Matthew G. LaPorte

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.

Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators

Unique chemical methodology enables the synthesis of innovative and diverse scaffolds and chemotypes and allows access to previously unexplored “chemical space.” Compound collections based on such new synthetic methods can provide small-molecule probes of proteins and/or pathways whose functions are not fully understood. We describe the identification, characterization, and evolution of two such probes. In one example, a pathway-based screen for DNA damage checkpoint inhibitors identified a compound, MARPIN (ATM and ATR pathway inhibitor) that sensitizes p53-deficient cells to DNA-damaging agents. Modification of the small molecule and generation of an immobilized probe were used to selectively bind putative protein target(s) responsible for the observed activity. The second example describes a focused library approach that relied on tandem multicomponent reaction methodologies to afford a series of modulators of the heat shock protein 70 (Hsp70) molecular chaperone. The synthesis of libraries based on the structure of MAL3-101 generated a collection of chemotypes, each modulating Hsp70 function, but exhibiting divergent pharmacological activities. For example, probes that compromise the replication of a disease-associated polyomavirus were identified. These projects highlight the importance of chemical methodology development as a source of small-molecule probes and as a drug discovery starting point.

Structure-Activity Study of Bioisosteric Trifluoromethyl and Pentafluorosulfanyl Indole Inhibitors of the AAA ATPase p97.

Exploratory SAR studies of a new phenyl indole chemotype for p97 inhibition revealed C-5 indole substituent effects in the ADPGlo assay that did not fully correlate with either electronic or steric factors. A focused series of methoxy-, trifluoromethoxy-, methyl-, trifluoromethyl-, pentafluorosulfanyl-, and nitro-analogues was found to exhibit IC50s from low nanomolar to double-digit micromolar. Surprisingly, we found that the trifluoromethoxy-analogue was biochemically a better match of the trifluoromethyl-substituted lead structure than a pentafluorosulfanyl-analogue. Moreover, in spite of their almost equivalent strongly electron-depleting effect on the indole core, pentafluorosulfanyl- and nitro-derivatives were found to exhibit a 430-fold difference in p97 inhibitory activities. Conversely, the electronically divergent C-5 methyl- and nitro-analogues both showed low nanomolar activities.

The discovery and structure-activity relationships of pyrano[3,4-b]indole based inhibitors of hepatitis C virus NS5B polymerase.

We describe the structure-activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12.

5-Hydroxyindoles by Intramolecular Alkynol–Furan Diels–Alder Cycloaddition

A convergent approach provides a convenient access to synthetically and biologically useful 3,4-disubstituted 5-hydroxyindoles. The one-pot procedure uses microwave heating to initiate an intramolecular [4 + 2]-cycloaddition of an alkynol segment onto a furan followed by a fragmentation, aromatization, and N-Boc deprotection cascade. Yields range from 15 to 74%, with aromatic substituents providing better conversions. 4-Trimethylsilylated analogues undergo a 1,3-silatropic rearrangement to give the O-TMS ethers.

Radical [3 + 2]-annulation of divinylcyclopropanes: Rapid synthesis of complex meloscine analogs

A radical [3 + 2]-divinylcyclopropane annulation cascade has been extended to encompass five D-ring variants of the meloscine/epimeloscine core structure. Representative ABCD tetracyclic intermediates were further elaborated with novel substituted E-rings through subsequent transformations of advanced intermediates that provided opportunities for late-stage variation of the B-ring (lactam) N-substituents which were also developed.

The Discovery of Pyrano[3,4-b]indole-Based Allosteric Inhibitors of HCV NS5B Polymerase with In Vivo Activity

The Discovery of Pyrano ACHTUNGTRENNUNG[3,4-b]indole-Based Allosteric Inhibitors of HCV NS5B Polymerase with In Vivo Activity Matthew G. LaPorte,* Randy W. Jackson, Tandy L. Draper, Janet A. Gaboury, Kristin Galie, Torsten Herbertz, Alison R. Hussey, Susan R. Rippin, Christopher A. Benetatos, Srinivas K. Chunduru, Joel S. Christensen, Glen A. Coburn, Christopher J. Rizzo, Gerry Rhodes, John O’Connell, Anita Y. M. Howe, Tarek S. Mansour, Marc S. Collett, Daniel C. Pevear, Dorothy C. Young, Tiejun Gao, D. Lorne J. Tyrrell , h] Norman M. Kneteman, i] Christopher J. Burns, and Stephen M. Condon*

HCS Campaign to Identify Selective Inhibitors of IL-6-Induced STAT3 Pathway Activation in Head and Neck Cancer Cell Lines

Signal transducer and activator of transcription factor 3 (STAT3) is hyperactivated in head and neck squamous cell carcinomas (HNSCC). Cumulative evidence indicates that IL-6 production by HNSCC cells and/or stromal cells in the tumor microenvironment activates STAT3 and contributes to tumor progression and drug resistance. A library of 94,491 compounds from the Molecular Library Screening Center Network (MLSCN) was screened for the ability to inhibit interleukin-6 (IL-6)-induced pSTAT3 activation. For contractual reasons, the primary high-content screening (HCS) campaign was conducted over several months in 3 distinct phases; 1,068 (1.1%) primary HCS actives remained after cytotoxic or fluorescent outliers were eliminated. One thousand one hundred eighty-seven compounds were cherry-picked for confirmation; actives identified in the primary HCS and compounds selected by a structural similarity search of the remaining MLSCN library using hits identified in phases I and II of the screen. Actives were confirmed in pSTAT3 IC50 assays, and an IFNγ-induced pSTAT1 activation assay was used to prioritize selective inhibitors of STAT3 activation that would not inhibit STAT1 tumor suppressor functions. Two hundred three concentration-dependent inhibitors of IL-6-induced pSTAT3 activation were identified and 89 of these also produced IC50s against IFN-γ-induced pSTAT1 activation. Forty-nine compounds met our hit criteria: they reproducibly inhibited IL-6-induced pSTAT3 activation by ≥70% at 20 μM; their pSTAT3 activation IC50s were ≤25 μM; they were ≥2-fold selective for pSTAT3 inhibition over pSTAT1 inhibition; a cross target query of PubChem indicated that they were not biologically promiscuous; and they were ≥90% pure. Twenty-six chemically tractable hits that passed filters for nuisance compounds and had acceptable drug-like and ADME-Tox properties by computational evaluation were purchased for characterization. The hit structures were distributed among 5 clusters and 8 singletons. Twenty-four compounds inhibited IL-6-induced pSTAT3 activation with IC50s ≤20 μM and 13 were ≥3-fold selective versus inhibition of pSTAT1 activation. Eighteen hits inhibited the growth of HNSCC cell lines with average IC50s ≤ 20 μM. Four chemical series were progressed into lead optimization: the guanidinoquinazolines, the triazolothiadiazines, the amino alcohols, and an oxazole-piperazine singleton.

Allosteric Indole Amide Inhibitors of p97: Identification of a Novel Probe of the Ubiquitin Pathway

A high-throughput screen to discover inhibitors of p97 ATPase activity identified an indole amide that bound to an allosteric site of the protein. Medicinal chemistry optimization led to improvements in potency and solubility. Indole amide 3 represents a novel uncompetitive inhibitor with excellent physical and pharmaceutical properties that can be used as a starting point for drug discovery efforts.

Optimization of pyrazole-containing 1,2,4-triazolo-[3,4-b]thiadiazines, a new class of STAT3 pathway inhibitors.

Structure-activity relationship studies of a 1,2,4-triazolo-[3,4-b]thiadiazine scaffold, identified in an HTS campaign for selective STAT3 pathway inhibitors, determined that a pyrazole group and specific aryl substitution on the thiadiazine were necessary for activity. Improvements in potency and metabolic stability were accomplished by the introduction of an α-methyl group on the thiadiazine. Optimized compounds exhibited anti-proliferative activity, reduction of phosphorylated STAT3 levels and effects on STAT3 target genes. These compounds represent a starting point for further drug discovery efforts targeting the STAT3 pathway.