Ihab S. Darwish

Discovery and Characterization of Substituted Diphenyl Heterocyclic Compounds as Potent and Selective Inhibitors of Hepatitis C Virus Replication

ABSTRACT A novel small-molecule inhibitor, referred to here as R706, was discovered in a high-throughput screen of chemical libraries against Huh-7-derived replicon cells carrying autonomously replicating subgenomic RNA of hepatitis C virus (HCV). R706 was highly potent in blocking HCV RNA replication as measured by real-time reverse transcription-PCR and Western blotting of R706-treated replicon cells. Structure-activity iterations of the R706 series yielded a lead compound, R803, that was more potent and highly specific for HCV replication, with no significant inhibitory activity against a panel of HCV-related positive-stranded RNA viruses. Furthermore, HCV genotype 1 replicons displayed markedly higher sensitivity to R803 treatment than a genotype 2a-derived replicon. In addition, R803 was tested by a panel of biochemical and cell-based assays for on-target and off-target activities, and the data suggested that the compound had a therapeutic window close to 100-fold, while its exact mechanism of action remained elusive. We found that R803 was more effective than alpha interferon (IFN-α) at blocking HCV RNA replication in the replicon model. In combination studies, R803 showed a weak synergistic effect with IFN-α/ribavirin but only additive effects with a protease inhibitor and an allosteric inhibitor of RNA-dependent RNA polymerase (20). We conclude that R803 and related heterocyclic compounds constitute a new class of HCV-specific inhibitors that could potentially be developed as a treatment for HCV infection.

Abstract B060: Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK

Introduction: In normal tissue homeostasis, interaction of phosphatidylserine externalized on apoptotic cells (ACs) with the TAM (Tyro3, Axl MerTK) family RTK MerTK, via its ligands Gas6 and Protein S, leads to AC phagocytosis (efferocytosis). The resulting clearance of AC antigens, immunosuppressive M2 macrophage polarization, and suppression of pro-inflammatory cytokine production promotes tolerance to AC-derived self-antigens. This homeostatic response is coopted in tumors, which are abundant in both ACs and TAM ligands, leading to a blunted anti-tumor immune response. Syngeneic tumors implanted in MerTK -/- mice exhibit poor growth and metastasis, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10 compared with those implanted in WT mice. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens and promoting an anti-tumor immune response, or by directly blocking tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell number, apoptosis and proliferation. Effects on immune function were tested in human primary dendritic cells (LPS-induced IL-23 production) and human primary T cells (anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5). Efferocytosis of apoptotic Jurkat cells by human primary macrophages was detected by flow cytometry. For the PD assay, MerTK expressing tumors were grown in nude mice. 30-60 minutes post-compound dosing, MerTK was stimulated in vivo for 1hr. Tumors were snap frozen and tumor lysates were blotted with anti-phospho-MerTK antibodies. Anti-tumor efficacy was studied in syngeneic models. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both classes of compounds with EC50 in vivo . Anti-tumor activity of these novel MerTK inhibitors is under investigation in syngeneic mouse models both as single agents and in combinations. Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Note: This abstract was not presented at the conference. Citation Format: Sacha J. Holland, Alexander M. Owyang, Sylvia Braselmann, Chrystelle Lamagna, Sothy Yi, Chi Young, Roy Frances, Arthur Bagos, Meagan Chan, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Jiaxin Yu, Ihab Darwish, Somasekhar Bhamidipati, Donald G. Payan, Esteban Masuda. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B060.

Abstract 4869: Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK

Introduction: MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic myeloid and epithelial cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands, Gas6 or Protein S, bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome. Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK -/- mice exhibit impaired growth and metastasis compared with those implanted in WT mice, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor induced immunosuppression, or directly promoting tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell proliferation, DNA content and apoptosis. Immune effector assays were LPS-induced IL-23 production in human primary dendritic cells and anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5 in human primary T cells. Efferocytosis of CFSE-labeled apoptotic Jurkat cells by anti-CD14-labeled human primary macrophages was detected by flow cytometry. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both compounds with EC50 Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Citation Format: Sacha J. Holland, Alex M. Owyang, Sothy Yi, Chi Young, Sylvia Braselmann, Roy Frances, Arthur Bagos, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Somasekhar Bhamidipati, Ihab Darwish, Matthew Duncton, Rajinder Singh, Esteban Masuda, Donald G. Payan. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4869.