Christopher A. Benetatos

RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL

RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3–caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1β inflammatory responses independent of MLKL and necroptotic cell death.

The novel SMAC mimetic Birinapant exhibits potent activity against human melanoma cells

Purpose: Inhibitor of apoptosis proteins (IAP) promote cancer cell survival and confer resistance to therapy. We report on the ability of second mitochondria-derived activator of caspases mimetic, birinapant, which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as TNF-α in melanomas. Experimental Design: Seventeen melanoma cell lines, representing five major genetic subgroups of cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-α. Effects on cell viability, target inhibition, and initiation of apoptosis were assessed and findings were validated in 2-dimensional (2D), 3D spheroid, and in vivo xenograft models. Results: When birinapant was combined with TNF-α, strong combination activity, that is, neither compound was effective individually but the combination was highly effective, was observed in 12 of 18 cell lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth without adding TNF-α in in vitro resistant cell lines. Birinapant combined with TNF-α inhibited the growth of a melanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell line. Conclusions: Birinapant in combination with TNF-α exhibits a strong antimelanoma effect in vitro. Birinapant as a single agent shows in vivo antitumor activity, even if cells are resistant to single agent therapy in vitro. Birinapant in combination with TNF-α is effective in a melanoma cell line with acquired resistance to BRAF inhibitors. Clin Cancer Res; 19(7); 1784–94. ©2013 AACR.

Novel Nonnucleoside Inhibitor of Hepatitis C Virus RNA-Dependent RNA Polymerase

ABSTRACT A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 μM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.

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.

Sensitization of human bladder tumor cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis with a small molecule IAP antagonist

Urothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. The large majority of bladder tumors are non-muscle invasive at diagnosis, but even after local surgical therapy there is a high rate of local tumor recurrence and progression. Current treatments extend time to recurrence but do not significantly alter disease survival. The objective of the present study was to investigate the tumoricidal potential of combining the apoptosis-inducing protein TNF-related apoptosis-inducing ligand (TRAIL) with a small molecule inhibitor of apoptosis proteins (IAP) antagonist to interfere with intracellular regulators of apoptosis in human bladder tumor cells. Our results demonstrate that the IAP antagonist Compound A exhibits high binding affinity to the XIAP BIR3 domain. When Compound A was used at nontoxic concentrations in combination with TRAIL, there was a significant increase in the sensitivity of TRAIL-sensitive and TRAIL-resistant bladder tumor lines to TRAIL-mediated apoptosis. In addition, modulation of TRAIL sensitivity in the TRAIL-resistant bladder tumor cell line T24 with Compound A was reciprocated by XIAP small interfering RNA-mediated suppression of XIAP expression, suggesting the importance of XIAP-mediated resistance to TRAIL in these cells. These results suggest the potential of combining Compound A with TRAIL as an alternative therapy for bladder cancer.

Newly Synthesized Hepatitis C Virus Replicon RNA Is Protected from Nuclease Activity by a Protease-Sensitive Factor(s)

ABSTRACT Biochemical characterization of hepatitis C virus (HCV) replication using purified, membrane-associated replication complexes is hampered by the presence of endogenous nuclease activity that copurifies with the replication complex. In this study, pulse-chase analyses were used to demonstrate that newly synthesized replicon RNA was protected from nuclease activity by a factor(s) that was sensitive to 0.5% NP-40 or protease treatment. Nuclease susceptibility was not related to disruption of lipid membranes, since NP-40 did not significantly affect the buoyant density of HCV replication complexes or protease susceptibility of HCV NS3 and NS5A proteins. These results suggest that a protease-sensitive factor(s) protects newly synthesized RNA from nuclease degradation.

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*

Abstract A25: Phase 1 PK/PD analysis of the Smac-mimetic TL32711 demonstrates potent and sustained cIAP1 suppression in patient PBMCs and tumor biopsies.

The Smac mimetic TL32711 differentially targets members of the inhibitor of apoptosis proteins (cIAP1, cIAP2 and XIAP) involved in the blockade of apoptosis. TNFα-activated pro-survival signaling pathway is maintained by the cIAPs. TL32711 causes rapid autoubiquitinylation and proteosomal degradation of the cIAP1 converting TNFα signaling from pro-survival to pro-apoptotic and potently inhibiting TNFα driven canonical NF-kB signaling. TL32711 also relieves inhibition of caspase-3 and -7 by XIAP to further potentiate apoptosis. The pharmacokinetics (PK) and pharmacodynamics (PD) of TL32711 have been studied in human tumor xenografts, patient plasma/PBMCs and Phase 1 tumor biopsy samples. In mice bearing the MDA-MB-231 xenograft, TL32711 is rapidly and extensively taken up into the tumor (tumor/plasma AUC ratio >22) and is eliminated slowly with a half-life of 96 hrs (20 hrs in plasma). A PK/PD link model was used to characterize the relationship between TL32711 tumor concentrations and cIAP1 suppression. cIAP1 suppression was dose and time dependent with cIAP1 levels reduced to 70% inhibition maintained 7–14 days post treatment following a single IV bolus dose (5 mg/kg). TL32711 had a potent effect on tumor cIAP1 levels (EC50 24 ng/g) and caused significant tumor growth inhibition and regressions at doses ≥2.5 mg/kg q3D. Efficacy has also been evaluated in primary human melanoma tumors, recently derived from patients and transplanted into nude mice. Significant tumor growth inhibition was observed in 5/6 primary melanoma tumor xenografts with mean Day 7 tumor concentrations of 187, 579 and 2658 ng/g at 15, 30 and 60 mg/kg respectively. TL32711 PK/PD (drug concentration analysis and cIAP1 degradation in PBMCs and tumor biopsies) has also been investigated in patients as part of the single agent Phase I study. Following weekly, 30 min IV infusions TL32711 plasma PK was dose proportional and non-accumulating (0.18 to 47 mg/m2). Plasma PK was tri-exponential with a long terminal t1/2 (73–79 hrs). The target AUC in plasma for therapeutic activity (71 h.ng/mL) based on the MDA-MB-231 model was achieved in patients at dose >2.88 mg/m2 (Mean AUC 86 h.ng/mL). This exposure was associated with marked uptake and retention in PBMCs (t1/2 = 29–35hrs) and resulted in prolonged cIAP1 suppression over 7 days. A dose related increase in PBMC PARP cleavage and plasma caspase-3 activity was also observed indicative of apoptosis pathway activation. TL32711 PK/PD was also assessed in tumor biopsy samples from patients 4 hrs to 6 days post treatment (11.5 to 17.2 mg/m2). TL32711 is extensively taken up into the tumor with levels >350 ng/g on day 6, significantly in excess of the EC50 for cIAP1 inhibition. Estimated tumor exposure at 35 to 47 mg/m2 was also in excess of the measured drug levels observed at 15 to 30 mg/kg in the primary human tumor xenograft models in mice. Together these PK/PD data show that TL32711 results in potent and sustained cIAP1 suppression over 7 days at tolerable dose levels with evidence of apoptosis pathway activation and promising early signs of efficacy in patients with solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A25.

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1–TRAF2 complex to sensitize tumor cells to TNFα

Vince et al. 2008. J. Cell Biol. doi:10.1083/jcb.200801010 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft.jtitle%253DJ.%2BCell%2BBiol.%26rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200801010%26rft_id%253Dinfo%253Apmid%252F18606850%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%