Yijun Deng

Identification and characterization of potent small molecule inhibitor of hemorrhagic fever New World arenaviruses.

Abstract Category A arenaviruses as defined by the National Institute of Allergy and Infectious Diseases (NIAID) are human pathogens that could be weaponized by bioterrorists. Many of these deadly viruses require biosafety level-4 (BSL-4) containment for all laboratory work, which limits traditional laboratory high-throughput screening (HTS) for identification of small molecule inhibitors. For those reasons, a related BSL-2 New World arenavirus, Tacaribe virus, 67–78% identical to Junín virus at the amino acid level, was used in a HTS campaign where approximately 400,000 small molecule compounds were screened in a Tacaribe virus-induced cytopathic effect (CPE) assay. Compounds identified in this screen showed antiviral activity and specificity against not only Tacaribe virus, but also the Category A New World arenaviruses (Junín, Machupo, and Guanarito). Drug resistant variants were isolated, suggesting that these compounds act through inhibition of a viral protein, the viral glycoprotein (GP2), and not through cellular toxicity mechanisms. A lead compound, ST-294, has been chosen for drug development. This potent and selective compound, with good bioavailability, demonstrated protective anti-viral efficacy in a Tacaribe mouse challenge model. This series of compounds represent a new class of inhibitors that may warrant further development for potential inclusion in a strategic stockpile.

Birinapant (TL32711), a Bivalent SMAC Mimetic, Targets TRAF2-Associated cIAPs, Abrogates TNF-Induced NF-κB Activation, and Is Active in Patient-Derived Xenograft Models

The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient–derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion. Mol Cancer Ther; 13(4); 867–79. ©2014 AACR.

Electrophilic Oxidation and [1,2]-Rearrangement of the Biindole Core of Birinapant

Birinapant/TL32711 (1) is a bivalent antagonist of the inhibitor of apoptosis (IAP) family of proteins and was designed to mimic AVPI, the N-terminal tetrapeptide of the second mitochondria-derived activator of caspases (Smac/DIABLO). Birinapant bound to the BIR3 domains of cIAP1, cIAP2, and XIAP with K i values of 1, 36, and 45 nM, respectively. Birinapant-mediated activation of cIAP1 resulted in cIAP1 autoubiquitylation and degradation and correlated with inhibition of TNF-mediated NF-κB activation, induction of tumor cell death in vitro, and tumor regression in vivo. Birinapant is being evaluated in Phase 1/2 trials for the treatment of cancer and hepatitis B virus (HBV) infection. After one year at accelerated storage conditions, a formulation of 1 afforded four degradants in >0.1% abundance by HPLC analysis. The primary degradants (2 and 3) were formed via oxidation of the biindole core, while the secondary degradants (5 and 6) arose via [1,2]-rearrangement of 3 and 2, respectively. Forced degradation conditions were developed, which allowed the isolation of 2 and 3 in multigram quantities. Novel deuterated analogues of 1 were prepared to determine the site of oxidation, and NMR experiments confirmed the chemical structures of 5 and 6. The de novo synthesis of 2, 3, 5, and 6 confirmed these experimental findings.

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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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