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HCV NS5A inhibitors are the backbone of directly acting antiviral treatments against the hepatitis C virus (HCV). While these therapies are generally highly curative, they are less effective in some specific HCV patient populations. In the search for broader-acting HCV NS5A inhibitors that address these needs, we explored conformational restrictions imposed by the [7,5]-azabicyclic lactam moiety incorporated into daclatasvir (1) and related HCV NS5A inhibitors. Unexpectedly, compound 5 was identified as a potent HCV genotype 1a and 1b inhibitor. Molecular modeling of 5 bound to HCV genotype 1a suggested that the use of the conformationally restricted lactam moiety might have resulted in reorientation of its N-terminal carbamate to expose a new interaction with the NS5A pocket located between amino acids P97 and Y93, which was not easily accessible to 1. The results also suggest new chemistry directions that exploit the interactions with the P97–Y93 site toward new and potentially improved HCV NS5A inhibitors.

Background: Agents that activate innate and adaptive immunity have the potential for transformative immunotherapeutic approach in cancer treatment. Recent evidence suggests that the induction of innate immunity via the activation of RIG-I pathway in tumor cells, as well as immune cells in the tumor microenvironment (TME), can produce type I Interferons and cytokines for the induction of adaptive immunity as well as tumor cell death, thereby synergizing antitumoral effects. We describe here the discovery of highly potent first-in-class nucleotide compounds as RIG-I agonists. Methods: Synthesis: Using published crystal structure of 5´ppp-dsRNA bound to RIG-I in conjunction with structure-guided drug design, a focused library of more than 100 nucleotide compounds was generated and evaluated in multiple assays: (i) RIG-I binding affinity: Differential scanning fluorimetry was used to screen compounds for determining binding affinity to RIG-I (ii) Induction of IRF3: Compounds were assessed for IRF3 induction in doses ranging from 1 ug/mL to 100 ug/mL, using wild-type- and RIG-I-knockout mouse RAW cells, as well as, THP-1 cells carrying lucia reporter gene under the control of ISG54 minimal promoter and lead compounds were identified; (iii) Induction of cytokines: Lead compounds @ 100ug/ml were further evaluated for the induction of IFN-β and other cytokines in PBMCs and mouse bone-marrow-derived dendritic cells (mBMDCs) by Luminex analysis; (iv) Induction of Interferon Stimulated Genes (ISGs): ISGs such as DDX58, IFIT1, IFIT12, and IFIT13 were assessed by quantitative RT-PCR following treatment of the compounds in THP1 cells, PBMCs, and mBMDCs; (v) Induction of apoptosis in CT26 colon carcinoma cells: For determining apoptotic activity, CT26 cells were treated with various concentrations of the lead compounds, pppdsRNA or vehicle control and the apoptotic activity was measured using Caspase-Glo® 3/7 Assay (Promega). (vi) Release of DAMPs in CT26 colon carcinoma cells: For evaluating release of ATP and HMGB1, CT26 cells were treated with various concentrations of the lead compounds and supernatants were tested for presence of ATP and HMGB1 by ELISA. Results: Through in vitro assays in conjunction with SAR studies, we have identified several potent and selective first-in-class nucleotide compounds as RIG-I agonists. Two lead nucleotide compounds, SB 1 and SB 2, activated RIG-I in a dose-dependent manner to induce IRF3 signaling pathway in various cells. SB 1 and 2 also showed potent in vitro antitumor activity in multiple cell lines via both RIG-I dependent apoptosis of tumor cells and through the induction of IFNs, ISGs, and other cytokines, along with the expression of PRRs and ISGs including DDX58 IFIT1, IFIT2, and IFIT3. Conclusion: Highly potent RIG-I agonists that show excellent selectivity in the induction of IFN, NF-KB, ISGs, and PRRs and apoptosis of tumor-derived cell lines have been identified. The lead nucleotide compounds are being advanced for preclinical studies for applications in immuno-oncology. Citation Format: Sreerupa Challa, Ireneusz Nowak, Geeta Meher, Rayomand Gimi, Seetharamaiyer Padmanabhan, Dillon Cleary, Leena Suppiah, Diane Schmidt, Sam Baskaran, Lakshmi Bhagat, Radhakrishnana Iyer. Discovery of novel first-in-class nucleotide analogs as RIG-I agonists for immuno-oncology [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B76.

Rapid clinical progress of hepatitis C virus (HCV) replication inhibitors, including these selecting for resistance in the NS5A region (NS5A inhibitors), promises to revolutionize HCV treatment. Herein, we describe our explorations of diverse spiropyrrolidine motifs in novel NS5A inhibitors and a proposed interaction model. We discovered that the 1,4-dioxa-7-azaspiro[4.4]nonane motif in inhibitor 41H (GSK2236805) supported high potency against genotypes 1a and 1b as well as in genotype 1b L31V and Y93H mutants. Consistent with this, 41H potently suppressed HCV RNA in the 20-day RNA reduction assay. Pharmacokinetic and safety data supported further progression of 41H to the clinic.