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

National Center For Advancing Translational Sciences

Investigators

Linked publications, trials & patents

Abstract

The 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. -Inhibitors of Bunyaviral endonucleases: A Frster resonance energy transfer (FRET)-based biochemical assay for RVFV and LACV endonucleases, and a cell-based Split-luciferase cellular thermal shift assay (CETSA) will be used for HTS to identify inhibitors of Bunyavirales endonucleases. -Inhibitors of Flaviviral NS2B-NS3 proteases: A high-throughput FRET-based biochemical assays for WNV, ZIKV, DENV2, and YWF NS2B-NS3 proteases will be used identify novel inhibitors of key flaviviral proteases. A cell-base protease assay using a Split Nanoluciferase reporter will be used as a complementary approach. -Inhibitors of viral Methyltransferases: An HTS platform utilizing recombinant Mtases from families including flaviviral, henipaviral and coronaviral Mtases is being developed. We are developing assays for NS5 (DENV, ZIKV, WNV), NSP14 and NSP16 (SARS-CoV-2), and L protein (CEDV) Mtases. The assay uses the MtaseGlo system to monitor SAM to SAH conversion. -Inhibitors of viral nucleocapsid-RNA interactions: Biochemical proximity assays are used to measure henipah and bunyaviral N protein-RNA interactions. By modifying RNAs to contain a fluorescent dye or a biotin tag, FRET or AlphaLISA can be used to monitor interactions between N protein and RNA. The Kales group has developed assays to facilitate the study of mutations in key CoV-2 therapeutic targets and leverage those technologies to target other viral families of pandemic potential. We have initiated the design and optimization of assays related to the following: -Inhibitors of SARS-CoV-2 PLPro Protease: We developed a Papain-like protease (PLPro) fluorescent-based enzymatic activity to screen for inhibitors of SARS-CoV-2, and a hit validation assay employing a luciferase-based reporter to support Structure-Activity Relationships (SAR). To support hit triage, we are developing a cell-based nanoluciferase assay to assess the effects of novel CoV-2 mutations. -Inhibitors of SARS-CoV-2 3CL/MPro Protease: We developed two enzymatic assays for HTS of novel CoV-2 3CL inhibitors, and to assess effects of mutations on current therapeutics, including Paxlovid. We identified several amino acid mutations in the SARS-CoV-2 3CL protease that may impact Paxlovid efficacy through in silico modeling and resistance studies. In collaboration with our Protein Expression and Purification Group at NCI Frederick, we are studying mutant forms of SARS-CoV-2 3CL to assess activity and efficacy of existing therapeutics. We developed a cell-based reporter assay incorporating novel mutations to study therapeutic resistance and identify novel inhibitors that may overcome escape variants. -Non-Nucleoside Inhibitors (NNIs) of SARS-CoV-2 RNA-dependent RNA Polymerase:. To identify non-nucleoside inhibitors (NNIs) of RNA-dependent RNA (RdRp), we are developing proximity-based enzymatic activity assays to support screens for inhibitors against recombinant CoV-2 RdRp. To support hit triage, and to study effects of mutations on drug efficacy, we are developing a cell-based luciferase reporter of RdRp activity. The Lee group has begun development of over a dozen live virus (2D or 3D) assays to screen for therapeutic agents against a range of viruses, including SARS-CoV-2, HCoV-229E, HCoV-NL63, & HCoV-OC43 (Coronaviridae); Rift Valley Fever virus MP-12 (RVFV)(Phenuiviridae), Hazara virus (HAZV)(Nairoviridae), Junin virus (JV)(Arenaviridae), California Encephalitis virus (CEV)(Peribunyaviridae) (Bunyavirales); Dengue virus (DENV), WNV-Kunjin virus (KUNV), Yellow Fever virus 17D (YFV-17D), and Zika virus (ZIKV) (Flaviviridae); Parainfluenza virus 1 (PIV-1), Parainfluenza virus 3 (PIV-3)(Paramyxoviridae); Respiratory Synctial virus (RSV)(Pneumoviridae); Enterovirus D68 (EV-D68)(Picornaviridae); Chikungunya virus (CHIKV), Mayaro virus (MAYV)(Togaviridae). -2D Live Virus HTS Assays We have generated P1 viral stocks of additional SARS-CoV-2 variants (across major variants; through SVC), HCoV-NL63, HCoV-OC43, HCoV-229E; RVFV-MP12, HAZV, CEV; DENV (serotypes 1-4), WNV-KUNV, YFV-17D, ZIKV; PIV-3 GFP; RSV-GFP; and CHIKV. Unlisted viruses in section one are in-house and either actively in propagation or planned for propagation. We have identified permissive cell lines for further HTS assay development for RVFV-MP12, PIV-3, RSV, ZIKV, DENV, YFV-17D, CHIKV. We have HT imaging assays in late-stage development for PIV-3-GFP, RSV-GFP, and RVFV-MP12. We are currently optimizing a no-wash 1536w assay for GFP reporter viruses. -3D Live Virus Assays We have analyzed SARS-CoV-2 variant infectivity differences on air liquid interface (ALI) lung models including an ALI bronchial model and an ALI alveolar model. We have screened compounds of interest for SARS-CoV-2 infection in ALI lung models. We have established infection permissiveness and kinetics in an ALI bronchial model and an ALI alveolar model with a panel of respiratory viruses including RSV, PIV-3, HCoV-OC43, and HCoV-229E. We have established SARS-CoV-2 variant infectivity differences in a small intestinal tissue model. We have developed a neuronal spheroid SARS-CoV-2 infection model. We are currently optimizing RVFV and CHIKV infection of human iPSC-derived neuronal spheroids. We have preliminary data on a vascularized tissue model for DENV infection. We have contracted a non-human primate airway model (MatTek) and have begun initial characterizations of the model in terms of cellular composition and viral infectivity across a panel of human respiratory viruses. The Eastman group focuses on cell-based discovery and live virus high-throughput assay development, including novel assay and reagent design and development. Over the past year, we have set up a 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. We have also optimized and standardized two high-throughput SARS-CoV2 viral replication assays within the high-containment laboratory. One of these assays uses an integrated recombinant luciferase readout and the other is a immunofluorescence based high-content assay for screening of 30 SARS-CoV2 variants. We have also established multiple screening assays for Zika virus (Flaviviridae). -Bunyavirus: APP is collaborating for the identification and development of novel Bunyavirales endonuclease inhibitors. We have optimized and miniaturized multiple viral replication assays, including multiple strains of La Crosse and Punta Toro virus, and performed primary screening against a 12,000 compound library in concentration-response. We are optimizing and validating orthogonal high-content immunofluorescence assays and viral load ddPCR quantitation assays. -APP priority assays: Efforts are ongoing to develop and miniaturize multiple live virus replication assays for assessing direct antiviral activity of small molecule screens, including assays for HCoV-NL63 (Coronaviridae), Junin virus (JV, Arenaviridae), Dengue virus (DENV, Flaviviridae) and Enterovirus D68 (EV-D68, Picornaviridae). We will develop assays using recombinant GFP/EGFP expressing O'nyongnyong virus (ONNV), Ross River virus (RRV), Sindbis virus (SINV), and Venezulan equine encephalitis virus (VEEV).