Antiviral Program for Pandemics (APP) & NCATS: Accelerating Antiviral Development

National Center For Advancing Translational Sciences

Investigators

Linked publications, trials & patents

Abstract

1) The Target-Based Assays 1 (Martinez) Group has identified viral targets amenable for therapeutic intervention via small molecule modulation, including endonucleases, exonuclease, proteases, methyltransferases, and nucleocapsid-RNA interactions. Candidate compounds resulting from each assay will be screened in live-virus assays by APP colleagues, and in silico approaches are used to support screening efforts. These viral targets include: Inhibitors of Bunyaviral endonucleases: High-throughput Foster resonance energy transfer (FRET)-based biochemical assays for RVFV, LACV, HANV, and PTV endonucleases have been developed and screened against small molecule libraries. Cell-based Split-luciferase cellular thermal shift assay (CETSA) have been developed to further characterize inhibitor-endonuclease engagement. Inhibitors of viral proteases: High-throughput FRET-based biochemical assays for flaviviral NS2B-NS3 (WNV, ZIKV, DENV2, and YWF), Alphaviral NSP2 (CHKV), and Picornaviral 2A and 3C (EN68 and E71) proteases have been developed and screened. Counterscreen assays against host proteases have also been implemented to support hit triage. A complementary cell-base protease assay using a Split Nanoluciferase reporter has been developed for the above proteases to further characterize compound activity in cells. Inhibitors of viral Methyltransferases: A high-throughput screening platform utilizing recombinant Mtases from families including Flaviviral, Henipaviral and Coronaviral Mtases is being developed. The assay uses the MtaseGlo system to monitor SAM to SAH conversion. Assays for Flaviviral NS5 (DENV, WNV) and Coronavirus NSP14/10 and NSP16/10 (SARS-CoV2) have been developed and screened. Inhibitors of viral Exonuclease: a FRET-based assay was developed to identify inhibitors of Coronavirus NSP14/10 exonuclease activity. We have screened NCATS-sourced libraries and to support hit triage, we are developing target engagement assays 2) The Target-Based Assays 2 (Kales) group has developed assays to facilitate the study of mutations in key CoV-2 therapeutic targets and are leveraging those technologies and platforms to target other viral families of pandemic potential, including Alphaviruses. The team has initiated the design and optimization of assays related to the following: Inhibitors of SARS-CoV-2 PLPro Protease: the team has screened 91K compounds to date, and is currently developing a cell-based nanoluciferase assay to assess cellular efficacy of lead compounds and the effects of novel CoV-2 PLPro mutations. Inhibitors of SARS-CoV-2 3CL/MPro Protease: two enzymatic assays have been developed to screen for novel CoV-2 3CL inhibitors, and to assess effects of mutations on current therapeutics, including Paxlovid. The team is studying mutant forms of SARS-CoV-2 3CL to assess activity and efficacy of existing therapeutics. Non-Nucleoside Inhibitors (NNIs) of SARS-CoV-2 RNA-dependent RNA Polymerase: several HTS-amenable biochemical assays and multiple secondary assays have been developed to support screens for inhibitors against recombinant CoV-2 RdRp. Nearly 100K compounds have been screened to date. Inhibitors of Chikungunya virus (CHIKV) nsp3/macrodomain: several activity and binding-based assays towards nsp3/macrodomain of Chikungunya and Venezuelan Equine Encephalitis (VEEV) have been developed. 3) The Advanced Models and Cell-Based Discovery (Lee) group works across a large panel of BSL-2 and BSL-3 viruses in both 2D and 3D assays for antiviral screening and development. Live viruses: More than 25 species of full-length or isolate human viruses + 1 minigenome system have been onboarded across 11 viral families, including Bunyavirales, Arenaviridae JUNV, Peribunyaviridae CEV, LACV, Phenuiviridae RVFV-MP12 and PTV (strains -Balliet and -Adames), Nairoviridae HAZV; Coronaviridae SARS-CoV-2 (previous and current variants of concern), HCoV-OC43/GFP, HCoV-229E, HCoV-NL63; Filoviridae EBOV (minigenome system); Flaviviridae DENV (serotypes 1-4), KUNV, YFV-17D, ZIKV (strains -MR766 and -PRVAB59); Paramyxoviridae PIV-3-GFP, PIV-1-GFP; Picornaviridae EV-D68; Pneumoviridae RSV; Togaviridae CHIKV 181/25/nluc, MAYV/nluc, ONNV-GFP, RRV-GFP, SINV-GFP (neuroinvasive and non-neuroinvasive strains), and VEEV-Tc83-GFP. HTS Assays: the team has 22 live virus 1536w or 384w HTS assays developed and 8 in development across 23 viral species, including Coronaviridae, Phenuiviridae (Bunyavirales), Peribunyaviridae (Bunyavirales), Arenaviridae (Bunyavirales), Flaviviridae, Paramyxoviridae, Picornaviridae and Togaviridae. In addition, the team continues to optimize and support the BSL-3 high-containment lab on NIH main campus containing liquid handlers, an automated confocal plate reader and an integrated screening system to support APP screening pipelines. 3D Live Virus Assays: the team continues to utilize air liquid interface (ALI) lung models including an ALI bronchial model and an ALI alveolar model to investigate antivirals against SARS-CoV-2 as well as for examining differences in SARS-CoV-2 variant infectivity. Additional work includes: o ALI lung tissue mimics to include infectivity screening with a panel of additional respiratory viruses ( o SARS-CoV-2 contemporary omicron variant infectivity differences in lung and small intestinal tissue models o Neuronal spheroid infection and disease models for encephalitis viruses, including RVFV, CHIKV, CEV, JCV, LACV, MAYV, SINV, and VEEV o Liver spheroid model that is permissive to a panel of APP viruses including RVFV, CHIKV, DENV, and YFV o Brain endothelial vascular bed model that recapitulates damage induced by exposure to the viral protein NS1 from the flavivirus JEV 4) The Medicinal Chemistry (Shamim) group is currently optimizing a lead for Papain like-protease, an undrugged target for SARS-CoV-2. Med-chem efforts are directed towards improving enzymatic inhibition, antiviral activity, and pharmacokinetic (PK) properties. A reporter-based luciferase enzymatic assay and cell-based SARS-CoV-2 live virus nano luciferase reporter are being employed to drive the SAR in a structure-based discovery campaign. The medicinal chemistry team is also working towards targeting the cap snatching endonuclease for bunyavirus.