Structure-based design, synthesis and biochemical testing of novel and potent Smac peptido-mimetics

Structure-based design, chemical synthesis and biochemical testing of a series of novel Smac peptido-mimetics as inhibitors of XIAP protein are described. The most potent compound, 6j, has a binding affinity (K(i) value) of 24 nM to XIAP BIR3 protein and is 24 times more potent than the native Smac AVPI peptide. Further optimization of these potent Smac mimetics may ultimately lead to the development of a novel class of anticancer drugs for the treatment of human cancer by overcoming apoptosis-resistance of cancer cells through targeting the inhibitor of apoptosis proteins.     

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.     

Smac mimetics and TNFalpha: a dangerous liaison?

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. It has been thought that small-molecule mimetics of Smac, an endogenous IAP antagonist, might potentiate apoptosis in cancer cells by promoting caspase activation. However, three recent papers, two in Cell (Vince et al., 2007; Varfolomeev et al., 2007) and one in Cancer Cell (Petersen et al., 2007), now report that Smac mimetics primarily kill cancer cells via a different mechanism, the induction of autoubiquitination and degradation of cIAPs, which culminates in TNFalpha-mediated cell death.     

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.     

NF023 binding to XIAP‐BIR1: Searching drugs for regulation of the NF‐κB pathway

Inhibitor of Apoptosis Proteins (IAPs) are the target of extensive research in the field of cancer therapy since they regulate apoptosis and cell survival. Smac‐mimetics, the most promising IAP‐targeting compounds specifically recognize the IAP‐BIR3 domain and promote apoptosis, competing with caspases for IAP binding. Furthermore, Smac‐mimetics interfere with the NF‐κB survival pathway, inducing cIAP1 and cIAP2 degradation through an auto‐ubiquitination process. It has been shown that the XIAP‐BIR1 (X‐BIR1) domain is involved in the interaction with TAB1, an upstream adaptor for TAK1 kinase activation, which in turn couples with the NF‐κB survival pathway. Preventing X‐BIR1 dimerization abolishes XIAP‐mediated NF‐κB activation, thus implicating a proximity‐induced mechanism for TAK1 activation. In this context, in a systematic search for a molecule capable of impairing X‐BIR1/TAB1 assembly, we identified the compound NF023. Here we report the crystal structure of the human X‐BIR1 domain in the absence and in the presence of NF023, as a starting concept for the design of novel BIR1‐specific compounds acting synergistically with existing pro‐apoptotic drugs in cancer therapy. Proteins 2015; 83:612–620. © 2015 Wiley Periodicals, Inc.     

Structure-based design and synthesis of tricyclic IAP (Inhibitors of Apoptosis Proteins) inhibitors

The design and synthesis of a series of novel tricyclic IAP inhibitors is reported. Rapid assembly of the core tricycle involved two key steps: Rh-catalyzed hydrogenation of an unsaturated bicyclic ring system and a Ru-catalyzed ring closing alkene metathesis reaction. The final Smac mimetics bind to cIAP1 and XIAP BIR3 domains and elicit the desired phenotype in cellular proliferation assays. Dimeric IAP inhibitors were found to possess nanomolar potency in a cellular proliferation assay and favourable in vitro drug-like properties.     

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.     

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.     

Crystal structure of the BIR1 domain of XIAP in two crystal forms

X-linked inhibitor of apoptosis (XIAP) is a potent negative regulator of apoptosis. It also plays a role in BMP signaling, TGF-beta signaling, and copper homeostasis. Previous structural studies have shown that the baculoviral IAP repeat (BIR2 and BIR3) domains of XIAP interact with the IAP-binding-motifs (IBM) in several apoptosis proteins such as Smac and caspase-9 via the conserved IBM-binding groove. Here, we report the crystal structure in two crystal forms of the BIR1 domain of XIAP, which does not possess this IBM-binding groove and cannot interact with Smac or caspase-9. Instead, the BIR1 domain forms a conserved dimer through the region corresponding to the IBM-binding groove. Structural and sequence analyses suggest that this dimerization of BIR1 in XIAP may be conserved in other IAP family members such as cIAP1 and cIAP2 and may be important for the action of XIAP in TGF-beta and BMP signaling and the action of cIAP1 and cIAP2 in TNF receptor signaling.     

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.     

Targeting the X-linked inhibitor of apoptosis protein through 4-substituted azabicyclo[5.3.0]alkane smac mimetics. Structure, activity, and recognition principles

The X-linked inhibitor of apoptosis protein (XIAP) is overexpressed in several malignant cells where it prevents apoptosis by binding to, and blocking, the activation of caspase-3, -7, and -9. Human XIAP (479 residues) is composed of three tandem-repeated baculoviral IAP repeat (BIR) domains (BIR1-3), and by a C-terminal RING domain. Smac-DIABLO [second mitochondria-derived activator of caspases (Smac)-direct IAP binding protein with low pI (DIABLO)], the natural antagonist of XIAP, binds through its N-terminal sequence AVPI to the same surface groove, in the BIR domains, that binds caspases. Synthetic compounds mimicking such tetrapeptide motif effectively block the interaction between IAP and active caspases, thus triggering apoptosis. Peptidomimetics based on an azabicyclo[x.y.0]alkane scaffolds, have been shown to bind the BIR3 domain of XIAP with micromolar to nanomolar affinities, thus presenting attractive features for drug lead optimization. Here we report a study on three newly synthesized Smac mimetics, which have been characterized in their complexes with XIAP BIR3 domain through X-ray crystallography and molecular modelling/docking simulations. Based on analysis of the crystal structures, we show that specific substitutions at the 4-position of the azabicyclo[5.3.0]alkane scaffold results in sizeable effects on the peptidomimetic-BIR3 domain affinity. By means of functional, biophysical and simulative approaches we also propose that the same Smac mimetics can bind XIAP BIR2 domain at a location structurally related to the BIR3 domain AVPI binding groove. Details of the XIAP-Smac mimetic recognition principles highlighted by this study are discussed in light of the drug-like profile of the three (potentially proapoptotic) compounds developed that show improved performance in ADMET (adsorption, distribution, metabolism, excretion and toxicity) tests.     

Recognition of Smac-mimetic compounds by the BIR domain of cIAP1

Inhibitor of apoptosis proteins (IAPs) are negative regulators of apoptosis. As IAPs are overexpressed in many tumors, where they confer chemoresistance, small molecules inactivating IAPs have been proposed as anticancer agents. Accordingly, a number of IAP-binding pro-apoptotic compounds that mimic the sequence corresponding to the N-terminal tetrapeptide of Smac/DIABLO, the natural endogenous IAPs inhibitor, have been developed. Here, we report the crystal structures of the BIR3 domain of cIAP1 in complex with Smac037, a Smac-mimetic known to bind potently to the XIAP-BIR3 domain and to induce degradation of cIAP1, and in complex with the novel Smac-mimetic compound Smac066. Thermal stability and fluorescence polarization assays show the stabilizing effect and the high affinity of both Smac037 and Smac066 for cIAP1- and cIAP2-BIR3 domains.     

A dimeric Smac/diablo peptide directly relieves caspase-3 inhibition by XIAP. Dynamic and cooperative regulation of XIAP by Smac/Diablo

Caspase activation, the executing event of apoptosis, is under deliberate regulation. IAP proteins inhibit caspase activity, whereas Smac/Diablo antagonizes IAP. XIAP, a ubiquitous IAP, can inhibit both caspase-9, the initiator caspase of the mitochondrial apoptotic pathway, and the downstream effector caspases, caspase-3 and caspase-7. Smac neutralizes XIAP inhibition of caspase-9 by competing for binding of the BIR3 domain of XIAP with caspase-9, whereas how Smac liberates effector caspases from XIAP inhibition is not clear. It is generally believed that binding of Smac with IAP generates a steric hindrance that prevents XIAP from inhibiting effector caspases, and therefore small molecule mimics of Smac are not able to reverse inhibition of the effector caspases. Surprisingly, we show here that binding of a dimeric Smac N-terminal peptide with the BIR2 domain of XIAP effectively antagonizes inhibition of caspase-3 by XIAP. Further, we defined the dynamic and cooperative interaction of Smac with XIAP: binding of Smac with the BIR3 domain anchors the subsequent binding of Smac with the BIR2 domain, which in turn attenuates the caspase-3 inhibitory function of XIAP. We also show that XIAP homotrimerizes via its C-terminal Ring domain, making its inhibitory activity toward caspase-3 more susceptible to Smac.     

Dimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part II: Structural and biological characterization

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head–head (8), tail–tail (9) or head–tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2–BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail–tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.     

Smac/Diablo antagonizes ubiquitin ligase activity of inhibitor of apoptosis proteins

Inhibitor of apoptosis proteins (IAPs) can block apoptosis through binding to active caspases and antagonizing their function. IAP function can be neutralized by Smac/Diablo, an IAP-binding protein that is released from mitochondria during apoptosis. In addition to their ability to interact with caspases, certain IAPs also display ubiquitin-protein isopeptide ligase activity because of the presence of a RING domain. However, it is not known whether the ubiquitin-protein isopeptide ligase activities of human IAPs contribute to their apoptosis inhibitory activity or whether this IAP property can be modulated through association with Smac/Diablo. Here we demonstrate that the ubiquitin ligase activities of XIAP, and to a lesser extent c-IAP-1 and c-IAP2, are potently repressed through binding to Smac/Diablo. We also show that mutation of the XIAP RING domain rendered this IAP a less effective inhibitor of apoptosis, suggesting that the ubiquitin ligase activity of XIAP contributes to its anti-apoptotic function. These data suggest that Smac/Diablo potentiates apoptosis by simultaneously antagonizing caspase-IAP interactions and repressing IAP ubiquitin ligase activities.     

cIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome

Although early studies of inhibitor of apoptosis proteins (IAPs) suggested that cIAP1 directly binds and inhibits caspases similarly to X-linked IAP (XIAP), a recent one found that micromolar concentrations of cIAP1 only weakly inhibit caspase-3, -7, or -9. Here, we show that cIAP1 specifically and cooperatively blocks the cytochrome c-dependent apoptosome in vitro. Hence, cIAP1 prevented the activation of procaspase-3 but had no effect on the processing of procaspase-9 or the activity of prior activated caspase-3. Like cIAP1, XIAP had no effect on procaspase-9 processing and was a more potent inhibitor of procaspase-3 activation than of already activated caspase-3 activity. Inhibition of procaspase-3 activation depended on BIR2 and BIR3 of cIAP1 and was independent of BIR1, RING, CARD, and UBA domains. Smac prevented cIAP1 from inhibiting procaspase-3 activation and reversed the inhibition by prior addition of cIAP1. A procaspase-9 mutant (D315A) that cannot produce the p12 subunit was resistant to inhibition by cIAP1. Therefore, the N-terminal Ala-Thr-Pro-Phe motif of the p12 subunit of the caspase-9 apoptosome facilitates apoptosome blockade. Consequently, cIAP1 cooperatively interacts with oligomerized processed caspase-9 in the apoptosome and blocks procaspase-3 activation.     

ras Oncogene triggers up-regulation of cIAP2 and XIAP in intestinal epithelial cells: epidermal growth factor receptor-dependent and -independent mechanisms of ras-induced transformation

Detachment of normal epithelial cells from the extracellular matrix (ECM) triggers apoptosis, a phenomenon called anoikis. Conversely, carcinomas (cancers of epithelial origin) represent three-dimensional disorganized multicellular masses in which cells are deprived of adhesion to the ECM but remain viable. Resistance of cancer cells to anoikis is thought to be critical for tumor progression. However, the knowledge about molecular mechanisms of this type of resistance remains limited. Herein we report that ras oncogene, an established inhibitor of anoikis, triggers a significant upregulation of anti-apoptotic proteins cIAP2 and XIAP in intestinal epithelial cells. We also observed that the effect of ras on cIAP2 requires ras-induced autocrine production of transforming growth factor alpha (TGF-alpha), a ligand for epidermal growth factor receptor, whereas ras-triggered up-regulation of XIAP is TGF-alpha-independent. Moreover, overexpression of either cIAP2 or XIAP in nonmalignant intestinal epithelial cell was found to block anoikis. In addition, an established IAP antagonist Smac or Smac-derived cell-permeable peptide suppressed ras-induced anoikis resistance and subsequent anchorage-independent growth of ras-transformed cells. We conclude that ras-induced overexpression of cIAP2 and XIAP significantly contributes to the ability of ras-transformed intestinal epithelial cells to survive in the absence of adhesion to the ECM and grow in a three-dimensional manner.     

USP11-dependent selective cIAP2 deubiquitylation and stabilization determine sensitivity to Smac mimetics

Given their crucial role in apoptosis suppression, inhibitor of apoptosis proteins (IAPs) have recently become attractive targets for cancer therapy. Here, we report that cellular IAP2 (cIAP2) is specifically stabilized in several cancer cell lines, leading to resistance to Smac mimetics, such as BV6 and birinapant. In particular, our results showed that cIAP2 depletion, but not cIAP1 depletion, sensitized cancer cells to Smac mimetic-induced apoptosis. Ubiquitin-specific protease 11 (USP11) is a deubiquitylase that directly stabilizes cIAP2. USP11 overexpression is frequently found in colorectal cancer and melanoma and is correlated with poor survival. In our study, cancer cell lines expressing high levels of USP11 exhibited strong resistance to Smac mimetic-induced cIAP2 degradation. Furthermore, USP11 downregulation sensitized these cells to apoptosis induced by TRAIL and BV6 and suppressed tumor growth in a xenograft model. Finally, the TNFα/JNK pathway induced USP11 expression and maintained cIAP2 stability, suggesting an alternative TNFα-dependent cell survival pathway. Collectively, our data suggest that USP11-stabilized cIAP2 may serve as a barrier against IAP-targeted clinical approaches.Apoptosis is an inherent cell death program that is crucial for various physiological processes such as development, the immune response, and tumorigenesis.¹ This process is finely tuned by numerous cellular signaling pathways involving hundreds of pro-apoptotic and anti-apoptotic factors.^{2, 3} Inhibitor of apoptosis proteins (IAPs) are a conserved protein family containing the baculoviral IAP repeat (BIR) domain.⁴ There are eight human IAP proteins, including cellular IAP1 (cIAP1/BIRC2), cIAP2/BIRC3, X chromosome-linked IAP (XIAP/BIRC4), and melanoma IAP (ML-IAP/BIRC7).⁵ IAPs such as XIAP can exert their anti-apoptotic function through the BIR domain, which directly interacts with caspases.⁵ In addition, several IAPs contain a RING domain with E3 ubiquitin ligase activities, which are crucial for apoptosis suppression. In particular, the E3 ligase activities of cIAP1/2 are necessary to regulate tumor necrosis factor receptor (TNFR) signaling.⁶ Upon TNFR activation, cIAP1/2 is recruited to TNFR through TNFα receptor-associated factor 2 (TRAF2), leading to K63-linked polyubiquitylation of receptor interacting protein kinase 1 (RIPK1), which is essential for NF-κB-mediated cell survival.⁷ The lack of RIPK1 polyubiquitylation via cIAP1/2 depletion or the presence of CYLD deubiquitylase triggers TNFR complex IIa formation, thereby inducing caspase-8-dependent apoptosis.⁸ In addition, cIAP1/2 prevents the formation of the RIPK1-containing death complex ripoptosome in response to several stimuli including CD95, TNFα-related apoptosis-inducing ligand (TRAIL), genotoxic stress, and Toll-like receptor (TLR) activation.^{9, 10, 11, 12, 13}IAPs are frequently overexpressed in various human cancers, and their expression is associated with chemoresistance and poor clinical outcome.⁶ Therefore, inhibiting IAP function is an attractive strategy to treat cancer through the induction of apoptosis.^{5, 14} Upon apoptotic stimuli, IAPs are inhibited by the second mitochondria-derived activator of caspases (Smac),⁵ and this discovery led to the development of Smac mimetic peptides using the IAP binding motif containing four amino acids (Ala-Val-Pro-Ile). These peptides were shown to sensitize cells to apoptotic stimuli and efficiently suppress tumor growth in a xenograft model.^{15, 16} Subsequently, a number of small-molecule compounds mimicking the Smac mimetic peptide (Smac mimetics) were developed with improved pharmacological properties and IAP-binding affinity. Interestingly, Smac mimetics, such as BV6 and compound A, were found to induce autoubiquitylation and degradation of cIAP1/2.^{17, 18} Furthermore, cIAP1/2 depletion with Smac mimetics activates the non-canonical NF-κB pathway to induce autocrine TNFα production, which is essential for Smac mimetic-induced apoptotic cell death.^{18, 19}Because cIAP1 and cIAP2 show functional redundancy in TNFα-mediated survival, the depletion of both proteins is usually required for effective induction of cell death upon TNFα treatment.^{20, 21} However, there are several reports showing that cIAP2 expression, but not cIAP1 expression, renders cells resistant to Smac mimetic-induced cell death.^{20, 21} For example, cIAP2 upregulation via phosphoinositide 3-kinase (PI3K) upon compound 3 treatment in certain cell lines was shown to facilitate apoptosis evasion.²² In addition, treatment with compounds A and C led to cIAP1 dimerization, without cIAP2 dimerization, resulting in the autoubiquitylation and subsequent degradation of cIAP1. These findings may explain why cIAP1 is degraded more efficiently than cIAP2 upon treatment with Smac mimetics.²³ Alternatively, because cIAP2 degradation requires cIAP1, cIAP2 may become more stable when cIAP1 is depleted using Smac mimetics.²⁴ Direct cIAP deubiquitylation by OTUB1 or USP19 has been suggested to be responsible for cIAP stabilization;^{25, 26} however, these previous studies did not focus on the difference in stabilization between cIAP1 and cIAP2 and only provided general deubiquitylation-dependent mechanisms.^{25, 26}While several studies have supported hypotheses for how cIAP2 survives in the presence of Smac mimetics, numerous independent studies have also shown that cIAP2 can be efficiently degraded by Smac mimetics in various cell lines.^{27, 28, 29, 30, 31, 32} These observations suggest the existence of other factors that specifically regulate cIAP2 stability upon Smac mimetic treatment. In this study, we propose a new mechanism involving USP11-mediated cIAP2 regulation. We found that the differential destabilization of cIAP1 and cIAP2 is dependent on the presence of the cIAP2-specific deubiquitylase USP11. Mechanistically, USP11 can protect cIAP2 from Smac mimetic-mediated degradation, rendering cell lines with high USP11 expression unresponsive to Smac mimetic treatment. However, USP11 downregulation sensitized these cells to TNFα- or TRAIL-induced apoptosis in the presence of Smac mimetic and further suppressed tumor growth in a xenograft model. Corroborating these data, USP11 overexpression was observed in colon cancer and melanoma patients with poor clinical outcome. Finally, the TNFα/c-Jun N-terminal kinase (JNK) pathway induced USP11 expression, which was necessary for cIAP2 protein stabilization and its anti-apoptotic function. Thus, the identification of cIAP2-specific deubiquitylation indicates that more elaborate strategies should be developed for pharmaceutical therapies targeting cIAPs.     

Changing the Apoptosis Pathway through Evolutionary Protein Design

One obstacle in de novo protein design is the vast sequence space that needs to be searched through to obtain functional proteins. We developed a new method using structural profiles created from evolutionarily related proteins to constrain the simulation search process, with functions specified by atomic-level ligand–protein binding interactions. The approach was applied to redesigning the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP), whose primary function is to suppress the cell death by inhibiting caspase-9 activity; however, the function of the wild-type XIAP can be eliminated by the binding of Smac peptides. Isothermal calorimetry and luminescence assay reveal that the designed XIAP domains can bind strongly with the Smac peptides but do not significantly inhibit the caspase-9 proteolytic activity in vitro compared with the wild-type XIAP protein. Detailed mutation assay experiments suggest that the binding specificity in the designs is essentially determined by the interplay of structural profile and physical interactions, which demonstrates the potential to modify apoptosis pathways through computational design.