birinapant, a smac-mimetic with improved tolerability for the treatment of solid tumors and...

Birinapant, a Smac-Mimetic with Improved Tolerability for theTreatment of Solid Tumors and Hematological MalignanciesStephen M. Condon,*,† Yasuhiro Mitsuuchi,*,† Yijun Deng,† Matthew G. LaPorte,†,∞ Susan R. Rippin,†

Thomas Haimowitz,† Matthew D. Alexander,†,× Pavan Tirunahari Kumar,† Mukta S. Hendi,†

Yu-Hua Lee,† Christopher A. Benetatos,† Guangyao Yu,† Gurpreet Singh Kapoor,† Eric Neiman,†

Martin E. Seipel,† Jennifer M. Burns,† Martin A. Graham,† Mark A. McKinlay,†,● Xiaochun Li,‡

Jiawei Wang,‡ Yigong Shi,‡ Rebecca Feltham,§,○ Bodhi Bettjeman,∥ Mathew H. Cumming,∥

James E. Vince,⊥,# Nufail Khan,⊥,# John Silke,§,⊥,#,◇ Catherine L. Day,∥ and Srinivas K. Chunduru†

†TetraLogic Pharmaceuticals, Inc., 343 Phoenixville Pike, Malvern, Pennsylvania 19355, United States‡School of Life Science, Tsinghua University, Beijing, 100084, China§Department of Biochemistry, La Trobe University, Victoria 3086, Australia∥Biochemistry Department, University of Otago, Dunedin 9054, New Zealand⊥The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia#Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia

*S Supporting Information

ABSTRACT: Birinapant (1) is a second-generation bivalentantagonist of IAP proteins that is currently undergoing clinicaldevelopment for the treatment of cancer. Using a range ofassays that evaluated cIAP1 stability and oligomeric state, wedemonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation ofcIAP1 in vitro. Smac-mimetic 1-induced loss of cIAPscorrelated with inhibition of TNF-mediated NF-κB activation,caspase activation, and tumor cell killing. Many first-generationSmac-mimetics such as compound A (2) were poorlytolerated. Notably, animals that lack functional cIAP1,cIAP2, and XIAP are not viable, and 2 mimicked features oftriple IAP knockout cells in vitro. The improved tolerability of 1 was associated with (i) decreased potency against cIAP2 andaffinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P2′ position of 1 was criticalto this differential activity, and this improved tolerability has allowed 1 to proceed into clinical studies.

■ INTRODUCTION

Apoptosis, or programmed cell death, is required for normalcellular function, tissue development, and homeostasis and ismediated by caspases, a family of cysteine proteases withaspartate specificity.1 Caspases facilitate the orderly dismantlingof the cytoskeleton, nuclear fragmentation, and processing ofcellular proteins like poly (ADP ribose) polymerase (PARP), allhallmarks of the apoptotic process.2 In addition, caspase-mediated apoptosis is the primary means by which cancerousand otherwise damaged cells are eliminated from tissueswithout eliciting an inflammatory response.2

As uncontrolled apoptosis would be detrimental to healthytissue, caspase activity is tightly regulated. Notably, caspaseactivation depends on formation of procaspase-activatingmultiprotein platforms such as the apoptosome3 andripoptosome,4,5 and then once activated, caspase activity ismodulated by another family of proteins, referred to as the

inhibitor of apoptosis, or IAP, proteins.6 The observation thatmany cancer cell lines and tumors possessed increased IAPgene copy number, elevated IAP protein levels, or translocationof specific IAP genes supported the hypothesis that deregulatedIAP activity might result in tumor initiation, progression, and/or resistance to anticancer treatment via aberrant caspaseinhibition.7,8 Consistent with this hypothesis, selective ablationof X-linked IAP (XIAP) by genetic depletion or siRNAtreatment rendered tumor cells sensitive to a variety ofconventional chemotherapeutic drugs.9

The mammalian IAP family10 has several members that haveclearly defined antiapoptotic functions including XIAP,11 twocellular IAPs (cIAP1, cIAP2),12 and melanoma IAP (ML-IAP/livin, Figure 1).13 Although these IAPs are reported to bind

Received: October 22, 2013Published: March 31, 2014

Drug Annotation

pubs.acs.org/jmc

© 2014 American Chemical Society 3666 dx.doi.org/10.1021/jm500176w | J. Med. Chem. 2014, 57, 3666−3677

pubs.acs.org/jmc

caspases, only XIAP is reported to be capable of caspaseinhibition via direct interaction with caspase-3, -7, and -9.11,14

Once activated, the N-terminus of caspase-9 (ATPF) can bind ashallow groove located on the XIAP baculovirus IAP repeat 3,or BIR3 domain, and this interaction inhibits its proteolyticactivity.15 Active caspase-3 and -7 are inhibited by binding tothe linker region immediately preceding the XIAP BIR2domain.16,17 The second mitochondria-derived activator ofcaspases (Smac, also known as DIABLO)18,19 contains a similarN-terminal tetrapeptide (AVPI). Structural and biochemicalstudies have shown that Smac and caspase-9 compete for thesame binding site on XIAP BIR3.20,21 These studies led to theparadigm that caspase-9 inhibition by XIAP BIR3 (prosurvival)can be relieved by Smac (proapoptosis) and prompted thesearch for small-molecule IAP antagonists that could disruptthe XIAP BIR3:caspase-9 interaction.22

The proapoptotic activity of IAP antagonists was initiallyattributed to their antagonism of XIAP-mediated caspaseinhibition. However, we and others observed that thesemolecules induced the rapid proteasomal degradation ofcIAP1 and cIAP2.23−25 Loss of cIAPs simultaneously resultedin the autocrine production of TNF and sensitized tumor cellsto the cytotoxic activity of TNF. From these observations, thecentral role of cIAP1 and cIAP2 in IAP antagonist-inducedtumor cell apoptosis was realized.

■ RESULTS

Discovery of 1, a Novel Smac-Mimetic as a CancerTherapeutic. The discovery of both monovalent26 and

bivalent IAP antagonists27 is well-described, and severalcompounds have entered into clinical development.28 Ourdecision to deliberately pursue a bivalent IAP antagonist wasbased on the observation that dimerization is a common themeof proapoptotic proteins, e.g., Smac;20 and as observed in theG-protein-coupled receptor field, multivalency has beensuccessfully employed to increase the potency and selectivityof both agonistic and antagonistic ligands.29 However, manyfirst-generation Smac-mimetics from our laboratories, including2 (Figure 2),24 were poorly tolerated. Moulin et al. used geneticdeletion in mice to demonstrate that loss of cIap1 inconjunction with either cIap2 or Xiap gene deletion resultedin mid-embryonic lethality.30 This observation suggested thatgeneration of a cIAP1/cIAP2 or cIAP1/XIAP double knockoutphenotype might negatively impact cellular signaling or survivalpathways.Thus, we initiated a program to identify second-generation

Smac-mimetics with a well-defined therapeutic index. Tofacilitate this process, we sought to characterize the biophysicalproperties of Smac-mimetic activity to uncover those propertiesthat correlated with antitumor efficacy and in vivo tolerability.Using these assays, we discovered 1 (Figure 2), a symmetric,bivalent mimetic of the N-terminal tetrapeptide (AVPI) ofSmac.Like many Smac-mimetics, both IAP binding motifs (IBM)

of 1 possessed a weakly basic N(Me)Ala as the N-terminal P1′residue that was required to maintain a salt bridge with anacidic residue within the IAP BIR3 binding groove. However,unlike first-generation Smac-mimetics such as 2 (Figure 2) and

Figure 1. Domain organization of the inhibitor of apoptosis (IAP) family of proteins: BIR, baculovirus IAP repeat domain; UBA, ubiquitin associateddomain; CARD, caspase recruitment domain; RING, really interesting new gene domain; CC, coiled-coil domain; NOD, nucleotide oligomerizationdomain; LRR, leucine-rich repeat domain.

Figure 2. Chemical structures of 1 and 2.

Journal of Medicinal Chemistry Drug Annotation

dx.doi.org/10.1021/jm500176w | J. Med. Chem. 2014, 57, 3666−36773667

other reported Smac-mimetics,23,27,31,32 Smac-mimetic 1 had anonbranched α-aminobutyric acid (Abu) residue at the P2′position and a 4S-hydroxypyrrolidine moiety at P3′. The P4′position comprised a 6-fluoroindole group that bound to ahydrophobic pocket located on the IAP BIR3 surface. The twoIBMs of 1 were linked through a single covalent bondconnecting the fluoroindole groups at the 2-position. Thesyntheses of 1 and 2 have been reported.33,34

Smac-mimetic 1 demonstrated dose-dependent caspase-3activation in cellular lysates.35 Addition of 1 was also associatedwith potent induction of apoptosis in multiple tumor celllines,35 and treatment of MDA-MB-231 cells with 1 inducedcaspase-dependent apoptosis by 72 h as evidenced by cleavageof the caspase-3 substrate, poly (ADP ribose) polymerase(PARP), and cell death.35 These results correlated with the lossof cIAP1 protein by 1.35

In the MDA-MB-231-derived murine xenograft model,treatment with 1 induced tumor regression in a dose-dependentfashion with no net body weight loss (BWL) (Figure 3A andFigure 3B; Supporting Information). In contrast, as an exampleof a first-generation Smac-mimetic, treatment with 2 caused amean 10% BWL after a single 1 mg/kg dose and a mean 18%BWL at study termination on day 4. In addition, fourtreatment-related deaths were observed at 5 mg/kg (n = 7,Figure 3C and Supporting Information). These observationssuggested that 1 and 2 might antagonize IAPs in distinct ways,thus leading to divergent biological outcomes.30 We thereforeutilized a series of assays to define more precisely the nature ofthe biological differences between these molecules.Smac-Mimetics 1 and 2 Interacted with IAP BIR3

Domains with Differing Affinities. To establish protein−ligand binding affinity, we employed a fluorescence polarizationassay. This assay monitored the displacement of an IBM-containing peptide, Abu-RPFK(5-carboxyfluorescein)-NH2,from the type III BIR domains36 of cIAP1, cIAP2, XIAP, and

ML-IAP.37 The positive control for this experiment was theSmac N-terminal tetrapeptide amide (AVPI-NH2).

38 Both 1and 2 bound the BIR3 domains of XIAP, cIAP1, and cIAP2 andthe single BIR domain of ML-IAP (Table 1).24,35 However,while 2 bound tightly to each of these BIR domains (Ki ≈ 1nM), the affinity of 1 for the BIR3 domains from XIAP andcIAP2 was reduced approximately 40-fold.

The binding of monovalent or bivalent IAP antagonists isreported to activate autoubiquitylation and subsequentproteasomal degradation of cIAP1 and, in some instances,cIAP2.23−25,39 Cell-based assays were therefore developed tomonitor the degradation of green fluorescent protein (GFP)tagged cIAP1 (i.e., GFP-cIAP1) and GFP-cIAP2 in A375cells.35 We recently reported that these GFP-based assays werea reliable indicator of endogenous wild-type cIAP1 and cIAP2protein levels.35 We also established an NF-κB-luciferasereporter assay in HeLa cells that are resistant to IAPantagonist-induced cell death to identify Smac-mimetics thatpotently inhibited TNF-mediated NF-κB/p65 activation.35

In this system, degradation of GFP-cIAP1 was similarfollowing treatment with 1 or 2 (Table 2). Also, the NF-κB-luciferase assay indicated that both 1 and 2 inhibited TNFR1-dependent NF-κB activation with comparable potency. In

Figure 3. In vivo activity and/or percent body weight change in mice treated with 1 or 2 (compound A). Arrows indicate dosing with drug. (A)Female athymic nu/nuMDA-MB-231-tumor bearing mice were treated with the described doses of vehicle control, 1, or docetaxel (positive control).(B) Mean percent body weight change (n = 10) was measured over 20 days. (C) Mean percent body weight change in female CB.17 scid mice (n =7) following 4 days of treatment. Dosing of 2, as indicated by the arrows, is at 1 and 5 mg/kg. A total of 4/7 treatment-related deaths were recordedfor the 5 mg/kg dosing cohort by day 5.

Table 1. Mean Ki Values for 1 and 2 to Selected IAP BIRDomainsa

Ki (nM)

compd XIAP BIR3 cIAP1 BIR3 cIAP2 BIR3 ML-IAP BIR

1 50 ± 23 ∼1 36 ∼12 ∼1 ∼1 ∼1 ∼1AVPI-NH2 130 2 20 900

aResults are expressed as mean ± standard deviation from four orgreater independent assays unless otherwise indicated.



contrast, GFP-cIAP2 was not as efficiently degraded followingtreatment with 1. This might be related to the reduced affinityof 1 for BIR3 of cIAP2. However, cIAP2 levels are reported tobe regulated by the E3 ligase activity of cIAP1,40 and IAPantagonist-induced degradation of cIAP2 was shown to bedependent on the presence of cIAP1.41 In addition, it has beenreported that cIAP2 forms a more stable RING-mediated dimerthan cIAP1 and thus has higher intrinsic E3 ubiquitin ligaseactivity in the absence of Smac-mimetics.39 Therefore, oneinterpretation of these results was that 2 promoted the E3ubiquitin ligase activity of cIAP1, which better mediated cIAP2degradation, than 1.41 The observation that 1 was less activeagainst cIAP2 than 2 might explain its improved tolerabilityrecognizing the embryonic lethality of the cIap1−/−cIap2−/−

double knockout phenotype.30

Stereochemistry of the P2′ Abu Residue of 1 WasCritical for cIAP1 BIR3 Binding, GFP-cIAP1 Degradation,and Inhibition of TNF-Induced NF-κB Activation. Becauseboth monovalent and bivalent IAP antagonists induced cIAP1degradation,23−25,39 we investigated whether both IBMs of 1were required for 1-induced GFP-cIAP1 degradation andinhibition of TNFR1-dependent NF-κB activation. Epimer 3(or mono-D-Abu 3) contained stereoinversion at a single Aburesidue of 1 and thus would serve as a “monovalent” control for1 (infra). Diastereomer 4 (or, bis-D-Abu 4) possessed two D-

Abu residues and served as a negative control for both 1 and 3in the biophysical assays (infra), and ent-birinapant (5) wasprepared as another non-IAP-binding, negative control (Figure4).Mono-D-Abu 3, with only one fully intact IBM, bound to

cIAP1 BIR3 with comparable affinity to 1. Bis-D-Abu 4exhibited no binding to the isolated BIR3 domain of cIAP1,suggesting that stereoinversion of the central Abu residueprevented protein−ligand interaction. Functionally, two L-Abu-containing IBMs (as in 1) were preferred for efficientdegradation of GFP-cIAP1 and potent inhibition of TNF-induced NF-κB activation (Table 3). In contrast, however, 3was approximately 20-fold less effective at degrading GFP-cIAP1 and >50-fold less potent at inhibiting TNF-induced NF-κB activation. Additionally, 3 had 100-fold reduced ability todegrade GFP-cIAP2 relative to 1.39,40 These results indicatedthat 1 activated cIAP1 autoubiquitylation more efficiently than3, suggesting that two fully functional IBMs were more efficientat promoting the degradation of cIAP1.This result might have been anticipated, as the structure of

Smac-peptides bound to the BIR3 domain of cIAP1 revealedthat the P2′ backbone residue makes critical hydrogen bondingcontacts with R308 of BIR3.42 Inversion of this stereocenterwould be expected to disrupt this interaction. Diastereomers 4and 5 displayed negligible activity in the cIAP1 binding, GFP-

Table 2. In Vitro Comparison of 1 and 2 in GFP-cIAP1 Degradation and GFP-cIAP2 Degradation and Inhibition of TNF-Mediated NF-κB-Luciferase Assaysa

EC50, nM

ΔGFP-cIAP1 ΔGFP-cIAP2

compd cIAP1 BIR3 Ki, nM 2 h 24 h 2 h 24 h inhibition of TNF-mediated NF-κB-luc at 4 h

1 ∼1 17 ± 11 5 ± 3 108 ± 46 151 ± 86 9 ± 52 ∼1 13 ± 5 4 ± 2 43 ± 17 27 ± 17 8 ± 2

aΔGFP-cIAP1 and ΔGFP-cIAP2: loss of GFP-cIAP1 and GFP-cIAP2, respectively, as assessed by flow cytometry (see Experimental Section).Results are expressed as the mean ± standard deviation of four or greater independent assays performed in duplicate unless otherwise indicated.

Figure 4. Analogues 3−5 containing one or more stereoinversions of IAP binding motifs.

Table 3. In Vitro Comparison of 1 and Diastereomers 3−5 in GFP-cIAP1 Degradation and GFP-cIAP2 Degradation andInhibition of TNF-Mediated NF-κB-Luciferase Assaysa

EC50, nM


compd cIAP1 BIR3 Ki, nM 2 h 24 h 2 h 24 h inhibition of TNF-mediated NF-κB-luc at 4 h

1 ∼1 17 ± 11 5 ± 3 108 ± 46 151 ± 86 9 ± 53 ∼1 417 ± 92 79 ± 21 >10000 4929 ± 1598 1728 ± 9854 >10000 5220 ± 200 3457 ± 135 5718 ± 157 5537 ± 115 4981 ± 9965 >10000 >10000 >10000 >10000 >10000 >10000




cIAP-based, and NF-κB-luciferase assays, indicating thatinversion of both P2′ stereocenters inactivated 1. As the bis-D-Abu analogue (4) displayed no appreciable binding to cIAP1BIR3, the mono-D-Abu analogue (3) was effectively a formalmonovalent IAP antagonist because it possessed only a singlefunctional pharmacophore.29

Smac-Mimetics 1 and 2, but Not Mono-D-Abu 3,Stabilized cIAP1 Dimers toward Co-Immunoprecipita-tion and More Efficiently Promoted cIAP1 Autoubiqui-tylation. Previously we established that multiangle laser lightscattering coupled in-line to size exclusion chromatography andmass spectral analysis (MALLS-SEC) could be used to monitorthe oligomeric state of the truncated cIAP1 protein (cIAP1-BUCR, Figure 1).39 Notably, addition of 2 or members of astructurally diverse set of monovalent IAP antagonistsprompted cIAP1-BUCR dimerization and subsequent E3ubiquitin ligase activity.39 This is consistent with thesecompounds binding to BIR3 and disrupting a criticalinteraction between BIR3 and the RING domain that otherwisestabilized the autoinhibited cIAP1 BUCR monomer.39,43,44

With 2 as a positive control, we used MALLS-SEC analysis tocharacterize the oligomeric state of cIAP1-BUCR in thepresence of 1, the mono-D-Abu epimer (3), and the bis-D-Abu diastereomer (4). As described previously, in the absenceof compounds, even at ∼300 μM the monomeric form ofrecombinant cIAP1-BUCR predominated (MW ≈ 40 kDa),whereas addition of 1 or 2 resulted in formation of the highermolecular weight cIAP1-BUCR dimer (MW ≈ 80 kDa; Figure5A).39 Mono-D-Abu 3 also promoted cIAP1-BUCR dimeriza-

tion in this assay, while the nonbinding bis-D-Abu (4) did notsubstantially promote cIAP1-BUCR dimerization. Thus, bothmonovalent and bivalent IAP antagonists induced dimerizationof cIAP1-BUCR.To assess the E3 ligase activity of IAP antagonist-induced

cIAP1-BUCR dimers, we monitored cIAP1 autoubiquitylationin vitro.39 Addition of bis-D-Abu 4 did not prompt ubiquitin(Ub) transfer above background (Figure 5B), consistent withthe inability of 4 to bind cIAP1 BIR3 or form a stabilized cIAP1dimer (Figure 5A). However, Ub transfer by cIAP1-BUCR wasincreased upon addition of both bivalent ligand 1 or 2, ormono-D-Abu 3. This was consistent with their ability topromote protein dimerization. The increased higher molecularweight poly-ubiquitylated cIAP1-BUCR products formed uponaddition of 1 or 2, relative to treatment with mono-D-Abu 3,suggested that interaction of 1 or 2 with cIAP1 activated Ubtransfer to a greater extent than mono-D-Abu 3.To uncover further differences between 1, 2, or 3-induced

cIAP1 E3 complexes in a cellular context, we employed co-immunoprecipitation (co-IP) and differentially tagged cIAP1-BUCR constructs.39 Like the first-generation Smac-mimetic 2,1 allowed the co-IP of Flag-tagged cIAP1-BUCR with mbw-tagged cIAP1-BUCR39,45 (Figure 5C). Mono-D-Abu 3 wasineffective. This suggested that the 1- or 2-induced cIAP1-BUCR dimers were further stabilized by the simultaneousengagement of two cIAP1 BIR3 domains, while mono-D-Abu 3was unable to occupy the second cIAP1 BIR3 domain tostabilize the dimer. Together, these results suggested that theincreased stability of the cIAP1-BUCR dimer when cross-linked

Figure 5. Characterization of 1, 2, mono-D-Abu 3, and bis-D-Abu 4. (A) MALLS-SEC analysis of cIAP1-BUCR (25 μM) in the absence or presenceof a 2-fold excess of 1, 2, 3, or 4. For each, the refractive index (line) and the calculated molecular mass (squares) are shown. (B) Comparison of 1,2, 3, and 4 in an autoubiquitylation assay. (C) Co-immunoprecipitation of Flag- and mbw-tagged cIAP1-BUCR by 1 or 2. Treatment with eithermono-D-Abu 3 or bis-D-Abu 4 did not induce co-immunoprecipitation of cIAP1-BUCR proteins.



by Smac-mimetic 1 or 2 correlated with increased biologicalactivity (Tables 2 and 3).β-Branching at the P2′ Position of Smac-Mimetics

Enhanced cIAP2 Degradation. Having established that thestereochemistry at P2′ had a profound effect on Smac-mimeticactivity, we investigated the contribution of the P2′ residuetoward IAP functional activity. A series of symmetric P2′analogues were prepared using known methods (Figure 6).33,34

Glycine analogue 6 displayed reduced cIAP1 BIR3 bindingrelative to 1 and was inactive in the functional assays (Table 4).The P2′ alanine-containing derivative 7, despite reduced cIAP1BIR3 binding, displayed modest functional activity in the GFP-cIAP1 assay (Table 4). Smac-mimetic 8 (P2′ = Nve) wascomparable to 1 in the GFP-cIAP1 and GFP-cIAP2 assays andin its ability to inhibit TNF-induced NF-κB activation. The P2′β-branched analogues (9 and 10) were approximately 10-foldmore potent in the GFP-cIAP2 assays relative to nonbranchedanalogues 1 and 8. These results confirmed that, like 2, otherSmac-mimetics with β-branching at the P2′ position could exerta greater impact on cIAP2 levels than the P2′ Abu-containing 1.Co-Crystallographic Analysis Showed That 1, but Not

Mono-D-Abu 3, Can Simultaneously Bind Two IAP BIR3Domains. Crystallographic and NMR analysis of severalmonovalent IAP antagonist-bound XIAP BIR3 complexesindicated that the P2′ residue projected away from the proteinsurface. In fact, substitution was well-tolerated at thisposition.26,32,46,47 The cocrystal structures of 1 in complexwith two XIAP BIR3 domains, and the cocrystal structure of 3bound to a single cIAP1 BIR3 domain were solved. In theformer, the two IBMs of 1 presented themselves in a gaucheorientation relative to the biindole core which positioned thetwo P2′ Abu residues in proximity (Figure 7A). Overlay of the

BIR3-bound IBMs of 1 and 3 revealed similar protein−ligandcontacts, while the D-Abu-containing IBM of 3 was projectedaway from the protein surface and anti to the bound IBM(Figure 7B). Together with the MALLS-SEC analysis (supra),these cocrystallographic results suggested that Smac-mimeticshad the capacity to engage multiple IAP BIR3 domains46 withinhigher-order IAP complexes, e.g., dimerized E3 ligases, and thatthe central P2′ region might be contributing to the stability,selectivity, or activity of certain Smac-mimetic-induced IAP E3ubiquitin ligase complexes.

Smac-Mimetic 2 and Other P2′ β-Branched Smac-Mimetics, Unlike 1, Potently Inhibited XIAP-DependentNOD Receptor-Mediated NF-κB Activation. We reasonedthat a component of the improved tolerability of 1 versus first-generation bivalent compounds such as 2 might relate todifferential activity on XIAP-mediated signaling pathways. TheNOD1 and NOD2 receptors are intracellular patternrecognition receptors that sense the pathogenic bacterialfragments meso-diaminopimelic acid (DAP) and muramyldipeptide (MDP), respectively.48 NOD signaling has beenreported to depend on ubiquitylation of the receptorinteracting protein kinase 2 (RIPK2) by XIAP.49,50 TheXIAP-dependent caspase-3 activation assay adequately demon-strated the ability of 1 and other IAP antagonists to inducecaspase-3 activity. However, this assay was insufficient forevaluating XIAP antagonism in whole cells.To evaluate the effect of NOD antagonism by 1 and other

IAP antagonists, we established both DAP- and MDP-stimulated NF-κB-luciferase reporter gene assays (to reporton NOD1 and NOD2 receptor signaling, respectively). Wescreened IAP antagonists at a single 10 μM drug concentration.Recently, impaired degradation of IκBα was observed in MDP-stimulated cells following treatment with 2 in both aconcentration- and XIAP-dependent fashion.50 Consistentwith those results, 2 fully inhibited DAP- and MDP-mediatedNF-κB activation (99% at 10 μM) (Table 5).50 In contrast, 1exhibited only modest inhibition of these NOD-dependentsignaling pathways (35% at 10 μM). Mono-D-Abu 3 exhibitedreduced binding to XIAP BIR3 and was inactive in the NOD2-based assay (Table 5). Incorporation of β-branching at the P2′position (i.e., 9 and 10) afforded increased XIAP BIR3 bindingand inhibition of NOD-mediated NF-κB activation, comparableto 2.These results suggested that in addition to potent

antagonism of both cIAP1 and cIAP2, Smac-mimetics like 2which contained β-branched P2′ residues also antagonizedXIAP in the cellular setting. Smac-mimetic 1 was less effective

Figure 6. Chemical representation of bivalent Smac-mimetics 6−10with increasing size and hydrophobicity at P2′.

Table 4. In Vitro Comparison of 1 and P2′ Analogues 6−10 in GFP-cIAP1 Degradation and GFP-cIAP2 Degradation andInhibition of TNF-Mediated NF-κB-Luciferase Assaysa

EC50, nM


compd R cIAP1 BIR3 Ki, nM 2 h 24 h 2 h 24 h inhibition of TNF-mediated NF-κB-luc at 4 h

6 H 65 ± 2.4 2576 ± 1127 847 (n = 2) 4603 ± 1767 5298 (n = 1) 707 ± 2267 Me 92 ± 36 463 ± 183 65 ± 21 3129 ± 1265 3878 ± 522 584 ± 2681 Et ∼1 17 ± 11 5 ± 3 108 ± 46 151 ± 86 9 ± 58 n-Pr ∼1 9 ± 4 6 ± 4 67 ± 11 86 ± 28 5 ± 29 i-Pr ∼1 3 ± 2 1 ± 0.2 6 ± 2 4 ± 2 1 ± 110 t-Bu ∼1 5 ± 2 1 ± 0.2 6 ± 2 5 ± 2 4 ± 2




at inhibiting NOD-dependent NF-κB activation even at 10 μM.This differential XIAP-dependent activity between 1 and 2 andother P2′ β-branched Smac-mimetics may provide furtherexplanation for the improved tolerability of 1.30

Smac-Mimetic 2 and Other P2′ β-Branched Smac-Mimetics, but Not 1, Induced the Secretion of theInflammatory Cytokine IL-1β from LPS-Primed BoneMarrow-Derived Macrophages. Gene ablation studies havedemonstrated an embryonic-lethal phenotype in animals thatlack both cIAP1 and either cIAP2 or XIAP (i.e.,cIap1−/−cIap2−/− or cIap1−/−Xiap−/−).30 This lethality may bea consequence of an “activated inflammasome phenotype” thatwas observed in triple knockout (i.e., cIap1−/−cIap2−/−Xiap−/−)macrophages.51 Genetic pan-IAP depletion was shown totrigger NLRP3-caspase-1 inflammasome-dependent IL-1βprocessing and secretion, suggesting that these three IAPs acttogether to suppress inflammasome activation.51,52 Recently, itwas reported that 2 recapitulated the phenotype observed intriple knockout (cIap1−/−cIap2−/−Xiap−/−) murine bonemarrow-derived macrophages (BMDMs).51

Given the heightened activity of 2 versus 1 on cIAP2, wespeculated that the improved tolerability of 1 might also beassociated with reduced antagonism of XIAP functional activityin this system. We thus evaluated a selection of Smac-mimetics

in both LPS-primed wild-type (WT) and XIAP knockout(Xiap−/−) murine BMDMs to assess their capacity to inducethe secretion of IL-1β following treatment with Smac-mimetic(Figure 8).From this analysis, it was evident that the P2′ β-branched

Smac-mimetics 2, 9, and 10 were capable of inducing IL-1βsecretion from LPS-primed WT BMDMs in a dose-dependentfashion.51 In contrast, treatment with the nonbranched Smac-mimetics (1 and 8) caused no increase in IL-1β secretion.These results were consistent with the ability of 2 to antagonizeall three IAPs as measured in the GFP-cIAP1, GFP-cIAP2, andXIAP-dependent NOD assays.To test whether the inability of 1 and 8 to induce IL-1β

secretion in this model was because they did not antagonizeXIAP function, we treated Xiap−/− BMDMs with 1 or 8. Asexpected, compounds 2, 9, and 10 induced IL-1β secretion inthe Xiap−/− BMDMs. Similarly compounds 1 and 8 induced IL-1β secretion in the Xiap−/− BMDMs. This demonstrated thateach of these molecules was capable of inducing IL-1β in theabsence of XIAP function. The implication of these results wasthat neither 1 nor 8 was itself able to abrogate XIAP activity inWT BMDMs sufficiently to result in inflammasome activation.The reason for the apparent discrepancy between the

behavior of 1 in the caspase-3 activation assay (supra)35

compared to the NOD and IL-1β assays is unclear but mayrepresent differences in XIAP RING domain dependency inthese two systems. Caspase-3 inhibition has been demonstratedfor several RING-deleted XIAP constructs,53,54 while NODsignaling was reported to be XIAP RING-dependent.50

Together these results suggested that the contrasting IAPantagonism profiles exhibited by 1 versus 2 was likelyresponsible for the observation of differential tolerability.

■ DISCUSSION

In this report, we characterized the activity of 1 and P2′ β-branched Smac-mimetics exemplified by 2 in an attempt toidentify the features that confer unique properties on these twocompounds. Although the literature offers support for thefunctional differences between monovalent and bivalent IAPantagonists including (i) bivalent IAP antagonists inducing the

Figure 7. Representations of the cocrystal structure of 1 bound to two XIAP BIR3 domains and mono-D-Abu 3 bound to a single cIAP1 BIR3domain. (A) The electrostatic surface of the XIAP BIR3 is shown, highlighting the key hydrogen bonding interactions for Smac-mimetic recognitionby IAP proteins (1, yellow; second XIAP BIR3, turquoise; PDB code, 4KMP). (B) Mono-D-Abu 3 bound to a single cIAP1 BIR3 domain (1, yellow;XIAP BIR3, turquoise; 3, salmon; cIAP1 BIR3, gray; PDB code, 4KMN) is overlaid with 1 bound to two XIAP BIR3 domains (showing only a single1:XIAP BIR3 domain for clarity), demonstrating conserved binding mode of 1 or 3 with cIAP1 and XIAP BIR3.

Table 5. In Vitro Comparison of 1 and Selected Smac-Mimetics in XIAP BIR3 Binding and NOD1- and NOD2-Dependent NF-κB-Luciferase Assaysa

% inhibition of

compdXIAP BIR3Ki, nM

DAP-mediatedNF-κB-luc at 4 h

MDP-mediatedNF-κB-luc at 4 h

1 50 ± 23 35 ± 16 35 ± 142 ∼1 98 ± 1 99 ± 13 102 ± 17 ND no inhibition8 ND ND 24 ± 0.19 16 ± 0.6 86 (n = 2) 100 (n = 2)10 2.2 ± 0.3 99 ± 2 98 ± 2

aResults are expressed as the mean ± standard deviation of four orgreater independent assays unless otherwise indicated. ND: notdetermined.



degradation of TRAF2-associated cIAP1 and cIAP2 andinhibiting TNFR1-mediated NF-κB activation,35 (ii) bivalentIAP antagonists inhibiting the XIAP-dependent NF-κBactivation via NOD receptors,50 and (iii) bivalent IAPantagonists inducing the ubiquitylation and degradation ofML-IAP,55 little information is available on the functionaldifferences among structurally unique bivalent IAP antagonists.We demonstrated that 1 is much better tolerated in animals

than the first-generation Smac-mimetic 2 and that it causesdose-dependent tumor regression in the MDA-MB-231xenograft model (Figure 3). Compounds 1 and 2 arecomparable in their abilities to bind cIAP1, induce proteindimerization, promote ubiquitin transfer, and inhibit TNFR1-dependent NF-κB activation (Table 2 and Figure 5). Theseresults correlated with the ability of 1 or 2 to induce apoptosisin tumor cell lines.24,35

However, the poor tolerability of 2 is reminiscent of thelethal cIap1−/−cIap2−/− or cIap1−/−Xiap−/− double knockoutphenotypes30 and the systemic inflammatory phenotypeobserved in cIap1lysCrecIap2−/−Xiap−/− triple knockout mice.56

In contrast, 1 spared both cIAP2 protein40,41 and XIAPfunctional activity as assessed in the NOD and IL-1β assays.This unique profile of 1 demonstrated the key importance ofthe P2′ position: like 2, other P2′ β-branched Smac-mimetics(i.e., 9, 10) were potent inhibitors of XIAP-dependent NODsignaling49,50,57 and induced the secretion of IL-1β fromBMDMs in a dose- and XIAP-dependent fashion.51

Importantly, incorporation of the nonbranched Abu residueat P2′ was required to inhibit TNFR1-mediated NF-κBactivation and activate caspase-dependent apoptosis35 by 1while avoiding inhibition of XIAP-dependent NF-κB activation

as evidenced in the NOD assay. Smac-mimetic 8 exhibited asimilar in vitro profile to 1. Further work is ongoing to evaluateits tolerability profile. Whether the functional differencesbetween 1 and P2′ β-branched Smac-mimetics like 2 are theresult of different propensities to cross-link IAP proteinsremains unknown.The assays described herein and the conclusions drawn from

these experimental results may allow for the identification ofother Smac-mimetics with preferred tolerability profiles.However, on the basis of its unique in vitro profile andsupportive preclinical toxicology, 1 was selected for advance-ment into human clinical trials for the treatment of both solidtumors and hematological malignancies.

■ EXPERIMENTAL SECTIONBiological Assays. Plasmids and Mutagenesis. cIAP1-BUCR was

cloned into pGEX-6p3 and confirmed by sequencing as previouslydescribed.39,58 Purified proteins have seven additional N-terminalresidues, GPLGSGT, as a result of cloning. The QuikChange site-directed mutagenesis kit (Stratagene) was used to generate mutants.Ubiquitin was expressed in pQE80L with an N-terminal 6×His fusion;UbcH5b was expressed as a GST fusion protein. Complete sequenceof all constructs can be obtained upon request. For the BIR3 bindingassays, the residue boundaries for the protein constructs employed arecIAP1 BIR3, 255−364; cIAP2 BIR3, 236−342; and XIAP BIR3, 238−358.

Transfections, Antibodies, and Reagents. Transient transfectionstypically using 1 μg of plasmid DNA per 10 cm plate of cells wereperformed with Effectene according to the manufacturer’s instructions(QIAGEN) as previously described.39 Antibodies were sourced asfollows: monoclonal anti-β actin (Sigma), monoclonal anti-Flag1:2000 (Sigma), monoclonal anti-mbw (mbw), monoclonal anti-ubiquitin (Cell Signaling), and IL-1β (R&D Systems). IAP antagonists

Figure 8. Effect of selected Smac-mimetic treatment of the secretion of IL-1β from WT and XIAP knockout (Xiap−/−) murine bone marrow-derivedmacrophages. WT and Xiap−/− BMDMs were primed with LPS ± the indicated concentration of Smac-mimetics for 24 h. WT BMDM results arepresented as the mean + SEM (n = 3 biological independent repeats). Xiap−/− BMDM results are presented for n = 1 (this result is representative ofthree independent experiments performed on different days). Total levels of IL-1β induced by Smac-mimetics in these assays varied, but the trendwas identical.



were prepared at TetraLogic Pharmaceuticals (Malvern, PA). Allreagents were used according to the manufacturer’s protocols.Protein Expression and Purification. All cIAP1 proteins were

expressed as glutathione S-transferase (GST) fusions in E. coliBL21(DE3) as previously described.39 Briefly, protein expressionwas induced by addition of IPTG and cultures were incubated at 18 °Covernight. The recovered cell pellet was sonicated in lysis buffer (PBS,pH 7.4, containing 2 mM DTT), and the soluble GST-fusion proteinwas bound to glutathione sepharose. Resin bound GST-cIAP1 proteinswere then washed and treated with PreScission protease (Amersham).The soluble fraction was purified using either a Sephadex 75 column ora Superdex S200 column equilibrated in PBS. Fractions that containedpurified protein were combined, concentrated, and quantified.Ubc5Hb was expressed in E. coli at 18 °C and purified from clarifiedlysate by affinity chromatography and size exclusion in a similarmanner.MALLS Analysis. Concentrated cIAP1-BUCR that had been fully

reduced by addition of DTT was analyzed by multiple angle laser lightscattering (MALLS) (Wyatt Technology) when coupled to a Superdex200 HR 10/30 column (GE Healthcare) equilibrated in 1× PBS, pH7.4, as previously described.39 Data were analyzed using ASTRA V(Wyatt Technology). IAP antagonists were added to protein samplesat 2× the molar concentration of binding sites and left on ice for 30min prior to their separation on the S200 column.Ubiquitylation Assays. For ubiquitylation assays, ∼5 μM soluble

cIAP1-BUCR, which had been mixed with a 1.25 molar excess (basedon the number of binding sites) of the indicated IAP antagonists for 15min, was mixed with 7.5 μM UbcH5b and 100 nM E1 in 20 mM Tris,pH 7.5, 50 mM NaCl, 50 μM ubiquitin, 5 mM ATP, 2 mM MgCl2 and2 mM DTT as previously described.39 Briefly, samples were incubatedfor 60 min at 37 °C for the indicated times. Then following addition of2× SDS−PAGE sample buffer the mixtures were resolved by SDS−PAGE on 4−12% BisTris gels and stained using Coomassie Blue.Smac-mimetics (1, 2, 3, and 4) were prepared as 10 mM stocks inDMSO and then diluted in water as required.Western Analysis and Immunoprecipitation. Samples were lysed

in DISC lysis buffer containing 1% Triton X-100 supplemented withprotease inhibitor cocktail (Roche) and N-ethylmaleimide (NEM) onice for 30 min and clarified by centrifugation as previously described.39

Samples were separated on precast 4−20% polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes for antibodydetection. All membrane blocking steps and antibody dilutions wereperformed with 5% skim milk in PBS containing 0.1% Tween 20(PTBS), and washing steps were performed with PTBS. Proteins onmembranes were visualized using ECL (Amersham, U.K.) followingincubation of membranes with HRP-coupled secondary antibodies.Green Fluorescent Protein-Linked cIAP1 and cIAP2 Assays.

Human melanoma A375 cells were established to stably expressgreen fluorescence protein (GFP) fusion cIAP1 (or cIAP2).35 Cellswere treated with various concentrations of IAP antagonists in 96-wellplates for 2 h, and GFP intensity was measured by fluorescence-activated cell sorting (FACS) analysis at 488 nm excitation and 530nm emission. Results are expressed as the mean ± standard deviationof four or greater independent assays unless where indicated.TNF-Induced NF-κB-Luciferase Reporter Assay. HeLa cells that

carry an NF-κB-luciferase reporter gene were established and utilizedfor quantitative measurement of activation of NF-κB by TNF.35 Thecells, seeded (50 000 cells/50 μL/well) in white-wall 96-well plates,were treated with various concentrations of IAP antagonists 2 h priorto TNF stimulation for 4 h. The luciferase activity was measured byusing Steady-Glo luciferase assay system (Promega Corp.). The platewas left in the dark for 15 min, and then the luminescence wasmeasured by using a Victor2 microplate reader (PerkinElmer Life andAnalytical Science, Shelton, CT). Results are expressed as the mean ±standard deviation of four or greater independent assays unless whereindicated.DAP- and MDP-Induced NF-κB-Luciferase Reporter Assay. The

stable cells (HCT-116 3-3) were seeded (50 000 cells/50 μL/well) inwhite-wall 96-well plates followed by overnight incubation at 37 °C.Cells were treated with IAP antagonists at 10 μM for 2 h and then

treated with 10 μg/mL DAP (or MDP) for 4 h at 37 °C. Theluciferase activity in each well was measured as described above.Results are expressed as the mean ± standard deviation of four orgreater independent assays unless where indicated.

Bone Marrow-Derived Macrophage (BMDM) Preparation,Stimulation, and IL-1β Analysis. The IL-1β analysis of Smac-mimetics was performed as previously described.50 Briefly, bonemarrow myeloid progenitor cells were differentiated into BMDMs for7 days in DME (Invitrogen) (37 °C, 5% CO2) supplemented with10% FBS and 20% L929-conditioned medium. After differentiation,adherent BMDMs were replated at 2 × 105 cells/well (96-well plates)or (5−6) × 105 cells/well (24-well plates) and primed with 20 ng/mLultrapure LPS for 3 h. Cells were stimulated for 24 h as indicated, andcell supernatant was collected for ELISA (IL-1β) and immunoblotanalysis. Cells lysates and supernatants were separated on 8%, 12%, orgradient 4−12% polyacrylamide gels (Invitrogen), and protein wastransferred to nitrocellulose (Amersham) membranes. Membraneswere blocked with 5% skim milk in PBST (PBS containing 0.05%Tween 20), and all primary antibody incubations were performedovernight and secondary antibody incubations were performed for 1−2 h. Membranes were washed 5−8 times in PBST after all antibodyincubations. Antibody dilutions were performed with 5% skim milk inPBST. Mice were treated in accordance with the Swiss FederalVeterinary Office guidelines (Switzerland) and under conditionsapproved by the Walter and Eliza Hall Institute Animal EthicsCommittee (Australia).

Crystallography. Crystallization and Data Collection. XIAPcontaining residues 252−350 (XIAP-BIR3) and cIAP1 containingresidues 256−358 (cIAP1-BIR3) were cloned into pET-15b(Novagen). Expression of protein was induced in LB medium with0.2 mM isopropyl-β-D-thiogalactopyranoside at OD600 of 1.2 at 22 °Cfor 12 h. Cells were resuspended in a buffer containing 25 mM Tris-Cl,pH 8.0, and 150 mM NaCl and lysed using sonication. Followinganother centrifugation step, the supernatant was loaded to Ni2+-NTAresin (Qiagen). The protein was released from the column bythrombin cleavage for 4 h at room temperature in a buffer containing25 mM Tris-Cl, pH 8.0, and 150 mM NaCl. The protein was appliedto Source-15Q (GE Healthcare), and the peak fraction wasconcentrated to ∼40 mg/mL and applied to Superdex-200 (GEHealthcare) in a buffer containing 10 mM Tris-Cl, pH 8.0, 150 mMNaCl, and 5 mM DTT. The peak fraction was collected forcrystallization. All of the crystals were grown at 18 °C by thehanging-drop vapor-diffusion method. Before crystallization, proteinwas mixed with 1 or mono-D-Abu 3 as molar ratio 1:10. Well buffer ofXIAP BIR3 and 1 contained 100 mM Bis-Tris, pH 5.8, and 2 M(NH4)2SO4. Well buffer of cIAP1 BIR3 and mono-D-Abu 3 contained1 M Na/K phosphate, pH 6.9.

The diffraction images of crystal were collected at Rigaku Saturn944+ and Spring-8 BL41XU. All data were integrated and scaled usingthe HKL2000 package.59 Further processing was carried out usingprograms from the CCP4 suite.60 Data collection statistics aresummarized in Supporting Information Table S1. The structure wasdetermined by molecular replacement method with XIAP BIR3 (PDBaccession code 1NW9) as the search model using PHASER.61 Thestructure was refined using the program PHENIX.62 The manualmodel modification was performed in COOT.63 All structural imageswere generated using PyMOL.64

Chemistry. Smac-mimetics 1 and 2 were prepared at TetraLogicPharmaceuticals (Malvern, PA) following reported procedures.33,34

IAP antagonists 3−10 were prepared using standard modifications ofthose methods. 1H, 13C, and MS characterization of IAP antagonists3−10 is provided in Supporting Information. All compounds weredetermined to be >95% pure by HPLC analysis.

■ ASSOCIATED CONTENT

*S Supporting InformationSpectral characterization of IAP antagonists 3−10 as well asdata refinement for all crystallographic representations. This



material is available free of charge via the Internet at http://pubs.acs.org.

Accession CodesThe codes for compound 1 bound to two XIAP BIR3 domainsare the following: PDB code, 4KMP; RCSB code, rcsb079530.The codes for compound 3 bound to bound to a single cIAP1BIR3 are the following: PDB code, 4KMN; RCSB code,rcsb079528.

■ AUTHOR INFORMATIONCorresponding Authors*S.M.C.: phone, 1-610-889-9900; fax, 1-610-889-9994; e-mail,[email protected].*Y.M.: phone, 1-610-889-9900; fax, 1-610-889-9994; e-mail,[email protected].

Present Addresses∞M.G.L.: Department of Chemistry, Chemical Methodologiesand Library Development, University of Pittsburgh, Pittsburgh,PA 15260.×M.D.A.: Celgene Corp., 10300 Campus Point Drive, Suite100, San Diego, CA 92121.●M.A.M.: The Task Force for Global Health, Center forVaccine Equity, 325 Swanton Way, Decatur, GA 30030.○R.F.: The Institute of Cancer Research, 237 Fulham Road,London SW3 6JB, U.K.◇J.S.: The Walter and Eliza Hall Institute of Medical Research,1G Royal Parade, Parkville, Victoria 3052, Australia.

Author ContributionsThe named authors all contributed to the preparation of thismanuscript. S.M.C., Y.D., M.G.L., S.R.R., T.H., M.D.A., P.T.K.,M.S.H., and Y.-H.L. contributed to the design and synthesis ofIAP antagonists. Y.M., C.A.B., G.Y., G.S.K., E.N., M.E.S., andS.K.C. contributed to the development and/or application ofthe binding, GFP-cIAP, TNF-NF-κB-luciferase, and NODassays. J.M.B., M.A.G., and M.A.M. contributed to the designand interpretation of the in vivo studies. J.W., X.L., and Y.S.generated and solved the IAP cocrystal structures of 1 and 3.R.F., B.B., M.H.C., J.S., and C.L.D. designed and performed theMALLS-SEC, ubiquitin transfer assays, and co-IP experiments.J.S., J.E.V., and N.K. designed and performed the IL-1β assay.

NotesThe authors declare the following competing financialinterest(s): S.M.C., Y.M., Y.D., M.G.L., S.R.R., T.H., M.D.A.,P.T.K., M.S.H., Y.L., C.A.B., G.Y., G.S.K., E.N., M.E.S., J.M.B.,M.A.G., M.A.M., and S.K.C. are current or former employeesand/or stockholders of TetraLogic Pharmaceuticals. Y.S. is aco-founder and stockholder in TetraLogic Pharmaceuticals. J.S.is a stockholder and serves on the Scientific Advisory Board ofTetraLogic Pharmaceuticals.

■ ACKNOWLEDGMENTSThe authors acknowledge the support from our colleagues atTetraLogic Pharmaceuticals (Malvern, PA) and our co-workersat the START Preclinical Research Group (San Antonio, TX)and the Piedmont Research Center, LLC (Morrisville, NC) forconducting the animal experiments described in this paper. J.S.was supported by NHMRC grants (Grants 433013, 541902,461221, 1046986) and fellowships (Grant 541901). C.L.D.acknowledges financial support from the Health ResearchCouncil of New Zealand. We thank C. Glenn Begley for criticalreading of this manuscript.

■ ABBREVIATIONS USED

Abu, α-aminobutyric acid; BIR, baculovirus inhibitor ofapoptosis protein repeat domain; BMDM, bone marrow-derived macrophage; BUCR, BIR3-UBA-CARD-RING cIAP1construct; BWL, body weight loss; CARD, caspase recruitmentdomain; cIAP, cellular inhibitor of apoptosis protein 1 orcellular inhibitor of apoptosis protein 2; co-IP, co-immunopre-cipitation; DAP, diaminopimelic acid; DIABLO, direct inhibitorof apoptosis protein binding protein with low pI (also, Smac);DMSO, dimethylsulfoxide; ELISA, enzyme-linked immuno-sorbent assay; FACS, fluorescence-activated cell sorting; FBS,fetal bovine serum; Flag, FLAG (DYKDDDDK) octapeptideepitope tag; GFP, green fluorescent protein; IAP, inhibitor ofapoptosis protein; IBM, inhibitor of apoptosis protein bindingmotif; LPS, lipopolysaccharide; MALLS-SEC, multiangle laserlight scattering coupled in-line to size exclusion chromatog-raphy; mbw, monomeric, nonfunctional Bcl-w ΔC10 carrierprotein epitope tag; MDP, muramyl dipeptide; ML-IAP,melanoma inhibitor of apoptosis protein; NOD, nucleotideoligomerization domain receptor 1 or nucleotide oligomeriza-tion domain receptor 2; NLRP3, nucleotide-oligomerization-domain-like receptor family, pyrin domain containing 3; PARP,poly (ADP ribose) polymerase; PBS, phosphate-buffered saline;RING, really interesting new gene domain; RIPK, receptor-interacting kinase 1 or receptor-interacting kinase 2; Smac,second mitochondria-derived activator of caspases (also,DIABLO); Tle, tert-leucine; TNF, tumor necrosis factor;TNFR1, tumor necrosis factor receptor 1; TRAF2, tumornecrosis factor receptor-associated factor 2; Ub, ubiquitin;UBA, ubiquitin-associated domain; XIAP, X-linked inhibitor ofapoptosis protein

■ REFERENCES(1) Riedl, S. J.; Shi, Y. Molecular mechanisms of caspase regulationduring apoptosis. Nat. Rev. Mol. Cell Biol. 2004, 5, 897−907.(2) Hanahan, D.; Weinberg, R. A. The hallmarks of cancer. Cell 2011,144, 646−674.(3) Mace, P. D.; Riedl, S. J. Molecular cell death platforms andassemblies. Curr. Opin. Cell Biol. 2010, 22, 828−836.(4) Tenev, T.; Bianchi, K.; Darding, M.; Broemer, M.; Langlais, C.;Wallberg, F.; Zachariou, A.; Lopez, J.; MacFarlane, M.; Cain, K.;Meier, P. The ripoptosome, a signaling platform that assembles inresponse to genotoxic stress and loss of IAPs.Mol. Cell 2011, 43, 432−448.(5) Feoktistova, M.; Geserick, P.; Kellert, B.; Panayotova Dimitrova,D.; Langlais, C.; Hupe, M.; Cain, K.; MacFarlane, M.; Hacker, G.;Leverkus, M. cIAPs block ripoptosome formation, a RIP1/caspase-8containing intracellular cell death complex differentially regulated bycFLIP isoforms. Mol. Cell 2011, 43, 449−463.(6) Gyrd-Hansen, M.; Meier, P. IAPs: from caspase inhibitors tomodulators of NF-kappaB, inflammation and cancer. Nat. Rev. Cancer2010, 10, 561−574.(7) Jager, R.; Zwacka, R. M. The enigmatic roles of caspases in tumordevelopment. Cancers 2010, 2, 1952−1979.(8) Fulda, S.; Vucic, D. Targeting IAP proteins for therapeuticintervention in cancer. Nat. Rev. Drug Discovery 2012, 11, 109−124.(9) McManus, D. C.; Lefebvre, C. A.; Cherton-Horvat, G.; St-Jean,M.; Kandimalla, E. R.; Agrawal, S.; Morris, S. J.; Durkin, J. P.; Lacasse,E. C. Loss of XIAP protein expression by RNAi and antisenseapproaches sensitizes cancer cells to functionally diverse chemo-therapeutics. Oncogene 2004, 23, 8105−8117.(10) Vaux, D. L.; Silke, J. IAPs, RINGs and ubiquitylation. Nat. Rev.Mol. Cell Biol. 2005, 6, 287−297.



http://pubs.acs.org

http://pubs.acs.org

mailto:[email protected]

mailto:[email protected]

(11) Eckelman, B. P.; Salvesen, G. S.; Scott, F. L. Human inhibitor ofapoptosis proteins: why XIAP is the black sheep of the family. EMBORep. 2006, 7, 988−994.(12) Varfolomeev, E.; Goncharov, T.; Maecker, H.; Zobel, K.;Komuves, L. G.; Deshayes, K.; Vucic, D. Cellular inhibitors ofapoptosis are global regulators of NF-κB and MAPK activation bymembers of the TNF family of receptors. Sci. Signaling 2012, 5, ra22.(13) Oberoi-Khanuja, T. K.; Karreman, C.; Larisch, S.; Rapp, U. R.;Rajalingam, K. Role of melanoma inhibitor of apoptosis (ML-IAP)protein, a member of the baculoviral IAP repeat (BIR) domain family,in the regulation of C-RAF kinase and cell migration. J. Biol. Chem.2012, 287, 28445−28455.(14) Suzuki, Y.; Nakabayashi, Y.; Nakata, K.; Reed, J. C.; Takahashi,R. X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3and -7 in distinct modes. J. Biol. Chem. 2001, 276, 27058−27063.(15) Srinivasula, S. M.; Hegde, R.; Saleh, A.; Datta, P.; Shiozaki, E.;Chai, J.; Lee, R. A.; Robbins, P. D.; Fernandes-Alnemri, T.; Shi, Y.;Alnemri, E. S. A conserved XIAP-interaction motif in caspase-9 andSmac/DIABLO regulates caspase activity and apoptosis. Nature 2001,410, 112−116.(16) Silke, J.; Ekert, P. G.; Day, C. L.; Hawkins, C. J.; Baca, M.;Chew, J.; Pakusch, M.; Verhagen, A. M.; Vaux, D. L. Direct inhibitionof caspase 3 is dispensable for the anti-apoptotic activity of XIAP.EMBO J. 2001, 20, 3114−3123.(17) Riedl, S. J.; Renatus, M.; Schwarzenbacher, R.; Zhou, Q.; Sun,C.; Fesik, S. W.; Liddington, R. C.; Salvesen, G. S. Structural basis forthe inhibition of caspase-3 by XIAP. Cell 2001, 104, 791−800.(18) Du, C.; Fang, M.; Li, Y.; Li, L.; Wang, X. Smac, a mitochondrialprotein that promotes cytochrome c-dependent caspase activation byeliminating IAP inhibition. Cell 2000, 102, 33−42.(19) Verhagen, A. M.; Vaux, D. L. Cell death regulation by themammalian IAP antagonist Diablo/Smac. Apoptosis 2002, 7, 163−166.(20) Wu, G.; Chai, J.; Suber, T. L.; Wu, J. W.; Du, C.; Wang, X.; Shi,Y. Structural basis of IAP recognition by Smac/DIABLO. Nature 2000,408, 1008−1012.(21) Huang, Y.; Rich, R. L.; Myszka, D. G.; Wu, H. Requirement ofboth the second and third BIR domains for the relief of X-linkedinhibitor of apoptosis protein (XIAP)-mediated caspase inhibition bySmac. J. Biol. Chem. 2003, 278, 49517−49522.(22) Deshayes, K.; Murray, J.; Vucic. D. The development of small-molecule IAP antagonists for the treatment of cancer. In Topics inMedicinal Chemistry; Bernstein, P. R., Buschauer, A., Georg, G. J., Kiso,Y., Lowe, J. A., Stilz, H. U., Eds.; Springer-Verlag: Berlin, 2012; Vol. 8,pp 81−104.(23) Varfolomeev, E.; Blankenship, J. W.; Wayson, S. M.; Fedorova,A. V.; Kayagaki, N.; Garg, P.; Zobel, K.; Dynek, J. N.; Elliott, L. O.;Wallweber, H. J. A.; Flygare, J. A.; Fairbrother, W. J.; Deshayes, K.;Dixit, V. M.; Vucic, D. IAP antagonists induce autoubiquitination of c-IAPs, NF-κB activation, and TNFα-dependent apoptosis. Cell 2007,131, 669−681.(24) Vince, J. E.; Wong, W. W-L.; Khan, N.; Feltham, R.; Chau, D.;Ahmed, A. U.; Benetatos, C. A.; Chunduru, S. K.; Condon, S. M.;McKinlay, M.; Brink, R.; Leverkus, M.; Tergaonkar, V.; Schneider, P.;Callus, B. A.; Koentgen, F.; Vaux, D. L.; Silke, J. IAP antagonists targetcIAP1 to induce TNFα-dependent apoptosis. Cell 2007, 131, 682−693.(25) Gaither, A.; Porter, D.; Yao, Y.; Borawski, J.; Yang, G.; Donovan,J.; Sage, D.; Slisz, J.; Tran, M.; Straub, C.; Ramsey, T.; Iourgenko, V.;Haung, A.; Chen, Y.; Schlegel, R.; Labow, M.; Fawell, S.; Sellers, W. R.;Zawel, L. A Smac-mimetic rescue screen reveals roles for inhibitor ofapoptosis proteins in tumor necrosis factor-alpha signaling. Cancer Res.2007, 67, 11493−11498.(26) Oost, T. K.; Sun, C.; Armstrong, R. C.; Al-Assaad, A.-S.; Betz, S.F.; Deckwerth, T. L.; Ding, H.; Elmore, S. W.; Meadows, R. P.;Olejniczak, E. T.; Oleksijew, A.; Oltersdorf, T.; Ronsenberg, S. H.;Shoemaker, A. R.; Tomaselli, K. J.; Zou, H.; Fesik, S. W. Discovery ofpotent antagonists of the antiapoptotic protein XIAP for the treatmentof cancer. J. Med. Chem. 2004, 47, 4417−4426.

(27) Li, L.; Thomas, R. M.; Suzuki, H.; De Brabander, J. K.; Wang,X.; Harran, P. A small molecule Smac mimic potentiates TRAIL- andTNFalpha-mediated cell death. Science 2004, 305, 1471−1474.(28) Dubrez, L.; Berthelet, J.; Glorian, V. IAP proteins as targets fordrug development in oncology. OncoTargets Ther. 2013, 6, 1285−1304.(29) Portoghese, P. S. From models to molecules: opioid receptordimers, bivalent ligands, and selective opioid receptor probes. J. Med.Chem. 2001, 44, 2259−2269.(30) Moulin, M.; Anderton, H.; Voss, A. K.; Thomas, T.; Wong, W.W.-L.; Bankovacki, A.; Feltham, R.; Chau, D.; Cook, W. D.; Silke, J.;Vaux, D. L. IAPs limit activation of RIP kinases by TNF receptor 1during development. EMBO J. 2012, 31, 1679−1691.(31) Sun, H.; Liu, L.; Bai, L.; Li, X.; Nikolovska-Coleska, Z.;McEachern, D.; Yang, C.-Y.; Qiu, S.; Yi, H.; Sun, D.; Wang, S. Potentbivalent Smac-mimetics: effect of the linker on binding to inhibitor ofapoptosis proteins (IAPs) and anticancer activity. J. Med. Chem. 2011,54, 3306−3318.(32) Cossu, F.; Milani, M.; Vachette, P.; Malvezzi, F.; Grassi, S.;Lecis, D.; Delia, D.; Drago, C.; Seneci, P.; Bolognesi, M.; Mastrangelo,E. Structural insight into inhibitor of apoptosis proteins recognition bya potent divalent Smac-mimetic. PLoS One 2012, 7, e49527.(33) Condon, S. M.; Deng, Y.; Laporte, M. G.; Rippin, S. R. Smac-mimetic. U.S. Patent 8283372, 2012.(34) Condon, S. M.; Deng, Y.; Laporte, M. G.; Rippin, S. R. DimericIAP inhibitors. U.S. Patent 7517906, 2009.(35) Benetatos, C. A.; Mitsuuchi, Y.; Burns, J. M.; Neiman, E. M.;Condon, S. M.; Yu, G.; Seipel, M. E.; Kapoor, G. S.; LaPorte, M. G.;Rippin, S. R.; Deng, Y.; Hendi, M. S.; Kumar, P. T.; Lee, Y.-H.;Haimowitz, T.; Alexander, M. D.; Graham, M. A.; Weng, D.; Shi, Y.;McKinlay, M. A.; Chunduru, S. K. Birinapant, a bivalent Smac-mimetic, targets TRAF2-associated cIAPs, abrogates TNF-inducedNF-kB activation, and is active in patient-derived xenograft models.Mol. Cancer Ther. 2014, 13, 867−879.(36) Eckelman, B. P.; Drag, M.; Snipas, S. J.; Salvesen, G. S. Themechanism of peptide-binding specificity of IAP BIR domains. CellDeath Differ. 2008, 15, 920−928.(37) Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita,Y.; Li, P.; Roller, P. P.; Krajewski, K.; Saito, N. G.; Stuckey, J. E.; Wang,S. Development and optimization of a binding assay for the XIAPBIR3 domain using fluorescence polarization. Anal. Biochem. 2004,332, 261−273.(38) Sharma, S. K.; Straub, C.; Zawel, L. Development ofpeptidomimetics targeting IAPs. Int. J. Pept. Res. Ther. 2006, 12, 21−32.(39) Feltham, R.; Bettjeman, B.; Budhidarmo, R.; Mace, P. D.;Shirley, S.; Condon, S. M.; Chunduru, S. K.; McKinlay, M. A.; Vaux, D.L.; Silke, J.; Day, C. L. Smac-mimetics activate the E3 ligase activity ofcIAP1 protein by promoting RING domain dimerization. J. Biol. Chem.2011, 286, 17015−17028.(40) Conze, D. B.; Albert, L.; Ferrick, D. A.; Goeddel, D. V.; Yeh, W.-C.; Mak, T.; Ashwell, J. D. Posttranscriptional downregulation of c-IAP2 by the ubiquitin protein ligase c-IAP1 in vivo. Mol. Cell. Biol.2005, 25, 3348−3356.(41) Darding, M.; Feltham, R.; Tevev, T.; Bianchi, K.; Benetatos, C.;Silke, J.; Meier, P. Molecular determinants of Smac-mimetic induceddegradation of cIAP1 and cIAP2. Cell Death Differ. 2011, 18, 1376−1386.(42) Kulathila, R.; Vash, B.; Sage, D.; Cornell-Kennon, S.; Wright, K.;Koehn, J.; Stams, T.; Clark, K.; Price, A. The structure of the BIR3domain of cIAP1 in complex with the N-terminal peptides of Smacand caspase-9. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 58−66.(43) Dueber, E. C.; Schoeffler, A.; Lingel, A.; Elliot, J. M.; Fedorova,A. V.; Giannetti, A. M.; Zobel, K.; Maurer, B.; Varfolomeev, E.; Wu, P.;Wallweber, H. J. A.; Hymowitz, S. G.; Deshayes, K.; Vuvic, D.;Fairbrother, W. J. Antagonists induce a conformational change incIAP1 that promotes autoubiquitination. Science 2011, 334, 376−380.



(44) Lopez, J.; John, S. W.; Tenev, T.; Rautureau, G. J. P.; Hinds, M.G.; Francalanci, F.; Wilson, R.; Broemer, M.; Santoro, M.; Day, C. L.;Meier, P. CARD-mediated autoinhibition of cIAP1’s E3 ligase activitysuppresses cell proliferation and migration. Mol. Cell 2011, 42, 569−583.(45) Silke, J.; Kratina, T.; Chu, D.; Ekert, P. G.; Day, C. L.; Pakusch,M.; Huang, D. C.; Vaux, D. L. Determination of cell survival by RING-mediated regulation of inhibitor of apoptosis (IAP) proteinabundance. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16182−16187.(46) Nikolovska-Coleska, Z.; Meagher, J. L.; Jiang, S.; Yang, C.-Y.;Qiu, S.; Roller, P. P.; Stuckey, J. A.; Wang, S. Interaction of a cyclic,bivalent Smac-mimetic with the X-linked inhibitor of apoptosisprotein. Biochemistry 2008, 47, 9811−9824.(47) Kipp, R. A.; Case, M. A.; Wist, A. D.; Cresson, C. M.; Carrell,M.; Griner, E.; Wiita, A.; Albiniak, P. A.; Chai, J.; Shi, Y.; Semmelhack,M. F.; McLendon, G. L. Molecular targeting of inhibitor of apoptosisproteins based on small molecule mimics of natural binding partners.Biochemistry 2002, 41, 7344−7349.(48) Inohara, N.; Nunez, G. NODs: intracellular proteins involved ininflammation and apoptosis. Nat. Rev. Immunol. 2003, 3, 371−382.(49) Damgaard, R. B.; Nachbur, U.; Yabal, M.; Wong, W. W.-L.; Fiil,B. K.; Kastirr, M.; Rieser, E.; Rickard, J. A.; Bankovacki, A.; Peschel, C.;Ruland, J.; Bekker-Jensen, S.; Mailand, N.; Kaufmann, T.; Strasser, A.;Walczak, H.; Silke, J.; Jost, P. J.; Gyrd-Hansen, M. The ubiquitin ligaseXIAP recruits LUBAC for NOD2 signaling in inflammation and innateimmunity. Mol. Cell 2012, 46, 746−758.(50) Damgaard, R. B.; Fiil, B. K.; Speckmann, C.; Yabal, M.; zurStadt, U.; Bekker-Jensen, S.; Jost, P. J.; Ehl, S.; Mailand, N.; Gyrd-Hansen, M. Disease-causing mutations in the XIAP BIR2 domainimpair NOD2-dependent immune signaling. EMBO Mol. Med. 2013,5, 1278−1295.(51) Vince, J. E.; Wong, W. W.-L.; Gentle, I.; Lawlor, K. E.; Allam, R.;O’Reilly, L.; Mason, K.; Gross, O.; Ma, S.; Guarda, G.; Anderton, H.;Castillo, R.; Hacker, G.; Silke, J.; Tschopp, J. Inhibitor of apoptosisproteins limit RIP3 kinase-dependent interleukin-1 activation.Immunity 2012, 36, 215−227.(52) Kearney, C. J.; Sheridan, C.; Cullen, S. P.; Tynan, G. A.; Logue,S. E.; Afonina, I. S.; Vucic, D.; Lavelle, E. C.; Martin, S. J. Inhibitor ofapoptosis proteins (IAPs) and their antagonists regulate spontaneousand tumor necrosis factor (TNF)-induced proinflammatory cytokineand chemokine production. J. Biol. Chem. 2013, 288, 4878−4890.(53) Takahashi, R.; Deveraux, Q.; Tamm, I.; Welsh, K.; Assa-Munt,N.; Salvesen, G. S.; Reed, J. C. A single BIR domain of XIAP sufficientfor inhibiting caspases. J. Biol. Chem. 1998, 273, 7787−7790.(54) Suzuki, Y.; Nakabayashi, Y.; Nakata, K.; Reed, J. C.; Takahashi,R. X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3and -7 in distinct modes. J. Biol. Chem. 2001, 276, 27058−27063.(55) Varfolomeev, E.; Moradi, E.; Dynek, J. N.; Zha, J.; Fedorova, A.V.; Deshayes, K.; Fairbrother, W. J.; Newton, K.; Le Couter, J.; Vucic,D. Characterization of ML-IAP protein stability and physiological rolein vivo. Biochem. J. 2012, 447, 427−436.(56) Wong, W. W.; Vince, J. E.; Lalaoui, N.; Lawlor, K. E.; Chau, D.;Bankovacki, A.; Anderton, H.; Metcalf, D.; O’Reilly, L.; Jost, P. J.;Murphy, J. M.; Alexander, W. S.; Strasser, A.; Vaux, D. L.; Silke, J.Unpublished results.(57) Krieg, A.; Correa, R. G.; Garrison, J. B.; Le Negrate, G.; Welsh,K.; Huang, Z.; Knoefel, W. T.; Reed, J. C. XIAP mediates NODsignaling via interaction with RIP2. Proc. Natl. Acad. Sci. U.S.A. 2009,106, 14524−14529.(58) Mace, P. D.; Linke, K.; Feltham, R.; Schumacher, F. R.; Smith,C. A.; Vaux, D. L.; Silke, J.; Day, C. L. Structures of the cIAP2 RINGdomain reveal conformational changes associated with ubiquitin-conjugating enzyme (E2) recruitment. J. Biol. Chem. 2008, 283,31633−31640.(59) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction datacollected in oscillation mode. Methods Enzymol. 1997, 276, 307−326.(60) Collaborative Computational Project, Number 4. The CCP4suite: programs for protein crystallography. Acta Crystallogr. 1994,D50, 760−763.

(61) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl.Crystallogr. 2007, 40, 658−674.(62) Adams, P. D.; Grosse-Kunstleve, R. W.; Hung, L.-W.; Ioerger, T.R.; McCoy, A. J.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.;Sauter, N. K.; Terwilliger, T. C. PHENIX: building new software forautomated crystallographic structure determination. Acta Crystallogr.2002, D58, 1948−1954.(63) Emsley, P.; Cowtan, K. Coot: model-building tools formolecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,60, 2126−2132.(64) DeLano, W. L. PyMOL; DeLano Scientific LLC: South SanFrancisco, CA, 2002; http://www.pymol.org.



http://www.pymol.org

birinapant, a smac-mimetic with improved tolerability for the treatment of solid tumors and...

Documents