Bivalent Smac Mimetics Research Articles

Design, in silico evaluation, and in vitro verification of new bivalent Smac mimetics with pro-apoptotic activity

Bivalent Smac mimetics have been shown to possess binding affinity and pro-apoptotic activity similar to or more potent than that of native Smac, a protein dimer able to neutralize the anti-apoptotic activity of an inhibitor of caspase enzymes, XIAP, which endows cancer cells with resistance to anticancer drugs. We design five new bivalent Smac mimetics, which are formed by various linkers tethering two diazabicyclic cores being the IAP binding motifs. We built in silico models of the five mimetics by the TwistDock workflow and evaluated their conformational tendency, which suggests that compound 3, whose linker is n-hexylene, possess the highest binding potency among the five. After synthesis of these compounds, their ability in tumour cell growth inhibition and apoptosis induction displayed in experiments with SK-OV-3 and MDA-MB-231 cancer cell lines confirms our prediction. Among the five mimetics, compound 3 displays promising pro-apoptotic activity and deserves further optimization.

The TwistDock workflow for evaluation of bivalent Smac mimetics targeting XIAP.

Purpose: Mimetics based on Smac, the native inhibitor of XIAP, are promising drug-candidates for the treatment of cancer. Bivalent Smac mimetics inhibit XIAP with even higher potency than monovalent mimetics, but how to optimize the linker that tethers the two monovalent binding motifs remains controversial.Methods: To construct an ensemble of bivalent complex structures for evaluating various linkers, we propose herein a workflow, named TwistDock, consisting of steps of monovalent docking and linker twisting, in which the degrees of freedom are sampled focusing on the rotation of single bonds of the linker.Results: The obtained conformations of bivalent complex distribute randomly in the conformational space with respect to two reaction coordinates introduced by the linker, which are the distance of the two binding motifs and the dihedral angle of the two planes through the linker and each of the binding motifs. Molecular dynamics starting from 10 conformations with the lowest enthalpy of every complex shows that the conformational tendency of the complex participated by compound 9, one of the compounds with the largest binding affinity, is distinct from others. By umbrella sampling of the complex, we find its global minimum of the free energy landscape. The structure shows that the linker favors a compact conformation, and the two BIR domains of XIAP encompass the ligand on the opposite sides.Conclusion: TwistDock can be used in fine-tuning of bivalent ligands targeting XIAP and similar receptors dimerized or oligomerized.

IAP Proteins Antagonist: An Introduction and Chemistry of Smac Mimetics under Clinical Development.

Smac mimetics (also known as IAP antagonist) are a new class of targeted drugs having a goal to suppress the IAPs, reestablishing the apoptotic pathways and inducing cancer cell death. Therefore, development of Smac mimetics was considered an attractive strategy for the development of new anticancer drugs. Lots of reviews have come in yesteryears which mainly discussed the biology of IAPs and their role in cancer development. None of these reviews focused on the chemical synthesis of Smac mimetics. Literature study was done by using standard bibliographic search engines like scifinder, pubmed etc. The characteristic features of screened articles were described in the review. The review gives an introduction of IAP proteins and Smac mimetics. Readers will gain an overview of the development of Smac mimetics with representative examples of both monovalent and bivalent Smac mimetics as anticancer agents and an understanding of their structure-activity relationships. Chemical synthesis of biologically important Smac mimetics was discussed briefly in this review. Small molecules that mimic Smac are continuously progressing towards clinical development. Smac mimetics are generally well tolerated and have demonstrated rapid suppression of their target (the IAPs), activation of apoptosis and anti-tumor activity. Continuous research has been done to generate even more insight into the function of IAP proteins to significantly enhance the therapeutical potential of Smac mimetics.

Abstract 1806: Birinapant, a bivalent SMAC-mimetic, promotes efficient cellular IAP E3 ligase activity and formation of a pro-apoptotic RIPK1:caspase-8 complex while monovalent IAP inhibitors are less efficient - implications for therapeutic utility

Abstract The second mitochondria-derived activator of caspases (SMAC) is thought to exert its pro-apoptotic activity as a homodimeric protein. Both monovalent and bivalent peptidomimetics of the SMAC tetrapeptide are being developed for cancer therapy. Birinapant/TL32711 is a bivalent SMAC-mimetic that targets the inhibitor of apoptosis (IAP) proteins whose gene abnormalities have been implicated in various cancers. Owing to structural differences between bivalent SMAC-mimetics and monovalent IAP-inhibitors, we sought to compare and contrast the biochemical activity of birinapant with several monovalent IAP-inhibitors including a monovalent- version of birinapant/TL32711, MV711. Previous studies have shown that both bivalent and monovalent agents promote auto-ubiquitylation and subsequent degradation of cIAP1 and cIAP2, which triggers tumor necrosis factor receptor (TNFR)-mediated cell death in certain tumor cell lines. However, birinapant showed substantial differences from IAP-inhibitors in degrading TRAF2-associated cIAP1 and cIAP2. Here we show that MV711 was less efficient at degrading cIAP1 by a factor of 7-fold (16 vs. 118 nM, birinapant vs. MV711, respectively) and inhibiting TNF-mediated NF-κB activation by 220-fold (9 vs. 1985 nM, respectively). In addition, a linker-lengthened variant of birinapant was less able to inhibit NF-κB activation by 71-fold (9 vs. 642 nM, respectively). We also studied the effect of birinapant or IAP-inhibitor treatment on SKOV-3, MDA-MB-231 and EVSA-T cancer cell lines in vitro. Comparable cIAP1 BIR3 domain binding constants and IC50 values for the degradation of cIAP1 and cIAP2 (ΔcIAP1/2) were observed for these two classes of agents, and both birinapant and IAP-inhibitors showed dose-dependent induction of cell death. However, despite such comparable potencies, the IAP-inhibitors did not completely kill SKOV-3 or MDA-MB-231 cells even with concentrations >100-times their ΔcIAP1/2 IC50 values. Birinapant revealed the highest suppression of cancer cell growth in the cell lines tested, even after the agent was removed, whereas the cell lines treated with the IAP-inhibitors showed rapid restoration of cell proliferation within 24 h following removal of the agents. These results suggested that monovalent IAP-inhibitors require maintenance of high steady state levels of drug to effectively suppress tumor growth in vivo. In agreement with their inability to induce cell death, IAP-inhibitors were less efficient in stimulating the formation of a RIPK1:caspase-8 complex when compared to birinapant in EVSA-T or SKOV-3 cells. These observations may be partly attributed to the reduced ability of IAP-inhibitors to degrade TRAF2-associated cIAP1 which serves a central role in the activation of NF-κB via TNFR. Citation Format: Yasuhiro Mitsuuchi, Christopher A. Benetatos, Thomas Haimowitz, Yijun Deng, Angeline C. Mufalli, Martin E. Seipel, Jennifer M. Burns, Gurpreet S. Kapoor, C. Glenn Begley, Stephen M. Condon. Birinapant, a bivalent SMAC-mimetic, promotes efficient cellular IAP E3 ligase activity and formation of a pro-apoptotic RIPK1:caspase-8 complex while monovalent IAP inhibitors are less efficient - implications for therapeutic utility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2014-1806

A Potent Bivalent Smac Mimetic (SM-1200) Achieving Rapid, Complete, and Durable Tumor Regression in Mice

We have designed, synthesized, and evaluated a series of new compounds based upon our previously reported bivalent Smac mimetics. This led to the identification of compound 12 (SM-1200), which binds to XIAP, cIAP1, and cIAP2 with Ki values of 0.5, 3.7, and 5.4 nM, respectively, inhibits cell growth in the MDA-MB-231 breast cancer and SK-OV-3 ovarian cancer cell lines with IC50 values of 11.0 and 28.2 nM, respectively. Compound 12 has a much improved pharmacokinetic profile over our previously reported bivalent Smac mimetics and is highly effective in induction of rapid and durable tumor regression in the MDA-MB-231 xenograft model. These data indicate that compound 12 is a promising Smac mimetic and warrants extensive evaluation as a potential candidate for clinical development.

Abstract 3333: Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2.

Abstract A variety of Smac-mimetic drugs have been developed to target the inhibitor of apoptosis (IAP) proteins including X chromosome-linked IAP (XIAP), cellular IAP proteins (cIAP1 and cIAP2) and ML-IAP whose gene mutations, amplifications and chromosomal translocations have been implicated in various malignancies. The IAPs also play an important role in multiple signaling pathways including the TNFα-mediated NF-kB and MAPK pathways (inflammatory responses) and pattern recognition receptor signaling pathways (innate immunity). Smac-mimetics have been designed to mimic the IAP-binding motif of the second mitochondria-derived activator of caspase (Smac). The IAP-binding motif consists of four amino acids (AVPI) that serve as an endogenous IAP antagonist. Structurally different Smac-mimetic compounds have been published and showed different binding affinity to IAP proteins. These differences in structure and binding have important consequences in terms of biological activity. Here, we present evidence that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in their ability to inhibit NF-kB in cells stimulated with TNFα. Over 300 monovalent and 300 bivalent Smac-mimetics, including compounds whose structures have been published, were tested for ability to degrade cIAP1 and cIAP2, and inhibit NF-kB in cell-based assays. Inhibition of NF-kB by bivalent Smac-mimetics correlated (R²=0.78) with the degradation of cIAP1 over a range of four logs, while monovalent Smac-mimetics did not (R²=0.20). Furthermore, monovalent Smac-mimetics degraded TRAF2-bound cIAP1 and cIAP2 less effectively (1/10-1/100 fold) compared to bivalent Smac-mimetics. While bivalent Smac-mimetics effectively degraded cIAP1 and cIAP2, birinapant (TL32711), a bivalent Smac-mimetic currently in Phase 2 clinical trials, was unique in its ability to preferentially degrade TRAF2-bound cIAP1 and cIAP2. Inhibition of NF-kB by birinapant was further characterized by the ImageStream cytometry, which showed that the nuclear translocation of p65 in response to TNFα stimulation was blocked in both HeLa and HL-60 cells. These data demonstrate that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in degrading TRAF2-bound cIAPs, in inhibition of NF-kB and efficiently mediating cell death. Furthermore, the unique profile of birinapant versus other bivalent Smac-mimetics may explain its preclinical and clinical safety profile. Citation Format: Yasuhiro Mitsuuchi, Stephen M. Condon, Eric M. Neiman, Christopher A. Benetatos, Martin E. Seipel, Gurpreet Singh Kapoor, Angeline C. Mufalli, Guangyao Yu, Orla Maguire, Hans Minderman, Mark A. Mckinlay, Martin Graham, David Weng, Srinivas Chunduru. Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3333. doi:10.1158/1538-7445.AM2013-3333

Bivalent Smac Mimetics with a Diazabicyclic Core as Highly Potent Antagonists of XIAP and cIAP1/2 and Novel Anticancer Agents

Nonpeptidic, bivalent Smac mimetics designed based upon monovalent Smac mimetics with a diazabicyclic core structure bind to XIAP, cIAP1, and cIAP2 with low to subnanomolar affinities and are highly effective in antagonizing XIAP in cell-free functional assays. They efficiently induce the degradation of cIAP1 and cIAP2 in cancer cells at concentrations as low as 1 nM, activate caspase-3 and -8, and cleave PARP at 3-10 nM. The most potent compounds in the series have IC(50) of 3-5 nM in inhibition of cell growth in both MDA-MB-231 and SK-OV-3 cell lines and are promising lead compounds for the development of a new class of cancer therapy.

Smac Mimetics Activate the E3 Ligase Activity of cIAP1 Protein by Promoting RING Domain Dimerization

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.

Abstract 3254: Novel bivalent Smac mimetics as a new class of anticancer agents

Abstract Second mitochondria-derived activator of caspase (Smac) is a protein released from mitochondria in response to apoptotic stimuli which functions as an endogenous inhibitor of several members of inhibitor of apoptotic proteins (IAPs), including XIAP, cIAP-1 and cIAP-2. Smac interacts with IAPs by binding of its N-terminal AVPI tetrapeptide to one or more BIR domains in IAPs. We designed and synthesized a series of non-peptidic, cell-permeable, bivalent small-molecules which mimic the dimeric Smac protein for targeting IAPs (bivalent Smac mimetics) and performed extensive evaluations of these bivalent Smac mimetics for their interaction with IAP proteins and their activity and mechanism of function in cancer cells. Our studies show that these Smac mimetics bind to XIAP, cIAP-1 and cIAP-2 with low nano-molar affinities. They function as extremely potent XIAP antagonists by concurrently targeting both the BIR2 and BIR3 domains of XIAP and nullify its inhibition to both caspase-9 and caspase-3/-7 and potently and effectively induce rapid degradation of these two proteins in cancer cells. The most potent bivalent Smac mimetics inhibit cell growth with IC50 values at low nanomolar and effectively induce apoptosis in a subset of cancer cell lines through TNFα dependent mechanism. Furthermore, bivalent Smac mimetics can dramatically enhance the antitumor activity of TRAIL in both TRAIL-sensitive and -resistant cancer cell lines. The combination of one such bivalent Smac mimetic, SM-164 with TRAIL induces rapid tumor regression in vivo in a breast cancer xenograft model in which either agent is ineffective. Mechanistic studies demonstrate that while RIP1 plays a minimal role in TRAIL's activity as a single agent, it is required for the synergistic interaction between TRAIL and SM-164. XIAP and cIAP1 work in concert to attenuate the activity of TRAIL and SM-164 strongly sensitizes TRAIL by concurrently targeting XIAP and cIAP1. Taken together, our data suggest that bivalent Smac mimetics may have a great therapeutic potential for the treatment of human cancer by targeting multiple IAPs, either alone or in combination with TRAIL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3254. doi:10.1158/1538-7445.AM2011-3254

Potent Bivalent Smac Mimetics: Effect of the Linker on Binding to Inhibitor of Apoptosis Proteins (IAPs) and Anticancer Activity

We have synthesized and evaluated a series of nonpeptidic, bivalent Smac mimetics as antagonists of the inhibitor of apoptosis proteins and new anticancer agents. All these bivalent Smac mimetics bind to full-length XIAP with low nanomolar affinities and function as ultrapotent antagonists of XIAP. While these Smac mimetics bind to cIAP1/2 with similar low nanomolar affinities, their potencies to induce degradation of cIAP1/2 proteins in cells differ by more than 100-fold. The most potent bivalent Smac mimetics inhibit cell growth with IC(50) from 1 to 3 nM in the MDA-MB-231 breast cancer cell line and are 100 times more potent than the least potent compounds. Determination of intracellular concentrations for several representative compounds showed that the linkers in these bivalent Smac mimetics significantly affect their intracellular concentrations and hence the overall cellular activity. Compound 27 completely inhibits tumor growth in the MDA-MB-231 xenografts while causing no signs of toxicity in the animals.

Characterization of bivalent peptides that bind to X‐linked inhibitor of apoptosis protein

X‐linked inhibitor of apoptosis protein (XIAP) is a regulator of programmed cell death and an important target in the development of cancer therapeutics. Biochemical studies reveal that the homodimeric protein Smac, the natural binding partner of XIAP, binds to XIAP via simultaneous contact to the BIR2 and BIR3 domains. Furthermore, the interaction is mediated primarily via the first four N‐terminal amino acids Ala‐Val‐Pro‐Ile. We and others have shown that dimeric peptides and peptidomimetics based upon the Ala‐Val‐Pro‐Ile motif also bind to XIAP through both the BIR2 and BIR3 domains. Despite the high structural and sequence similarities between the BIR2 and BIR3 domains, Ala‐Val‐Pro‐Ile binds to BIR2 with substantially lower affinity than to BIR3. Herein we explore the potential for asymmetric bivalent Smac mimetics wherein one binding motif has been optimized for the BIR2 domain. We show that the tetrapeptide Ala‐Glu‐Ala‐Val has a higher affinity for BIR2 than Ala‐Val‐Pro‐Ile, and we have incorporated this motif into dimeric peptides with both BIR2 and BIR3 targeting motifs. We also will present mutagenesis experiments aimed at restoring the tight binding of the BIR3 domain to BIR2. The implications of these results in the design of XIAP antagonists will be discussed. We acknowledge the Research Corporation (Cottrell College award to K.E.S.) and the Beckman Scholars Program (undergraduate research fellowship to K.F.S.) for support of this research.

Small-molecule pan-IAP antagonists: a patent review

Importance of the field: The inhibitor of apoptosis (IAP) proteins are critical regulators of cancer cell survival that have become important targets for therapeutic intervention in human malignancies. One strategy for targeting IAP proteins involves agents that mimic the amino terminus of the endogenous IAP protein antagonist second mitochondria-derived activator of caspases (Smac)/direct IAP-binding protein with low pI (DIABLO) and thus block critical IAP protein interactions.Areas covered in this review: This review of the IAP antagonist patent literature covers the period from 2000 to mid-2009. Over 50 patents and patent applications pertaining to IAP antagonists have been published over the past 10 years. In the case of several filings, only the original source is reviewed in this analysis.What the reader will gain: Readers will gain an overview of IAP protein antagonist scaffolds, with representative examples including monovalent and bivalent Smac mimetics, and an understanding of their structure–activity relationships.Take home message: The feasibility of disrupting IAP protein interactions with pro-apoptotic proteins using monovalent and bivalent Smac-derived peptidomimetic compounds has been broadly established. Four such compounds have entered or been approved to enter human clinical trials, which will hopefully allow the utility of this potential therapeutic approach to be evaluated in cancer patients.

Comparison of in Vitro Apoptotic Response of Chronic Lymphocytic Leukemia (CLL) Cells to Bcl-2 Antagonist ABT-737 and IAP Antagonist BV6.

Abstract 4386Altered expression of Bcl-2-family and IAP-family proteins has been considered to play central roles in apoptosis dysregulation in cancer and leukemia, promoting malignant cell expansion and contributing to chemoresistance. Anti apoptotic Bcl-2-family proteins Bcl-2 and Bcl-XL as well as IAP proteins, including XIAP, have been thoroughly validated as drug discovery targets for cancer and strategies for inhibiting these proteins have been devised based on mimicking their endogenous antagonists, as represented by ABT-737, a fully synthetic Bcl-2/Bcl-XL antagonist developed at Abbott Laboratories, and BV6, bivalent SMAC mimetics developed at Genentech Inc. Chronic lymphocytic leukemia (CLL) is a quintessential example of a human malignancy caused by defective programmed cell death. Over-expression of the Bcl-2 protein is one of the most consistent and prominent etiological factors associated with this disease, whereas over-expression of XIAP protein has been demonstrated in a majority of CLL patients. In this study, we evaluated the pro-apoptotic effects of ABT-737 and BV6 on CLL cells in side-by-side comparisons. We found that the CLL cells of 31 of 42 patients were highly sensitive to ABT-737, inducing potent dose-dependent killing of cells with an IC50 = 52.5 ± 22.5 (mean ± std error). In contrast, the CLL cells of 11 of 42 patients were relatively resistant to ABT-737, requiring higher concentrations to induce apoptosis (IC50 >1 μM). Preliminary statistical analysis was performed to correlate ABT737-sensitive and -resistant CLL with various other characteristics, including patient treatment status, ZAP-70 expression, and VH mutation status, but no significant correlation was observed. In contrast, the CLL cells from all 42 patients examined were relatively resistant to the IAP antagonist BV6, which required high concentrations to induce apoptosis (IC50 > 1 μM). Similar results were obtained using other SMAC mimetic compounds. Analysis of CLL cells for endogenous SMAC protein expression and for the subcellular location of SMAC protein demonstrated that the leukemia cells of 18 of 23 patients expressed high-levels of SMAC, as assessed by immunoblotting. Furthermore, SMAC was located in the cytosolic fraction of these leukemia cells in 7 of 18 cases examined. However, CLL cells with high-levels of cytosolic SMAC had little evidence of concomitant release of cytochrome c from mitochondria and demonstrated little spontaneous apoptosis in vitro. These results suggest that the leukemia cells of some CLL patients may already express cytosolic SMAC, which might explain the relative insensitivity of CLL cells to SMAC mimetics such as BV6. Taken together, these studies suggest that Bcl-2 antagonists such as ABT-737, but not SMAC mimetics such as BV6, may have promising therapeutic activity for most CLL patients. (NIH CA-081534) Disclosures:No relevant conflicts of interest to declare.

Structural Basis for Bivalent Smac-Mimetics Recognition in the IAP Protein Family

XIAP is an apoptotic regulator protein that binds to the effector caspases -3 and -7 through its BIR2 domain, and to initiator caspase-9 through its BIR3 domain. Molecular docking studies suggested that Smac-DIABLO may antagonize XIAP by concurrently targeting both BIR2 and BIR3 domains; on this basis bivalent Smac-mimetic compounds have been proposed and characterized. Here, we report the X-ray crystal structure of XIAP-BIR3 domain in complex with a two-headed compound (compound 3) with improved efficacy relative to its monomeric form. A small-angle X-ray scattering study of XIAP-BIR2BIR3, together with fluorescence polarization binding assays and compound 3 cytotoxicity tests on HL60 leukemia cell line are also reported. The crystal structure analysis reveals a network of interactions supporting XIAP-BIR3/compound 3 recognition; moreover, analytical gel-filtration chromatography shows that compound 3 forms a 1:1 stoichiometric complex with a XIAP protein construct containing both BIR2 and BIR3 domains. On the basis of the crystal structure and small-angle X-ray scattering, a model of the same BIR2-BIR3 construct bound to compound 3 is proposed, shedding light on the ability of compound 3 to relieve XIAP inhibitory effects on caspase-9 as well as caspases -3 and -7. A molecular modeling/docking analysis of compound 3 bound to cIAP1-BIR3 domain is presented, considering that Smac-mimetics have been shown to kill tumor cells by inducing cIAP1 and cIAP2 ubiquitination and degradation. Taken together, the results reported here provide a rationale for further development of compound 3 as a lead in the design of dimeric Smac mimetics for cancer treatment.

Design of small-molecule peptidic and nonpeptidic Smac mimetics.

Smac/DIABLO is a protein released from mitochondria into the cytosol in response to apoptotic stimuli. Smac promotes apoptosis at least in part through antagonizing inhibitor of apoptosis proteins (IAPs), including XIAP, cIAP-1, and cIAP-2. Smac interacts with these IAPs via its N-terminal AVPI binding motif. There has been an enormous interest in academic laboratories and pharmaceutical companies in the design of small-molecule Smac mimetics as potential anticancer agents. This task is particularly challenging because it involves targeting protein-protein interactions. Nevertheless, intense research has now generated potent, specific, cell-permeable small-molecule peptidomimetics and nonpeptidic mimetics. To date, two types of Smac mimetics have been reported, namely, monovalent and bivalent Smac mimetics. The monovalent compounds are designed to mimic the binding of a single AVPI binding motif to IAP proteins, whereas the bivalent compounds contain two AVPI binding motif mimetics tethered together through a linker. Studies from several groups have clearly demonstrated that both monovalent and bivalent Smac mimetics not only enhance the antitumor activity of other anticancer agents but also can induce apoptosis as single agents in a subset of human cancer cell lines in vitro and are capable of achieving tumor regression in animal models of human cancer. In general, bivalent Smac mimetics are 100-1000 times more potent than their corresponding monovalent Smac mimetics in induction of apoptosis in tumor cells. However, properly designed monovalent Smac mimetics can achieve oral bioavailability and may have major advantages over bivalent Smac mimetics as potential drug candidates. In-depth insights on the molecular mechanism of action of Smac mimetics have been provided by several independent studies. It was shown that Smac mimetics induce apoptosis in tumor cells by targeting cIAP-1/-2 for the rapid degradation of these proteins, which leads to activation of nuclear factor kappaB (NF-kappaB) and production and secretion of tumor necrosis factor alpha (TNFalpha). TNFalpha promotes formation of a receptor-interacting serine-threonine kinase 1 (RIPK1)-dependent caspase-8-activating complex, leading to activation of caspase-8 and -3/-7 and ultimately to apoptosis. For the most efficient apoptosis induction, Smac mimetics also need to remove the inhibition of XIAP to caspase-3/-7. Hence, Smac mimetics induce apoptosis in tumor cells by targeting not only cIAP-1/-2 but also XIAP. The employment of potent, cell-permeable, small-molecule Smac mimetics has yielded important insights into the regulation of apoptosis by IAP proteins. To date, at least one Smac mimetic has been advanced into clinical development. Several other Smac mimetics are in an advanced preclinical development stage and are expected to enter human clinical testing for the treatment of cancer in the near future.

Design and characterization of bivalent Smac-based peptides as antagonists of XIAP and development and validation of a fluorescence polarization assay for XIAP containing both BIR2 and BIR3 domains

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics.