71st icrea colloquium "intrinsically disordered proteins (id ps) the challenge of...

Xavier Salvatella ICREA and IRB Barcelona

[email protected]

Intrinsically disordered proteins (IDPs): the challenge of

druggability

Normal mode analysis, a theoretical tool that is comple-

mentary to MD, and its coarse-grained equivalents, theGaussian network model (Bahar et al. 2010) and the aniso-

tropic network model (Zheng 2010), have been useful to

determine the motion that occurs on potentially slower timescales (low-frequency modes). Normal mode analysis pro-

duces projections of the modes rather than conformational

ensembles but can, in principle, probe longer timescales.Although relating the frequencies of motion of biomolecules

to biological function has remained contentious (Kamerlin

and Warshel 2010; Karplus 2010) the motion of biomole-cules is likely to be related to their function.

The experimental evidence for protein motion comes

from a wide range of techniques covering different time-scales. Faure et al. (1994) were able to directly model the

motion of lysozyme in the crystalline form by using atomic

fluctuations around the mean atomic positions that give riseto diffuse scattering of the beam in diffraction experiments.

Neutron scattering has also been used, in a similar manner,

to detect motion in myoglobin (Kneller and Smith 1994;Frauenfelder and Mezei 2010), as has Mossbauer spec-

troscopy, by use of which it was observed that fluctuations

of the solvent cause internal protein motion (Frauenfelderet al. 2009). Further evidence of motion from X-ray crys-

tallography data comes in the form of the multiple con-formations of proteins and nucleic acids that are obtained

when the crystallization process is carried out several

times. These conventional experiments can be comple-mented by single-molecule techniques that enable the

observation of single states of a given molecule. Single

molecule fluorescence energy transfer (FRET) spectros-copy, for example, enables, in principle, observation of

distance distributions rather than average distances. These

methods have provided very solid evidence that, because ofmacromolecular dynamics, inter-atomic distances are not

fixed, either in proteins (Deniz et al. 2000) or in nucleic

acids (Deniz et al. 1999).The most detailed and exhaustive experimental studies

of protein motion have been conducted with NMR spec-

troscopy. This technique has the ability to probe structuralmotion with atomic detail over the entire range of time-

scales from picoseconds to seconds (Fig. 2). Since the first

comprehensive study of fast protein motion by NMR (Al-lerhand et al. 1971) it is has become routine to characterize

the motion of proteins (Kay et al. 1989) that is faster than

rotational diffusion by use of heteronuclear relaxationrates. For a recent review of the application of NMR to the

study of the rapid motion of biomolecules and their com-

plexes, the reader is directed to Jarymowycz and Stone(2006). In addition, Kay et al. have shown that it is possible

to use the Carr–Purcell–Meiboom–Gill (CPMG) NMR

measurements developed by Palmer et al. (2001) and Loriaet al. (1999) to identify and characterize conformations that

are present with a very low population (Korzhnev et al.

2010) when they are in relatively slow (ls–ms) exchangewith the most stable conformation of the macromolecule

(Mittermaier and Kay 2006). Structural fluctuations

occurring on the nanosecond to microsecond timescale canbe probed by measurement of residual dipolar coupling

(RDC) (Salmon et al. 2011).

Biomolecular dynamics from ensembles

Because the ability to visualize motion in macromolecules

can provide details on how this contributes to biological

function, a number of techniques have been developed forthis purpose. Below are presented three methods that

Fig. 2 Timescales of biological motion (above) and experimental andtheoretical methods (below). Protein and nucleic acid dynamic time-scales are shown in green and red, respectively. Timescales common toall biomolecules are shown in black. Experimental methods like small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering(WAXS) are shown over the range they can detect fluctuations. Motionon faster timescales averages during the experiments

Fig. 3 Dynamic content of BPTI from a 1 ms simulation run byD. E. Shaw Research, showing that motion of side chains ispronounced on the sub-sc timescale and that backbone motion issignificant on the supra-sc timescale. Taken from Shaw et al. (2010)

Eur Biophys J (2011) 40:1339–1355 1341

123

The structures of biomolecules fluctuate with a wide range of timescales and amplitudes.

Structures are very informative but a description of the associated dynamics is

important for understanding the relationship between structure and function

Fenwick et al Eur Biophys J 2014

To perform and analyze NMR experiments that provide information on protein flexibility in terms

of conformational ensembles.

Protein dynamics

Single domain Multidomain Disordered

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JACS 2010 PLoS CB 2014

PNAS 2013

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Nat Commun 2014

Intrinsically disordered proteins

Intrinsic disorder (ID) challenges the assumption that proteins must fold to become functional.

3.4. Continuum of Functional Features 66004. Structure 6601

4.1. Structural Continuum 66014.2. Conformational Ensembles 66014.3. Protein Quartet 66024.4. Supertertiary Structure 6602

5. Sequence 66025.1. Sequence−Structural Ensemble Relation-

ships 66025.2. Prediction Flavors 66045.3. Disorder−Sequence Complexity Space 66045.4. Overall Degree of Disorder 66045.5. Length of Disordered Regions 66045.6. Position of Disordered Regions 66045.7. Tandem Repeats 6604

6. Protein Interactions 66056.1. Fuzzy Complexes 66076.2. Binding Plasticity 6607

7. Evolution 66077.1. Sequence Conservation 66087.2. Lineage and Species Specificity 66087.3. Evolutionary History and Mechanism of

Repeat Expansion 66108. Regulation 6610

8.1. Expression Patterns 66118.2. Alternative Splicing 66118.3. Degradation Kinetics 66138.4. Post-translational Processing and Secretion 6613

9. Biophysical Properties 66139.1. Solubility 66139.2. Phase Transition 66149.3. Biomineralization 6615

10. Discussion 661510.1. Current Methods for Function Prediction of

IDRs and IDPs 661610.1.1. Linear Motif-Based Approaches 661610.1.2. PTM Site-Based Approaches 661710.1.3. Molecular Recognition Feature-Based

Approaches 661710.1.4. Intrinsically Disordered Domain-Based

Approaches 661710.1.5. Other Approaches 6617

10.2. Requirement for Annotation 661710.3. Integration of Methods for Finding IDR and

IDP Function 661810.4. Future Directions 6618

11. Conclusion 6619Author Information 6620

Corresponding Authors 6620Author Contributions 6620Notes 6620Biographies 6621

Acknowledgments 6625Abbreviations 6625References 6626

1. INTRODUCTION

1.1. Uncharacterized Protein Segments Are a Source ofFunctional Novelty

Over the past decade, we have observed a massive increase in theamount of information describing protein sequences from avariety of organisms.1,2 While this may reflect the diversity insequence space, and possibly also in function space,3 a largeproportion of the sequences lacks any useful functionannotation.4,5 Often these sequences are annotated as putativeor hypothetical proteins, and for the majority their functions stillremain unknown.6,7 Suggestions about potential proteinfunction, primarily molecular function, often come fromcomputational analysis of their sequences. For instance,homology detection allows for the transfer of information fromwell-characterized protein segments to those with similarsequences that lack annotation of molecular function.8−10

Other aspects of function, such as the biological processesproteins participate in, may come from genetic- and disease-association studies, expression and interaction network data, andcomparative genomics approaches that investigate genomiccontext.11−17 Characterization of unannotated and uncharac-terized protein segments is expected to lead to the discovery ofnovel functions as well as provide important insights into existingbiological processes. In addition, it is likely to shed new light onmolecular mechanisms of diseases that are not yet fullyunderstood. Thus, uncharacterized protein segments are likely

Figure 1. Structured domains and intrinsically disordered regions (IDRs) are two fundamental classes of functional building blocks of proteins. Thesynergy between disordered regions and structured domains increases the functional versatility of proteins. Adapted with permission from ref 50.Copyright 2012 American Association for the Advancement of Science.

Chemical Reviews Review

dx.doi.org/10.1021/cr400525m | Chem. Rev. 2014, 114, 6589−66316590

Van der Lee et al Chem Rev 2014


This class of proteins cannot be represented as rigid conformations … they instead must be represented by conformational ensembles.

...

vs.

JACS 2011 JACS 2010


ID proteins have structural properties resembling those of statistical coils devoid of long range

interactions.

Smith LJ … Dobson DM JMB 2005

Jha AK et al PNAS 2005; Bernadó et al PNAS 2005


Statistical coils can be improved to better reflect the underlying conformational ensemble by using experimental data and molecular simulations .

Jha AK PNAS 2005 JACS 2010


Statistical coils can be improved to better reflect the underlying conformational ensemble by using experimental data and molecular simulations .

Candotti … Orozco PNAS 2013

Intrinsic disorder is important for biomedicine

Intrinsic disorder is frequent in proteins that play roles in neurodegeneration and cancer.

transcript clearance, translation rate, and protein degra-dation [14]. The interactions of ID proteins with theirpartners, and the final output of that interaction, willprobably be sensitive to modulation by binding ofsmall molecules that can affect their affinity for bindingpartners.

Because of the absence of a stable three-dimensionalstructure, the primary sequence is truly the determinantof the ID binding sites. Disordered regions can be pre-dicted with approximately 90% accuracy [15]. Withinthese regions the sequences that undergo binding inter-actions, molecular recognition features (MoRFs), havebeen analyzed on the basis of the type of conformationadopted in their complex [16], and predictors of MoRFshave been developed [17]. MoRFs have been proposed indrug design where they would be used to identify struc-tured binding partners followed by determination of thecomplex structure. The MoRF would then be substitutedby a small molecule [17]. This strategy is complementaryto one considered here. Predictions of binding regions indisordered proteins have been carried out on multipleproteomes and their abundance is found to increase withincreasing organism complexity [18]. The likely strongoverlap in protein interaction sites and small-moleculebinding sites in ID proteins means there are already toolsto predict and compare potential small-molecule bindingsites on a proteome scale.

Targeting disordered c-Myc monomersThe c-Myc oncoprotein (hereafter referred to as Myc) is atranscription factor that regulates large numbers of genes

important in key cellular processes such as growth,differentiation, metabolism, and apoptosis [19–21]. Myc’sfunction requires association with its partner Max througha basic-helix-loop-helix-leucine zipper (bHLHZip)domain in each protein [22,23]. The individual monomersare disordered and undergo coupled folding and bindingupon dimerization (Figure 2a). Myc overexpressionoccurs in most human cancers [21,24] and the feasibilityof targeting Myc was recently demonstrated in a mousemodel [25]. Inhibition of its function has been sought by avariety of biochemical strategies (dominant negatives,antisense, and siRNA among others) [26–28]. Despitethe difficulties of targeting protein–protein interactions,the potential of targeting Myc in cancer therapy ledmultiple groups to search for small molecules that couldinhibit the Myc–Max interaction. After many of theinhibitors were discovered, several small molecules weredemonstrated to be unfolding the Myc–Max dimer andspecifically binding to the monomeric, disorderedHLHZip region [29!!,30!]. The number of small mol-ecules able to target Myc, the demonstration that theextensive (3206 A2) [23] Myc–Max protein interfacecould be disrupted by a small-molecule binding to onedisordered protein partner, and several other recentexamples of small molecules binding to short unstruc-tured peptide segments, have clearly established thefeasibility of using small molecules to target ID proteins.Unique to the Myc binding molecules is their ability toforce their target protein from a highly structured hetero-dimer into a monomeric, disordered form in order to bind(Figure 2). Examining the range of strategies, techniques,and outcomes used by multiple groups in finding andanalyzing inhibitors of Myc provides useful insight intosmall molecule–ID protein interactions. The presump-tion is that all molecules that disrupt the Myc–Max dimeralso bind to disordered monomers (either Myc, Max, orboth) but that has not been demonstrated in all cases.

The first small-molecule inhibitors of Myc–Max dimer-ization were found by screening 7000 compounds fromcombinatorial libraries. The initial screen yielded fourrelated molecules from a peptidomimetic library [31].Two of the molecules, IIA4B20 and IIA6B17(Figure 2), showed inhibition of Myc dependent cellgrowth. Both of these compounds also showed activityagainst cells transformed with Jun. Recently anothercombinatorial library was assembled using the same sub-stituents as the original but built from a racemic, trans-3,4dicarboxylic acid core. Two molecules, mycmycin-1 and2, were found that effectively inhibited Myc dependentcell growth; mycmycin-1 (Figure 2) also showed stronginhibition of Myc–Max dimerization [32!]. Neither myc-mycin inhibited Jun dependent cell growth. This gain inspecificity demonstrated an important aspect of IDproteins as targets: although they may interact with avariety of molecules, a subset of these molecules candisplay high specificity for the protein target.

482 Next Generation Therapeutics

Figure 1

Prevalence of disordered regions in disease-associated proteins.Proteins associated with signaling and the indicated diseases show amuch greater abundance of extended regions of disorder thaneukaryotic proteins from Swiss-Prot and PDB (PDB_S25).Figurereproduced from reference [10].

Current Opinion in Chemical Biology 2010, 14:481–488 www.sciencedirect.com

Uversky et al Annu Rev Biophys 2008; Metallo SJ Curr Opin Chem Biol 2010

IDPs often gain structure upon binding to their targets

The entropic cost of folding upon binding causes the protein protein interactions involving IDPs to

be weak and optimal for regulation.

200 | MARCH 2005 | VOLUME 6 www.nature.com/reviews/molcellbio

R E V I EW S

residues — as is the case for the KID of CREB — or anentire protein domain. For example, the 116-residue N-terminal domain of DNA-fragmentation factor 45-kDa subunit (DFF45) is unstructured in solution,but folds into an ordered globular structure on forminga heterodimeric complex with DFF40 (REF. 42).

A case study: transcriptional co-activatorsCBP and its PARALOGUE p300 are modular transcriptionalco-activators. They modify both chromatin and tran-scription factors through their intrinsic acetyltrans-ferase activity, and also function as scaffolds for therecruitment and assembly of the transcriptionalmachinery43–45. Of the 2,442 amino acids that comprisethe sequence of human CBP, more than 50% are inregions of the protein that are intrinsically disordered,according to many of the available prediction pro-grams. Compositional bias in intrinsically disorderedregions is exemplified by CBP, the sequence of which isshown in FIG. 2a. There are long amino-acid sequencesbetween the folded domains that contain predomi-nantly Gln, Pro, Ser, Gly, Thr and Asn residues. By con-trast, most of the interaction domains that have beenidentified have an amino-acid composition that ismore typical of globular proteins. Interestingly, thereare a number of segments within the ‘disordered’regions of CBP that contain relatively high proportionsof hydrophobic and charged residues; these regionsperhaps represent as-yet-unidentified interactionmotifs. There are only seven domains in CBP/p300 thatare capable of folding independently (FIG. 2b), and fourof them require zinc binding to stabilize their tertiarystructures (the transcriptional-adaptor zinc-finger-1(TAZ1) domain, the plant homeodomain (PHD), azinc-binding domain near the dystrophin WW domain(ZZ), and the transcriptional-adaptor zinc-finger-2(TAZ2) domain). The 3D structures of the foldeddomains of CBP/p300 — except the PHD and histoneacetyltransferase (HAT) domains — have been deter-mined by NMR methods, and their functions as tem-plates for coupled folding and binding processes arediscussed below.

TAZ domains: scaffolds for assembly. The TAZ domains46

of CBP/p300 are zinc-binding domains with a distinc-tive helical fold47. The TAZ1 and TAZ2 domains sharesignificant sequence homology and adopt similar 3Dstructures, and the only significant differences are in thelocation of the third zinc-binding site and the C-terminalhelix47,48. However, these subtle structural changesallow them to discriminate between different subsetsof transcription factors. The TAZ2 domain is the site ofinteraction both with transactivation domains of viraloncoproteins such as E1A and with the tumour sup-pressor p53 (REFS 49–52), whereas TAZ1 mediates keyinteractions with hypoxia-inducible factor-1α (HIF1α)and thereby regulates the hypoxic response53. Theseligands have all been found to be disordered in the freestate. The C-terminal transcription-activation domain ofHIF1α is unstructured in solution, but undergoes localfolding transitions to form three short helices on binding

by binding to misfolded proteins and RNA molecules,such that they function as recognition elements and/orhelp in the loosening and unfolding of kinetically-trapped folding intermediates.

Many intrinsically disordered proteins undergotransitions to more ordered states or fold into stablesecondary or tertiary structures on binding to theirtargets — that is, they undergo coupled folding andbinding processes5,37,38 (BOX 2). One of the most well-characterized examples is the kinase-inducible tran-scriptional-activation domain (KID) of CREB. TheKID polypeptide is intrinsically disordered, both as anisolated peptide and in full-length CREB39,40, but it foldsto form a pair of orthogonal helices on binding to itstarget domain in CBP41 (BOX 2). Interestingly, the intrin-sic disorder of the KID can be reliably predicted from itsamino-acid sequence, as can an inherent helical propen-sity in the region that undergoes the coupled foldingand binding transition. Indeed, the identification ofamphipathic elements embedded within regions of aprotein that are predicted to be disordered might pro-vide clues as to the location of potential functional sites.Coupled folding and binding might involve just a few

PROTEIN QUARTET

A similar division for proteinstructure as the protein trinity,but the quartet includes a pre-molten globule state as wellas the unfolded, molten-globuleand folded states.

RAMACHANDRAN PLOT

A plot of the backbone dihedralangles φ and ψ for a polypeptidechain. Areas of low energy(greater probability) encompassangles that are observed in α-helical and β-sheetstructures, and a part of thebroad β-minimum is defined asthe ‘polyproline II’ region.

TQISTIAESEDSQESVDSVTDSQKRREILSRRPSYRKILNDLSSDAPGVPRIEEEKSEEETSAP

a

b

Box 2 | Coupled folding and binding

Coupled folding and binding is the process in which an intrinsically disordered protein, orregion of a protein, folds into an ordered structure concomitant with binding to its target.There is an entropic cost to fold a disordered protein, which is paid for using the bindingenthalpy (BOX 3). For example, the phosphorylated kinase-inducible domain (pKID) of thetranscription factor cyclic-AMP-response-element-binding protein (CREB) isunstructured when it is free in solution39,40, but it folds on forming a complex with theKID-binding (KIX) domain of CREB-binding protein (CBP)41 (see figure, part a).

The amino-acid sequence of the KIX-binding region of CREB is shown in part b of thefigure. The colour coding for the amino acids is: green for small residues, unchargedhydrophilic residues and Pro; yellow for hydrophobic residues; red for acidic residues;and blue for basic residues. Helices that were predicted using The PredictProtein server117

(see the online links box), but which are not observed in the free peptide, are shown ingrey above the sequence. The locations of the helices that are observed in pKID on itsbinding to KIX are shown in pink below the sequence.

Dyson and Wright Nat Rev Mol Cell Biol 2005

IDPs often gain structure upon binding to their targets

The entropic cost of folding upon binding causes the protein protein interactions involving IDPs to

be weak and optimal for regulation.

Teilum et al. Thermodynamics of protein-protein interactions

with an ordered partner had in that work an overrepresentationof hydrophobic residues as leucine and isoleucine in the core ofthe interface, and the ordered binding partner had an increasednumber of charged residues. Thus, this apparent counter balanceis in full accordance with the overall sum of the interface wereport here. A decomposition of the distribution into individualresidues within the current set supports previous findings,although the effect is small (the largest difference is for Cys whichis 41% less abundant in the ORD-IDP complexes) (Figure 1A).Therefore, if specificity is embedded in interactions betweencharged and polar side-chains in the interface (Eaton et al., 1995;Wong et al., 2013), we find no indication to suggest that the IDPsbind to globular proteins with higher specificity than globularproteins do.

Recall the basic thermodynamic relation, !G0 = !H0−T!S0 in which the entropy-enthalpy compensation infers that!H0 and T!S0 are highly correlated (Brady and Sharp, 1997;Williams et al., 2004; Teilum et al., 2009). Thus, !G0 forthe complexes in the selected sets covers a narrow rangefrom −19.8 kcal mol−1 to −4.2 kcal mol−1 (corresponding toKd from 3 fM to 830µM) compared to !H0 and T!S0 thatare found in the ranges from −66.7 to 19.9 kcal mol−1 andfrom −56.1 to 28.5 kcal mol−1, respectively. The analysis of thethermodynamic parameters shows that the enthalpy (!H◦) andthe entropy (!S◦) for binding are not significantly differentbetween the two groups of proteins (t-test, P > 0.1). However,the average entropic contribution (−T!S◦) to the binding freeenergy for interactions between two ordered proteins is 2.5 ±1.6 kcal mol−1 smaller (more stabilizing) than for interactionsbetween an ordered and a disordered protein. Within bothgroups there is a linear correlation between T!S◦ and !H◦

(ORD-ORD: slope = 1.09 ± 0.03, r = 0.97; ORD-IDP: slope =1.06 ± 0.02, r = 0.98), which demonstrates a similar entropy-enthalpy compensation (Figure 2A). Thus, the same underlyingthermodynamic principles are true for both groups.

In contrast to !S◦ and !H◦, there is a significant differencein !G◦ between the groups (t-test, P < 0.0001). For the ORD-ORD complexes <!G◦> = −11.1± 0.4 kcal mol−1, and forthe ORD-IDP complexes <!G◦> = −8.5 ± 0.2 kcal mol−1.The difference in <!G◦> is 2.5 ± 0.4 kcal mol−1, which isprimarily accounted for by the difference in T!S◦ (vide supra).This number is close to the 2.6 kcal mol−1 recently publishedfrom a much smaller dataset based on mutation studies (Huangand Liu, 2013). Note that the distribution of !G◦ among thecomplexes for which a structure is available is similar to thedistribution in the full dataset, and that this is true for thedifference in <!G◦> too. As we see no differences in thesizes of the binding interfaces or the amount of hydrophobicresidues in the interfaces, and since the disordered proteinsin the ORD-IDP complexes rarely form extended hydrophobiccores in their folded conformations, the hydrophobic surfacearea buried in ligand binding process must be similar in thetwo classes of protein complexes. Consequently, the differencein T!S◦ is unlikely to arise from significant differences in thedesolvation entropy contribution. This conclusion is in contrastto a computational study of complexes involving extended IDPs,which were selected based on a radius-of-gyration criterion of the

FIGURE 2 | Thermodynamics of 196 protein-protein complexes. (A)Histogram of the binding free energy, !G◦, for complexes between twoordered proteins (red) and one ordered and one disordered protein (blue). Bothdistributions were fit to a Gaussian distribution (solid lines). (B) Plot of !H◦

versus T!S◦ for the same protein–protein complexes with the same colorcode as in (A). The solid lines represent the best linear fits to the data.

three-dimensional structure of the complex (Wong et al., 2013).However, in that work the energetic terms were not decomposedinto enthalpic and entropic contributions. Nevertheless, theexperimental data for the large group of complexes that wehave compiled suggest to us that the less favorable entropiccontribution for the ORD-IDP complexes primarily originatesfrom loss in conformational entropy. Indeed, it agrees withthe mechanistic difference between binding an ordered and adisordered ligand. The disordered polypeptide has to fold to form

Frontiers in Molecular Biosciences | www.frontiersin.org 3 July 2015 | Volume 2 | Article 40

Teilum et al Front Mol Biosci 2015

Ordered Disordered

Why has evolution favoured intrinsic disorder

One intriguing proposal is that intrinsic disorder makes low affinity compatible with high

specificity. convoluted and extended interaction surface than a struc-tured protein (Figure 1), which allows for a precise fit to thetarget and hence high specificity (see below for furtherdiscussion). However, rigidification (akin to a disorder-to-order transition) of the protein on binding to a nucleic acidcosts free energy. Hence, the overall binding affinity is notexcessive. Schulz’s concern was that an overly strongassociation constant would mean that the nucleic acid isalways in the bound state, so that binding would effectivelybe irreversible. However, it should be noted that the boundfraction depends both on the association constant and onthe protein concentration (Box 1). Regardless of the mag-nitude of the association constant, any desired boundfraction can be obtained by tuning the protein concentra-tion.

Intrinsic disorder is similar to flexibility, and the result-ing high specificity with low affinity was proposed as abenefit of IDPs [2–4]. However, does an IDP really have anadvantage in this regard over a hypothetical ordered pro-tein (Figure 1b), which would have both high specificityand high affinity? Potentially, the low affinity associatedwith intrinsic disorder could present a problem, becausethis would mean that the IDP has to be maintained at ahigh cellular concentration to achieve a significant boundfraction for its cognate target (Box 1). As noted above, cellswork against such high concentrations of IDPs. It is worthnoting that whereas Schulz viewed the low affinity due tothe free-energy cost of an disorder-to-order transition as anadvantage, Spolar and Record viewed it as a necessaryexpense for achieving high specificity [24].

Extended interaction surfacesFollowing Schulz’s observation for flexible nucleic-acid-binding proteins [23], others have recognized that IDPsoften form extended interaction surfaces with their cellulartargets [2–5,25]. Gunasekaran et al. compared the areas ofbinding interfaces involving IDPs and those involvingordered proteins, and concluded that to achieve the sameinterface area, IDPs require much smaller protein sizesthan ordered proteins do [25]. They suggested that smallerprotein size allows a decrease in cellular macromolecularcrowding, which significantly affects the thermodynamicand kinetic properties of biological processes [26].

What is the benefit of an extended interaction surface?Obviously, the resulting extensive, specific intermolecularinteractions allow the IDP to overcome the free-energy costof the disorder-to-order transition, so that the overallbinding affinity is not excessively low [27]. As alreadyalluded to, the disorder-to-order transition allows a precisefit of the IDP to its target, which leads to high specificity.However, it should be noted that if the argument of bindingpromiscuity holds, the malleability of IDPs could also allowthem to fit with non-cognate targets, and thereby losespecificity. Nussinov further suggested that extended in-teraction surfaces facilitate efficient signal propagation[28].

Enhanced association ratesBased on somewhat different lines of reasoning, severalstudies have argued or predicted that intrinsic disorder (orflexibility) can speed up protein association [29–32]. Thatdisorder should lead to an increase in ka is really notunexpected, as illustrated by the simple example in whichflexible loops of a protein close up the binding pocket afterligand binding (a scenario referred to as ‘‘gating binding-pocket’’ [33]). In this case, the hypothetical ordered proteinhas loops that close the binding pocket, and hence theligand cannot enter the binding pocket at all. By contrast,a protein with flexible loops allows the ligand to enter someof the time and binding becomes possible. In cases in whichIDPs wrap around their targets, steric clashes can similar-ly make it impossible for hypothetical ordered proteins tobind their targets [2].

Pontius [29] proposed that attachment of weakly inter-acting, disordered polymers to ordered macromoleculeswould enhance the association rate of the latter. Hisargument was that the polymers would hold the macro-molecules together, which would allow them to exploredifferent separations and relative orientations to find astereospecific fit. In some sense this argument is similar tothe idea of reduction in dimensionality proposed by Adamand Delbruck many years ago to rationalize the possibilitythat nonspecific DNA sequences flanking a specific sitefacilitate the search by a protein for the specific site [34].This possibility was later confirmed by experiments[35,36].

Shoemaker et al. recognized that compared to an or-dered protein, an IDP can have a greater capture radius(see Glossary) for a specific site on the target [30]. In theirview, the IDP binds the target weakly at a relatively largedistance, followed by folding as the protein approaches thebinding site. Their calculation, using separation as the

Rigid protein(a)

(b)

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Hypotheticalprotein

TiBS

Figure 1. Binding affinity and specificity of a rigid (or ordered) protein and aflexible (or disordered) protein. (a) A rigid protein tends to form a simple, relativelysmooth interaction surface with the target. (b) Left: a flexible protein, whenunbound, can sample different conformations, especially around the binding site(as illustrated by dashed curves representing alternative conformations). Right: inthe bound state, flexibility allows the protein to wrap around protrusions andindents of the target, which gives rise to a convoluted interaction surface and highspecificity. Middle: a hypothetical protein that adopts the bound conformationeven when unbound would have a much higher free energy than a flexible protein.However, the hypothetical protein would have the same high specificity but amuch higher binding affinity than the flexible protein.

Opinion Trends in Biochemical Sciences February 2012, Vol. 37, No. 2

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Zhou H-X TIBS 2011

convoluted and extended interaction surface than a struc-tured protein (Figure 1), which allows for a precise fit to thetarget and hence high specificity (see below for furtherdiscussion). However, rigidification (akin to a disorder-to-order transition) of the protein on binding to a nucleic acidcosts free energy. Hence, the overall binding affinity is notexcessive. Schulz’s concern was that an overly strongassociation constant would mean that the nucleic acid isalways in the bound state, so that binding would effectivelybe irreversible. However, it should be noted that the boundfraction depends both on the association constant and onthe protein concentration (Box 1). Regardless of the mag-nitude of the association constant, any desired boundfraction can be obtained by tuning the protein concentra-tion.







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High affinity + high specificity

Low affinity + high specificity

Why has evolution favoured intrinsic disorder

This is because in many scenarios it is desirable that protein protein interactions are of low

affinity e.g. in signaling. convoluted and extended interaction surface than a struc-tured protein (Figure 1), which allows for a precise fit to thetarget and hence high specificity (see below for furtherdiscussion). However, rigidification (akin to a disorder-to-order transition) of the protein on binding to a nucleic acidcosts free energy. Hence, the overall binding affinity is notexcessive. Schulz’s concern was that an overly strongassociation constant would mean that the nucleic acid isalways in the bound state, so that binding would effectivelybe irreversible. However, it should be noted that the boundfraction depends both on the association constant and onthe protein concentration (Box 1). Regardless of the mag-nitude of the association constant, any desired boundfraction can be obtained by tuning the protein concentra-tion.







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Zhou H-X TIBS 2011








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TiBS



45

High affinity + high specificity

Low affinity + high specificity

Transactivation domains

resemble the denatured states of globular proteins (for example, the p160 steroid receptor coactivator ACTR; ref. 12) to partially struc-tured (“pre-molten globules”) or more compact ensembles (“molten globules”)13 that may have some secondary structure (for example, p27Kip1 (ref. 14) or MeCP2 (ref. 15)). Disorder can also be present in locally disordered N- or C-terminal tails or internal linkers11.

The disordered state of IDPs is intimately linked to their unusual amino acid composition— they are enriched in polar and charged residues (lysine, arginine, glutamate, glutamine and serine) and depleted in hydrophobic residues (tryptophan, phenylalanine,

results conflict with the generally accepted idea that a well-defined structure is a prerequisite for protein function and have led to a reas-sessment of the structure-function paradigm7. Currently, there are over 500 IDP examples assembled in the DisProt database8 whose disordered state is experimentally supported by biophysical data. The relevance of structural disorder in vivo has been corroborated experimentally—for example, by in-cell NMR measurements, which indicate the persistence of a disordered state in crowding conditions9,10.

IDPs comprise a variety of broad structural categories11. These range from completely unstructured proteins (“random coils”) that

Box 1 Basic elements of the eukaryotic transcription machineryTranscription of protein-encoding genes is one of the first steps in deciphering the genetic material. Fundamentally, it is the synthesis of mRNA from a DNA template, carried out in eukaryotes by the enzyme RNA polymerase II. Transcription is a hierarchical process involv-ing many different macromolecular assemblies regulated by numerous protein-protein and protein-DNA interactions (Fig. 1).

Chromatin and chromatin modifying enzymes function to regulate accessibility of the DNA at the global (that is, multiple-kilobase-pair) level (Fig. 1a). The subunit of chromatin is the nu-cleosome, which is a complex of 146-base-pair chromosomal DNA with an octamer of core his-tones. Arrays of nucleosomes spaced at roughly 200-base-pair intervals make up chromatin fibers, which are structurally dynamic and can condense locally and globally into chromosomal domains. There are two major classes of chro-matin modifying enzymes: those that add or remove specific post-translational modifications (for example, the Gcn5 acetyltransferase) and those that use the energy of ATP hydrolysis to alter the structure of nucleosomes and specific chromatin regions (for example, SWI/SNF). Chromatin modifying enzymes are large multi component assemblies that have elongated, flexible shapes (Fig. 1a). Collectively, chromatin and chromatin modifying enzymes operate at the epigenetic level and coordinate global ac-cessibility of promoter DNA.

Once the chromatin environment becomes accessible, regulation of the promoter involves the action of regulatory transcription factors, co-activators/co-repressors and the basal transcription machinery. Upstream DNA sequences are targeted by specific DNA binding proteins, called transcription factors (Fig. 1b), which are responsible for controlling (activation/inhibi-tion) specific gene expression. Transcription factors have a modular architecture with a DNA binding domain and a transactivator domain (TAD). TADs communicate with other regulatory transcriptional proteins and have an important role in orchestrating the transcriptional as-semblies. Co-activators provide a link between chromatin, transcription factors and the basal transcription machinery (Fig. 1c). Co-activators generally have multiple transcription factor binding sites and are thus able to process multiple transcriptional regulatory inputs. The co-activator p300 is known to interact with over 50 proteins and has histone acetyltransferase activity. Co-activators can also adopt a variety of structural forms needed for different stages of transcription. For example, the Mediator co-activator complex undergoes a large confor-mational transition from a closed form to an open form to be able to accommodate RNAP II. The actual recruitment of RNAP II to promoter DNA is accomplished by the general transcription factors (GTFs): TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH (Fig. 1d). The TATA DNA element at the promoter is recognized by the TATA box binding protein (TBP), which significantly bends DNA. This TBP-DNA association provides a platform for the assembly of associated factors. The assembly of RNAP II, GTFs and co-activators is called the pre-initiation com-plex (PIC). The C-terminal domain (CTD) of RNAP II is heavily phosphorylated during transcription (for example, by TFIIH or by the kinase subunit of the Mediator), and its phosphorylation stage has a critical role in pre-mRNA processing and termination of transcription.

UAS UAS

TATA TATA

a b

c d

Mediator

Activator/repressor

RNAP II

IIE IIHIIA

IIB

IIF RNAP IICTD

P P P

Activator/repressor

TAD

TATA

UAS

UAS

ISWI WSTF

TATA

Figure 1 Schematic representation of the four main (highly interdependent) components of the eukaryotic transcription machinery. (a–d) Chromatin remodeling (a), transcription factors (b), co-activators (c) and basal machinery (d). Disordered histone (yellow) tails (dashed line) assist the assembly of nucleosomes and provide platforms for chromatin remodeling complexes. Chromatin remodeling complexes such as SWI/SNF regulate the accessibility of the DNA, and their mobility is enhanced by disordered regions. Transcription factors decipher regulatory information encoded in enhancer regions (UAS, upstream activating sequence), and they interact with other proteins via disordered TADs. Large co-activator complexes such as the Mediator (head, middle and tail modules are shown in orange, green and yellow, respectively) transmit signals from enhancer- and repressor-bound factors to the core machinery. Transcription initiation is achieved by RNAP II assisted by five general transcription factors (TFIIA, TFIIB, TFIIE, TFIIF and TFIIH). The disordered CTD of RNAP II serves as a scaffold for a range of complexes involved in different stages of transcription, and it functions in a phosphorylation-dependent manner.

NATURE CHEMICAL BIOLOGY VOLUME 4 NUMBER 12 DECEMBER 2008 729

R E V I E W

The regulation of transcription involves interactions between transcription activation domains (TADs) and

the basal transcription machinery

Fuxreiter, Tompa et al Nat Chem Biol 2008


Transactivation domains

IDPs also seem to have important roles in the function of ATP-dependent chromatin remodeling complexes, such as ISWI, CHRAC, NURF, RSC and SWI/SNF, which regulate accessibility of the genomic DNA (Fig. 1a). All these complexes facilitate sliding and transfer of histone octamers along the DNA and expose targeted DNA segments to nucleases and other probes. Gel mobility analysis suggests that most of these remodeling complexes exhibit significant deviations from globularity49,55–58, as they migrate at higher molecular weight than that expected from the mass of the complex. IDRs in chroma-tin remodeling complexes likely mediate low-affinity interactions with DNA and establish variable contact patterns required for slid-ing. Thus disordered regions enhance mobility along DNA (ref. 55), and their removal significantly impairs the sliding process50,55,56. Structural characterization by electron microscopy of the yeast SWI/SNF remodeler revealed a structure composed of eight subunits assembled into a modular and highly irregular structure57 (Fig. 1). Gel mobility analysis and disorder predictions also suggest that the Snf5 and Swi3 subunits of the SWI/SNF remodeling complex are rich in IDRs. Removal of the N-terminal domain of Swi3 (predicted to be poorly ordered) results in the remainder of the protein migrating at its actual molecular weight (C. Peterson, University of Massachusetts Medical School, personal communication). Swi3 serves as an assem-bly scaffold and is involved in histone binding using IDRs to interact with multiple partners. Snf5 has a role in recruitment of SWI/SNF to specific genomic regions—another process with coordinated changes in macromolecular interactions. We hypothesize that IDPs allow mal-leability in the structure of chromatin remodeling and modifying complexes, which facilitates their interaction with the equally struc-turally malleable chromatin fiber.

Modification of chromatin structure may also be facilitated by increased DNA distortion induced by “architectural transcription

many interactions involving the N-terminal tails are likely of tran-sient nature and require rapid association and dissociation with their partners. Although both core and linker histone tails are predomi-nantly (~40%) composed of positively charged residues, their func-tions cannot be recapitulated by simply neutralizing DNA charges at high ionic strength45. These observations might imply that local organization of IDRs can be a functional determinant for histone proteins that can be perturbed by post-translational modifications (for example, methylation and acetylation)52. These covalent modifi-cations have been proposed to function as a “histone code” that influ-ences and regulates specific macromolecular interactions and cell functions48,53. Although in most cases the structural consequences of these covalent modifications have not been elucidated, they might alter secondary structure preferences of IDRs, which could abolish nucleosome-nucleosome interactions or modulate interactions with chromatin remodeling complexes49 and other chromatin proteins.

The intrinsically disordered C-terminal tail of linker histones under-goes a disorder-to-helix transition upon binding to DNA (ref. 54) and contributes to stabilization of condensed chromatin structures. The linker histone H1 °CTD also binds to and activates the DFF40 (also known as CAD) apoptotic nuclease. Interestingly, different CTD regions are used for chromatin condensation and nuclease binding. These H1 °CTD regions have distinct, highly conserved amino acid composi-tions18. Accordingly, scrambling the sequence of these segments does not alter CTD-dependent stabilization of higher order chromatin structures (X. Lu and J.C. Hansen, Colorado State University, personal communication). Although possible changes in the interaction and activation mechanism have not been elucidated, the IDR had to be suf-ficiently long (at least 47 residues). These results emphasize that there is no specific sequence requirement for the IDRs—only some spatial and electrostatic conditions have to be fulfilled.

a bDNA

Tetramer Cyclin A

Sirtuin

CBP

S100SET9

tGcn5

Tfb1

MDM2

Rpa70

1.0

0.5

0.0

PO

ND

R s

core

0 50 100 150 200 250 300 350 400

Sequence index

Figure 2 Molecular recognition by intrinsically disordered proteins. (a) Binding of IDPs to their partners is often coupled to disorder-to-order transition. IDP segments might have transient secondary structures that are preserved in the bound state (preformed element, orange), whereas other segments may exhibit different conformations (yellow) with different partners (promiscuity). Owing to this segmental mode of binding, some parts of IDPs may remain disordered (fuzzy, dashed lines) in the bound state, which contributes to the multifunctionality (for example, moonlighting) of these proteins. (b) Disorder profile of the p53 transcription factor. p53 is at the center of a large signaling network regulating expression of genes involved in a variety of cellular processes, such as cell cycle progression, apoptosis induction, DNA repair and response to cellular stress. p53 interacts with a large number of other proteins, and the interaction sites are signaled by downward spikes in the plot of disorder. Disorder predictions were performed by PONDR VL-XT (http://www.pondr.com/); segments with scores above 0.5 correspond to disordered regions, while those below 0.5 correspond to ordered regions or binding sites. The structures of the complexes representing illustrative examples of various molecular recognition features (MoRFs) are displayed around the predicted disorder pattern.


R E V I E W

IDPs also seem to have important roles in the function of ATP-dependent chromatin remodeling complexes, such as ISWI, CHRAC, NURF, RSC and SWI/SNF, which regulate accessibility of the genomic DNA (Fig. 1a). All these complexes facilitate sliding and transfer of histone octamers along the DNA and expose targeted DNA segments to nucleases and other probes. Gel mobility analysis suggests that most of these remodeling complexes exhibit significant deviations from globularity49,55–58, as they migrate at higher molecular weight than that expected from the mass of the complex. IDRs in chroma-tin remodeling complexes likely mediate low-affinity interactions with DNA and establish variable contact patterns required for slid-ing. Thus disordered regions enhance mobility along DNA (ref. 55), and their removal significantly impairs the sliding process50,55,56. Structural characterization by electron microscopy of the yeast SWI/SNF remodeler revealed a structure composed of eight subunits assembled into a modular and highly irregular structure57 (Fig. 1). Gel mobility analysis and disorder predictions also suggest that the Snf5 and Swi3 subunits of the SWI/SNF remodeling complex are rich in IDRs. Removal of the N-terminal domain of Swi3 (predicted to be poorly ordered) results in the remainder of the protein migrating at its actual molecular weight (C. Peterson, University of Massachusetts Medical School, personal communication). Swi3 serves as an assem-bly scaffold and is involved in histone binding using IDRs to interact with multiple partners. Snf5 has a role in recruitment of SWI/SNF to specific genomic regions—another process with coordinated changes in macromolecular interactions. We hypothesize that IDPs allow mal-leability in the structure of chromatin remodeling and modifying complexes, which facilitates their interaction with the equally struc-turally malleable chromatin fiber.

Modification of chromatin structure may also be facilitated by increased DNA distortion induced by “architectural transcription

many interactions involving the N-terminal tails are likely of tran-sient nature and require rapid association and dissociation with their partners. Although both core and linker histone tails are predomi-nantly (~40%) composed of positively charged residues, their func-tions cannot be recapitulated by simply neutralizing DNA charges at high ionic strength45. These observations might imply that local organization of IDRs can be a functional determinant for histone proteins that can be perturbed by post-translational modifications (for example, methylation and acetylation)52. These covalent modifi-cations have been proposed to function as a “histone code” that influ-ences and regulates specific macromolecular interactions and cell functions48,53. Although in most cases the structural consequences of these covalent modifications have not been elucidated, they might alter secondary structure preferences of IDRs, which could abolish nucleosome-nucleosome interactions or modulate interactions with chromatin remodeling complexes49 and other chromatin proteins.

The intrinsically disordered C-terminal tail of linker histones under-goes a disorder-to-helix transition upon binding to DNA (ref. 54) and contributes to stabilization of condensed chromatin structures. The linker histone H1 °CTD also binds to and activates the DFF40 (also known as CAD) apoptotic nuclease. Interestingly, different CTD regions are used for chromatin condensation and nuclease binding. These H1 °CTD regions have distinct, highly conserved amino acid composi-tions18. Accordingly, scrambling the sequence of these segments does not alter CTD-dependent stabilization of higher order chromatin structures (X. Lu and J.C. Hansen, Colorado State University, personal communication). Although possible changes in the interaction and activation mechanism have not been elucidated, the IDR had to be suf-ficiently long (at least 47 residues). These results emphasize that there is no specific sequence requirement for the IDRs—only some spatial and electrostatic conditions have to be fulfilled.

a bDNA

Tetramer Cyclin A

Sirtuin

CBP

S100SET9

tGcn5

Tfb1

MDM2

Rpa70

1.0

0.5

0.0

PO

ND

R s

core

0 50 100 150 200 250 300 350 400

Sequence index

Figure 2 Molecular recognition by intrinsically disordered proteins. (a) Binding of IDPs to their partners is often coupled to disorder-to-order transition. IDP segments might have transient secondary structures that are preserved in the bound state (preformed element, orange), whereas other segments may exhibit different conformations (yellow) with different partners (promiscuity). Owing to this segmental mode of binding, some parts of IDPs may remain disordered (fuzzy, dashed lines) in the bound state, which contributes to the multifunctionality (for example, moonlighting) of these proteins. (b) Disorder profile of the p53 transcription factor. p53 is at the center of a large signaling network regulating expression of genes involved in a variety of cellular processes, such as cell cycle progression, apoptosis induction, DNA repair and response to cellular stress. p53 interacts with a large number of other proteins, and the interaction sites are signaled by downward spikes in the plot of disorder. Disorder predictions were performed by PONDR VL-XT (http://www.pondr.com/); segments with scores above 0.5 correspond to disordered regions, while those below 0.5 correspond to ordered regions or binding sites. The structures of the complexes representing illustrative examples of various molecular recognition features (MoRFs) are displayed around the predicted disorder pattern.


R E V I E WIntrinsic disorder : P53


Androgen receptor

This nuclear hormone receptor is a therapeutic target in prostate cancer as well as in spinal bulbar

muscular atrophy, a rare neurodegenerative disease.

919 residues

Androgen receptor

36 | OCTOBER 2001 | VOLUME 1 www.nature.com/reviews/cancer

R E V I EW S

the development of AIPC24. If treatment providesselective pressure for mutations that cause AIPC,intermittent treatment might reduce or delay the ten-dency towards development of mutant cells thatbecome androgen independent. This important issuewarrants further study.

The specific types of mutation that lead to AIPCwill be discussed in the subsequent sections. We havecategorized five potential mechanisms by which AIPCcan develop (TABLE 1; FIG. 2). Some of these mecha-nisms also apply to other forms of steroid-hormone-independent cancer, such as breast cancer (BOX 3).

Type 1: the hypersensitive pathwayOne possible mechanism by which a prostate cancer cir-cumvents the effects of androgen ablation therapy is byincreasing its sensitivity to very low levels of androgens.Prostate cancers that use this mechanism are not, strictlyspeaking, androgen independent — their responses stilldepend on AR and androgen — but they have a loweredthreshold for androgens.

AR amplification. There are several potential mecha-nisms that would allow increased tumour-cell prolif-eration, despite low circulating androgens in thepatient. One mechanism to accomplish this is byincreasing the expression of the AR itself. IncreasedAR abundance leads to enhanced ligand-occupiedreceptor content, even in the face of reduced androgenconcentration. Approximately 30% of tumours thatbecome androgen independent after ablation therapyhave amplified the AR gene, resulting in increased ARexpression, whereas none of the primary tumoursfrom the same patients before androgen ablation hadan AR gene amplification15,25. These results indicatethat amplification was probably the result of clonalselection of cells that could proliferate, despite verylow levels of circulating androgens. Interestingly,patients with tumours that had AR amplification sur-vived longer than patients with tumours that wererefractory to ablation therapy but did not have ampli-fication of the AR gene15. One possible explanation isthat these amplified tumours are more differentiatedthan other prostate cancers, perhaps allowing thepatients to have a better outcome.

Although tumours with AR amplification haveincreased levels of AR, the signal to proliferate presum-ably continues to require androgen15,25. This is an exam-ple of how tumours that seem clinically to be androgenindependent could simply have increased their sensitivi-ty to androgens so that they continue to proliferate in alow androgen environment. AR gene amplification thatis detected in tumours that are progressing duringandrogen deprivation monotherapy with gonadotropin-releasing hormone (GnRH) analogues (BOX 1) might beassociated with a favourable treatment response to sec-ond-line combined total androgen ablation with addedanti-androgens26. This finding indicates that at leastsome AR-amplified tumours retain a high degree ofdependency on residual androgens that remain in serumafter monotherapy26.

However, many studies have found only a few ARmutations in primary prostate cancer14; in compari-son, metatastic prostate cancer frequently has muta-tions in the AR — possibly with a frequency as high as50% (REFS 14–18). Mutations also might be common inother crucial pathways10. Recent investigations there-fore support the theory that androgen ablation thera-py provides selective pressure to target the androgensignalling pathway16,18–20. For example, therapy withthe anti-androgen flutamide might select for mutantARs in which flutamide acts as an agonist rather thanan antagonist18. Even in the TRAMP (transgenic ade-nocarcinoma of mouse prostate) model of prostatecancer, in which SV40 large T antigen is overexpressedin the prostate luminal epithelial cells, mutations inthe AR frequently develop, and different types ofmutation are found in castrated versus intact mice21,22.So, the timing of the development of mutations thatcause AIPC remains uncertain. Intermittent androgenablation is considered a possible means of delaying

AR

SHBG Testosterone

5α-reductaseDHT

Ligandbinding

Dimerization andphosphorylation

AR

Androgen-responsive cell

DNA binding

ARA70

Androgen-response element

Target gene activation

PSA Growth Survival

Biological responses

P

AR

AR

AR AR

HSP

HSP

Co-activatorrecruitment

P

AR

P P

GTA

Figure 1 | Androgen action. Testosterone circulates in the blood bound to albumin (notshown) and sex-hormone-binding globulin (SHBG), and exchanges with free testosterone.Free testosterone enters prostate cells and is converted to dihydrotestosterone (DHT) by theenzyme 5α-reductase. Binding of DHT to the androgen receptor (AR) induces dissociationfrom heat-shock proteins (HSPs) and receptor phosphorylation. The AR dimerizes and canbind to androgen-response elements in the promoter regions of target genes6. Co-activators (such as ARA70) and corepressors (not shown) also bind the AR complex,facilitating or preventing, respectively, its interaction with the general transcription apparatus(GTA). Activation (or repression) of target genes leads to biological responses including growth,survival and the production of prostate-specific antigen (PSA). Potential transcription-independent actions of androgens are not shown.

ANDROGEN RESPONSE

ELEMENT

(ARE). Site composed ofhexanucleotide repeats and aspacer, usually in the promoterregions of target genes, thatcontains the androgen receptorzinc-finger-binding region.

GENERAL TRANSCRIPTION

APPARATUS

(GTA). A complex of proteinswith the potential to facilitatetranscription of genes. In vivospecificity of gene transcriptionby the GTA is regulated byinteracting transcriptionfactors.

© 2001 Macmillan Magazines Ltd

Feldman & Feldman Nat Rev Cancer 2001

•  DHT binds to AR in cytosol

•  AR translocates to nucleus

•  AR binds to DNA

•  AR recruits transcription machinery

anti-androgens

luteinizing hormones

detectable wild type (Fig. 1C), indicating that a clonal populationcarrying AR-V716M accounted for all three metastases. No othermutations recurred in this sample. Given that AR-V716M isactivated by a wide array of ligands (26), its predominance in thispatient’s cancer supports its role in treatment resistance.The splice variant AR23 was only in antiandrogen-treated

cases. A variant generated by the use of a cryptic splice site inintron 2 was identified in one or more clones in five of eight tumorsfrom treated patients, but in none of the hormone-naıve tumors.Alternative splicing inserted 69 bp of intron 2 in frame to add 23amino acids between the zinc fingers of the DNA binding domain.This variant, AR23, was previously found in androgen insensitivity

syndrome due to a mutation upstream of exon 3 that alteredsplicing (30). Recently, AR23 was identified in a prostate metastasisfrom a bicalutamide-treated patient (31). AR23 was engineered intoan expression plasmid, and its activity assayed after transfection.As also shown by Jagla and colleagues (31), AR23 was incapable ofnuclear localization on hormone addition but rather formedcytoplasmic speckles (Fig. 2A) and failed to activate androgen-responsive reporters (Fig. 2B). Previously, AR23 was shown toincrease endogenous AR-T878A activity when overexpressed inLNCaP cells (31). In Fig. 2B , AR23 also increased wtAR activation(2-fold greater PSA-luc activity) following coexpression in PC-3cells. Moreover, in the presence of AR23, wtAR was less inhibited by

Figure 1. Recurring AR mutations from prostate cancer metastases. A, mutations found in multiple cases. For codons carrying mutations to different aminoacids, both changes are shown. B, mutations in multiple clones per sample. Only DQ86 was shared among groups. AR domains and repeats are boxed. Mutationsabove the map are silent or nonsense; mutations below are missense. Codon color indicates treatment group. Q, polyglutamine tract; NTD, NH2-terminal domain;G, polyglycine tract; DBD, DNA binding domain; H, hinge region; LBD, ligand binding domain. C, V716M was the only AR sequence in three metastases frompatient 28 but did not occur in normal kidney. Electropherograms (left to right ): amplified cDNA clone from metastasis 1 with G3261A (numbering from GenBankNM_000044) resulting in V716M; wild-type sequence from normal kidney genomic DNA; cDNA and genomic DNA of metastasis 2. Green arrow, mutation; black arrow,wild-type base.

Treatment-Selected AR Mutations in Prostate Cancer

www.aacrjournals.org 4437 Cancer Res 2009; 69: (10). May 15, 2009

American Association for Cancer Research Copyright © 2009 on March 1, 2012cancerres.aacrjournals.orgDownloaded from

Published OnlineFirst April 14, 2009; DOI:10.1158/0008-5472.CAN-08-3605

Steinkamp et al Cancer Res 2009

AR transactivation domain


18 Chapter 1. Introduction

healthy tissue, and can emerge as an adaptive response to therapies targeting the

androgen signaling axis, in particular to the recently approved abiraterone acetate and

enzalutamide. [47, 53, 68, 133] Since these splice variants lack the LBD, therapeutic

strategies that target the binding of hormone to the LBD (either by inhibiting hormone

synthesis, such as abiraterone acetate, or by using potent competitors of androgens to

bind to the LBD, such as enzalutamide) appear not to have an e↵ect on the constitutive

activity of these splice variants.

(2%). Mutations are rarely found in untranslated regions [27].Mutations in the LBD could potentially affect the ligand specificityof AR, allowing it to be activated by non-androgenic steroids, oranti-androgens, in a promiscuous manner.

The T877A mutation, which has been described by multipleinvestigators, expands AR ligand binding to estrogen, progestin, se-lected corticosteroids, and selected anti-androgens [32]. Anothermutation in the ligand-binding pocket, H874Y, was identified inCRPC patients treated with flutamide. This mutation also increasesligand promiscuity, allowing DHEA, estradiol, progesterone, andhydroxyflutamide to activate transcription in various model sys-tems [33]. Mutations outside of the LBD could cause gain-of-func-tion or loss-of-function of the receptor by influencing on nuclearlocalization, co-regulator binding, protein stability, and promoterselectivity [34]. Constitutively active mutants have been describedin the regulatory NTD (G142V, M523V, G524D, and M537V) [35].

When certain steroid or steroid binding treatments are with-drawn (flutamide, bicalutamide, nilutamide, megestrol, cyproter-one acetate, prednisone, or estramustine), there is potential forimprovement in PSA and/or other parameters of disease progres-sion [36]. Whether or not mutated AR is responsible for these with-drawal responses is not clear but laboratory-based experimentsclearly uphold the feasibility of such a hypothesis.

Taken together, AR mutations in CRPC potentially allow for con-tinued ligand dependent activation of AR by creating promiscuousligand binding, altered binding of co-regulators, and/or alterationsin genomic regulatory element binding. More studies are needed toassess the clinical impact of these mutations on disease progres-sion. Better categorization of these mutants in patients may pro-vide a greater degree of personalization of therapeutic selection.

AR splice variants

A large number of AR splice variants (AR-Vs) have been recentlyidentified and characterized in CRPC patients (see Fig. 3). Thesevariants have insertions of cryptic exons downstream of the se-quences encoding the DBD or deletions of the exons encodingthe AR-LBD, resulting in a disrupted AR open reading frame andthe expression of truncated AR-V proteins devoid of the functional

LBD [37–42]. The majority of the AR-Vs identified to date displaysconstitutive activity. Two major AR-Vs, AR-V7 (also named as AR3)and ARv567es, have been shown to be capable of regulating targetgene expression in the absence of the full-length AR (AR-FL) signal-ing. Profiling of gene expression changes after knockdown orectopic expression of AR-V7 or ARv567es suggests that AR-Vsand AR-FL regulate an overlapping yet distinctive set of targetgenes [43–45]. These studies are rapidly evolving and significantdifferences in the AR-V transcriptome have been identified in dif-ferent studies, possibly due to the use of different model systems.

AR-Vs are prevalently upregulated in CRPC compared to hor-mone-naïve cancers, and can emerge as an adaptive response totherapies targeting the androgen signaling axis, especially new po-tent drugs such as abiraterone and enzalutamide [46,47]. It isimportant to recognize the existence of discrepancy between theabundance of AR-V mRNAs and that of AR-V proteins reported inclinical specimens. Although the levels of AR-V mRNAs have beenreported to be relatively low, Western analyses of 13 CRPC bonemetastases demonstrate that the levels of AR-V proteins could con-stitute a median of 32% of the AR-FL protein level [39]. In 38% ofthese CRPC bone metastases, the AR-V proteins are expressed ata level comparable to that of the AR-FL protein [39].

There is now intriguing evidence supporting the important con-tribution of the constitutively-active AR-Vs to the development ofcastration resistance. Ectopic expression of AR-V7 or ARv567esconfers castration-resistant growth of LNCaP xenograft tumors[42], whereas specific knockdown of AR-V7 attenuates the growthof castration-resistant 22Rv1 xenograft tumors in castrated host[38]. In addition, AR-V7 or ARv567es expression level has beenshown to be associated with adverse clinical outcomes. Higherexpression of AR-V7 in hormone-naïve prostate tumors predictsincreased risk of biochemical recurrence following radical prosta-tectomy [38,40]. Patients with high AR-V7 or detectable ARv567esexpression have significantly shorter cancer-specific survival thanother CRPC patients [39]. Thus, the extensive in vitro and xenograftliterature on AR-V expression translates into clinically relevantobservations.

In addition to the role of the constitutively-active AR-Vs in pro-moting castration-resistant progression after first-line ADT, their

Fig. 3. Schematic representation of the structure of AR-FL and AR-V transcripts and proteins. H, hinge region; U, untranslated region; ZF, zinc finger.

A. Egan et al. / Cancer Treatment Reviews 40 (2014) 426–433 429

Figure 1.7: Schematic representation of the transcripts and proteins of the full-length AR(AR-FL) and various splice variants (AR-V). NTD: N-terminal domain, DBD: DNA-bindingdomain, H: hinge region, LBD: ligand-binding domain, ZF: Zn finger, U: untranslated region.From [68].

Several natural or synthetic compounds have been identified to inhibit the function

of AR splice variants. [134–140] Further studies are needed to understand their mode

of action and to explore the potential of such agents to inhibit PCa progression. Several

of these compounds are currently in preclinical or clinical trials.

Bypass mechanisms PCa cells also adopt survival mechanisms that bypass the AR

and the AR signaling axis completely, and promote growth and survival of the cancerous

cells by increasing cell proliferation and inhibiting apoptosis. Many of the upregulated

signaling pathways in CRPC, including the MAPK and AKT pathway, contribute to

cell survival in an AR-independent fashion. [16] The MAPK cascade may influence cell

Egan et al Cancer Treat. Rev. 2014

AR transactivation domain The domain has transient secondary structure in regions involved in

protein protein interactions

DeMol et al ACS Chem Biol 2016


R E V I EW S







AR

SHBG Testosterone

5α-reductaseDHT

Ligandbinding


AR


DNA binding

ARA70



PSA Growth Survival


P

AR

AR

AR AR

HSP

HSP


P

AR

P P

GTA


ANDROGEN RESPONSE

ELEMENT



APPARATUS




•  DHT binds to AR in cytosol

•  AR translocates to nucleus

•  AR binds to DNA

•  AR recruits transcription machinery

?


TFIIF

Interaction with TFIIF

The NTD has been reported to interact directly with subunit 1 of TFIIF; this general transcription

factor is tightly associated with RNAP II and is part of the transcription machinery.

Chen et al EMBO J 2010

12.64.8

15.412.6

11.9 13.6

14.1

21.016.819.6

11.511.6

8.7

27.0

18.7

9.5 10.5

20.7

22.015.213.9 13.6

15.9

12.014.6

13.411.9

16.4

1 244

1Tfg2

A

B C D

1 73598 167 305 400 510 673 728

Tfg1

N-ter Chargedregion

WHdomain

LinkerWH

domain

Tfg3

400

Dimerizationdomain

Dimerizationdomain

Insertion

Insertion

144 227 35455 192 292

Figure 3 TFIIF domain architecture. (A) Schematic representation of TFIIF subunits and domains. Links between TFIIF subunits (blue) andwithin TFIIF subunits (grey). (B, C, D) Cross-links confirm domain modelling of yeast sequences into the human crystal structures for (B) theTfg1 WH domain, (C) the Tfg2 WH domain, and (D) the dimerization domain of Tfg1 (blue) and Tfg2 (red). Lysine residues (sphere for c-aatom) and observed links (dashed lines, red for high confidence, grey for low confidence, green for inter-protein Tfg1–Tfg2) with distancefound in the respective homology model.

Tfg3

DimerizationdomainTfg1 N-ter

Tfg2 WH domain

Topview

Tfg2 linker

Tfg1 charged region

Dynamic binding patch of Tfg2 C-terminal part(Not possible in the PIC)

Tfg1 WH domain

Tfg2 linker

Tfg1 N-ter

Tfg2 WH domain

Sideview

90

Tfg2(211*)

Tfg2(138*)

Tfg1(394)

Tfg1(349*)

Rpb2(345*)

Rpb2(164*)

Rpb2(90*)

Rpb2(353)

Rpb2(422)

Rpb2(426)

Rpb2(133)

Rpb2(358)

Rpb2(134*)

Clamp

Jaw

Lobe

WallProtrusion

Rpb1Rpb2

Rpb5

Rpb9

Rpb4/7

A

B

Figure 4 Architecture of the Pol II–TFIIF complex. (A) The TFIIF dimerization domain has been positioned on the Pol II surface based on aseries of cross-links between Pol II and the dimerization domain. Cross-link sites on the Pol II surface (slate and pink, matching the colour codeof the dimerization domain), cross-link sites in TFIIF (sphere for C-a atom), cross-links used for positioning the dimerization domain (reddashed line) and for validation (green dashed line). For linkage sites that are absent from the Pol II structure or the model of the Tfg1–Tfg2dimerization domain the nearest residue that is present is highlighted and labelled together with an asterisk (compare with SupplementaryTable 3 and Supplementary Table 5). (B) Location of high-confidence cross-linking sites on Pol II surface coloured according to cross-linkedTFIIF domains (represented in Figure 3). The dimerization domain has been placed on the Pol II surface; the location of other TFIIF regions isindicated. Two views are used, the top view and the side view, related by a 901 rotation around the horizontal axis. For linkage sites that areabsent from the Pol II structure or the model of the Tfg1–Tfg2 dimerization domain the nearest residue that is present is highlighted (comparewith Supplementary Table 3 and Supplementary Figures S4 and S5).

Architecture of the Pol II–TFIIF complexZA Chen et al

&2010 European Molecular Biology Organization The EMBO Journal VOL 29 | NO 4 | 2010 721

•  Interaction with the CTD

•  Reports that TFIIF binding causes AR to fold

McEwan and Gustafsson PNAS 1997

and partially purified as described previously (24). Proteinconcentrations were measured against BSA standards usingthe Bradford reagent (Bio-Rad).

Protein–Protein Interaction Assay. The microtiter plateinteraction assay was essentially as described previously (23,24). Briefly, AR142–485 or BSA control in binding buffer [20mM Hepes, pH 7.6y10% (volyvol) glyceroly100 mM KCly0.2mM EDTAy5 mM MgCl2y5 mM 2-mercaptoethanol] wereallowed to adsorb to the surface of a scintillation-microtiterplate (Wallac, Oy, Finland). Unoccupied surfaces were sub-sequently blocked with binding buffer containing 5 mgymlBSA, and the wells were incubated with binding buffer con-taining 1 mgyml BSA and radiolabeled human basal transcrip-tion factors, synthesized in a rabbit reticulocyte lysate system(Promega). After extensive washing with binding buffer � 1mgyml BSA, the bound radioactivity was measured directly ina micro ⇥ counter (Wallac, Oy, Finland), and bound proteinswere recovered in SDS sample buffer.

In Vitro Transcription and Squelching Assays. Preparationof yeast nuclear extracts for in vitro transcription, together withreporter genes and reaction conditions have all been describedin detail previously (24–26).

RESULTSThe N-Terminal Region of the Human AR Contacts Basal

Transcription Factors. The N terminus of the AR has beenshown to contain a complex transactivation function made upof multiple regions (see Introduction). In an attempt tounderstand the mechanism by which the AR activates tran-scription, a panel of basal transcription factors was screenedfor interactions with the receptor transactivation domain. A

polypeptide containing amino acids 142–485 of the humanreceptor was expressed and purified by metal chelation chro-matography (Fig. 1). The purified protein was allowed toadsorb onto the surface of a microtiter plate and incubatedwith 35S-labeled basal transcription factors TFIIB, TBP,TFIIE� and -⇥, TFIIF (RAP30 and RAP74), and two of thesubunits of TFIIH (p44 and p62). AR142–485 interacted selec-tively with the RAP74 subunit of TFIIF and showed modestbinding to the RAP30 subunit of TFIIF and to TBP (Fig. 2A).Little or no significant binding was observed with TFIIB,TFIIE, or the two subunits of TFIIH, as judged relative to theBSA control and binding to proteins lacking a transactivationfunction (Fig. 2 A and data not shown; see also refs. 23 and 24).To evaluate the significance of these observations, we com-pared the binding of AR142–485 with previously reported in-teractions between the serum response factor (SRF) andRAP74 (27) and the potent viral activator VP16 and TBP (seeref. 16). The binding of AR142–485 to RAP74 was as strong, ifnot stronger, compared with SRF245–508 (see references 23 and24; also data not shown). While the interaction with TBP wascomparable to that seen with VP16 (Fig. 2 A and refs. 23 and24). Recovery of the bound material and analysis by SDSyPAGE confirmed that the measured radioactivity representedintact labeled RAP74 binding to AR142–485, while little or no

FIG. 1. (A) Schematic representation of the human AR showingthe domain organization and the region of the N terminus used in thepresent study. (B) Purification of His-tagged AR142– 485 andAR142–485-Lex proteins. Proteins were estimated to be at least 75%pure from the Coomassie blue-stained gel.

FIG. 2. (A) Screening of 35S-labeled human general transcriptionfactors for binding to AR142–485. The measured radioactivity is plottedrelative to wells containing BSA only, which was set at 1. The bindingof TBP to the C-terminal transactivation domain of the herpes simplexviral activator protein VP16 is shown for comparison. The resultsrepresent the mean ⇥ SD of at least four observations from two ormore independent experiments, except TFIIH, subunits where themean of two observations only is shown. (B) SDSyPAGE analysis ofthe bound RAP74 subunit of TFIIF to AR142–485. The input represents5% of the material incubated per well.

8486 Biochemistry: McEwan and Gustafsson Proc. Natl. Acad. Sci. USA 94 (1997)

Interaction with TFIIF

TFIIF binds to a C-terminal motif of the NTD and induces in it a helical conformation

AR transactivation domain Binding epitope of AR on TFIIF defined by NMR is the same as that of FCP1, a phosphatase specific for RNA Pol II that plays a key role in transcription

termination.

Nguyen et al Biochemistry 2003

This PPI plays a role in CRPC It has been reported that the AR motif binding to

TFIIF is key for androgen-independent activation in CRPC cell lines [22Rv1, C4-2]

Dehm et al Cancer Res 2007 Figure 5. The AR 435WHTLF439 motif mediatesligand-independent TAU5 transcriptional activity.A, C4-2 cells were transfected with wild-type,DTAU5, and AHTAA mutant versions of hARGal4,as well as DTAU5 versions of hARGal4 with21-amino-acid inserts encompassing wild-type orAHTAA mutant versions of the 435WHTLF439

domain as indicated. PSAenh(GAL4)-LUC wasused as a reporter for these experiments. Cellswere maintained 48 h in the absence ofandrogens. Luciferase activity was determined.Columns, mean from at least three independentexperiments, each done in duplicate; bars, SE.Values are shown relative to the activity of theGAL4-based reporter construct in the absence ofandrogens and transactivator, which wasarbitrarily set to 1. Lysates were analyzed byWestern blot for hARGal4 protein expression withGal4-specific antibodies. B, C4-2 cells weretransfected with wild-type, DTAU5, or AHTAAversions of hARGal4 along with PSAenh(GAL4)-LUC. Cells were grown under androgen-freeconditions, cross-linked, lysed, and subjected toChIP with antibodies specific for Gal4 ornonspecific IgG. Immunoprecipitated DNAfragments were amplified by PCR using primersspecific for the PSAenh(GAL4)-LUC reporteras indicated in the schematic on the right. C,C4-2 cells were transfected with wild-type orDTAU5 versions of hARGal4, or DTAU5 versionsof hARGal4 harboring one, two, or three copiesof a 21-amino-acid insert encompassing the ARWHTLF motif. PSAenh(GAL4)-LUC was used asa reporter for these experiments. Assays weredone as described for A . D, LNCaP cells weretransfected with hARGal4-based constructs asdescribed for A and B . Cells were treated underserum-free conditions with 1 nmol/L miboleroneor ethanol for 24 h (*P V 0.05).

WxxLF Mediates Ligand-Independent AR Activity

www.aacrjournals.org 10073 Cancer Res 2007; 67: (20). October 15, 2007

American Association for Cancer Research Copyright © 2007 on December 20, 2011cancerres.aacrjournals.orgDownloaded from

DOI:10.1158/0008-5472.CAN-07-1267

W! L!F!

A! A!A!

433WHTLF437)

433AHTAA437)

433AHTAA437)

433WHTLF437)

detectable wild type (Fig. 1C), indicating that a clonal populationcarrying AR-V716M accounted for all three metastases. No othermutations recurred in this sample. Given that AR-V716M isactivated by a wide array of ligands (26), its predominance in thispatient’s cancer supports its role in treatment resistance.The splice variant AR23 was only in antiandrogen-treated

cases. A variant generated by the use of a cryptic splice site inintron 2 was identified in one or more clones in five of eight tumorsfrom treated patients, but in none of the hormone-naıve tumors.Alternative splicing inserted 69 bp of intron 2 in frame to add 23amino acids between the zinc fingers of the DNA binding domain.This variant, AR23, was previously found in androgen insensitivity

syndrome due to a mutation upstream of exon 3 that alteredsplicing (30). Recently, AR23 was identified in a prostate metastasisfrom a bicalutamide-treated patient (31). AR23 was engineered intoan expression plasmid, and its activity assayed after transfection.As also shown by Jagla and colleagues (31), AR23 was incapable ofnuclear localization on hormone addition but rather formedcytoplasmic speckles (Fig. 2A) and failed to activate androgen-responsive reporters (Fig. 2B). Previously, AR23 was shown toincrease endogenous AR-T878A activity when overexpressed inLNCaP cells (31). In Fig. 2B , AR23 also increased wtAR activation(2-fold greater PSA-luc activity) following coexpression in PC-3cells. Moreover, in the presence of AR23, wtAR was less inhibited by

Figure 1. Recurring AR mutations from prostate cancer metastases. A, mutations found in multiple cases. For codons carrying mutations to different aminoacids, both changes are shown. B, mutations in multiple clones per sample. Only DQ86 was shared among groups. AR domains and repeats are boxed. Mutationsabove the map are silent or nonsense; mutations below are missense. Codon color indicates treatment group. Q, polyglutamine tract; NTD, NH2-terminal domain;G, polyglycine tract; DBD, DNA binding domain; H, hinge region; LBD, ligand binding domain. C, V716M was the only AR sequence in three metastases frompatient 28 but did not occur in normal kidney. Electropherograms (left to right ): amplified cDNA clone from metastasis 1 with G3261A (numbering from GenBankNM_000044) resulting in V716M; wild-type sequence from normal kidney genomic DNA; cDNA and genomic DNA of metastasis 2. Green arrow, mutation; black arrow,wild-type base.

Treatment-Selected AR Mutations in Prostate Cancer

www.aacrjournals.org 4437 Cancer Res 2009; 69: (10). May 15, 2009

American Association for Cancer Research Copyright © 2009 on March 1, 2012cancerres.aacrjournals.orgDownloaded from

Published OnlineFirst April 14, 2009; DOI:10.1158/0008-5472.CAN-08-3605

Steinkamp et al Cancer Res 2009


433WHTLF437

Tau-5

ca 120 res

Androgen receptor


R E V I EW S







AR

SHBG Testosterone

5α-reductaseDHT

Ligandbinding


AR


DNA binding

ARA70



PSA Growth Survival


P

AR

AR

AR AR

HSP

HSP


P

AR

P P

GTA


ANDROGEN RESPONSE

ELEMENT



APPARATUS




•  The monomeric NTD is intrinsically disordered

•  It binds weakly to TFIIF

•  Helix induction and phosphorylation activate the interaction of the NTD with TFIIF

TFIIF

Androgen receptor


R E V I EW S







AR

SHBG Testosterone

5α-reductaseDHT

Ligandbinding


AR


DNA binding

ARA70



PSA Growth Survival


P

AR

AR

AR AR

HSP

HSP


P

AR

P P

GTA


ANDROGEN RESPONSE

ELEMENT



APPARATUS




•  Is it going to be possible to inhibit the protein protein interactions established by the NTD ?

•  In short is the NTD druggable ?

? TFIIF

Castration resistant prostate cancer Current treatments succeed for two to three years

but eventually prostate cells activate AR in an androgen-independent fashion to lead to the

refractory stage of the disease for which there is no treatment.

context of chromatin structure. Both PSA and TMPRSS2 areandrogen-regulated genes with well-characterized AREs.R1881 treatment significantly increased AR binding to AREs inpromoter (PSA-PR-ARE) and enhancer (PSA-EN-ARE) of PSA(Figure 2C) and TMPRSS2-ARE (Figure 2D), which was signifi-cantly inhibited by EPI-001. CBP was also recruited to PSA-EN-ARE, PSA-PR-ARE, and TMPRSS2-ARE in response to

androgen (p < 0.01). Consistent with a decrease in AR bindingto AREs, recruitment of CBP was also significantly decreasedby EPI-001 (p < 0.05). The decrease in androgen-induced ARinteraction with AREs by EPI-001 was not due to decreasedlevels of AR protein, general prevention of serine phosphoryla-tion of AR, or prevention of AR nuclear translocation (FiguresS2B –S2E).

Figure 1. EPI-001 Blocks AR Transcriptional Activity(A) Chemical structures of EPI-001 (BADGE.H2O.HCl) or the inactive analog 185-9-1 (BADGE.2H2O).

(B) Transactivation assays of the AR NTD were performed in LNCaP cells cotransfected with p5 3 Gal4UAS-TATA-luciferase and either AR-(1-558)-Gal4 DBD,

Gal4DBD-CREB prior to incubation with FSK or IL-6 or vehicle for 24 hr. Bicalutamide (BIC), EPI-001, or 185-9-1 were added 1h before the addition of FSK or IL-6.

(C) IC50s of EPI-001 versus 185-9-1 on AR NTD transactivation were examined in LNCaP cells with varying concentrations of EPI-001 and 185-9-1.

(D) EPI-001 inhibits both ligand-dependent and ligand-independent activation of the AR. LNCaP cells transfected with the PSA(6.1kb)-luciferase reporter and

treated with R1881, FSK, or IL-6 under serum-free conditions for 24 hr.

(E) 22RV1 cells transfected with the PSA(6.1kb)-luciferase reporter, pretreated for 1 hr with BIC or EPI-001, prior to addition of R1881 for an additional 24 hr.

(F) LNCaP cells stably transfected with ARR3-luciferase reporter were pretreated for 1 hr with EPI-001 (5 mg/ml) or 185-9-1 (5 mg/ml) before the addition of R1881

for an additional 24 hr. Bars represent the mean and standard deviation (± SD).

(G) Cos-1 cells transfected with ARR3-luciferase reporter and an expression vector for AR1-653 were treated with R1881or EPI-001 for 24 hr.

(H) PC3 cells transfected with ARR3-luciferase reporter and an expression vector for wild-type AR or AR1-653 were treated with R1881 or EPI-001. Concentra-

tions: R1881 (1 nM), FSK (50 mM), IL-6 (50 ng/ml), bicalutamide (BIC, 10 mM), EPI-001 (10 mg/ml), or 185-9-1 (10 mg/ml) unless stated otherwise. Error bars repre-

sent the mean and standard error of the mean (± SEM) in (B)–(E), (G), and (H). Student’s t test: *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.

Cancer Cell

EPI-001 Inhibits Transactivation of the AR NTD

Cancer Cell 17, 535–546, June 15, 2010 ª2010 Elsevier Inc. 537

*" *"

Andersen et al Cancer Cell 2010

Skinner M Nat Rev Cancer 2010

indication of tumor burden prior to castration and was measuredagain at the duration of the experiment. Initial serum PSA levelswere similar in both treatment groups (69 ± 11 versus 60 ± 13 ng/ml, p = 0.36) prior to castration. Serum PSA was decreased byonly 22.1% ± 29.4% at the duration of the experiment in controlmice compared to 67.2 ± 24.8% in EPI-001-treated mice(Figure 7A). These significant differences in serum PSA betweenthe two groups correlated to differences in tumor volume(Figure 7B). Prostatic tumors harvested from hosts receivingEPI-001 were approximately 62% smaller than tumors fromcontrol mice (122.6 ± 104.9 mm3 versus 326.5 ± 125.6 mm3,respectively, p = 0.009).

Subcutaneous PC3 Xenograft ModelsThe LNCaP and LTL313 xenograft models reflect the majority ofclinical CRPC that express AR and are dependent on endoge-nous AR for growth and survival.We employed PC3 human pros-tate cancer cells that are insensitive to androgen and do notexpress functional AR to provide an indication of specificity ofEPI-001. In vitro, EPI-001 did not significantly affect proliferationof PC3 cells (Figure 4E). Male mice were castrated and random-ized into two groups when PC3 s.c. tumors were approximately50 mm3 (tumor volume = 56.5 ± 5.8 mm3, n = 14). Four days aftercastration, the animals were treated every other day with an i.v.

dose of 50 mg/kg body weight EPI-001 or matching volume ofvehicle (control, DMSO). In contrast to LNCaP xenografts, EPI-001 did not reduce tumors (Figures 8A and 8B). These datasupport that EPI-001 is specific to the AR and does not affectcells that do not depend on functional AR for growth and survival.

DISCUSSION

Current therapies that target ARLBDprovide transient responsesbut do not provide cures for patients with CRPC and presumablyfail due to gain-of-function mutations in the LBD or to expressionof constitutively active splice variants lacking the LBD. The AF-1in the NTD contributes most of the transcriptional activity to theAR. AF-1 mutations in the NTD do not affect ligand-binding butobliterates transcriptional activity of the AR (Jenster et al.,1991). Drugs that target the AR NTD would block the activity ofthe AR regardless of ligand. Thus, while conventional therapyhas concentrated on androgen-dependent activation of the ARthrough its C-terminal LBD, our concept of blocking transactiva-tion of the NTD for therapy of CRPC has not been previously ad-dressed. Here we identified a lead compound for developingdrugs that can be used to delay or potentially cure CRPC. Weshow that EPI-001: (1) inhibited transactivation of the AR NTD;(2) was specific for inhibition of the AR without inhibiting PR orGR transcriptional activities; (3) blocked induction of androgen-regulated genes; (4) interacted with the AF-1 region in the NTD;(5) reducedprotein-protein interactionswith theNTD; (6) inhibitedconstitutively active AR1-653 that lacks the LBD; (7) reduced ARinteraction with well-characterized AREs on target genes; (8)blocked androgen-induced proliferation; and (9) caused cytore-duction of CRPC in xenografts dependent on AR for growthand survival without causing toxicity.EPI-001-related compounds were isolated from the marine

sponge Geodia lindgreni and have structural resemblance toBADGE (Bisphenol A Diglycidic Ether), which indicates thatthey may be of industrial origin. The collected sponge presum-ably bioaccumulated the BADGE derivatives from contaminatedseawater. Approximately 20 BADGE analogs were tested in cell-based assays to obtain additional Structure Activity Relationshipfor the pharmacophore. The compound BADGE.HCl.H2O, orEPI-001, had the most potent activity. EPI-001 and 185-9-1(BADGE.2H2O) are harmless metabolites of bisphenol A with noestrogenic or androgenic effects (Poole et al., 2004; Volkel et al.,2002; Tsai, 2006; Stroheker et al., 2004). Metabolic systemsare unable to transform BADGE into bisphenol A (Poole et al.,2004). BADGE and its chlorohydrins were examined with conclu-sions that they are not carcinogenic or genotoxic at daily doseswell in excess of those used here (EFSA, 2004). There are no

Figure 7. EPI-001 Inhibits Serum PSA and Growth of OrthotopicLNCaP Xenografts in Castrated Mice(A) Mice were administered 50 mg/kg body weight EPI-001 by i.v. every other

day for a total of 8 doses. Serum PSA was measured 2 days after the last dose

when the prostates were harvested and tumor volume measured. Initial serum

PSA: 69 ± 11 (control) and 60 ± 13 ng/ml (EPI-001), p = 0.36. SerumPSA is rep-

resented as the percentage drop from the start of the experiment. Error bars

represent the mean ± SEM.

(B) Tumor volumes and photographs of representative prostates with LNCaP

tumors from mice administered DMSO or EPI-001. Bars represent the

mean ± SEM.

Figure 8. EPI-001 Does Not Cause Cytoreduc-tion of PC3 Xenografts(A) PC xenograft growth in SCID mice bearing s.c.

tumors receiving i.v. injections of EPI-001 (n = 7) or

vehicle control (n = 7). Four days after castration,

animals received 50 mg/kg body weight by tail vein

injection every other day for a total of five injections.

Tumors were harvested 2 days after the last injection.

Bars represent the mean ± SEM.

(B) Photographs of harvested tumors. Scale bars

represent 10 mm.

Cancer Cell

EPI-001 Inhibits Transactivation of the AR NTD

Cancer Cell 17, 535–546, June 15, 2010 ª2010 Elsevier Inc. 543

Skinner M Nat Rev Cancer 2010 Sadar MD Cancer Res 2011

Castration resistant prostate cancer 7.58.08.59.0

110

115

120

125

δ1H/ppm

δ15 N

/ppm

8.128.138.148.15

119.4

119.5 C404

7.937.947.957.967.977.98

121.2

121.3

121.4A402

A398

7.937.947.957.96

123.8

123.9

Small chemical shift changes when incubated with 10 eq of EPI-001, especially in the weakest peaks.

-‐50 -‐40 -‐30 -‐20 -‐10 0 10 20

142 162 182 202 222 242 262 282 302 322 342 362 382 403 423 443


Δδ1

5 N/p

pb

* * *

Castration resistant prostate cancer

Tau-5 Tau-5

Fast

Castration resistant prostate cancer

In vivo EPI-001 covalently binds to the NTD of AR via nucleophylic attack of the C-Cl bond by

an AR side chain.

Muyung et al J Clin Invest 2013

Tau-5 Tau-5

Slow

Fast

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-‐50 -‐40 -‐30 -‐20 -‐10 0 10 20

142 162 182 202 222 242 262 282 302 322 342 362 382 403 423 443


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Cys 404

IDPs as drug targets

•  Play key roles in neurodegenerative diseases and cancer

•  Most often involved in weak protein protein interactions

•  Do not form persistant secondary and tertiary structures and therefore a challenge for structural biology and structure-based drug discovery

•  Whether it will be possible to drug ID proteins with small molecules is uncertain

Laboratory of Molecular Biophysics