ray cocrystal structure of a peptide boundto PSD-95 PDZ3. The backbone peptide−protein hydrogen bondsunder investigation are indicated by gray and black dotted lines.Individual sites for hydrogen bond mutation are labeled (0, −1), (−1,−2), (−2, −3), and (−3, −4), from C-to-N terminus. (b) An outlineof the synthesis of a dansyl-labeled amide-ester mutant peptide. Estermutations were introduced though the coupling of α-hydroxy acids tothe amide chain.103 Reprinted with permission from ref 103. Copyright2013 American Chemical Society.



from naturally occurring protein partners. A dansyl group wasattached to the N-terminus to enable binding studies by a FPassay, and to measure binding kinetics by stopped-flowfluorimetry. The ester mutants bound with lower affinity thanthe amide analogs, though it is not yet clear to what extent thedrop in affinity is influenced by changes in peptideconformation induced by the amide-to-ester mutation.While still in the early stages of evolution, hotspot residue

theory in its present form has provided important guidelines forthe design of PPI inhibitors, which, as the forthcomingsubsections will demonstrate, have delivered some importantdesigned molecules targeting a range of different PPI targets.3.1.2. Conformationally Constrained Peptides and

Miniproteins. Knowing that PPIs are mediated by constitutingpeptide sequences, the use of inhibitory peptides directlyderived from binding epitopes of participating proteins wasenvisioned. However, inhibitory effects of such peptides highlydepend on their structural characteristics both in solution andbound to the target protein.104 Generally, isolated peptidesadopt a defined three-dimensional structure upon binding to aprotein target contrasting with their flexible nature in theunbound state. This restriction in conformational freedomupon binding leads to an entropic penalty affecting the affinityfor the target. Consequently, peptide modifications and non-natural amino acids were developed that restrict the conforma-tional freedom and induce a particular bioactive structure. Inaddition to increased target affinity, the constraint of conforma-tional freedom was reported to support proteolytic stability,selectivity and in some cases cellular uptake of bioactivepeptides.105 Different secondary structure motifs have beenstabilized such as loops, β-sheets, and helices.106 Thissubsection focuses on recent reports of PPI inhibitors (forearlier examples see previous reviews)107,108 and does not give ageneral overview of conformationally constrained peptides. Thepeptide-derived inhibitors are grouped according to the type ofstabilized secondary structure, starting with loops in cyclicpeptides followed by β-sheets and helices. Finally, switchablesecondary structure motifs are described. Often, more than onestabilization approach was utilized for the inhibition of aparticular protein−protein interaction. To avoid redundancy,the figures in this subsection summarize different stabilizationapproaches for a given target protein or protein family: MDM2,BCL-2 family proteins, β-catenin, and estrogen receptors.3.1.2.1. Cyclic Peptides. The term cyclic peptide refers to

relatively short macrocyclic peptides that adopt looplikestructures (in contrast to cross-links which stabilize β-sheetsor helices). A variety of macrocyclization strategies have beendeveloped either directly connecting the termini of a peptide orinvolving a cross-linking of side chains.109 Only a subset ofthese cyclization strategies has been used to generate inhibitorsof PPIs. A very early example pointing toward an increasedbioactivity of cylic peptides was reported by Ruoslahti and co-workers.110 Using phage display they identified a disulfidecross-linked peptide that acts as α5β1 integrin ligand andshowed 10-fold higher affinity that its linear analog. Recently,Tavassoli and co-workers employed a bacterial reverse two-hybrid system111 in combination with the split intein circularligation of peptides.112 They identified various cyclicpeptides113−115 targeting protein complexes such as Gag−TSG101113 or HIF-1α−HIF-1β.114 An important stage in thelife cycle of HIV is the release of viral particles from the hostcell. This step requires the virally encoded Gag protein tointeract with host protein TSG101. The authors report the

selection of the cyclic peptide cyclo-IYWNVSGW from apeptide library containing about 3.2 million members.113 Aftertagging the peptide with a cell permeable Tat sequence,inhibition of virus-like particle secretion was observed in cellculture. As another example, the heterodimeric transcriptionfactor hypoxia inducible factor-1 (HIF-1) was targeted. HIF-1regulates the cellular response to reduced oxygen levels playingan important role in tumor survival and progression.Consequently, inhibition of HIF-1 dimerization is consideredan attractive therapeutic approach. The bacterial reverse two-hybrid system was used to screen for HIF-1α−HIF-1βdimerization inhibitors.114 The selected peptide cylco-CLLFVYwas again linked to the Tat sequence and showed inhibitoryeffects on HIF-1 dimerization in cell culture when used at lowmicromolar concentrations. Grossmann, Ottmann and co-workers reported a macrocyclization strategy that uses ring-closing olefin metathesis to conformationally constrainirregularly structured peptides.116 In this approach, a bindingepitope of the virulence factor exoenzyme S was stabilized byreplacing two lipophilic side chains with a hydrophobic cross-link. The resulting cyclic peptides proved efficient in inhibitingthe interaction between a domain of exoenzyme S and thehuman target protein 14-3-3 in vitro. Another efficientapproach that allows the generation of cyclic peptide librariesand their selection for protein binders is the phage-displaybased identification of bicyclic peptide ligands which hashowever not been used to select for PPI inhibitors so far.117

This technique provides access to large libraries of conforma-tionally constrained peptides and can be expected to yieldpotent PPI inhibitors in the future.

3.1.2.2. Stabilized β-Sheets. β-Sheets are composed of atleast two β-strand sequences that are aligned in a parallel orantiparallel fashion. Artificially stabilized β-sheets generallyconsist of a single amino acid sequence with a central β-hairpinmimetic that aligns the flanking sequences thereby nucleatingthe formation of an antiparallel β-sheet.118 Such hairpin-likestructures can be further stabilized by a cross-linking of C- andN-terminus, using an approach developed by scientists atPolyphor termed peptide epitope mimetics (PEM)118 and byadditional intrastrand bridges.119

The interaction between tumor suppressor p53 and itsnegative regulator proteins MDM2 or MDMX is a well-studiedtarget in anticancer therapy and a commonly used modelsystem for the evaluation of novel approaches for PPIinhibition. This interaction is mediated by a short α-helicalpeptide sequence of p53 binding to the globular domains ofMDM2 or MDMX. Within the p53 helix phenylalanine (F19),tryptophan (W23), and leucine (L26) align along one face andwere identified as hot spots for MDM2 binding (Figure 9a).120

Interestingly, phage display selections against MDM2 and/orMDMX tend to yield helical peptides.121,123 For example, Luand co-workers performed a phage display with the syntheticD-enantiomer of MDM2 resulting in the selection of a peptidesequence that binds the protein in a helical conformation.Figure 9b shows the crystal structure of MDM2 in complexwith the corresponding left handed helical D-peptide.121

Nonetheless, Robinson and co-workers were able to design acyclic antiparallel β-sheet utilizing an L-/D-proline dimer ashairpin mimetic and incorporated the three hot spot residues atappropriate positions along one β-strand.118 The first macro-cycle showed only weak inhibitory activity and was evolved intopeptide 78A (Figure 9c) exhibiting submicromolar affinities forMDM2.118



3.1.2.3. Stabilized α-Helices. α-Helices participate in manyPPIs and can serve as starting point for the design of PPIinhibitors.124 A number of approaches utilize the helical bindingepitope itself introducing moieties that stabilize the secondarystructure.105,108 Early examples for helix stabilizing elementsinvolve the cross-linking of side chains,125,126 the use of helixinducing natural127 and α-methylated amino acids,128,129 andthe installation of N-terminal modifications (N-caps) capable ofnucleating an α-helix by the formation of intramolecularhydrogen bonds.130 Recently, metal ion coordination,131,132

supramolecular interactions133 and multimer assembly134 wereused to stabilize the α-helical conformation of peptides. In thecontext of PPI inhibition three major stabilization approacheswere developed (Figure 10): One utilizes the covalent cross-linking of amino acids located along one face of an helix withlactam125 and disulfide bridges126 being the most frequently

used examples. In another strategy the side chain macro-cyclization of all-hydrocarbon α-methylated building blocks isused to enforce an α-helical conformation (stapled pep-tides).135 Finally, a particular N-cap, the so-called hydrogenbond surrogate (HBS), was reported to support nucleation ofα-helices therefore increasing their conformational stability(Figure 10).136 In this subsection examples for stabilized α-helical interaction motifs are grouped based on the nature ofthe utilized stabilizing scaffold.

Cross-Linked Side Chains. Covalent side chain tethers havebeen widely used for the stabilization of α-helical structures. Afirst proof of concept has been provided by the cross-linking ofnatural amino acids.105,108 Early examples involve the tetheringof lysine and glutamate by lactam formation and disulfidebridges between two cysteine residues.126 Fairlie and co-workers showed that the simultaneous use of two lactam tethersprovides a general approach for the stabilization of interfacial α-helical peptides.137 Disulfide bridges were evolved intothioether linkages to avoid reductive cleavage associated witha use of disulfides in biological systems.138 An alternativeapproach for the cross-linking of amino acid side chains wasreported by Lin and co-workers, who use bisarylmethylenebromide for the covalent linkage of two cysteine residues atposition i and i + 7, respectively (Figure 11a).139 This approachwas utilized to stabilize an α-helical peptide known to competewith p53 for the binding of MDMX and MDM2 (Figure 9a).Compared to the unmodified sequence, the covalently cross-linked peptide showed modest increases in helicity andbioactivity and most importantly an enhancement in cellpermeability. The cellular uptake of this inhibitory peptide was

Figure 9. Tumor suppressor p53 is negatively regulated by MDM2.p53 inhibition occurs via multiple mechanisms, including directbinding and thereby masking the p53 transactivation domain. Crystalstructures of MDM2 (gray) in complex with different peptides areshown: (a) MDM2/MDMX binding sequence of p53 (blue, PDB1ycr);120 (b) Left-handed helix DPMI-α (green, PDB 3lnj),121 (c)Cyclic antiparallel β-sheet 78A (orange, PDB 2axi),118 and (d) stapledpeptide SAH-p53−8 (purple, PDB 3v3b),122 Panels b−d showsuperimposed helix of p53 in transparent blue (selected interactingresidues are highlighted gray in the sequence and are shown explicitlyin structures).

Figure 10. Schematic drawing of different approaches for the covalentstabilization of α-helixes with relative spacing between cross-linkedamino acids (i,i+n).



further improved by conjugation of spermine to the C-terminus.140 Later, the same group reported the use ofbisphenylmethylene bromide for a stabilization of the NOXA-derived α-helical peptide targeting the BCL-2 family proteinMCL-1.141 In this study, D-cysteine is introduced at position iand the natural L-enantiomer at position i + 7. The crystalstructure of the resulting peptide inhibitor Bph-NOXA-2 withMCL-1 verifies the α-helical conformation of the peptide in thebound state (Figure 11a). To achieve activity in cell-culture, thepeptide sequence was further optimized by replacing polarresidues with alanine thereby increasing the overall hydro-phobic character of the peptide which supported its tendencyto cross cell membranes and reach its intracellular target.141

Focusing on cysteine residues at positions i and i + 4,Greenbaum and co-workers describe a screening of 24 differentcross-linkers regarding their ability to stabilize an α-helicalpeptide.142 This study indicates that structurally rigid linkersappear to provide the strongest stabilizing effect, with dibromo-m-xylene representing the most suitable cross-linking agent.Using glutamate side chains as chelate ligands, Ball and co-workers report p53-derived α-helical dirhodium metallopep-tides that bind MDM2.131 This approach represents an

alternative transition metal mediated cross-linking of aminoacid side chains.

Cross-Linked α-Methylated Amino Acids (Stapled Pep-tides). Verdine and co-workers have introduced the hydro-carbon stapling of α-helical peptides.135,145 This strategycombines two features previously employed to stabilize the α-helical structure of peptides: (I) the use of α-methylated aminoacids128 and (II) the formation of olefin containing cross-linksvia ring-closing olefin metathesis introduced by Blackwell andGrubbs.146 So far, two cross-link architectures proved mostuseful for the generation of stapled PPI inhibitors: an eight-carbon tether with modified amino acids at positions i and i + 4,and an 11-carbon bridge with i,i + 7 spacing (Figure 10). In aground-breaking study, the peptide stapling approach was usedto develop an inhibitor that targets Bcl-2 protein familymembers and showed efficacy in a mouse model of Bcl-2 drivenlymphoma.147 Bcl-2 family proteins represent key regulators ofapoptosis and control the cellular survival via a complexnetwork of interactions between pro- and antiapoptotic familymembers. Antiapoptotic BH (Bcl-2 homology) domainproteins (e.g., Bcl-2 and Mcl-1) promote cellular survival bytrapping critical pro-apoptotic BH3 proteins (e.g., BID, BAD,and BIM) thereby inhibiting activation of pro-apoptotic multiBH domain proteins (e.g., BAX and BAK). Using different BH3helices as starting point (BID,147,148 BAD,148,149 BIM,150 andMcl-1143) a variety of “stabilized alpha-helices of Bcl-2domains” (SAHBs) was designed by Walensky and co-workers.SAHB peptides proved to be helical, protease resistant and cell-permeable. However, not every stapled BH3 helix exhibitsimproved bioactivity143,151 which requires the synthesis andtesting of a set of modified peptides to identify suitablecandidates.143 For example, the generation of a potent Mcl-1inhibitor involved the synthesis of about 20 different SAHBpeptides finally yielding Mcl-1 SAHBD as the most activecompound.143 The crystal structure of Mcl-1 SAHBD incomplex with Mcl-1 (Figure 11b) reveals an α-helicalconformation of the SAHB peptide and a direct involvementof the hydrocarbon cross-link in target binding.143

Stapled peptides were also used to generate inhibitors of theinteraction between tumor suppressor p53 and its negativeregulators MDMX and MDM2 (Figure 9a). The p53 derived i,i+ 7 stapled peptide SAH-p53−8,152 developed by Walenskyand co-workers, proved to have nanomolar affinities for bothMDMX and MDM2 with a preference for MDMX.153 A crystalstructure of SAH-p53−8 in complex with MDM2 (Figure 9d)verifies the α-helical conformation and reveals an intimateinteraction between staple and target protein providing anexplanation for the high stability of this peptide−proteincomplex.122 Further improved stapled peptides were derivedfrom phage display selections154 and structure based sequenceoptimization aiming for the development of dual specificMDM2-MDMX inhibitors.155 In a xenograft cancer model thedual specific inhibitor ATSP-7041 was effective in thesuppression of p53-dependent tumor growth verifying thepotential of stapled peptides as therapeutically relevantinhibitors of PPIs.Estrogen receptors (ERs) belong to a class of transcription

factors that can be activated by the binding of estrogen orpeptide coactivators (Figure 12a). These activating peptideligands can adopt an α-helical structure thereby presentingthree conserved leucine residues along one face of the helix.This hydrophobic patch is recognized by a groove on thesurface of ERs. The α-helical coactivator ligands served as

Figure 11. BCL-2 family proteins are key regulators in apoptosis.MCLl-1 and BcL-x(L) are antiapoptotic Bcl-2 family members thatbind to α-helixes of antiapoptotic BH3 (Bcl-2 homology domain 3)proteins thereby inhibiting their apoptosis-inducing effect. Aninhibition of these PPIs is considered an attractive anticancer strategy.Structures show antiapoptotic proteins (gray) bound to stabilized BH3helices. Crystal structure of Mcl-1 (gray) with (a) bisaryl-bridged Bph-NOXA-2 (blue, PDB 4g35)141 and (b) stapled Mcl-1 SAHBD (orange,PDB 3mk8).143 (c) NMR-derived solution structure of Bcl-x(L) incomplex with photoswitchable BAKI81F

i,i+11 (green, PDB 2lp8).144

Selected interacting residues are highlighted gray (in sequence) andshown explicitly (in structures).



model system for the evaluation of various stabilizing scaffolds.Early examples involve the use of lactam156,158 and disulfidetethers (PERM-1, Figure 12b).156 Phillips et al. describe the useof i,i+4 staples for the design of stabilized ER ligands and reportthe crystal structure of two stapled peptides (SP1 and SP2)bound to ER (Figure 12c and 12d).157 These structures showdirect interactions between the staple and surface residues ofER. In the case of SP1 (Figure 12c), these interactions induce arotation of the helix thereby shifting the register by one aminoacid relative to the starting sequence (Figure 12a). Thisdramatic rearrangement indicates a significant contribution ofthe hydrocarbon staple to the binding affinity.The inhibition of certain transcriptional activator complexes

represents an appealing strategy for the inactivation of signalingpathways that appear essential for the survival of cancer cells.Due to the involvement of numerous PPIs, a targeting of thesecomplexes proved to be particularly challenging.159 Notably,the stapling of α-helical interaction motifs provided inhibitorsof the Notch and Wnt signaling pathway, respectively. Theheterotrimeric NOTCH1 transcription factor complex wasdirectly inhibited by the stapled peptide SAHM1 that triggersreduced expression of NOTCH1 target genes in cell culture

and shows antiproliferative effects in a mouse model ofNOTCH1-dependent lymphoblastic leukemia.160 The activa-tion of Wnt signaling depends on the formation of a complexbetween coactivator protein β-catenin (gray) and transcriptionfactors of the LEF/TCF family such as TCF4 (blue, Figure 13).Verdine and co-workers report the stapling of the α-helical β-catenin binding domain (CBD) of Axin sharing a binding sitewith TCF4 and the subsequent affinity optimization of thispeptide using phage display.161 Cell penetration and local-ization properties of β-catenin targeting peptides are crucial forthe desired biological activity.161,162 Therefore, an introductionof arginine residues, known to promote cellular uptake, wasperformed. The resulting active peptides StAx-35 and −35Rcompete with TCF4 for β-catenin binding and selectivelyreduce the expression of Wnt target genes. A crystal structure ofStAx-35 bound to β-catenin (Figure 13) verified occupancy ofthe expected binding site163 overlaying with the one ofTCF4.161 Walensky and co-workers used the CBD of BCL9(green, Figure 13) as starting point for the design of stapledpeptide SAH-BCL9B.

164 The same sequence was previouslystabilized by triazole containing cross-links yielding peptides

Figure 12. (a) Estrogen receptor (ER) α (gray) bound to coactivatorpeptide NRCA (blue, PDB 2qgt). (b) ERα (gray) in complex withdisulfide cross-linked PERM-1 (green, PDB 1pcg).156 (c) ERβ (gray)bound to stapled SP1 (orange, PDB 2yjd).157 (d) ERα (gray) incomplex with stapled SP2 (purple, PDB 2yja).157 (b−d) Super-imposed helix of NRCA is show in transparent blue (selectedinteracting residues are highlighted gray in the sequences and areshown explicitly in structures).

Figure 13. β-Catenin is a central hub in the Wnt signaling pathway. Abinding of β-catenin to transcription factors of the LEF/TCF familysuch as TCF4 is a prerequisite for the activation of canonical Wntsignaling. For expression of a subset of Wnt target genes a binding ofBCL9 is necessary. Middle: Crystal structure of β-catenin (gray)bound to the β-catenin binding domains (CBDs) of TCF4 (blue) andBCL9 (green, PDB ID: 2gl7),166 Top: crystal structure of the StAx-35−β-catenin complex (PDB ID: 4djs) with the sequence of StAx-35and −35R, respectively.161 Bottom: sequence of stapled peptide SAH-BCL9B

164 (selected interacting residues are highlighted gray insequences and shown explicitly in structures).



with increased affinity for β-catenin.165 SAH-BCL9B competeswith β-catenin−BCL9 dimerization in vitro and induces thereduction of Wnt reporter activity in cell culture. In additionthe authors report the peptide to inhibit proliferation andcellular migration of tumor cells.164 Waldmann, Grossmann andco-workers reported the generation of stapled peptides capableof binding Rab proteins,.167 Rab proteins are master regulatorsof intracellular vesicular transport and trafficking and belong tothe family Ras-related small GTPases that proove to beextremely difficult targets. In this study, the authors use astapled peptide to inhibit the interaction between a Rab-proteinand a corresponding effector protein in vitro.Another set of PPIs targeted by stapled peptides aims for the

inhibition of HIV. Debnath and co-workers report theinhibition of viral capsid formation using a stapled version ofa peptide sequence (CAI) that was earlier shown to inhibit theviral Gap poly protein essentially involved in this process.168

Later, Walensky and co-workers used a double staplingapproach for the stabilization of the gp41 peptide, a knownHIV-1 fusion inhibitor.169 Remarkably, stapled peptide SAH-gp41(626−662) exhibits increased protease resistance and oralavailability resulting in improved antiviral activity.169

Hydrogen Bond Surrogates (HBS). N-terminal caps arestabilizing scaffolds that do not require a modification of aminoacid side chains.130 Their helix inducing effect originates fromthe formation of hydrogen bonds at the N-terminus of apeptide thereby nucleating helix formation.170,171 Hydrogenbond surrogates (HBS) replace the very first hydrogen bond inan α-helical peptide by a covalent linkage, thereby representing

a special type of N-caps. An early HBS example was reported byCabezas and Satterthwait utilizing a hydrazone bridge.172 Later,Arora and co-workers designed all hydrocarbon surrogates thatwere cyclized by ring-closing olefin metathesis.136 HBSstabilized peptides proved useful for the generation of variouspeptide-derived PPI inhibitors.172,173 Arora and co-workersreported the stabilization of a gp41 peptide capable ofinhibiting the gp41 mediated cell fusion.173 In addition, anHBS stabilized p53 helix proved useful for the inhibition of thep53−MDM2 interaction.174 Arora and co-workers generatedhypoxia-inducible factor 1α (HIF-1α) derived HBS stabilizedpeptides that inhibit the interaction between HIF-1α and basaltranscriptional coactivator p300/CBP.175 The HBS helix bindsp300/CBP thereby inhibiting the expression of hypoxia-inducible genes leading to suppressed tumor growth in amurine xenograft model of renal cell carcinoma.176 Finally, thesame lab reported the design of an orthosteric inhibitor of theinteraction between the small GTPase Ras and its nucleotideexchange factor Son Of Sevenless (SOS).177 Using an SOS-derived α-helix it was possible to generate HBS stabilizedpeptides capable of inhibiting the nucleotide exchange in vitroand in cell culture. Taking into consideration that smallGTPases are considered as particularly challenging intracellulartargets, these results highlight the potential of the HBSapproach for the stabilization of α-helical peptides and their useas PPI inhibitors.

Miniproteins. A viable alternative to short cross-linkedpeptides is to graft the biologically active peptide epitope or hotspot residues to naturally occurring miniature (mini)proteins.

Figure 14. aPP-derived miniprotein library targeting the p53-MDM2 PPI (grafted p53 residues underlined and highlighted in bold).178 Adapted withpermission from ref 178. Copyright 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 15. (a) Structural comparison of the α-helical domain of BmBKTx1 and the helical segment of p53.180 Adapted with permission from ref 180.Copyright 2008 American Chemical Society. (b) Overlay of the X-ray cocrystal structures of Stingin 1 (green) and phage peptide, PMI (pink),bound to MDM2.189 Reprinted with permission from ref 189. Copyright 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.



Miniproteins are promising drug candidates due to their stable,well-defined secondary structures,178−183 synthetic tractability,and cell permeability.184,185 Miniprotein grafting has beenextensively used for directed protein targeting, for example toion channels.186,187 Early work on miniprotein graftingtargeting PPIs has been reported for p53-MDM2.188

Schepartz and co-workers developed inhibitors of the p53-MDM2 PPI using the miniprotein avian pancreatic polypeptide(aPP, 1) (Figure 14).178 In contrast to other disulfide-richminiproteins (see below), the tertiary structure of aPP isstabilized via multiple hydrophobic contacts between theeighteen-residue α-helix and an eight-residue PPII helix,which are linked via a type I β-turn. Here, the hotspot residuesof p53 were grafted to the solvent exposed face of theminiprotein’s α-helix and phage display screening performed tooptimize the folding and MDM2 binding properties. The mostpromising miniproteins were synthesized by solid-phasepeptide synthesis, and labeled with 5-iodoacetamidofluoresceinto enable analysis of MDM2 affinity in an FP assay. Theseefforts resulted in potent, low-nanomolar affinity inhibitors ofthe p53-MDM2 PPI (e.g., 2). Subsequent remodeling of theminiprotein’s fold via strategic replacement of a helix breakingproline residue disfavored dimer formation, which enabled PPIstudies across a broader miniprotein concentration range.179 Asimilar approach was used by Lu et al. starting from a differentdisulfide-rich miniprotein − the potassium ion channel toxinBmBKTx1 (Figure 15a),180 which afforded a series of modifiedminiprotein-based inhibitors with submicromolar affinity toboth MDM2 and MDMX. The same group also grafted thehotspot residues of a phage peptide, PMI, to the disulfide-rich18-mer miniprotein, apamin (Figure 15b).189

The choice of starting miniprotein is an importantconsideration as it predetermines the topology of the proteininteracting surface. To highlight this point, four structurallydifferent disulfide-rich α-helical miniproteinsapamin, κ-hefutoxin1, a scyllatoxin analog, CD4M3, and Om-toxin3,each consisting of different length α-heliceswere compared asscaffold structures for the development of androgen receptor(AR)-coactivator inhibitors (3−6, Figure 16).183 AR binds tocoactivator proteins via a highly conserved helical FXXLFmotif. The phenylalanine and leucine hotspot residues weregrafted onto the different miniprotein scaffolds guided bycomputational design, and the target miniproteins synthesizedby solid phase peptide synthesis.182 Significant differences inaffinity were observed between the different miniprotein,including a 10-fold improvement in activity compared tocompetitior phage peptides. Miniprotein-derived inhibitors ofthe estrogen receptor (ER) have also been developed by usingphage display screening, starting from the apamin miniproteinscaffold.181

Cyclotides. Cyclotides are plant-derived disulfide-richminiproteins with an intriguing circular cystine knot (CCK)topology, which enhances the metabolic stability and cellpenetrating properties of these peptide sequences.190 Just as fornatural miniproteins, the tertiary structure of cyclotides

tolerates substantial sequence variation, and can thereforefunction as a scaffold structure for the grafting of biologicallyactive peptide epitopes. Li and co-workers engineered acyclotide to specifically inhibit the MDM2/MDMX/p53−PPIby grafting a nanomolar potent α-helical phage peptide, PMI(bearing conserved p53 hot spot residues Phe19, Trp23, andLeu26) into the peptide sequence of the cyclotide Momordicacochinchinensis trypsin inhibitor I (MCoTI-I, Figure 17).191

Figure 16. Miniproteins 3−6 targeting the androgen receptor (AR)-coactivator interaction.183 Adapted with permission from ref 183.Copyright 2009 Royal Society of Chemistry.

Figure 17. (a) Solution (NMR) structure of cyclotide MCo-PMI(purple) in complex with hDM2 (cyan blue) represented in ribbonformat. b) Backbone superposition of the cocrystal structures of PMIpeptide (green) (PDB id: 3EQS) and cyclotide (purple) in complexwith MDM2 (blue) and hDM2 (light blue), respectively. The keypocket-forming side chains represented in stick format.191 Reprintedwith permission from ref 191. Copyright 2013 American ChemicalSociety.



The peptide was inserted into the flexible loop 6 region ofMCoTI-I, between residues Ser31and Gly33, to minimize stericinterference with the cyclotide scaffold. The N-terminal regionof the apamin miniprotein was inserted between the N-terminus of the PMI peptide and the cyclotide to favor thebiologically active α-helical conformation. The linear MCoTI-derived sequence was prepared by either chemical synthesis(SPPS) or bacterial recombinant expression (modified Mxegyrase A intein and a TEV protease recognition sequence) andprotein folding induced by reduced glutathione (GSH). Theresulting cyclotides bound to MDM2 and MDMX with lownanomolar affinities, and, importantly, induced cytotoxicity inp53 wild-type human cancer cells in a p53-dependent mannerboth in vitro and in vivo.Self-Assembled Peptide Nanostructures. Self-assembling

protein-like nanostructures, which combine peptide graftingwith supramolecular chemistry, are a viable alternative to thenatural scaffold structures described previously. Recently, a p53-derived peptide targeting MDM2 was inserted into amacrocyclic peptide scaffold capable of forming self-assembledpeptide nanostructures also known as αSSPNs (Figure 18).192

The driving force for self-assembly in this case is β-sheetformation, and the self-assembled state is thought to stabilizethe helical secondary structure of the p53 peptide, thusmimicking the influence of protein folding on secondarystructure formation. The p53-grafted αSSPN inhibited bindingof a fluorescently labeled p53 peptide to MDM2 with lowmicromolar affinity, and demonstrated improved stabilitycompared to the unmodified p53 peptide.3.1.2.4. Switchable Secondary Structures. The design of

stabilizing scaffolds that allow the temporal control overpeptide secondary structures can provide access to novelapplications. So far, different stimuli were implemented

including metal sensitive193 and photo-144,194,195 or redox-switchable moieties.132,196 One example involves the photo-induced and irreversible cycloaddition of a p53-derived peptidegenerating a cell-permeable dual inhibitor of MDM2-MDMX.197 The reversible photocontrol of an α-helix wasachieved by Woolley and co-workers who installed anazobenzene based cross-linker designed to stabilize the helicalstructure in its trans conformation.194 A similar approach wasused by Allemann and co-workers who reported the photo-control of a BAK BH3 helix by installation of an azobenzenelinker.195 The solution structure of the controllable peptideBAKI81F

i,i+11 in its helical conformation was determined incomplex with the target protein Bcl-x(L) (Figure 11c) usingNMR.144 The structure clearly shows the azobenzene linker inits trans-conformation supporting the bioactive α-helicalconformation of the peptide.

3.1.3. Peptidomimetics. An important and frequentlychallenging step in structure-based design is the translation oftheoretical and structural information about protein−proteininterfaces into bona fida small molecule inhibitors. Arguably themost straightforward approach is to optimize the amino acidsequence of a peptide derived from the natural binding epitopeor a phage-derived peptide (section 3.4.1). This approach isfrequently the first validatation of PPI targetability, providesuseful structural indicators for the design of nonpeptidicinhibitors, and can result in useful biochemical and cellularprobes for chemical biology studies. Recent examples of thisinclude a structure-based design of p53 transactivation domains(TAD) mimetics,198 and inhibitors of ubiquitin E3 ligaseSCFFbx4.199

However, while the opportunities for amino acid sequencediversity are great, side-chain diversity is limited to the twentystandard natural amino acids, which places a ceiling on the lead

Figure 18. Molecular model of a p53-derived peptide bound to MDM2, and MDM2 bound to a self-assembled nanoparticle bearing p53-derivedpeptide grafted to the surface.192 Reprinted with permission from ref 192. Copyright 2013 American Chemical Society.



optimization process. Furthermore, peptide inhibitors com-posed of natural amino acids are prone to proteolyticdegradation and exhibit poor cell penetrating properties. Theuse of non-natural amino acids and other artificial modificationsdramatically increases the opportunities for structural diversity,and can be used to favor the binding properties and metabolicstability of the peptide-derived modulators.Ciulli and co-workers adopted a rational design approach to

develop peptidomimetic small molecule inhibitors of the VHL-HIF-1α PPI. Hypoxia-inducible factor 1α (HIF-1α) is atranscription factor which regulates the expression of a hostof gene products in the hypoxic state, for instance in chronicanemia associated with chronic kidney disease and cancer.Under normoxic conditions, the intracellular concentration ofHIF-1α is kept low by VHL-mediated ubiquitination. Critical tothis regulatory process is hydroxylation at position P564 ofHIF-1α by prolyl hydroxylase domain (PHD) enzymes, whichis essential for the binding of HIF-1α to the VHL complex. AsPHD inhibition strategies often suffer from off target effects,inhibition of VHL-HIF-1α is a highly attractive alternativeapproach to restore the transcriptional activity of HIF-1α.200,201

For this work, hydroxy-proline (Hyp) was used as the chemicalstarting point (the minimal recognition unit), considering itsimportance for HIF-1α binding to VHL, and de novo designsoftware used to guide the design of Hyp analogs. Acompetitive FP assay was used to evaluate the compoundsability to bind to the VHL complex, and verified usingWaterLOGSY NMR spectroscopy. A structure-guided medic-inal chemistry approach was adopted, in which analogs weresynthesized using an efficient solid-phase synthesis strategy inan effort to improve on the binding affinity. Finally, X-raycocrystallographic data verified that the small molecule indeedbinds to VHL at the HIF-1α binding site. The active compoundfrom the initial studies was 7 (Figure 19), with an IC50 = 117 ±10 μM. After structure-guided medicinal chemistry optimiza-tion, the activity of the compound was initially increased to 4.1± 0.4 μM (compound 8, Figure 19). More extensive structure−activity relationship (SAR) studies were performed to expandon the promising initial activity, culminating in the firstsubmicromolar inhibitor of the VHL-HIF-1α interaction (9,Figure 19), with a binding mode distinct from that ofcompound 8.4 This work is an important example of thestrength of using rational design instead of a fragment-baseddrug discovery approach (section 3.4.4).202

Wang and co-workers recently developed peptidomimeticinhibitors of the MLL1-WDR5 interaction.203 Mixed lineageleukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4)methyltransferase (HMT), responsible for the mono-, di-, andtrimethylation of histones. The H3K4 HMT activity of MLL1 isregulated by the complex between MLL1, WDR5, RbBP5, andASH2L. Disruption of the MLL1-WDR5 PPI through pointmutations at WDR5 caused complex dissociation and a loss ofMLL1 H3K4 HMT activity. This makes small moleculeinhibition of the MLL1-WDR5 PPI a highly promising strategyfor the treatment of certain forms of leukemia. In previousstudies the Ala-Arg-Ala motif of MLL1 corresponding toresidues 3764−3766 was shown to be the minimum bindingepitope, and tripeptide Ac-ARA-NH2 identified as a potentbinder of WDR5 (Ki = 120 nM). The authors systematically

evaluated the binding of Ac-ARA-NH2 at the five subpockets ofthe WDR5 binding site focusing on the N and C-terminus aswell as the three amino acid residues, through a combination ofnatural and unnatural modifications. For peptide generation, asolid-phase peptide synthesis-based approach was employedand analogs were compared initially by FP. An importantoutcome was a more than 100-fold gain in binding affinity(Figure 20). The cocrystal structure was solved for compoundsMM-101 and MM-102. Both compounds bind to the centralchannel of the WD40 propeller, precisely where the MLL1peptide binds. The increase in activity compared to the MLL1WIN peptide could be explained by additional hydrophobic

Figure 19. Development of peptiodmimetic inhibitors (7−9) of the VHL-HIF-1α interaction using a rational design approach by Ciulli and co-workers.201

Figure 20. Potent inhibitors of the MLL1-WDR5 interaction reportedby Wang and colleagues.203



interactions at the WDR5 surface not made by the MLL1 WINpeptide.Importantly, MM-102 − the most promising compound of

the series − effectively reduced the expression of two MLL1targeted genes, HoxA9 and Meis-1, which are essential forMLL1 mediated leukemogenesis. Furthermore, the samecompound was effective at inhibiting cell growth and inducingapoptosis in leukemia cells bearing MLL1 fusion proteins. Thisoutcome highlights the potential of structure-based approachesfor the development of small molecule PPI inhibitors.Macrocyclic inhibitors of the menin-MLL1 PPI have alsobeen developed, starting from an octapeptide derived from themenin-binding motif of MLL1.204

3.1.3.1. Bioisostere Replacement. An important property ofdrug molecules is their cell permeability given that proteintargets are frequently intracellular. Amino acid phosphorylationis an ubiquitous post-translational modification at protein−protein interfaces, with the phosphate group contributing asignificant portion of the binding energy. However, phosphory-lated peptides are challenging lead structures given theinstability of the phosphate ester group towards phosphatasesand due to the negative charge of the phosphate ester group atphysiological pH, which prevents cellular uptake.205 Amolecularly efficient solution to these problems is bioisosterereplacement. A bioisostere is a substituent or functional groupwith similar physical or chemical properties, which elicits thesame or improved affinity/selectivity, ideally with reducedtoxicity, increased metabolic stability and improved bioavail-ability.205 There is currently considerable interest in thedevelopment of bioisostere replacements for the phosphateester group.205 A recent example of this applied to PPIinhibtion can be seen in the combined work of Yao206 andArrendale207 on small molecule inhibitors of 14-3-3-mediatedPPIs. Yao and co-workers laid the important foundations byadopting a small molecule micro array to discover the firstreported nonpeptidic inhibitor of 14-3-3 mediated PPIs.206 In asimilar way to Ciulli, Crews et al.,201 in their work on the VHL-HIF-1α PPI (Figure 19), Yao uses a phosphorylated peptidesequence, the minimal recognition motif for binding to 14-3-3(Figure 21, 10), as the chemical starting point for theconstruction of the peptidomimetic library, leading tocompound 11 as the most potent inhibitor molecule (IC50 =2.6 μM, in a competitive fluorescence polarization assay).Arrendale and colleagues used 10, and combined bioisosterereplacement with a pro-drug strategy to improve metabolicstability and increase cellular uptake (Figure 21, 12), with anIC50 of 5.0 μM against human DG75 leukemia cells.207

Ji et al. used bioisostere replacement to design inhibitors ofthe β-catenin/T-cell factor (Tcf) PPI.208 Preliminary alaninescanning and surface plasmon resonance (SPR) studiesquantified the contribution of different hotspot residues ofthe β-catenin protein surface. The side chain residues of K435/K508 and the backbone amide nitrogen of N430 and theirinteraction with the D16/E17 residues of Tcf4 (through chargeand H-bonding interactions) were discovered to be the mostimportant elements of β-catenin/Tcf binding. Importantly,though, close inspection of the cocrystal structures of β-cateninwith Tcfs, suggested further room for optimization by targetingadditional charge and H-bonding bonding elements in thevicinity of the binding interface. Virtual docking studies wereperformed and imdazole and tretrazole ring systems chosen tomimic the pKa of the carboxylic acid groups of Glu-Asp (pKa4.5−5.6), which in addition to interactions with Lys435/

Lys508/Asn430 could make favorable cation-π interactionswith R469 not seen between Tcf4 and β-catenin (Figure 22,13). While the Glu-Asp dipeptide, 14, measured a Ki = 370 μM,the inhibitor molecule, 15, was a satisfying 100-fold more activetoward inhibition of Tcf4 binding to β-catenin (Figure 22).Site-directed mutagenisis and SAR studies confirmed the modeof action, though the activity of 15 could not be improved and

Figure 21. Peptidomimetic inhibitors of the 14-3-3 protein−proteininteractions reported by Yao206 and Arrendale.207

Figure 22. Development of small molecule inhibitors by bioisosterereplacement targeting the β-catenin/T-cell factor PPI.208



the important issue of cell permeability is the subject ofongoing research. A related structure-based design approachwas used targeting two proximal hotspot regions at the CD4-gp120 interface to develop small molecule inhibitors of HIV-1entry.209

Nonpeptidic Peptidomimetics. A structure-based designapproach, combining chemical synthesis, X-ray crystallography,and conformational analysis, was used to identify novelnonpeptidic inhibitors of the p53-MDM2 interaction.210 Arigorous optimization of the lead compound, piperidone 16,was performed, focusing on the piperidone ring conformationand side-chain groups, in particular, the N-alkyl substituent(Figure 23). Most notably, the introduction of an α-methylsubstituent, stabilized a favorable trans-diaryl conformation(Figure 23, compound 17). Interestingly, in-depth biophysicaland structural analysis of 16 (also known as Pip-1) and itsstructural analog 18 (a.k.a. Pip-2) identified a ligand-dependentordering of MDM2 (Figure 23). Direct contacts between them-chlorophenyl substituent of piperidinone 18 and amino acidresidues Val14 and Thr16 assist in stabilizing a β-strandstructure in the N-terminal region (Figure 23),211 which isotherwise unstructured in the apo form, or in complex with p53or Nutlin-3a (19, Figure 23). Thio-benzodiazepines have alsobeen recently reported as inhibitors of p53-MDM2.212 Othernonpeptidic peptidomimetic PPI inhibitors include those forthe nuclear receptor (NR)-coactivator interaction.213−218

3.1.4. Mimetics of Protein Secondary Structure. Allsmall molecule inhibitors of PPIs are to some degree mimeticsof protein secondary structure. In contrast to conformationallyconstrained peptides and miniproteins (3.1.2) and the previoussection, peptidomimietic (3.1.3), this section focuses on thedevelopment of artifical oligomeric small molecule mimetics,which try to capture the backbone and side chains topology ofthe native α-helical secondary structure. For accounts on smallmolecule mimetics of other protein secondary structures, pleaserefer to the following reviews: β-strand/β-sheets,219−223 β-hairpin,117,224 α-turn,225 and β turn.226,227

3.1.4.1. Foldamers. Foldamers are artificial oligomers, which,under the guidance of noncovalent interactions, fold into stablesecondary structures with a well-defined display of side chainresidues.228−231 Foldamer is a general term encompassing arange of different molecular classes. Foldamers have beendesigned to mimic a number of different protein secondarystructures, including α helices,105,107,232−236 β-strand/β-sheets,107,233,235 which are exhaustively reviewed else-where.105,107,232−236

β-Peptide Foldamers. β-peptide foldamers mimic a range ofdifferent protein secondary structures,237,238 some of whichhave been successfully targeted to PPIs.239 For example, β-peptide inhibitors of p53-MDM2 were developed in which thep53 hotspot residuesF19, W23, and L26were introducedat three residues intervals along one face of the β3-peptide 14-

Figure 23. Lead compound, piperidinone, 16 (Pip-1) and the discovery of AM-8553 (compound 17), a potent and selective inhibitor of the p53-MDM2 PPI with superior in vitro and in vivo properties and a promising drug profile.210 Piperidinone analog 18 (Pip-2) and Nutlin 3a (19). (a) X-ray cocrystal structure of 18 bound to human MDM2.211 (b) A novel ligand-induced β-strand structure at the N-terminus region of the protein ishighlighted yellow. Reprinted with permission from ref 211. Copyright 2012 American Chemical Society.



helix.240 In this case, a significant improvement in MDM2binding affinity was achieved through the introduction of non-natural α-amino acids.241 Promising activity toward the relatedMDMX protein was also reported,242 which raises the potentialof β-peptide foldamers as dual MDM2/MDMX inhibitors forthe treatment of tumors overexpressing one or both of thesenegative suppressors. The cellular uptake efficiency of β-peptidefoldamers has also been improved through judiciousmodification of the side chain residues.243−245 High-resolutioncocrystallographic data of β-peptide foldamers bound to targetprotein surfaces are urgently needed though before structure-guided rational design-based approaches to PPI modulation canbegin in ernest.246

α/β-Peptide Foldamers. In contrast to β-peptide foldamers,α/β-peptide foldamers are composed of mixtures of α- and β-amino acids residues, which means that their secondarystructures more closely mimics that of native α-helicalpeptides.247,248 Therefore, α/β-peptides foldamers more closelymatch the binding profile of α-helical peptides and can berationally modified to fine-tune target affinity and selectivity.While the presence of natural α-amino acid residues does makefoldamers more susceptible to enzymatic degradation, thistendency can be mitigated through a careful distribution of the“right” β-content along the α/β-oligomer backbone.249 A seriesof high resolution cocrystal structures of α/β-peptide bound todifferent protein surfaces have paved the way for the structure-based rational design of foldamer modulators of PPIs.The high-resolution crystal structure of a 15-mer α/β-peptide

foldamer (20), bound to the antiapoptotic protein, Bcl-x(L)was reported by Fairlie and Gellman and colleagues (Figure24).250 The N-terminal region of foldamer 20, modeled on theα-helical region of the BH3 domain of pro-apoptotic Bim andBak, consists of α amino acids in alternation with β residuesbearing five-membered (APC) ring constraints, while the C-terminal region contains exclusively α amino acids. The β-amino acid residues promote helical folding of the foldamer,while the α amino acids residues contain side chain groups

important for protein recognition at the Bcl-x(L) surface(Figure 24a). A foldameric α/β-peptide mimetic of the PUMABH3 domain has also been cocrystallized with Bcl-x(L) (Figure24b),251 and a rational design approach used to improve affinitytoward Bcl-x(L) and the pro-related apoptotic protein Mcl-1.252

Similarly, inhibition of HIV-cell fusion was achieved usinggp41-mimetic α/β-peptides.253 Taken together, this workprovides convincing evidence of the capacity of foldamers tonot only mimic the key interactions of native α-helicalstructures, but also to contribute to protein binding throughadditional non-native protein interactions.Attempts have been made to develop α/β-peptide inhibitors

of topologically irregular protein interfaces, such as therecognition surface of vascular endothelial growth factor(VEGF).254 Aside from introducing ring constraints into theα/β-peptide peptide backbone, the use of Coulombicinteractions between acidic and basic side chain residues hasproved to be another effective way to stabilize the helicalsecondary structure in work on the CHR domain of HIVprotein gp41.255 A systematic evaluation of binding to twodifferent protein partners Bcl-x(L) and Mcl-1 using structurallydiverse α/β-peptide foldamers derived from Bim BH3 domainwas also performed.256

3.1.4.2. Peptoids. Peptoid inhibitors of PPIs share many ofthe characteristics and much of the potential of their β-peptideand α/β-peptide foldameric counterparts.257 As oligomers ofunnatural, N-substituted glycine bulding blocks, they areresistant to proteolytic degradation and, though intrinsicallymore flexible than β-peptide and α/β-peptides due to the lossof H-honding capacity, their backbones can be induced to foldinto well-defined helical structures through the careful choice ofN-substituent groups. Peptoid inhibitors of p53-MDM2,258 19SRP,259 VEGF−VEGFR2,260 and Apaf1261 have been reported,and are all discussed in detail as part of a dedicated andcomprehensive review on the structure−function of pep-toids.257

Figure 24. α/β-peptide foldamer 20; (a) comparison of the different binding modes of a foldamer (green) and the BimBH3 peptide (blue) bound tothe Bcl-x(L) surface.250 Reprinted with permission from ref 250. Copyright 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (b) overlayof the foldamer:Bcl-x(L) cocrystal structure (navy:white) with the cocrystal structure of PUMA:Mcl-1 (PDB ID: 2ROC; green:red).251 Reprintedwith permission from ref 251. Copyright 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.



3.1.4.3. Proteomimetic Oligomeric Scaffolds. In contrast tofoldamers, whose structures are more closely resembling thenative polypeptide chain, proteomimetic oligomeric scaffoldsare instead composed of conformationally rigid artificialoligomeric structures such a terphenyls and oligobenzamides.Proteomimetic oligomeric scaffolds targeting a range ofdifferent PPIs have been extensively reviewed else-where,105,107,232−235 including most recently for oliga-mides262,263 targeting p53-MDM2264−269 and Bcl-x(L)-medi-ated PPIs.270,271

3.2. Natural-Product Inspired PPI Modulation

Self-medication is commonplace throughout the animalkingdom.272−274 Just as organisms instinctively search thesurrounding flora and fauna for food in reponse to malnourish-ment, a state of disease caused by imbalances in homeostatis, sothey search for the most effective pharmaceutical agent to treatother diseases. Recent data suggests that the Neanderthals, whodied out over 24 000 years ago, self-medicated with plantextracts through an awareness of the nutritional and medicinalvalue of the surrounding vegetation.275 Thus, by instinct or bylearning, man has traditionally turned to nature as a source ofmedication. The pharmaceutically active agent in naturalextracts is often a single secondary metabolite, a naturalproduct, which is typically biosynthesized in minute quantitiesby the host organism (e.g., plant or bacteria). In the context ofthis review, we thus define a natural product as a nonpeptidiclow molecular weight metabolite of biosynthetic origins.Natural products have proved to be a tremendously rich and

profitable source of drug molecules for the pharmaceuticalindustry. Despite this though, interest in natural product drugdevelopment waned at the end of the last century, owing to thetechnical difficulties of screening natural product extracts andthe rise of combinatorial chemistry (combichem). In theintervening period, many new synthetic chemotypes werediscovered and applied to drugs, through combichem did notfully live up to the heightened expectations and withaccompanying technological advances in HTS,276 there is arenewed interest in natural products.277−279 However, thesupply from their natural sources is often unsustainable, andnotwithstanding some impressive total synthesis efforts,280−282

the chemical complexity of natural products frequentlyprohibits chemical access to sufficient quantities for advancedbiological testing. Nevertheless, their unrivaled structuralcomplexity, diversity, and biological relevance means thatnatural products will continue to provide a rich source of leadstructures and important guiding principles for the design ofcompound libraries for screening campaigns.3.2.1. Natural Products. This section of the review

provides an overview of natural products characterized as PPImodulators in the time period from 2008 to the present day, asan illustration of their rich structural and functional diversity.For a comprehensive review of natural products as a source ofnew drug molecules in the period 1981−2010, including manytargeting PPIs, the readers are encouraged to read Newman andCragg’s excellent review,283 and all previous reviews of theirscited therein. Where possible, natural products are groupedaccording to a common PPI target.3.2.1.1. Pro-Apoptotic Proteins. Current inhibitors of the

Bcl-2 family of antiapoptotic proteins bind to Bcl-2 and Bcl-x(L), but target Mcl-1 with only low affinity, rendering themless effective for the treatment of cancer types overexpressingMcl-1. Thus, there is a need for Mc1−1-selective small

molecule inhibitors. The marine natural product, marinopyrroleA, 21 (Figure 25), was recently reported to inhibit BIM bindingto Mcl-1 resulting in degradation of Mcl-1 via the proteasomesystem. The mode of action of 21 differes from other knowninhibitors, such as the pan-Bcl-2 inhibitor obatodax, and whileactive towards Mcl-1-dependent leukemia cells, 21 could notinduce cell death in Bcl-2- or Bcl-x(L)-dependent leukemiacells.284 Oxy-polyhalogenated diphenyl ethers such as 22(Figure 25) recently identified by Crews et al. also exhibitedmoderate inhibitory effects toward the Mcl-1 mediated PPI(Mcl-1/Bak) in a FRET based screen of marine extracts.285

Rigorous studies on the total synthesis of p53-MDM2 PPIinhibitor, chlorofusin (23, Figure 25), have been key todetermining the absolute stereochemistry, and to enableimportant structure−activity relationship studies286 in theabsence of high resolution X-ray cocrystallography data.

3.2.1.2. Phosphorylation-Mediated PPIs. Phosphorylation-dependent protein−protein interactions are important intra-cellular drug targets given their involvement in manyintracellular signaling pathways. Very few natural products areknown which selectively inhibit phosphorylation-dependentPPIs. Noteworthy, therefore, are reports by Berg and co-workers of salvianolic acid A (24) and B (25), components ofthe roots of Salvia miltiorrhiza (Danshen), as inhibitors of SH2domain binding to phosphotyrosine-containing peptides(Figure 26).287 A small panel of disease-relevant SH2domainsLck, Src, STAT1, STAT2, and STATwereevaluated in a FP assay against a small library of naturalproducts isolated from plant extracts, and 24 and 25 bothemerged as the most active compounds against Lck and SrcSH2 domains, with midmicromolar IC50 values. Molecularmodeling studies (Lck SH2 domain/pYEEI cocrystal structure)suggested above all that the catechol group A (Figure 26) canfunction as a bioisostere for the phosphotyrosine functionalgroup, forming hydrogen bonds with Arg134 and Glu157. Thisresult may lead to more potent lead compounds targeting Src-

Figure 25. Natural products targeting pro-apoptotic PPIs, Mcl-1-BIM-Bak and p53-MDM2.



family SH2 domains. Berg and colleagues combine the powerfuldirecting effect of the phosphate group with the biologicalrelevance of natural products to identify inhibitors of theSTAT5b SH2 domain (26, Figure 26) and the Pin1 substratebinding domain (27, Figure 26).288

Poloxin (28) and thymoquinone A (29) were recentlyreported as the first nonpeptidic inhibitors of polo-box domain(PBD) of serine/threonine kinase Polo-like kinase 1 (Plk1)binding to phosphoserine/phosphothreonine-containing pep-tides.289 PBD functions as a site for intracellular anchoring ofPlk1, and it is thought that inhibiting the PPI formed betweenPDB and target phosphorylated peptide motifs can disruptintracellular localization and would represent a viablealternative to inhibiting kinase activity rather than targetingthe ATP binding site of Plk1.Gambogic acid, 30 (Figure 27), inhibits the chaperone

protein Hsp90 (90 kDa heat shock protein). According to SPRspectroscopic analysis, 30 binds to the N-terminal domain ofHsp90 with a Kd in the micromolar range, and does notcompete with geldanamycin, an established Hsp90 inhibitor,

but instead targets a site remote from the ATP binding pocketof the Hsp90 protein.290 A high-throughput screening of a123,599-strong natural product library of marine naturalproducts, microbial metabolites and plant extracts againstthree different PPIs, proteasome assembling chaperone 3(PAC3) homodimerization, T-cell factor-7/β-catenin, andPAC1-PAC2, identified the fungal metabolite JBIR-22 (31) tobe a submicromolar inhibitor of PAC3 homodimerization(Figure 27).291 Molecular docking studies suggested that 31inhibits protein homodimerization through binding at thePAC3 dimerization surface. Pateamine, 32, a marine metabolitefrom Mycale sp., and simplified analog 33 (Figure 27), des-methyl-des-amino pateamine (DMDA-PatA) have recently beenreported to inhibit translation initiation through targeting ofeIF4AI and eIF4AII, two isoforms of the RNA-dependentATPase and ATP-dependent helicase, eIF4A, a component ofthe eIF4F complex.140 X-ray crystallography data are stilllacking, though 32 and 33 are both believed to exert theireffects by inducing global changes in the eIF4A protein.Compound 33 was later reported to prevent cachexia-inducedmuscle wasting in mice caused by the cytokines interferon γand tumor necrosis factor α or by C26-adenocarcinomatumors.292,293 Prieurianin/endosidin 1, 34 (Figure 27) − asecondary metabolite isolated from tropical trees − wasidentified by Coupland, Waldmann and colleagues from thescreening of a natural product library against the model plantArabidopsis thaliana in search of circadian clock effectors.294

While the precise target of 34 remains elusive, it is known tostabilize the actin cytoskeleton and affect endosome traffickingin vivo but not in vitro, indicating the need for actin-associatedproteins. Furthermore, early indications are that 34 wouldappear to interact with actin filaments via a unique molecularmechanism, potentially involving actin binding proteins such asprofilins, formins, Actin Related Protein 2/3s (ARP2/3s),ADF/cofilin, Actin Interacting Protein 1 (AIP1) or gelsolin/villins. A common difficulty with targeting PPIs is the lack ofcontrol over selectivity, as one of the two proteins at theinterfaces commonly recognizes a number of different proteinpartners. Two different compound libraries were screened forinhibition of transcriptional activator, MLL, binding to theGACKIX domain of coactivator CBP/p300.295 Whereas a 50000-strong commercial library composed of small drug-likemolecules did not produce any hit compounds in this assay, astructurally diverse library of over 15 000 marine-derivednatural product extracts did. Lobaric acid, 35, and sekikaicacid, 36 (Figure 27), were characterized as a novel class ofGACKIX inhibitors, capable of modulating two distinct proteinactivator binding sites through allosteric binding to a dynamicsurface within the CBP/p300 coactiavtor protein.

3.2.2. Approaches to Diversifying Natural Products.Natural products are excellent lead structures for PPI drugdiscovery in view of their diverse and biologically relevantstructures.279,283 However their usefulness is restricted by thefact that natural products are commonly available in onlyminute quantities from often unsustainable natural sources.Furthermore, natural products typically require a degree ofchemical optimization before arriving at a druggable entity.Therefore, sustainable chemical or genetic methods are needed,which can deliver biologically active natural products to scale,or which can produce structurally diverse analogs libraries ofnatural products or natural-product-like compounds in a timeefficient manner.

Figure 26. Natural products targeting phosphorylation-dependentPPIs.



3.2.2.1. Total Synthesis. Total synthesis in the broader senseis the art of synthesizing complex molecular structures fromsimple chemical building blocks.280 The total synthesis of anunknown natural product is frequently an important con-firmation of its predicted structure296 and can sometimesdeliver sufficient quantities of material for initial targetvalidation studies. Once successfully charted, the syntheticroute opens up new synthetic opportunities including access tostructural analogs for structure−activity relationship studies,and the diverted total synthesis of chemical biology probes(refer to section 3.2.2.3).297 With a few most notableexceptions,281,282 the scalability of a natural product synthesisis inversely proportional to its structural complexity, whichtherefore puts greater emphasis on other chemical approachesto access scalable quantities of compound, such as semisyn-thesis (section 3.2.2.2) or mutagenesis (section 3.2.2.4), orapproaches which recapitulate the bioactivity of complexnatural products in a more molecularly efficient manner, forinstance the biology-oriented synthesis (BIOS) of smallcompound libraries (section 3.2.2.5).3.2.2.2. Semisynthesis. A typical semisynthesis campaign

begins with an advanced intermediate or a mature naturalproduct isolated from a natural source, and makes use ofchemical methods to complete the targeted synthesis of abiologically active compound or functional probe.298 Thus,much or all of the compound’s structural complexity isassembled biosynthetically by the host organism, in starkcontrast to total synthesis, which builds up molecular

complexity in a stepwise manner starting from simple chemicalbuilding blocks. For PPI drug discovery semisynthesis can be asignificantly more efficient way to access a target naturalproduct than competing methods (e.g., the semisynthesis ofmicrotubule stabilizing paclitaxel from 10-deacetylbaccatinIII)299 and can lead to clinically useful analogs, which wouldbe difficult to prepare via other synthetic or genetic routes (e.g.semisynthesis of rapamycin analogs targeting mTOR).300

Where natural product semisynthesis has proved particularlyuseful is in the molecular interrogation of stabilizing proteininterfaces. Ohkanda and colleagues prepared semisyntheticanalogs of fusicoccin A capable of labeling the 14-3-3 adapterprotein in a ligand and site-selective manner.301 Ottmann andKato et al. also rationally designed a semisynthetic fusicoccinanalog, which selectivity stabilized the 14-3-3/TASK3 PPI.302

3.2.2.3. Diverted Total Synthesis. The expression “divertedtotal synthesis” implies a rationale-driven diversion from theoriginal total synthesis route to a target natural product, withthe purpose of validating a specific chemical or biologicalhypothesis.297 Analog studies for structure activity relationshipstudies is one example of diverted total synthesis. Danishefsky’swork on the diverted total synthesis of migrastatin analogs aspotent inhibitors of cell migration,303,304 and Furstner’s onactin-targeting latrunculin analogs305 are early demonstrationsof the approach.Recently, a diverted total synthesis approach was adopted by

Waldmann, Arndt and co-workers to target molecularly efficientcell-permeable molecular probes of intracellular actin filaments

Figure 27. Natural product modulators of diverse PPIs.



based on the bipartite cyclodepsipeptide natural products,jasplakinolide, 37, and chondramide C, 38 (Figure 28).306 Bothnatural products bind to three actin monomers simultaneously.At low concentrations this leads to stabilization of actinfilaments, while at higher concentrations it causes massdisruption of actin dynamics and a breakdown of the actincytoskeleton. Earlier work had established a unified syntheticroute to both 37 and 38: a novel and efficient solid-phase-basedsynthesis using ring-closing metathesis to forge the depsipep-tide macrocycle.307 An analog library was synthesized to probethe actin pharmacophore, which gave rise to simplified analog39 (Figure 28), lacking a methyl substituent in the polyketideregion and the bromo-substituent on the indole ring of the D-tryptophan. Analog 39 was found to be as cytotoxic as 37 and38 in cell culture. The modifications also improved the overallefficiency of the synthesis and paved the way to gram quantitiesof the compound and the preparation of molecular probes.

In later work, results from structure−activity relationshipstudies of natural analogs of 37 and 38 suggested that thedepsipeptide macrocycle would tolerate structural variation ofthe L-Ala methyl substituent, as it was hypothesized that themethyl substituent points away from the actin binding site intosolvent.307 The alanine residue was replaced with a lysine (40)and was found to retain the potent actin-targeting properties ofthe parent compound. Various dye molecules were attached to40 via the lysine side-chain, and the conjugates used to stainactin filaments in fixed cells. A BODIPY analog of 40 was foundto be cell permeable and could be used to stain static actinfilaments in live cells (BODIPY-40, Figure 28).

3.2.2.4. Mutagenesis. An alternative approach to thechemical synthesis of natural products has been to elucidatethe biosynthetic gene cluster encoding the enzymes responsiblefor the assembly of the target natural product in the hostorganism. Information of this kind can then be used toreprogramme the dedicated biosynthetic pathways, or engineerartificial cellular synthetic production systems in a plug-and-

play fashion.308 In this way the natural product and associatedanalogs can be synthesized to scale,,309−313 either in the nativehost or in a stable heterologous host.314 The potentialadvantages of this approach include a significantly reducedenvironmental impact, increased source sustainability and theenormous unlocked potential for structural diversity.Wilkinson and co-workers rationally optimized the anticancer

properties of the complex polyketide natural product rapamycin(41, Figure 29), produced by Streptomyces rapamycinicusNRRL5491, via a re-engineering of the polyketide synthaseresponsible for its production and by referral to the X-raycocrystal structure of FKBP12−rapamycin−FRAP.315 Inparticular, compounds rapalog 42, lacking the methylsubstituent at C17 and rapalog 43, lacking the OMe andmethyl substituents at C16 and C17, respectively. By inspectionof the X-ray crystal structure, it was reasoned that replacing themethoxy substituent with a secondary alcohol functionality atC16 might lead to a favorable hydrogen-bond interaction withthe protein polyamide backbone while at the same timemitigating potentially destabilizing steric interactions at theprotein surface (43, Figure 29). Removal of the methyl group atthe C17 position might also facilitate improved binding of thetriene region (C17−C22) of 42 at the rapamycin bindingdomain of mTOR. Importantly, these rapalogs displayed

Figure 28. Diverted total synthesis of 39 and 40, actin-targetinganalogs of bipartite natural products jasplakinolide/jaspamide (37)and chondramide C (38).306 Overlay of fluorescence microscopyimages of live U2OS cells: (left), expressing mCherry-actin, whichvisualizes monomeric G-actin; (middle) cell-permeable BODIPY-labeled jasplakinolide/chondramide C analog, BODIPY-40 whichselectively labels actin filaments (F-actin); (right), overlay of the leftand middle images, which distinguishes dynamic from static actinstructures. Reprinted with permission from ref 306. Copyright 2012American Chemical Society.

Figure 29. Chemical structure of rapamycin, 41, and rationallydesigned rapalogs 42 and 43 generated by re-engineering therapamycin polyketide synthase.315



enhanced inhibition of cancer cell lines as compared torapamycin 1.An impressive 1 g/L biosynthesis of taxadiene was recently

reported (about 15 000-fold improvement over the wild-type)by Stephanopoulos et al.. Taxadiene is noted as the firstcommitted intermediate along the biosynthetic route to Taxol,a potent anticancer natural product first isolated from the Taxusbrevifolia Pacific yew tree.316

Significant progress has been made toward elucidating thebiosynthesis gene cluster of the diterpene glucosides, fusicoccinA (44) and cotylenin A (45), produced by Phomopsis amygdaliand Cladosporium sp. 501−7W (Figure 30). Analogs 44 and 45both selectively stabilize different 14-3-3-mediated PPIs, whichwould suggest that selectivity in 14-3-3 PPI stabilization isencoded in subtle structural differences between fussicocinanalogs, which warrants further investigations into theirbiosynthetic gene cluster. Key enzymes responsible for themolecular tailoring of the aglycon317 and sugar moieties offusicoccin A318 have recently been characterized. A preparativescale fermentative production of the shared 5-8-5 fusicoccanetricyclic diterpene precursor has recently been described (46,Figure 30).319 The bimodular fusicoccadiene synthase, the

enzyme responsible for the biosynthesis of 46 could beexpressed equally in both prokaryotic E. coli as well aseukaryotic A. nidulans and S. cerevisiae cells, for heterologousfermentation. The yeast strain produced the highest yields ofthe desired metabolite in this case.

3.2.2.5. Biology-Oriented Synthesis (BIOS). Past successesof natural products as drug molecules has sustained interest intheir use as lead structures for PPI drug discovery. However,their prohibitively complex structures have motivated thesearch for alternative sources of leads for PPI drug discoverycampaigns. By virtue of their biosynthetic origins, naturalproducts occupy a chemical space, which is quite unique fromall other classes of small molecule.320−322 The concept of‘natural product chemical space’ has inspired new approaches tothe design of small compound libraries, with as their aim therecapitulation of the structural complexity and biologicalrelevance common to natural products.323−325 The approachwhich comes most to the fore is biology-oriented synthesis(BIOS),326−328 which aims to generate focused collections ofstructurally diverse compounds built around biologicallyrelevant and prevalidated scaffold structures, such as thosefound recurrently in drug molecules or as fragments of

Figure 30. Tailoring of the aglycon317 and gluoside moieties318 of fusicoccin A, 44, and the structurally related brassicicene C help to define acommon biosynthetic logic, which connects cotylenin A, 45 (biosynthetic modifications elucidated highlighted in bold red, responsible enzymes andhost organism highlighted in gray bold italics).



bioactive natural products. Waldmann and colleagues used aBIOS approach to develop inhibitors of the SH2-domainfunctions of STAT, with activity in vitro and in cells (46−48,Figure 31).329

The same research group also identified 3,3′-pyrrolidinyl-spirooxindoles as disruptors of microtubule polymerization (49,Figure 32).330 Spirooxindoles are a recurrent chemical motif inbioactive natural products, for example the tubulin polymeriz-ing spirotryprostatin B1. Non-natural spirooxindoles have alsobeen reported as potent peptidomimetic inhibitors of p53-MDM2.331 For the preparation of 49, a metal-catalyzed crosscoupling methodology was used, which generated structurallydiverse analogs with excellent enantiopurity.330 Perhaps themost intriguing aspect of this BIOS library is the identificationof a novel mode-of-action for this compound class: namely, theinterference with microtubule polymerization and the for-mation of multipolar spindles and more than one microtubuleorganizing center per cell, which contrasts with the biologicalprofile of spirooxindoles reported previously. The authors cite

differences in the spatial arrangement of side chain functionalitybetween analogous spirooxindole scaffolds as the reason fortheir contrasting biological profiles.A focused BIOS library based on the oxepane scaffold

structure yielded Wntepane 1 (50, Figure 32) − an activator ofthe Wnt signaling via reversible binding to van-Gogh-likereceptor protein 1 (Vangl1).332 Tetrahydroisoquinoline inhib-itors of tubulin polymerization have also been reported (51,Figure 32), which target a site distinct from the colchicine andthe vinca alkaloid binding sites.333 Finally, tubulexin A, atetrahydropyran derivative (52, Figure 32), was characterized asan inhibitor of mitosis through targeting of the CSE1L proteinand the vinca alkaloid binding site of tubulin.334

3.3. Supramolecular-Induced PPI Modulation

Supramolecular chemistry is the study of chemical systemsgoverned by reversible noncovalent interactions and inspired bythe assembly of biomolecules in nature.335,336 A supramolecularPPI modulator disrupts its protein target via noncovalentinteractions. In this sense, all small molecules discussed in thisreview are to some extent supramolecular PPI modulators.However, in this section 3.3, supramolecular-induced PPImodulation comes to signify a role reversal compared toclassical small molecule host-interactions, where the protein(more specifically a side chain residue of the protein) plays therole of the guest, and a synthetic small molecule the role of thehost.Modern drug discovery and supramolecular chemistry are

overlapping research fields, which apply many of the samemolecular design principles. In modern drug discovery, drugmolecules disrupt their protein targets via a lock-and-keymechanism in which the protein lock plays host to the smallmolecule key. For classical drug targets, such as enzymaticactive sites or ligand-activated transcription factors, the energyof binding is typically channelled through noncovalentinteractions between a single low molecular weight moleculeand protein side chain residues buried deep within a solvent-excluded pocket. For PPIs, the lock-and-key mechanism stillapplies, though the binding energy is this time focused atmultiple hotspot residues, which are heterogeneously dis-tributed across a more expansive protein interface. In modernsupramolecular chemistry, supramolecules are assembled vianoncovalent interactions between host and guest molecules,which function via a similar lock-and-key mechanism, andthrough which much of the binding energy is channelled,similar to hotspot residues at protein−protein interfaces.

3.3.1. Supramolecular-Induced Protein Dimerizationand Oligomerization. Protein dimerization is a ubiquitousclass of PPI, which adds stability and function to proteins in thecell. Chemical methods for controlling protein dimerization aretherefore of benefit to the chemical biology and drug discoveryfields.337 Supramolecular host−guest chemistry is one methodfor inducing dimerization in intrinsically nondimeric or dimericproteins. The host−guest elements are either geneticallyencoded or chemically inserted via expressed protein ligation,and dimerization controlled in a spatiotemporal fashion byvarying the concentration of host−guest constructs. Theadvantage of this approach is its orthogonality to other moredirect chemical methods.Supramolecular induced protein assembly mediated by high-

affinity host−guest binding to β-cyclodextrin338,339 have beenreported.340−344 Cucurbit[8]uril (Q8)345−349 forms 1:2 ternarycomplexes with two Phe-Gly-Gly or Trp-Gly-Gly tripeptides350

Figure 31. Small molecule BIOS library targeting the SH2-domainfunctions of STAT.

Figure 32. Different bioactive small molecules (49−52) identifiedfrom biological screening of focused BIOS compound libraries.



or 1:1:1 ternary complexes with methylviologen and dihydrox-ynapthalene, thus enabling supramolecular-induced homo-351

and heterodimerization of intrinsically nondimeric proteins.352

Q8 has also been used to induce tetramerization of dimericproteins,353 and significantly augment (50-fold) the catalyticactivity of dimeric caspases.354 Hou and co-workers also usedQ8:FGG binding to generate nanowires of glutathione S-transferase (GST).355

3.3.2. Supramolecular-Modulation of the ProteinSurface. A seven-membered cucurbituril (Q7) has recently

been shown by isothernal titration calorimetry and protein X-ray crystallography to bind to the N-terminal phenylalanine ofinsulin.356 The Ka of the interaction is in the same range asmany physiologically relevant protein−protein interactions andshould in principle suffice to confer PPI modulating activity. Inthe Q7-insulin crystal structure the aromatic side chain of theN-terminal phenylalanine of insulin is accommodated in thecavity of Q7 (Figure 33), covering around 200 Å2 solvent-accessible surface area of insulin. A considerable number ofpolar contacts is established between Q7′s carbonyl oxygensand main-chain nitrogens of Phe1, Val2, and Asn3 alsoincluding the side-chain nitrogen of the latter. Whereas theoverall conformation of insulin shows no change upon Q7binding, the first four N-terminal amino acids unfolded andmoved away from the protein s main body to interact with thesupramolecular ligand. Importantly, Q7 showed no measurableaffinity for insulin lacking the N-terminal phenylalanine,impressivly demonstrating the specificity of this compound.Another family of supramolecular ligands that have been

shown to interact with proteins are the Calixarenes.357−360 For

example, these molecules were used to enhance the functionallyimportant tetramerization of the tumor suppressor p53357 or apotassium channel.358 In 2012, the crystal structure ofcytochrome c in complex with a Calixarene was reported(Figure 33).361 In the asymmetric unit of the protein crystaltwo copies of cytochrome c and three Calixarene moleculescould be identified. One Calixarene molecule bound to eachLys4 and Lys22 of one cytochrome c and to Lys89 of thesecond cytochrome. The lysine side-chains are enclosed by thehydrophobic interior of the Calixarene molecules with thesulfonate-bearing rings establishing electrostatic contacts withthe γ-amino group of the enclosed lysine as well as additionalpolar parts of the protein. The Calixarene molecule binding tothe Lys89 site for example establishes further polar contacts ofits sulfonate rings to the backbone amide of Lys89 and a salt-bridge to the side-chain of Lys5. The existence of multiplelysine binding sites of Calixarene on the cytochrome c surfaceobserved in the crystal structure and related NMR mappingstudies inspired the authors to define a model of theCalixarene/cytochrome c interaction where the supramolecularligand continuously explores and camouflages the surface of theprotein by sequentially binding to up to five high-probabilitybinding sites. The reported promotion of cytochrome cassembly together with the activities toward tetramerizationof p53 and potassium channels suggests a valuable role forCalixarenes in PPI modulation in the future.A third class of supramolecular ligands whose binding to

proteins have been structurally elucidated are MolecularTweezers, that in contrast to the two former ring structuresconstitute an open belt-like organization with a molecular cavityformed by alternating norbornadiene and benzene rings.362 Inenzymatic assays the Molecular Tweezers were shown to inhibitseveral hydrolases in a manner reversible by the addition of freelysine derivatives363,364 which prompted the assumption(supported by in silico analyses) that the Molecular Tweezerspreferentially bind to exposed lysines to confer their activity. In2013, the crystal structure of a Molecular Tweezer bound to theadapter protein 14-3-3 was published (Figure 33).365 Here,corresponding electron density for the supramolecular ligandcould only be observerd associated with one surface exposedlysine, Lys214. In addition to direct binding to 14-3-3 asmeasured by SPR and ITC, the Molecular Tweezer inhibits thebinding of fluorescently labeled peptides derived from theprotein kinase C-RAF and the pathogenic protein ExoS. ThisPPI inhibiting activity can be explained by the location ofLys214 which is situated at the rim of the central binding cleftof 14-3-3, the primary site of interaction with their partnerproteins including C-RAF and ExoS. Judging by the excellentinitial electron density the molecular tweezer wraps around theside chain of Lys214 and embraces it almost completely,electrostatically engaging the terminal NH2 with one of itsphosphate groups while the other points away from the aminoacid. One explanation for the fact that only Lys214 wasobserved bound to the Molecular Tweezer could lie in itsunique environment in the protein. By binding to Lys214, thehydrophobic corpus of the Tweezer is shielded from theaqueous environment by a ring of nonpolar residues (Tyr213,Thr217, and Leu218). This “binding preference” of theMolecular Tweezer to certain lysines with a rather hydrophobicdirect environment is in perfect agreement with QM/MMcalculations of possible interactions of the supramolecularligands with all 17 surface-exposed lysines in 14-3-3. These

Figure 33. Supramolecular ligands whose interactions with proteinshave been solved by X-ray crystallography. For details, see main text.



results could help in the design of Molecular Tweezers withmore specific binding modes.

3.4. Compound Library Generation

The high-throughput screening (HTS) of compound libraries isa highly effective way to discover new PPI modulators.11,12 Inthe case of well-defined PPIs with established modulatorcompounds, HTS is an opportunity to serendipitously discovernew classes of modulator with different modes of interaction.For new or less well-defined PPIs, which lack endogenous leadstructures (e.g., a peptide binding epitope), compoundscreening is arguably the only reliable way to make theimportant breakthrough. The dynamic and heterogeneousbehavior of PPIs makes them more challenging drug targetsthan classical enzymatic drug targets. Therefore, success withPPI drug discovery is particularly reliant on methods capable ofgenerating and screening large high-quality (diverse, complexand biological relevant) compound libraries. Increasing thelibrary size is challenging from the perspective of compoundhandling, scale, and the method of hit detection. Increasing thediversity, complexity and biological relevance of compoundlibraries challenges the synthetic chemist to devise new andmore efficient synthetic methodologies fit for the purpose. Themethods for generating compound libraries discussed hereinmeet these challenges in elegant and molecularly efficient ways.3.4.1. Biological Techniques. Phage display366−368 and

ribosome display369−371 are robust and highly efficienttechniques for screening large peptide (and protein) librariesfor novel binders of protein surfaces or to optimize the affinityand selectivity of known peptide binders. In the case of phagedisplay, the peptide sequence is expressed on the surface of thephage particle; phage libraries of diverse peptide sequences arethen simultaneously screened against a surface-immobilizedprotein target during an initial panning phase. The optimalphage binders are then amplified and sequenced. The size anddiversity of phage libraries are restricted though by thetranformation efficiency of the bacterial cell. In the case ofribosome display the transcription and translation processes areall performed in vitro, which means the technique is in principlecapable of handling larger and more structurally diverse peptidelibraries. The advantage of both techniques lies with theirexquisite efficiency: compound library synthesis (translation)and compound screening are combined within the sametechnique, while repeated panning, leads to a Darwinianselection of the best binding peptide sequences.3.4.1.1. Yeast “N” Hybrid. Classical yeast two-hybrid (Y2H)

systems372,373 have proven to be highly effective tools for thediscovery and characterization of PPIs. Recent adaptations toY2H, termed “yeast three-hybrid systems’’, are showing someapplications for the charaterizing of small molecule-proteininteractions and PPI drug discovery.374

Phage Display. Phage display has been extensively used todissect the protein interface of PPIs. Important examplesinclude the nuclear receptor-coactivator interaction375−377 andp53-MDM2.378 The rational optimization of phage-derivedpeptides guided by high-resolution X-ray cocrystallography is ahighly effective way to engineer potent and selective PPIinhibitors. A phage display screen of the p53-MDM2 and p53-MDMX PPIs identified pDI − LTFEHYWAQLTS as a potentdual MDM2/MDMX inhibitor.123 The cocrystal structures ofpDI bound to MDM2 and MDMX helped to steer the designof more potent inhibitors (Figure 34), culminating in pDIQ −ETFEHWWSQLLS, a pDI analog with four mutations in the

amino acid sequence (underlined) which was 5-fold morepotent than pDI. pDIQ unwinds at the C-terminus of the α-helix on binding to MDMX, and makes contacts with ahydrophobic patch, which is unique to MDMX. A detailedatomistic picture of peptide−protein interactions of this sort isfundamental to the development of new and more potentinhibitors targeting the p53-MDM2/MDMX PPI.The structural diversity of phage display has until recently

been limited to the screening of structurally diverse linearpeptide libraries based on standard natural amino acids. Thecoupling of site-specific unnatural amino acid mutagenesis(section 3.4.1.4) or the post-translation chemical modificationof amino acids side-chains creates opportunities to enhance thestructural diversity of compound libraries. Macrocyclic peptidesare a recurring theme in bioactive natural products and PPIdrug discovery. In general, macrocyclization correlates withincreased potency as it lowers the entropic cost of proteinbinding by preorganizing the peptide into its bioactiveconformation. Macrocyclization can also increase cell perme-ability and resistance to enzymatic degradation (section 3.1.2).An elegant phage display-based strategy was used to generateand screen artificial bimacrocyclic peptides.379 A random linearphage peptide library (>4 × 109 members) bearing threereactive cysteine residues was chemically postmodified with amesitylene linker to induce peptide bicycle formation (Figure

Figure 34. X-ray cocrystal structure of pDIQ (orange) bound toMDM2 (top) and MDMX (bottom).123 The C-terminal region ofpDIQ is helical when bound to MDM2, but unwinds partially whenbound to MDMX. Reprinted with permission from ref 123. Copyright2010 American Society of Biochemistry and Molecular Biology.



35). This approach has so far yielded a number of enzymeinhibitors, including a potent inhibitor of human plasmakallikrein. The broad applicability of phage display to the studyof PPIs coupled with the promise of screening macrobicyclicpeptide libraries (see section 3.4.3), means that phage-selectedbicyclic peptides116 will be a valuable tool in future PPI drugdiscovery campaigns.For Haberkorn and colleagues phage display screening of a

combinatorial peptide library based on the Min-23 miniproteinscaffold was essential for identifying SFTI-1 inhibitors of thedelta-like ligand 4 (Dll4)-NOTCH1 PPI.380 The chemicalsynthesis of Min-23 inhibitors of Dll4-NOTCH1 and the phagedisplay screening on the SFTI-I scaffold were eitherunsuccessful or yielded unsatisfactory results. The bindingepitope of a Min-23 phage peptide was successfully grafted tothe variable loop region of the SFTI-I using a solid-phase basedsynthesis, with retention of specificity for Dll4 binding andtumor-targeting activity in vitro and in cell culture. Importantly,the grafted SFTI-I peptides also showed proteolytic stability.Phage display screening is also useful for optimizing the bindingproperties of rationally designed PPI inhibitors. For example,Schepartz and Brunsveld both used phage display screening tooptimize modified α-helical miniproteins targeting p53-MDM2178 and the ER-coactivator PPI,181 respectively. Othercell-based display techniques not covered in this review, whichhave been used to study PPIs, include yeast display381−383 andbacterial cell surface display.384

3.4.1.3. Ribosome Display. The diversity of compoundlibraries generated by cell-based techniques such as phage iscurrently limited by cellular transformation efficiency, and thesubstrate scope of the transcription/translation machinery. Innoncellular display techniques, however, such as ribosomedisplay and mRNA display, all steps are performed in vitro.26 Inprinciple therefore ribosome display facilitates the identificationof highly evolved peptide binders beyond the reach of phagelibraries.385,386

Initially developed by Hanes and Pluckthun for use as aprotein evolution tool,369 ribosome display was first applied tothe study of PPIs by Taussig and colleagues to characterize theinteraction between lymphocyte signaling protein Grb2 and theN-terminal SH3 domain of Vav1.387 Recently, Brunsveld andcolleagues used ribosome display to re-evaluate the AF-2 of the

estrogen receptor (ER) in search of new coactivator peptideconsensus motifs.388 Earlier phage display studies375 hadconcluded that both the α- and β-isoform of ER preferentiallybind short helical LXXLL peptides at the ER coactivatorbinding site (AF-2), where L = Leu and X = any other aminoacid. Important for potent peptide binding are surface Glu andLys residues (Figure 36), which align with the helix dipole.Eight panning rounds of ribsome display screening wereperformed against both ERα and ERβ. The canonical LXXLLconsensus motif emerged on sequencing and cluster analysis ofpeptides enriched over four panning rounds. Intriguingly, theLXXLL motif was seen to ‘mature’ into PXLXXLLXXP by theeighth round. Subsequent biochemical and biophysical studiesof a series of prolinyl-peptides identified them as potent, lownanomolar inhibitors of ER-coactiavator binding, and that theflanking proline residues in particular were important fordetermining the precise length of the α-helix and the strengthof the surface binding through additional conformationalconstraints (e.g., His N-terminal to the flanking proline Pro-2, Figure 36).Other recent applications of ribosome display screening have

been in the optimization of the binding affinity of a human IL-13-neutralizing antibody for the potential treatment ofasthma,389 the generation of protein nanoarrays,390 and theselection of photoresponsive peptide aptamers.391

A related in vitro display technique useful for the screening ofpeptide and protein binders is mRNA-display.392,393 In contrastto ribosome display, where the nascent peptide (or protein)and progenitor mRNA are held in a metastable noncovalentassembly with the ribosome, the mRNA and peptide moleculesare covalently linked via a puromycin linker.394 This approachhas been used to screen for binders of calmodulin.395 A moretime-efficient variant of mRNA display, termed transcription−

Figure 35. General strategy for the generation and enrichment ofbicyclic peptide PPI modulators via post-translational chemicalmodification of phage peptides with mesitylene.379

Figure 36. Ribosome display screening identifies proline derivedpeptides as potent ER binders (Top). High resolution X-raycocrystallography demonstrates a clear role for the flanking prolineresidues in precisely determining the helix length (Bottom).388

Reprinted with permission from ref 388. Copyright 2013 AmericanChemical Society.



translation coupled with association of puromycin linker(TRAP) display, has recently been reported.396

3.4.1.4. Flexible tRNA-Acylation Ribozymes (Flexizymes).Phage and ribosome display techniques are limited by thesubstrate scope of the transcription/translation machinery. Site-specific unnatural amino acid mutagenesis of proteins in vitroand in cells397 is possible using orthogonal tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pairs.398 The genetic code israpidly expanding,399 which so far includes post-translationalmodifications400 multisite-specific mutagenesis,401 and theincorporation of nonproteinogenic amino acids into wholeorganisms.402 The coupling of unnatural amino acid muta-genesis to in vitro display techniques represents a powerful wayto expand the structural diversity of peptide-based libraries.Suga et al. engineered flexible tRNA-acylation ribozymes

(flexizymes)403,404 coupled to a flexible in vitro translation(FIT) system integrated with mRNA display for in vitroselection. Referred to as random nonstandard peptideintegrated discovery (RaPID), this system has been used togenerate and screen large libraries of natural product-likemacrocyclic peptides incorporating unnatural nonproteinogenicamino acids.405−407 The approach was recently used to identifypotent macrocyclic peptide inhibitors of E6AP408 Akt2,409 andVEGFR2 activity.410

3.4.2. Microarrays. Peptide microarrays have been moreextensively used to study PPIs, for instance in epitope mappingof protein surfaces,411−414 rather than as a tool to identify PPImodulators. Similar to on-bead strategies, microarrays are moreeffective than biological display techniques for assembling andscreening large, structurally diverse nonpeptidic compoundlibraries. For example, a microarray approach was used toimprove the binding affinity of a six residue peptoid, to the KIXdomain of transcription coactivator CREB-binding protein(CBP).415

3.4.3. One-Bead-Two-Compound Approach (OBTC). Aneat chemical alternative to the generation and screening ofbicylic peptides by phage display379 is to use the one-bead-two-compound (OBTC) approach, as illustrated by Pei andcolleagues.416 The advantage of OBTC compared to biological

display methods is the control over structural diversity: in factthe one-bead-one-compound approach also works well for thegeneration and screening of nonpeptidic or peptidemimeticcompound libraries.417−419 Where OBTC falls down though(and where phage display really excels) is in the evolutionaryoptimization of active compounds. Nevertheless, the OBTC

approach has been applied with success to identify potentinhibitors of Tumor Necrosis Factor-α (TNF-α), for instanceAnticachexin C1 (53, Figure 37). In this case, a mesitylenelinker was introduced to induce bicycle formation. Noteworthyfor this method is the ability to topologically separate inner andouter regions of each bead through the careful choice ofreaction conditions, which enables the cosynthesis of linear andbicyclic forms of each peptide on the same bead, thus aiding inthe decoding of active compounds by MS analysis.

3.4.4. Fragment-Based Drug Discovery (FBDD). Frag-ment-based drug discovery (FBDD) also referred to asfragment-based lead discovery (FBLD) uses a combination ofbiophysical techniques to detect the weak binding of smallfragment molecules, typically with a molecular weight (MW) <300 Da.93,420−422 The result is a highly efficient screening ofaccessible chemical space of a protein target, which leavessufficient room for the optimization of binding affinity throughchemical modifications. The most success until now has beentargeting enzyme active sites. But, more and more examples arebeginning to appear in the literature of PPI inhibitors

discovered by FBDD, as the field gets to grips with moredifficult and less tractable molecular targets. In this regard, workby Ciulli, Crews and colleagues on the pVHL-HIF-1αinteraction has been highly instructive (section 3.1.3).202

Friberg combined fragment-based methods with a structure-based design approach to discover potent inhibitors of BH3-Mcl-1 (Figure 38).423 An important outcome of this work is thestrong coherency of the SAR data for both the fragment hitsand the merged compounds.In a separate example, FDA-approved drug molecules were

employed as fragment molecules for the structure-based designof novel scaffold structures targeting Bcl-2 and Bcl-x(L)PPIs.424 A pharmacophore model was formulated, based onthe cocrystal structure of BAD BH3 binding to Bcl-x(L), againstwhich 1410 FDA-approved drugs were screened. Whereascompound 57 bound Bcl-2 and Bcl-x(L) weakly (Figure 39),covalent attachment to fragment molecules targeting a proximalbinding site yielded subnanmolar affinity binders of both Bcl-x(L) and Bcl-2. Here the choice of linker was key to theidentification of high affinity binders. Compound 58 wascytotoxic toward H146 and H1417 cancer cell lines in the lownanomolar range. The use of approved drug moelcules ensuresgood pharmacological and toxicological properties early on inthe drug discovery program.

Figure 37. Anticachexin C1 (53), a potent, nonprotein inhibitor ofTumor Necrosis Factor-α (TNF-α), identified by the OBTCapproach.416

Figure 38. Inhibitors of the BH3-Mcl-1 PPI (56) derived fromfragments 54 and 55 using a fragment-based drug discovery andrational structure-guided design approach.423



NMR spectroscopy was used to screen fragment binders ofthe GTPase K-Ras as inhibitors of Soc-mediated and K-Rasactivation and signaling.425 A fragment-based approachcombining ITC, NMR spectroscopic and X-ray crystallographictechniques has also been used to identify novel inhibitors ofBRCA2−RAD51.4263.4.4.1. Natural Product Fragments. The biological

relevance of natural products is equally well serving asinspiration for the design of high quality fragment librariesfor PPI FBDD.427 Fragment libraries are typically dominated bystructurally diverse, yet flat aromatic (sp2-rich) ring structures,which means that the chemical space surveyed by such librariesis relatively conservative. Natural product fragments of a similarsize and molecular weight as regular FBDD fragments (e.g.,commercially available and analogous libraries) by comparisonoccupy an entirely different region of chemical space. Thehypothesis is that just as natural products are consideredstructurally diverse, then so must the fragment molecules thatcollectively make up complex natural products (at leastcompared to regular fragment libraries). Structural diversity isassured by the increase in sp3-hybridized character andstereogenicity of natural product fragments compared toregular fragments. The potential of natural product fragmentlibraries for PPI drug discovery was demonstrated in recentwork by Waldmann and colleagues.427 They used adeconstructive chemoinformatic approach to analyze >180000 library of natural product structures to identify a series offragment molecules targeting p38α MAP kinase and inhibitorsof several phosphatases, which were validated by protein X-raycocrystallography. Amino acids or dipeptides would also appearto fit the mold of natural product fragments, as work on thestructure-based design of small molecule inhibitors of the VHL-HIF-1α PPI would indicate (see section 3.1.3 on peptidomi-metics). In this case, Ciulli and colleagues effectively transforma small yet structurally complex amino acid fragment with weakinitial activity into a potent PPI inhibitor through theintroduction of aromatic side-chain groups.428

3.4.4.2. Fragments Prepared by Diversity-Oriented Syn-thesis (DOS). Aside from biological relevance, anotherimportant criterion for the design of high quality fragment/compound libraries is structural diversity. The structuraldiversity of a compound library is expressed at differentmolecular levels: from the nature of the side chain functionality,to the configurational arrangement of stereogenic centers, andultimately the size and shape of the molecular framework orscaffold around which side chain functionaility is oriented (i.escaffold diversity). Side chain variation has for all intents andpurposes been mastered.429 How to maximize stereochemicaland skeletal variation within a compound library in amolecularly efficient manner has proved to be morechallenging.On the subject of structural diversity, BIOS430 and diversity

oriented synthesis (DOS)431 are similar approaches. Wherethey differ though is in the methodological approach to librarydesign and preparation. While BIOS focuses more on theclassification of scaffold structures of known natural productsand drug molecules as a means to generating guiding priniciplesfor the design of BIOS compound/fragment libraries,327,432,427

DOS aims to generate entirely new non-natural scaffoldstructures, which mimic the structural diversity of natural

products in a molecularly efficient manner.433 The trulyinnovative element of DOS thus lies in the methodologicalprocesses for generating especially skeletal diversity in smallcompound libraries using as few synthetic steps as possi-ble.434−439 Where PPI drug discovery stands to gain from DOSfragment libraries is in the degree of chemical space coveragenot seen in other fragment libraries.440−442 The potential of thisapproach is evident in work by Nelson and colleagues443 on thesynthesis of over eighty different scaffold structures in a singleDOS library (Figure 40).443−447 A number of other examples ofDOS libraries have recently been reported.440,448−452

A number of examples of PPI inhibition using smallmolecules DOS libraries have been reported.453,454 Schreiberand co-workers identified Robotnikin a small molecular binderof Sonic hedgehog (Shh), as modulator of Shh signaling.444

Figure 39. Lead compound 57, derived from the same core scaffold asFDA-approved drugs Lipitor and Celecoxib, and the structure basedesign of potent Bcl-x(L) and Bcl-2 inhibitor 58.424

Figure 40. Generation of over eighty structurally different scaffoldstructures within one compounds library using a diversity-orientedsynthesis (DOS) strategy by Nelson and colleagues using the build/couple/pair DOS strategy.443 Adapted with permission from ref 447.Copyright 2009 Nature Publishing Group.



Other small molecule modulators of Shh signaling resultingfrom DOS compound libraries have recently been disclosed.455

DOS libraries have also been designed targeting theantiapoptotic protein Bcl-x(L)456 DOS is thus an excellentstrategy for maximizing structural diversity within compoundlibraries and is set to play a leading role alongside BIOS indelivering high quality small molecule and fragment libraries forFBDD campaigns.440

3.4.4.3. Target-Guided Fragment Assembly. FBDD is anefficient way to identify weakly active PPI modulators for PPIdrug discovery. An often difficult step in this process though isin the optimization of these weak fragments to make strongerand more selective modulators. An efficient approach whichelegantly rolls interative screening and optimization into onestep is target-guided synthesis, which can be either underkinetic or thermodynamic control.Kinetic Target Guided Synthesis. In this case, small

molecule fragments are assembled in the presence of theprotein target using irreversible bond-forming reactions such asthe in situ 1,3-dipolar cycloaddition reaction between organicazides and alkynes.457,458 Recent applications of this approachinclude to develop inhibitors of mycobacterial transcriptionalregulator, EthR,459 and Abl tyrosine kinase.460 PPIs are

challenging targets for kinetic target-guided synthesis giventheir shallow extended pockets. Manetsch and co-workers claima first proof-of-concept for kinetic target-guided synthesis toPPI drug discovery in work on the template assembly of Bcl-x(L) acylsulfonamide inhibitors derived from fragments bearingthio acid or sulfonyl azide functional groups (so-called sulfo-click chemistry).461

A random library of nine different thio acids and ninedifferent sulfonyl azides were screened in the presence of Bcl-x(L), leading to exclusive formation of sulfonamides SZ4TA2,SZ7TA2, SZ9TA1, and SZ9TA5 (Figure 41) as judged by LC/MS-SIM analysis.462 Control experiments indicated that thetemplate-assembled compounds were the most active of all theeighty-one possible acylsulfonamide combinations. Investiag-tions into the Bcl-x(L) templation effect indicated thatassembly occurred at the BH3 binding site of Bcl-x(L). In arelated approach, semisynthetic epoxy-analogs of the 14-3-3-stabilizing natural product Fusicoccin were subjected to in vitroepoxide ring-opening by thiol-containing pentapeptides,templated by 14-3-3ζ.463

Dynamic Combinatorial Chemistry. The second target-guided strategy, termed dynamic combinatorial chemistry,works under thermodynamic control.464,465 Compound libra-ries formed under dynamic reversible conditions respond tochanges in the free energy of the system, for example onaddition of a template, through either amplification orabbreviation of the individual components. In protein-templated synthesis, the protein is intimately involved in theDarwinian assembly of its own best binder from a structurallydiverse pool of fragment molecules facilitated by reversiblechemical transformations.465 Just as for kinetic target-guidedsynthesis, dynamic combinatorial library (DCL) synthesis is auseful method for targeting enzymatic active sites. Methodo-logical breakthroughs have brought less tractable targets such asPPIs within reach. Greaney, Campopiano and colleaguesrecently demonstrated the use of aniline-catalysis for thegeneration of a DCL of acylhydrazones targeting two isozymesof glutathione S-transferase (GST).466 Crucial to the success ofthis approach was the ability of the DCL to reach equilibrationwithin a reasonable time frame (<6 h) and under conditionscompatible with the protein target. The same group lateremployed a DCL approach to identify bivalent inhibitors ofhomodimeric GST.467 This work indicates that DCLs capableof probing a larger protein surface area (in the latter casespanning two 28 kDa protein monomers) are also capable ofaddressing challenging molecular targets such as PPIs.

3.4.5. In Silico Screening. In silico screening has proved tobe a highly effective way to identify active PPI modulators, thuseliminating the waste associated with conventional screen-ing.87,468−470 A virtual screening-based approach was recentlyused to identify novel inhibitors of viral HIV-1 integrase (IN)binding to Lens epithelium-derived growth factor (LEDGF-p75). LEDGF-p75 is a cellular growth factor, which promotesviral integration through tethering of the viral preintegrationcomplex (PIC) to cellular chromatin.471,472 Small moleculeinhibitors of LEDGF/p75-IN are potential second-generationantivirals, which operate via allosteric interference HIVintegration and replication, compared to first generationantivirals, such as raltegravir, which target the catalytic activityof IN and other viral enzymes. Pharmacophore-based filtering,guided by the cocrystal structure of LEDGF-p75 bound toHIV-1 IN, effectively whittled a 200 000-sized commercialcompound library down to 25 candidate molecules, of whichone, 59 (Figure 42), achieved an encouraging 36% inhibition at100 μM in an AlphaScreen assay. Rational optimization of thishit compound yielded 2-(quinolin-3-yl)acetic acid derivative 60(Figure 42), which achieved a much improved IC50 =12.2 μMin the same AlphaScreen and promising antiviral activity (EC50= 41.9 μM). Importantly, though, the cocrystal structure of 60bound to the IN core domain helped to lower the activity of

Figure 41. Kinetic target-guided synthesis of small molecule inhibitorsof the Bcl-x(L)-BH3 using a sulfo-click chemistry reaction betweenfragments bearing sulfonyl azide and thio acid functional groups.462



this compound class even further leading to analog 70 (Figure42), with an IC50 = 1.4 μM in the AlphaScreen and a 10-foldimprovement in antiviral activity compared to 60. Analog 61also showed selectivity for LEDGF-p75-IN over other LEDGF-p75-mediated PPIs, did not interefere with integrase-DNAbinding, and exhibited only weak inhibition of the catalyticactivity of integrase. X-ray cocrystallograhic analysis showed thephenyl, carboxylic acid and chlorine functions of compound 61to be a good mimic of amino acid residues Ile365, Asp366 andLeu368 of the LEDGF-p75 IBD, respectively (Figure 42)The LEDGINS represent a novel class of antivirals, which

operate via an allosteric inhibitory mechanism, and theirdiscovery is a validation of the in silico screening approach forPPI drug discovery. A virtual screening-based approach was also

used to identify low MW inhibitors of the interferon-α (IFN-α)-IFNAR PPI. In this case, small molecule binding to IFN-αwas validated using NMR and SPR measurements.470 Smallmolecule inhibitors of 14-3-3-dependent PPIs have also beendiscovered with the help of virtual screening.53,473

3.4.6. Multicomponent Reactions (MCRs). Multicompo-nent reactions (MCRs) are an economic way to generatestructurally diverse compound libraries which occupy achemical space (MCR chemical space) distinct from thechemical space populated by BIOS, DOS, and commericalcompound libraries.474 The principle of MCRs is straightfor-ward: three or more simple building blocks are reacted in thesame vessel to produce a single more complex product in whichnearly all of the atoms are incorporated into the final product.Appendage group diversity is achieved through simple variationof the MCR building blocks. Scaffold diversity can be generatedby reacting the MCR products further using functional groupswhich are chemically orthogonal to the MCR process.474

Methodologically related to DOS, MCRs enable rapid access tostructurally diverse and biologically relevant scaffold structuresin one or two reaction steps, as well as the straighforwardchemical optimization of ‘hit’ compounds. Domling andcolleagues employ MCRs in the development of novelinhibitors of the p53-MDM2 PPI.475−477 In one notableexample,477 seven different scaffolds classes were identified via aprocess of anchor generation, virtual screening, molecularmodeling and compound library synthesis using MCRs. Anumber of other PPI targets have been explored using a MCR-based approach, including XIAP,478 Bcl-w/Bak,479 VEGF-neurophilin-1,480 and RGD-integrin,481 which are discussed inmore detail in a very recent and comprehensive reviewdedicated to the broader chemical biology applications ofMCRs.474

Figure 42. Inhibitors of LEDGF-p75-HIV-1 IN (LEDGINs), 59−61,identified using a virtual screening-based approach. Inset: X-raycocrystal structure of 2-(quinolin-3-yl)acetic acid derivative 61, boundto the integrase core domain: carbon (yellow), oxygen (red), nitrogen(blue), and chlorine (green).471 Adapted with permission from ref 471.Copyright 2010 Nature Publishing Group.

Figure 43. Immunosuppressants Cyclosporin A (CsA) and FK506 bound to their target proteins, the ternary complexes of the catalytic subunit(CnA) and the regulatory subunit of Calcineurin (CnB) plus either Cyclophilin (CyPA, in the case of CsA, PDB ID: 1M63) or FKPB12 (in the caseof FK506, PDB ID: 1TCO), respectively.



4. STABILIZATION OF PPIs

Compared to current efforts to develop inhibitors of PPIs, theopposite strategy of employing small-molecule stabilizers ofPPIs is strongly underrepresented in chemical biology and drugdiscovery. However, especially in the past few years, a numberof impressive examples were published where this generalstrategy led to molecules with this mode of action. Startingfrom the classical examples of the immunosuppressive naturalproducts Cyclosporin A, FK506, and Rapamycin, as well as theimportant cell biology tools Brefeldin A and Forskolin, we willdiscuss significantly less complex molecules like Inositoltriphosphate and the phytohormones Auxin, Jasmonat, andBrassinolide. Subsequently, we are highlighting molecules thatoriginated from drug development projects and that eitherstabilize a homo-oligomeric state of their target protein(Tafamidis, Phenanthoziane, and nucleoprotein dimerizers) orindeed stabilize a regulatory protein complex like the 1 EBIOclass of channel function potentiators, HDM2-HDMXinactivators, or inverse agonists of nuclear receptors. Finallywe will discuss stabilizers of a special class of adapter proteins:the 14-3-3 proteins. Here, not only is the fusicoccanes family ofnatural product described, as nature’s example of moleculesstabilizing 14-3-3 PPIs, but also molecules discovered by high-throughput screening. For the selection of molecules for thischapter the most important guide was the existence of clearstructural evidence for their proposed mode of actions, typicallya well-resolved crystal structure of the corresponding proteinpartners in complex with the small molecule.

4.1. Cyclosporin A, FK506 and Rapamycin

The prototypical natural products that confer their activity byPPI stabilization are the immunosuppressive molecules Cyclo-sporin A (CsA), FK506, and Rapamycin (Rp). CsA, ametabolite produced by the ascomycete Tolypocladiuminf latum, was identified from a Norwegian soil sample collectedin 1970482 and was shown by researchers of Sandoz to displayimmunosuppressive properties.483 The first patients weretreated with CsA in 1980484 and the drug was approved bythe FDA in 1983.482 The primary CsA-binding protein wasidentified in 1984 and was named Cyclophilin A (CyPA).485 Itscrystal structure in complex with a model tetrapeptide (N-acetyl-AAPA-amidomethylcoumarin) was solved in 1991486

followed by the structure in complex with CsA in 1994.487 CsAoccupies the active site of the peptidyl-prolyl isomerase CyPAand hence inhibits its enzymatic activity. Since inhibition of thepeptidyl-prolyl isomerase activity alone cannot explain thephysiological effects of CsA a key step to elucidate its truemolecular mechanism was the finding that the CyPA-CsAcomplex binds to and inhibits the phosphatase Calcineurin(Cn).488−490

The crystal structure of the ternary Cn-CyPA-CsA complexfinally revealed the mechanistic details of how CsA inhibits theenzymatic activity of Cn.491 Here, the CyPA-CsA complexbinds to the composite surface formed by the catalytic (CnA)and the regulatory (CnB) subunit of Cn (Figure 43). Thestructure can unambiguously explain how CsA inhibits thecatalytic activity of Cn by physically blocking access to theactive site of the phosphatase. Furthermore, a direct contact ofArg148 of CyPA with the catalytically important Arg122 ofCnA observed in the crystal structure might add to theinhibiting activity of CsA.491 The binding site for CsA iscomposed of a number of hydrophobic side chains contributedby the catalytic (CnA, Figure 43, marine) and the regulatory

(CnB, Figure 43, slate) subunits of Cn as well as CyPA (Figure43, green). Several polar contacts contribute to the interaction,for instance between CyPA Arg55 and MeLeu10 of CsA as wellas CnA Tyr341 and CsA Ala7. Importantly, only very fewresidues are found directly establishing contacts between theproteins: CyPA residues, Glu81, Lys82, Arg69 and Arg148 arecontacting Gln50, Glu359 and Arg122 of CnB (Figure 44).This qualifies CsA as a main contributor to the induction andstability of this remarkable protein−protein complex.The discovery of FK-506 as an immunosuppressive agent

produced by Streptomyces tsukubaensis was reported by scientistfrom Fujisawa Pharmaceutical in 1987.492 Two years later, theprimary protein receptor of FK-506 was identified (FKBP,FK506 Binding Protein) and was shown to be a peptidyl-prolylisomerase, similar but not identical to the Cyclosporin Abinding protein Cyclophilin.493 The crystal structure of theFKBP-FK506 complex was published in 1991.494 In the sameyear, Calcineurin was reported to be the common target for thebinary CyPACsA and the FKBP-FK506 complexes.488 Finally,researchers at Vertex published the crystal structure of theternary complex consisting of Cn, FKBP and FK-506 in1995.495 This structure showed how the FK506-FKBP12complex binds to Cn. Interestingly, FK506 is situated 25 Åaway from the active site of the phosphatase and thereforecannot directly inhibit the catalytic activity and even the morebulkier form of FKBP12 itself is not directly interacting withcatalytic residues of Cn. This observation agrees with thefinding that the phosphatase activity of Cn toward a small-molecule substrate (p-nitrophenyl phosphate) is not inhibitedby the FK506-FKBP12 complex.488 Instead, FK506-FKBP12inhibits Cn by physically hindering the macromolecular,peptide substrates to reach the active site.

Figure 44. Endogenous metabolite inositol tetraphosphate bound toits target proteins, the binary complex of the histone deacetlyaseHDAC3 and its activator SMRT. In the upper part, the binding pocketis shown in surface representation, the lower part displays in detail theinvolvement of single residues from HDAC3 (blue) and SMRT(green) in inositol tetraphosphate binding (PDB ID: 4A69).



The actual binding pocket of FK506 consists of a number ofhydrophobic side chains from the catalytic (CnA, Trp352, andPhe356) and the regulatory (CnB, Leu115, Met118, andLeu123) subunit of calcineurin, as well as from FKBP12(Phe46, Val55, Ile56, and Trp59). Polar contacts can be foundbetween the C13-OMe and C15-OMe of FK506 and the side-chain nitrogen of Trp352 from CnA, as well as the C1-, C8-,C9-, and C10-OHs and Tyr82, Ile56, and Asp37 from FKBP12.Rapamycin was initially described in 1975 as a an antifungal

metabolite from Streptomyces hygroscopicus isolated from a soilprobe from an Easter Island (Rapa Nui)496 and was foundshortly later to have inhibitory activity toward the mammalianimmune system.497 In the late 1980s/early 1990s it wasdiscovered that Rapamycin binds to the same protein asFK506498 but that the physiologically relevant protein target isa protein kinase,499 mammalian target of rapamycin (mTOR),which is inhibited upon binding of the Rapamycin-FKBPcomplex. The structural details the stabilizing activity ofRapamycin toward the FKBP-mTOR complex were revealedby the crystal structure of the ternary complex in 1996.500

Rapamycin was approved by the FDA in 1999 under its brandname Rapamune (Wyeth, now Pfizer).

4.2. Forskolin and Brefeldin A

Forskolin was isolated from Coleus forskohlii in 1977 byresearchers from Hoechst as a cardioactive and blood-pressurelowering compound.501 A few years later (1981), Forskolin wasshown to confer its effect by activating membrane-boundadenylyl cyclase leading to an increase of the second messengermolecule cAMP.502 Adenyly cyclase is a transmembraneenzyme with two cytoplasmic domains, C1 and C2503 andthe binding affinity between these domains is increased byForskolin.504 In 1997, two crystal structures revealed howForskolin bound to the dimer interface505 and promotes theformation of an active complex including its stimulatoryheterotrimeric G protein subunit Gsα.

506 These structuresshow how Forskolin binds to a composite, strongly hydro-phobic pocket that is formed at the rim of the interface of IIC2and VC1506 or the C2 homodimer.505

Brefeldin A (BFA) is a fungal metabolite isolated fromEupenicillium brefeldianum that disrupts protein secretion. Itfunctions by inhibiting golgi function through stabilization ofthe interaction of the small G protein ARF1 with its guanineexchange factor ARF-GEF.507 This activity gained the moleculea high popularity as a cell biology tool for the study ofmembrane trafficking.508 The structural basis of BFA action onthe ARf1-ARF-GEF complex was revealed in 2003 with thepublication of the crystal structure of BFA in complex withARF1 and the SEC7 domain of the human ARF-GEFARNO509 or the yeast ARF-GEF Gea1.510

4.3. Inositol Tetraphosphate

Histone acetylation is an important mechanism for theepigenetic control of gene expression. Here, acetylation oflysines in the tails of histones leads to chromatin opening andenabling gene expression; deacetylation of these lysines resultsin chromatin condensation and consequently the shutdown ofgene transcription. Since epigenetic control plays a role in manydiseases, among them many cancers, pharmacological modu-lation of the respective enzymes, HAT (Histone acetylases) andHDACs (Histone deacetylases), is of interest. In particular,several HDACs are involved in repressing (“silencing”) thetranscription of tumor suppressor genes likes p21 and currently2 inhibitors of this class of enzymes are approved (Vorinostat,

Zolinza; Romidepsin, Istodax) and more than further 10 are inlate clinical testing.511,512

HDAC1, 2, and 3 achieve only full enzymatic activity whencomplexed with corepressors. For example, HDAC3 needs tobind to SMRT via its deacetylase activating domain (DAD) tobe fully active. Recently, the crystal structure of the HDAC3-SMRT-DAD complex, expressed in and purified from HumanEmbryonic Kidney cells (HEK293), was solved.513 To theirsurprise, Watson and colleagues noticed unaccounted electrondensity between HDAC3 and SMRT-DAD. Fortunately, thequality of the density (2.1 Å resolution) was sufficiently high toidentify Inositol tetraphosphate (IP4) as the small moleculesandwiched between the two proteins. IP4 is accommodated ina highly basic pocket concomitantly built by HDAC3 andSMRT-DAD and makes extensive contacts to both proteins(Figure 44). There are each five hydrogen bonds or salt bridgesformed between IP4 and HDAC3 (His 17, Gly 21, Lys 25, Arg265, and Arg 301) and SMRT-DAD (Lys 449, Tyr 470, Tyr471, Lys 474, and Lys 475). Similar to Cyclosporin A, FK506and Rapamycin, but different from Brefeldin A, Forskolin andFusicoccin, IP4 seems to be essential for the interaction of thepartner proteins in the complex.In the absence of IP4, the numerous basic residues on either

side of the binding pocket would most probably preventcomplex formation due to charge repulsion. This assumption issupported by mutational studies of residues lining the bindingpocket of the ligand and the fact that a stable complex couldnot be obtained in the absence of sufficient concentrations ofIP4. Since this molecule is an endogenous metabolite ineukaryotic cells and has furthermore been implicated inregulation of gene expression and chromatin remodeling it isvery likely that this molecule represents a means ofphysiological regulation that − together with the examples ofthe phytohormones Auxin and Jasmonate employs the“molecular glue” mode-of-action of stabilizing an importantregulatory PPIs.

4.4. Phytohormones Auxin, Jasmonate, and Brassinolide

4.4.1. Auxin. Auxin (indole-3-acetic acid, IAA) is animportant plant hormone that plays pivotal roles in plantgrowth and developmental processes.514 How this hormone isperceived and confers its manifold physiological functions wasan unsolved issue in plant biology for many years. In the early2000s it became clear that a protein degradation system isinvolved in sensing and transducing Auxin signaling toward theactivation of transcriptional programmes.515 Here, the ubiq-uitin-ligase complex, SCFTIR1, promotes the ubiquitin-dependent proteolysis of a family of transcriptional regulatorsknown as Aux/IAAs in an Auxin-dependent manner.516

Proteolytic degradation of the Aux/IAA proteins activates afamily of transcription factors, the Auxin responsive factors(ARFs), that are in the nonstimulated state inhibited by theAux/IAA proteins.517 It has been shown that the TIR1component of the SCFTIR1 complex is directly bindingAuxin which promotes the interaction of SCFTIR1 with Aux/IAAs.518,519 The exact molecular mechanism behind thismediation was clarified in 2007 when the crystal structure ofTIR1-Aux/IAA-Auxin complex was reported.520 The structureshowed that the leucine-rich repeat (LRR) region of TIR1 isharboring the binding pocket for Auxin as well as represents thedocking surface for the Aux/IAA recognition motif. Deep in thepocket Auxin is accommodated and immediately above thebound Auxin molecule the highly coiled Aux/IAA peptide is



docked and completely covers the plant hormone. In this way,Auxin actually contributes significantly to the binding surface ofthe Aux/IAA peptide and enhances the interaction betweenTIR1 and Aux/IAA which ultimately leads to the degradation ofAux/IAA and the liberation of ARF transcription factors.4.4.2. Jasmonate. The phytohormone Jasmonate is

implicated in the regulation of a plethora of processes inplant physiology, growth, development, and defense againstpathogens.521 The bioactive entity of Jasmonate is a conjugatewith the amino acid L-isoleucine (Ile), resulting in (3R,7S)-jasmonoyl-L-isoleucine (JA-Ile).522 The activity of JA-Ile ismimicked by the phytotoxin Coronatine that is produced bythe pathogen Pseudomonas syringae.523 The search forCoronatine-resistant mutants identified the protein COI1(Coronatine insensitive 1) as a key element in the Jasmonatesignal perception.524 Similar to TIR1 of the Auxin recep-

tor518,519 COI1 is a F-box protein that recruits proteinsubstrates to the SCF ubiquitin ligase with members of theJAZ (JAsmonate ZIM domain) family of transcriptionalrepressor discovered as the direct binding partners ofCOI1.525 In the absence of Jasmonate JAZ proteins inactivateMYC2, a transcriptional regulator that binds to promotors ofJasmonate-responsive genes and activates their expression.526

Proteolytic degradation of JAZ initiated by ubiquitinationthrough the SCFCOI1 complex relieves repression of MYC2resulting in expression of Jasmonate target genes.527 In 2010,Sheard et al. reported the crystal structure of the COI1-JAZ1-JA-Ile complex (Figure 45).528 Although the overall fold of thecomplex is similar to that of the TIR1-Aux/IAA-auxincomplex,520 not surprisingly the architecture of the ligandbinding pocket shows features specific for the accommodation

of JA-Ile. Whereas Auxin engages also main-chain contacts, JA-Ile exclusively contacts side chains of its protein receptor.The keto group of the cyclopentanone ring of JA-Ile is

forming hydrogen bonds with Tyr444 and Arg496 whereas therest of this ring is sandwiched between Phe89 and thehydrocarbon moeity of the Tyr444 side chain (Figure 45). Thepentenyl side chain of JA-Ile is accommodated in a hydro-phobic pocket formed by Ala86, Phe89, Leu91, Leu469, andTrp519. Deep in the pocket the carboxyl group forms a saltbridge with Arg348 and Arg409, the amide establishes a contactto Tyr386 and the carbonyl to Arg85. Very importantly, alsodirect contacts are observed between JA-Ile and JAZ peptide.Here, Ala204 donates a hydrogen bond from its backboneamide to the keto group of JA-Ile. This residue also packs withits side chain between the keto group and Phe89 of COI1. Theguanidinium group of Arg206 of JAZ points deep into theJasmonate binding pocket and interacts directly with thecarboxyl group of JA-Ile. Finally, an important, albeit indirect,contact is formed via a hydrogen bond between the backbonecarbonyl of Pro 202 in JAZ1 and the ligand-interacting COI1residue Arg 496.

4.4.3. Brassinolide. Brassinosteroids are a class ofimportant plant hormones that direct many processes inplant development.529 One of the genes involved in perceptionand transduction of brassinosteroid signaling encodes theleucine-rich repeat receptor (LRR) kinase BRI1 (BRassinoste-roid Insensitive 1).530 In contrast to animal steroid receptorsthat are predominantly localized in the cytosol and thenucleus,531 BRI1 and other receptors for brassinosteroids are

Figure 45. Plant hormone Jasmonate bound to its target proteins, thebinary complex of the ubiquitination complex protein COI1 and thetranscriptional repressor JAZ1. In the upper part, the binding pocket isshown in transparent surface representation and cartoon, the lowerpart displays in detail the involvement of single residues from COI1(green) and JAZ1 (blue) in Jasmonate binding (PDB ID: 3OGL).

Figure 46. Plant hormone Brassinolide bound to its target proteins,the binary complex of the LRR receptor kinase BRI1 and the somaticembryogenesis receptor kinase SERK1. In the upper part, the bindingpocket is shown in transparent and solid surface representation, thelower part displays in detail the involvement of single residues fromBRI1 (green) and SERK1 (blue) in Brassinolide binding (PDB ID:4LSX).



found at the plasma membrane or endosomes.532 The somaticembryogenesis receptor kinase (SERK) is a genetic componentin brassinosteroid signaling,533 has been shown to directly bindto BRI1 with these two kinases transphosphorylating each otherin a brassinosteroid-dependent manner.534 This then fullyactivated receptor triggers multiple downstream events thatlead to major changes in gene expression profiles.529,535

How brassinosteroids act on this system in mechanistic detailhas been shown very recently by elucidation of the crystalstructure of BRI1 and SERK1 in complex with thephysiologically most active brassinosteroid, Brassinolide(BL).536 BL binds in a hydrophobic groove at the innersurface of the LRR of BRI1 (Figure 46). Residues lining thisrelatively shallow canyon are Trp564, Tyr597, Tyr599, Tyr642,Ser647, Met657, Phe681, and Thr729. Phe61 of SERK1 stacksagainst ring C of the BL thereby establishing an importanthydrophobic anchoring point for this residue that in theuncomplexed structure of SERK1 is not visible due todisorder.536 Furthermore, His62 of SERK1 is engaging theplant hormone via main-chain as well as side-chain hydrogenbonds to the diol moiety of BL, nicely explaining the crucialroles of the 2α and 3α hydroxyl groups.537 A recently reported2,3-acetonide derivative of BL was shown to be a brassinoste-roid pathway antagonist.538 This 2,3-acetonide BL might bindto BRI1 but is most probably no longer able to mediate andstabilize the interaction with SRK1 which would result indisruption of the pathway.

4.5. Small-Molecule Stabilizers of Oligomeric-StateHomocomplexes

4.5.1. Tafamidis. Amyloid diseases are believed to becaused by the extracellular accumulation of protein depositsaggregates, the so-called amyloid fibrils.539 The transthyretinamyloidoses (ATTR) are characterized by progressive neuro-or cardiomyopathy and are fatal within 10 years of onset.540,541

ATTR are caused by aggregation of transthyretin (TTR) that inits functional, physiological form is a tetramer involved in thetransport of thyroxine and the vitamin A-retinol bindingcomplex.542,543 Dissociation of the TTR tetramer into dimersand monomers, misfolding of the monomers, misassembly intosoluble oligomers, and aggregation have been implicated inATTR pathogenesis.544 A substantial number of smallmolecules that stabilize the TTR tetramer have beenreported.545−561 One of them, Tafamidis, was developed byresearchers at Pfizer and recently shown to bind to the twonormally unoccupied thyroxine binding sites in the TTRtetramer, to kinetically stabilize TTR and to inhibit the amyloidcascade.562 The crystal structure of Tafamidis was published in2012562 and showed the molecule binding to a symmetricpocket in the dimer interface (Figure 47). The 2,3-dichlorosubstituents are accommodated by the two symmetric halogenbinding sites of the thyroxine pocket formed by the hydrophicenvironment of Ala108, Leu110, and Thr112. The carboxylsubstituent of the benzoxazole is engaged in water-mediated H-bonds with Lys15 and Glu54 of both TTR protomers. Thissimultaneous, symmetric binding of Tafamidis to this interfacepocket therefore explains the stabilizing activity of thecompound toward the TTR dimer. The authors used exchangeexperiments with unlabeled versus FLAG-tagged TTRtetramers to show that Tafamidis indeed stabilized TTRtetramers in solution by substantially decreasing dissociationkinetics with no exchange of monomers after 96 h.562

Importantly, Tafamidis not only stabilizes wild-type TTR but

also the clinically important mutated forms V30 M and V122Iand a range of further, regularly appearing mutations found inATTR.562 A phase II/phase III-clinical trial with patientssuffering from ATTR caused by the V30 M version of TTRshowed the efficacy of Tafamidis with patients displaying 52%less neurologic impairment, a 80%-preservation of small nerve-fiber function and an overall improved physiological status.563

4.5.2. Phenothiazines. The S100 proteins were namedafter their ability to stay soluble in 100% ammonium sulfate.564

They harbor two Ca2+-binding loops and undergo a substantialconformational change upon calcium-binding exposing ahydrophobic interaction surface for their partner proteins.565

Increased expression of individual members of the S100 proteinfamily is correlated with a number of human diseases, amongthem cancer, neurodegeneration and inflammation.566 Forexample, increased levels of S100A4 is associated with a highincidence of metastasis and an overall poor prognosis indifferent cancers.567 Furthermore, high S100A4 expressioncontributes to rheumatoid arthritis, kidney fibrosis, and cardiachypertrophy.568,569 In light of these important pathogenicactivities of S100A4, the group of Brenick reported in 2008 theidentification of several phenothiazines that blocked the activityof S100A4.570 One of these compounds, Trifluoroperazine(TFP), was later shown in crystallographic and biochemicalstudies to inhibit S100A4 function by stabilizing an inactive

Figure 47. Antiamyloidosis agent Tafamidis bound to its targetproteins, the dimeric complex of transthyretin. In the upper part, thebinding pocket is shown in surface representation, the lower partdisplays in detail the involvement of single residues from protomer 1(green) and protomer 2 (blue) of transthyretin in Tafamidis binding(PDB ID: 3TCT).



pentamer.571 Backing the relevance of the crystallographicoligomer the occurrence of the pentamer in the presence ofTFP was corroborated by analytical sedimentation as well ascross-linking studies.In the presence of TFP S100A4 crystallized as a pentamer

with two copies of the molecule sitting adjacent to each otherin the interface between two protomers (Figure 48). Thetrifluoro-substituted phenothiazine moiety of TFP is bound to ahydrophobic patch comprising the side chains of Ile82, Met85,and Cys86, from one protomer and Phe89 as well as Phe93from the other. The methylated piperazine ring establishesfurther contacts with the side chains of Ser44, Phe45, Leu46and Gly47 and additionally one potential hydrogen bond isformed between the carbonyl oxygens of Phe45 of theS100A4_A protomer and N3 of the piperazine ring.Importantly, the authors of this study could show that thepentameric state of S100A4 is also induced and stabilized insolution, presenting another nice example of small moleculethat inhibits the function of a proto-pathogenic protein bystabilizing an inactive oligomeric state.4.5.3. Influenza Nucleoprotein (NP). Influenza nucleo-

protein (NP) is an essential viral protein that is involved in viralreplication, ribonucleoprotein (RNP) formation, vRNase trans-port to the nucleus, and virion assembly.572−574 Consequently,inhibitors of NP function can be expected to display antiviralactivity. Recently, Kao et al. reported the identification of

nucleozin, an isoxazole compound with antiviral activity.575

Similar results were reported elsewhere.576 One year later, arelated compound (Compound 3) identified from a HTS atBristol-Myers Squibb was shown by protein crystallography tobind in the interface of a NP oligomer (Figure 49). Induction ofsuch higher-order NP oligomers by Compound 3 in solutionwas shown by dynamic light scattering (DLS). In the crystalstructure, two copies of Compound 3 bridge two NP protomersand thus stabilizes the interface of this dimer.Tyr289, Phe291, Tyr296, and Leu306 from NP_A together

with Tyr52 and Tyr313 from NP_B build an intermonomerhydrophobic pocket that accommodates the ligand. BetweenTyr289 of NP_A and the nitro-aryl moiety of Compound 3 aface-to-face π-stacking can be observed. Tyr52 of NP_Bestablishes a hydrophobic interaction with the piperazinemoiety of Compound 3. The side-chain OH of Ser376 ofNP_B makes a hydrogen bond with the amide carbonyl of theligand and the γ-nitrogen of Arg99 from NP_B establishes anelectrostatic interaction with the phenolic oxygen of Com-pound 3. Finally, the main-chain nitrogen of Glu53 makes apolar contact with the oxazole ring oxygen of Compound 3.

4.5.4. MDMX Homodimer Stabilizers. Enhancement ofp53 activity by blocking p53 binding to its negative regulatorMDM2 is a feasible approach in the development of novelanticancer agents for tumors displaying wild type p53background. This strategy is currently exploited by thedevelopment of small-molecule inhibitors of the p53-MDM2complex that are effective in cells overexpressing MDM2(Nutlins etc.). However, in cancer cells with normal levels ofMDM2, p53 inactivation seems to be performed by MDMXand p53-MDM2 disruptors show only marginal efficiency inthese cancers. Hence, the simultaneous inhibition of both

Figure 48. S100A4 inhibitor Trifluoroperazine bound to its proteintarget, the pentameric complex of S100A4. For reasons of clarity, onlytwo adjacent S100A4 monomers and their contact interface are shown.In the upper part, the binding pocket is depicted in surfacerepresentation, the lower part displays in detail the involvement ofsingle residues from protomer 1 (green) and protomer 2 (blue) ofS100A4 in Trifluoroperazine binding (PDB ID: 3KO0).

Figure 49. Influenza nucleoprotein (NP) inhibitor Compound 3bound to its protein target, the dimeric complex of NP. In the upperpart, the binding pocket is depicted in surface representation, thelower part displays in detail the involvement of single residues fromprotomer 1 (green) and protomer 2 (blue) of NP in Compound 3binding (PDB ID: 3RO5).



MDM2 and MDMX binding to p53 would be desirable.Researchers at Roche screened for such dual-activity inhibitors

and identified a series of indolyl-hydantoin compounds thateffectively inhibited the interaction of both proteins withp53.577

Surprisingly, biochemical experiments and the cocrystalstructure of MDMX or MDM2 in complex with one of thesecompounds, RO-2443, revealed that this molecule was bindingin a 1:1 stoichiometry to the interface of the homodimer andcontacted both protomers simultaneously. The crystal structureof MDM2 in complex with a p53-derived peptide showed thatthis PPI is mainly mediated by the accommodation of threehydrophobic side chains from p53 (Phe19, Trp23, and Leu26)to subpockets in a groove of MDM2 (Figure 50).120 RO-2443occupies two of these sites “in trans” with the indolyl-hydantoinmoiety occupying the Phe pocket (lined by Ile60, Met61, andVal92) of one of the protomers and the difluoro phenyl the Trppocket (lined by Met53, Leu56, Phe90, and Leu98) of the otherprotomer. Very importantly, a substantial portion of the contactsurface is provided by the mutual, antiparallel stacking of thedrug molecules. By size-exclusion chromatography with staticlight scattering (SEC-SLS) the dimer-inducing activity of RO-2443 was confirmed also in solution.577

4.5.5. Topoisomerase II/ICRF-187. Anthracyclines likeDoxorubicin are among the most effective chemotherapy agentsin leukemias and solid tumors.578 However, their use isrestricted due to cardiotoxic effects during treatment.579

Cardiotoxicity is believed to be caused by the development ofreactive oxygen species in the presence of anthracyclines andcellular Fe-ions.580 The bisdioxypiperazine ICRF-187 (Dexra-

zoxane) is an approved cardioactive protectant that is able toact as a chelator of free Fe-ions when converted in its metabolicproduct ADR-295.581 ICRF-187 is also an inhibitor of

topoisomerase II (topo II) by blocking its turnover througharrest of the dimeric state582 and its cardioprotective andantitumor activities could be explained based on this activity.583

The structural basis for the activity of ICRF-187 action on thetopo II dimer was elucidated in 2003 with the S. cerevisiaenzyme.584 In this study it was shown how ICRF-187 stabilizeda nucleotide-bound, dimeric state of the enzyme by bindingsimultaneously to both promoters and bridging the dimerinterface.ICRF-187 binds to a 350-Å3 cavity that comprises in total 14

residues with each of the subunits contributing 7 amino acids(Figure 51). Similar to the binding mode of Fusicoccin,Forskolin and Brefeldin A, binding of ICRF-187 does notsignificantly change the position and orientation of the 14residues directly involved in binding of the drug molecule(rmsd = 0.302 Å over all atoms).584 A considerable entropicbenefit is suggested by the fact that six of the eight coordinatedwater molecules in the binding pocket are displaced uponaccommodation of ICRF-187. The two piperazinedione ringsare each surrounded by a, as the authors stated it, “tyrosine-dome” whose cealings and walls are built by Tyr144/Tyr144′and Tyr28/Tyr28′ (Figure 51). The two cyclic N-acyl amidesof the piperazinedione rings engage each the side-chaincarbonyl oxygens of Gln365 and Gln365′ in hydrogen bonds(Figure 51). In addition to nicely explaining the mechanistic

Figure 50. MDMX inhibitor RO-2443 bound to its protein target, theMDMX homodimer. In the upper part, the binding pocket is depictedin surface representation, the lower part displays in detail theinvolvement of single residues from protomer 1 (blue) and protomer 2(green) of NP in RO-2443 binding (PDB ID: 3U15).

Figure 51. Topoisomerase II inhibitor ICRF-187 bound to its proteintarget, the Topoisomerase II homodimer. In the upper part, thebinding pocket is depicted in transparent surface and cartoonrepresentation, the lower part displays in detail the involvement ofsingle residues from protomer 1 (blue) and protomer 2 (green) ofTopoisomerase II in ICRF-187 binding (PDB ID: 1QZR).



basis of ICRF-187 action and the observations of a number ofICRF-187-resistant mutants, this structure also identified twounoccupied subpockets in the binding site of the drug moleculethat could be used for the rational design of some novel topo IIdimerizing molecules.4.5.6. 1-EBIO Class Stabilizers of SK-Channel-CaM

Interaction. Ca2+-activated potassium channels like the smallconductance K+ channel (SK) play important roles in thenervous and cardiovascular system.585−587 Calmodulin (CaM)binds to SK and functions as a high-affinity calcium sensorleading to opening of the channel when occupied by Ca2+.588

Since impairment of SK channel function seems to contributeto hypertension,587 potentiation of SK channel activity might be

neuroprotective589 and SK channels have been implicated incancer development. Significant efforts have been directed tothe development of small molecules that target SKchannels.586−590 The 1-EBIO (1-ethyl-2-benzimidazolinone)class of compounds potentiate SK channel activity591 canexperimentally reduce seizure incidence,592 enhance survival ofhippocampal neurons after cerebral ischemia,593 and can lowerblood pressure.594

In 2012, Zhang and co-workers reported the crystal structureof 1-EBIO-class compounds binding to the interface of theCalmodulin-binding domain (CaMBD) of the SK2 channel andCaM (Figure 52).595 The binding pocket of 1-EBIO iscomprised of A477, L480, and V481 of CaMBD2-a, and F19,I27, L32, M51, I52, V55, I63, F68, M71, and M72 of CaM(Figure 52). Compared with the binary SK2-CaMBD-CaMstructure (PDB ID: 1G4Y), coordination of 1-EBIO results in

rearrangement of some side-chains in this pocket. Mostimportantly M71 of CaM loses its contact to Ala77 ofCaMBD and Met51 of CaM undergoes a slight rotationalmovement to accommodate 1-EBIO.Whereas 1-EBIO itself is at best a very weak stabilizer of the

SK2-CaMBD/CaM interaction, the situation seems differentwith NS309, the most potent member of the 1-EBIO class ofmolecules (EC50 1-EBIO = 395 μM; EC50 NS309 = 0.44μM).595 Here, molecular docking calculations predicted abinding mode slightly different from 1-EBIO,595 possiblyexplaining the facilitation of the assembly of CaM and SKchannels by stabilizing this PPI.596

4.5.7. RAR-NCoR. Several nuclear receptors (NRs) areinvolved in both activation and repression of target-genetranscription. The retinoic acid receptor (RAR) for exampleconveys its physiological functions in the control of develop-ment, reproduction and homeostasis by employing bothroles.597 Inhibition of gene function is mediated by therecruitment of the corepressors SMRT (Silencing mediatorfor retinoid and thyroid hormone receptors) and NCoR

(Nuclear receptor corepressor) and activation necessitatesagonist-driven interaction with coactivators.598,599 Inverseagonists can promote the interaction with corepressors andthereby decrease the basal activity of apo receptors.600 BMS493has been defined as an inverse pan-RAR agonist that stronglyfacilitates corepressor interaction.601 In the complex of RAR/NCoR and BMS493 helices H1−H10 of the RAR LBD foldinto the typical NR helical sandwich that resembles the agonist-bound state (Figure 53).602 However, in contrast to the

Figure 52. Channel potentiator 1-EBIO bound to its protein target,the complex of the Calmodulin-binding domain of the small-conductance K+ channel (SK-CaMBD) and Calmodulin (CaM). Inthe upper part, the binding pocket is depicted in surfacerepresentation, the lower part displays in detail the involvement ofsingle residues from CaM (blue) and SK-CaMBD (green) in 1-EBIObinding (PDB ID: 4G28).

Figure 53. Inverse agonist BMS493 bound to its protein target, thecomplex of the nuclear receptor RAR and the corepressor NCoR. Inthe upper part, the binding pocket is depicted in surfacerepresentation, the lower part displays in detail the involvement ofsingle residues from RAR (green) and NCoR (blue) in BMS493binding (PDB ID: 3KMZ).



agonist- or neutral antagonist-bound state H11 and H12 arestrongly disordered when BMS493 occupies the ligand-bindingpocket.The most surprising difference, however, was the fact that the

C-terminal part of H10 folds into a well-defined extended β-strand allowing for an antiparallel β-sheet with the N-terminalresidues of NCoR. This conformational feature was not seenwith other previously described NR-co-repressor struc-tures.603−605 van der Waals contacts of the phenyl D ring ofBMS493 with Ile396 and Leu398 of RXR stabilize the β-strandconformation and thus favor binding of NCoR (Figure 53).Furthermore, direct hydrophobic contacts of BMS493 toNCoR residues Leu2051, His2054, Ile2055 and Ile2058 canbe observed qualifying this compound as another example of atrue protein−protein interface glue. Further examples of NRligands that directly bind include 4-hydroxytamoxifen (4-OHT)that acts as an inverse agonist in the complex of estrogen-related receptor-γ (ERRγ) and SMRT,605 Apoprisnil bound toprogesterone receptor (PR) and NCoR,604 and GW6471stabilizing the interaction of peroxisome proliferator-activatedreceptor-α (PPARα) with SMRT (Figure 54).603

4.6. PPI Stabilizers of 14-3-3 Protein−Protein Interactions

14-3-3 proteins are a class of adapter proteins that have beendiscovered in a systematic cataloging of mammalian brainproteins.606 They interact with several hundred partner proteinsin eukaryotic cells by binding to short, phosphorylatedsequences harboring a central serine or threonine.607 Physicalinteraction of 14-3-3 can directly modulate the activity status ofthe target protein, both in an activating (p53, Wee1, FT,AANAT) as well as an inhibiting manner (CRaf, Beclin 1,Rnd3). An important role for 14-3-3 proteins is the control ofsubcellular localization of their protein partners. They forexample prevent nuclear import of the dual-phosphataseCdc25C and the oncogenic transcription factor TAZ, therebyfunctionally inactivating these proteins. On the other hand theypromote forward trafficking of plasma membrane proteins likethe potassium channel TASK.608

4.6.1. Fusicoccin A. A number of small-moleculestabilizers, natural products as well as HTS hits, have beenreported in recent years. The molecule that is known longest tostabilize a 14-3-3 protein-protein interaction is the naturalproduct Fusicoccin (FC).609 This wilt-inducing phytotoxin isproduced by the pathogenic fungus Phomopsis amygdali(earlier Fusicoccum amygdali). FC conveys its toxic effects bystabilizing the activated complex of the plasma membrane H-ATPase and 14-3-3 proteins. The 14-3-3 proteins arephysiological regulators of H-ATPase function and bind to

the pump’s autoinhibitory C-terminus thereby activating theenzyme.610,611

The structural details of FC action on the 14-3-3/H-ATPasecomplex were revealed when the corresponding ternarycomplex was crystallized.612,613 In the structure of the 14-3-3dimer in complex with the last C-terminal 52 amino acids of theH-ATPase, FC fills a gap in the protein−protein interface(Figure 55). The hydrophobic 5-8-5 terpene ring system insertsdeeply in the central 14-3-3 binding channel with especially themethyl-methoxy substituent being essential to contact thehydrophobic bottom of this channel, lined by Val53, Phe126and Met130. The 5-8-5 ring system establishes extensivehydrophobic interactions with Pro174, Ile175, Gly178, Leu225,and Ile226 from 14-3-3 and with Ile956 from the H+-ATPase(Figure 55). In addition, a number of mainly water-mediated

Figure 54. Further example of inverse agonist of nuclear receptors that stabilize the interaction of the receptor with the corepressors NCoR orSMRT (PDB IDs: 2OVM, 1KKQ, and 2GPV).

Figure 55. Fungal metabolite Fusicoccin bound to its protein target,the complex between dimeric 14-3-3 and the regulatory domain of theH+-ATPase (last 52 C-terminal residues, CT52). In the upper part, thebinding pocket is depicted in surface representation, the lower partdisplays in detail the involvement of single residues from 14-3-3 (blue)and the H+-ATPase (green) of NP in Fusicoccin binding (PDB ID:2O98).



polar interactions is established between Fusicoccin and 14-3-3,namely with Asn49, Lys56, Ser219, and Asp222. Importantlysuch an interaction can also be observed with His930 of the H+-ATPase C-terminus, determining a second direct contactbetween the natural product and the H+-ATPase.Lately, we have also shown that FC-A can stabilize the

interaction of 14-3-3 with the F-domain of estrogen receptor α(ERα).614 Binding of 14-3-3 proteins to ERα inhibits thetranscriptional activity of the nuclear receptor by interferingwith its dimerization and presumably nuclear import. SinceERα is the driving force of proliferation in 75% of breastcancers and treatment with ER antagonist like tamoxifen areprone to development of resistance, new approaches forpharmacological intervention in ERα function are needed.615

Even if Fusicoccin may not be the ultimate molecule to inhibitERα function by stabilizing the ERα/14-3-3 interaction, itproofs the “druggability” of this protein−protein interface andcould help to guide drug discovery efforts to developtherapeutic agents for recurrent breast cancers.Another Fusicoccin-related molecule that has been used to

stabilize a 14-3-3 PPI is FC-THF, a semisynthetic derivative ofFusicoccin J. Here we have added a tetrafuran ring to the C12position of Fusicoccin to create a 14-3-3 PPI stabilizer that isspecific for mode III 14-3-3 recognition motifs where thepolypeptide chain ends at position +1 C-terminal to thephosphorylated serine or threonine residue.302 This moleculewas used to stabilize the binding of 14-3-3 proteins to thepotassium channel TASK3, whose plasma membrane insertionis promoted by 14-3-3 proteins.616 Stabilization of thisinteraction resulted in an increase of channel number andactivity in Xenopus oocytes.302

4.6.2. Cotylenin A. Cotylenin A (CN-A) was discovered inthe early 1970s as a metabolite from Cladosporium sp. 501−7Wthat showed cytokinin-like activity in plants.617 In the plantcontext CN-A shows a very similar phytotoxicity as Fusicoccinand is believed to target the same protein complex, theactivated H+-ATPase/14-3-3 complex.618,619 However, inhuman acute myeloid leukemia cells, CN-A induces differ-entiation both in cell culture620,621 and in a mouse model.622

The best antitumor results could be achieved when CN-A wascombined with rapamycin or interferon α to stop tumor growthin breast cancer623 or tumor regression in non-small cell lungcarcinoma622 or ovary carcinoma.623 Previously we have solvedthe structure of Cotylenin A bound to the 14-3-3 complex witha C-terminal peptide of the H+-ATPase624 and the N-terminal14-3-3 binding motifs (pS233/pS259) of the protein kinase C-RAF.625 Binding of 14-3-3 to these sites results in inhibition ofC-RAF.626 The crystal structure of the binary 14-3-3-C-RAFcomplex627 showed that the two C-RAF phosphorylation sitesbound simultaneously to one 14-3-3 dimer with each site beingaccommodated in one of the two binding grooves. Coopera-tivity of these two binding events resulted in a-fold increase ofaffinity to 14-3-3, when compared to binding of the individualsites pS233 and pS259, respectively.Soaking of the binary 14-3-3-C-RAFpS233pS259 complex

with CN-A led to binding of the natural product to theinterface of both N-terminal 14-3-3 sites in C-RAF and to a 17-fold stabilization of the interaction (Figure 56).625 CN-Aestablished a number of polar contacts to 14-3-3 (Asn42,Lys120, Asp231) as well as the C-RAF peptide (e.g., the sidechain oxygen of Thr234 and the carbonyl oxygen of Pro235)but similar to Fusicoccin A binding to 14-3-3 is conferredmainly by hydrophobic contacts to residues like Val46, Phe117,

Pro165, Ile166, Gly169, and Leu216. In comparison to themode III motif/14-3-3 complex to which Fusicoccin A bindsthe interface of CN-A with the mode II C-RAFpS233 motifseems to be more extensive. Here, we can observe hydrophobiccontacts to the side chain of Thr234 and Pro235 as well as tomain-chain carbons of His237 and Ala237. Since FC-A due toits C-12 hydroxylation (compare Figures 55 and 56) cannotstabilize the 14-3-3-C-RAF interaction this is a very interestingexample showing that a single, relatively minor chemicalmodification can convey specificity toward the stabilization of agiven 14-3-3 PPI.

4.6.3. Pyrrolidone1 and Epibestatin. The phytotoxinFusicoccin A which displays an effective weed killing activitycould in principle be a promising candidate for the develop-ment of a total herbicide. However, its chemical complexity andas a consequence its price of production (either byfermentation or synthesis) precludes its use as a massagrochemical agent. Due to our detailed knowledge of thestructural and mechanistic features of its mode-of-action wewondered if it might be possible to identify compounds thatcould bind to the well-characterized PPI interface and stabilizethe 14-3-3 binding to the H+-ATPase. To this end we screenedthe library of the Chemical Genomics Centre (at that time 37000 compounds) in an ELISA-like assay and identified two hits,Epibestatin and Pyrrolidone1, that mediated stabilization of thecomplex.628 Stabilization of the 14-3-3/H+-ATPase interactionwas corroborated with surface plasmon resonance where the

Figure 56. Cladosporium metabolite Cotylenin A bound to its proteintarget, the complex between dimeric 14-3-3 and the two N-terminal14-3-3 binding sites in C-RAF, pS233 and pS259. In the upper part,the binding pocket is depicted in surface representation, the lower partdisplays in detail the involvement of single residues from 14-3-3 (blue)and C-RAF (green) in Cotylenin A binding (PDB ID: 4IHL).



two molecules conferred quite distinct binding kinetics.Whereas the association of 14-3-3 proteins in the flow phaseto immobilized H+-ATPase CT52 was very fast in the presenceof Pyrrolidone1, Epibestatin mediated a much slower initialinteraction of the two proteins. However, once the Epibestatin-stabilized 14-3-3/H+-ATPase complex was formed, it wasextraordinarily stable with a very slow dissociation kinetic. Incontrast, dissociation of the Pyrrolidone1-stabilized complexwas fast.Crystallization of Epibestatin and Pyrrolidone with 14-3-3

and a 30 amino-acid fragment of the C-terminus of the H+-ATPase (CT30) revealed that the two compounds bound toadjacent but different and nonoverlapping sites in the rim of the14-3-3/H+-ATPase interface. Epibestatin is binding to arelatively narrow channel, is sandwiched between the twoproteins, and shares roughly the same contact surface with 14-3-3 and the H+-ATPase (Figure 57). In contrast, Pyrrolidone1binds to a much more “open” site and establishes more than70% of its contact surface to 14-3-3. The considerabledifferences in the binding sites of Epibestatin and Pyrrolidone1can nicely explain why these two compounds can chemically beso different from each other and yet both stabilize the same 14-3-3 complex. In this context it is important that both moleculesstabilize among a dozen tested 14-3-3 PPIs (e.g., with p53, C-RAF, Mlf1, and Cdc25C) only the interaction with the H+-ATPase. From the known 14-3-3 complex structures that arecurrently available one can nicely explain this specificity by theconsiderably different PPI interfaces. It is therefore notunreasonable to assume that in principle it should be possibleto identify a specific stabilizer for each 14-3-3 PPI, a prospectthat holds tremendous potential for the development of newchemical biology tools or even drug development candidates.

5. CONCLUDING REMARKS

PPIs are operative at all levels of cellular organization andfunction. The constantly expanding knowledge about cellularinteractomes will promote further awareness of the importantrole of PPIs for pathology and foster the identification of novelpotential targets that can be expected to contribute towardnext-generation therapeutics. Chemical methods for accessingsmall molecular modulators of PPIs are maturing: for PPIinhibition, the role of peptide binding epitopes as leadstructures will grow in importance, in light of the expandingtoolbox of chemical approaches for constraining peptidesecondary structure. Peptide stapling and hydrogen-bondsurrogates, among other approaches, were used to improvethe metabolic stability and cell membrane permeability ofpeptides, which is an important step toward PPI targetingtherapeutics. Oligomeric structures such as foldamers representhighly promising, metabolically stable secondary structuremimics, while the structural diversity and biological relevanceof natural products will continue to be a rich source for PPIdrug discovery, either as drug molecules themselves, particularlythrough semisynthesis and mutagenesis techniques, or asinspiration for the design of compound libraries for PPIscreening campaigns. The efficient generation and screening oflarge and structurally diverse compound libraries is an essentialaspect of PPI drug discovery. Given the growing importance ofpeptides as lead structures, biological techniques such as phageand ribosome display should prove highly effective as screeningtools. Virtual screening can offer a cost-effective alternative toHTS, while supramolecular approaches represent novelorthogonal entries for PPI modulation. PPI stabilization isstill an underexplored mode of PPI modulation, despite thegreat success stories of drug molecules such as Rapamycin and

Figure 57. Epibestatin and Pyrrolidone1, identified from high-throughput screening (HTS), bound to its protein target, the complex betweendimeric 14-3-3 and the C-terminal regulatory domain of the H+-ATPase. In the upper part, the binding pocket is depicted in surface representationwith the central overview showing an overlay of both compounds binding to the complex. The lower part displays in detail the involvement of singleresidues from 14-3-3 (blue) and the H+-ATPase (green) in binding of the two compounds (PDB IDs: 3M50 and 3M51).



Taxol. For the future, it is our hope that this strategy will bemore strongly taken up by academia as well as industry.

AUTHOR INFORMATIONCorresponding Author

*Tel. +31 40-247 2835. E-mail: [email protected]

The authors declare no competing financial interest.

Biographies

Lech-Gustav Milroy (1981) is an assistant professor of chemicalbiology at the Biomedical Engineering Department and at the Instituteof Complex Molecular Systems of the Eindhoven University ofTechnology. He received his Ph.D. in 2008 in the research group ofProf. Steven V. Ley CBE FRS at the University of Cambridge, workingon the design synthesis and biological evaluation of natural-productinspired small molecules. Lech then received an Alexander vonHumboldt fellowship in 2008 and moved to the Max Planck Institutefor Molecular Physiology in Dortmund, Germany to work with Profs.Herbert Waldmann and Hans-Dieter Arndt on the synthesis ofmolecular tools derived from complex natural products targeting theactin cytoskeleton. Since 2010 Lech has been based in the ChemicalBiology group at the TU/e. His research focuses on small moleculesynthesis for drug development, as chemical biology tools and for thedevelopment of new nanomaterials.

Tom N. Grossmann is a group leader at the Technical University andthe Chemical Genomics Centre in Dortmund, Germany. His labfocuses on the development of strategies for the chemical modificationof proteins and the stabilization of peptide secondary structures. Since2013, his research group is supported by the Emmy Noether programof the German Research Foundation (DFG). He performed hisChemistry studies at the Humboldt University in Berlin, Germanyfinishing with a research project in the lab of Peter Vollhardt at the

University of California, Berkeley. In 2004, he joined the group ofOliver Seitz at the Humboldt University in Berlin working on nucleicacid templated reactions. In 2009, he started his postdoctoral researchin the lab of Gregory L. Verdine at Harvard University focusing onstabilized α-helical peptides.

Sven Hennig is a group leader at the Chemical Genomcis Centre(CGC) of the Max Planck Society in Dortmund, Germany. He isbiochemist by training and received his Ph.D. in 2008 for his work instructural biology on the PERIOD clock proteins at the Max PlanckInstitute for Molecular Physiology. Afterwards, he did his postdoctoralresearch at the CGC in the group of Christian Ottmann working on14-3-3 HTS assay design for stabilization of PPIs. For the next twoyears Sven Hennig joined the group of Dr. Archa H. Fox at theWestern Australian Institute for Medical Research (WAIMR) in Perth,Australia. He was awarded a Research Development Award of theUniversity of Western Australia (UWA) as an Honorary Fellow for hiswork on nuclear protein−protein and protein−RNA complexes calledParaspeckles. In 2012 he returned to the CGC in Dortmund and wasawarded a 5-years CGC funding of his independent research groupfocusing on the modulation of protein-long-noncoding RNAcomplexes.

Luc Brunsveld (1975) is professor of chemical biology at thebiomedical engineering department and at the Institute of ComplexMolecular Systems of the Eindhoven University of Technology. Afterobtaining a Ph.D. in supramolecular chemistry with E. W. Meijer hewas a Humboldt Fellow at the Max Planck Institute of MolecularPhysiology with H. Waldmann and medicinal chemistry group leaderat Organon. In 2005 he started as group leader in Dortmund andsubsequently moved to Eindhoven in 2008. His interests are incombining supramolecular chemistry with chemical biology and inprotein−protein interactions with a specific focus on nuclear receptors.



mailto:[email protected]

Christian Ottmann is Associate Professor for Molecular Cell andStructural Biology at Eindhoven University of Technology, TheNetherlands. He works on small-molecule modulation of protein−protein interactions (PPIs) with a special focus on stabilization of 14-3-3 adapter protein PPIs. Before taking up his current position inEindhoven he was a group leader at the Chemical Genomics Centre(CGC) of the Max Planck Society in Dortmund, Germany. Heobtained his Ph.D. (summa cum laude) in 2003 from the University ofTubingen with Claudia Oecking. In 2012 he was recipient of theInnovation Award in Medicinal/Pharmaceutical Chemistry of theDCh/DPhG and in 2013 of the Young Chemical Biologist Award ofthe International Chemical Biology Society (ICBS).

ACKNOWLEDGMENTS

We are grateful to our colleagues working in our groups nowand in the past and to those working in the field of PPIs forstimulating discussions. Funded by the Ministry of Education,Culture and Science (Gravity program 024.001.035).

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