SBIR-STTR Award

The HIV host restriction factor APOBEC3G (A3G) can inhibit HIV by inducing catastrophic mutations in the viral genome. The HIV Vif protein protects HIV by binding to A3G and directing it to the proteasomal degradation pathway. The significance of the proposed SBIR phase I research is that Vif remains a new antiviral target whose clinical potential has yet to be fully explored. Despite much academic research on Vif, only a limited effort has gone toward identifying small molecule antagonists of Vif. OyaGen, Inc. is the only commercial entity currently pursuing the identification of chemical scaffolds that specifically interact with Vif in meaningful ways for drug development. An inherent limitation of prior anti-Vif drug discovery efforts has been the use of primary drug screens in which ‘hits’ may be due to more than one mechanisms of action. We designed a live cell quenched FRET (FqRET) reporter assay that enables selection of membrane- permeable antagonists of protein-protein interactions between Vif and A3G using a Vif mutant that retains wild type binding to A3G but no longer binds to Elongin C and no longer facilitates A3G degradation. From a screen of a 110K small molecule library of drug-like compounds we identified 165 prioritized his for further analysis. Although a few compounds were dose-dependent antagonists of Vif binding of A3G, one compound, hereafter referred to as C5, was non-cytotoxic and displayed dose-dependent: i) inhibition of Vif-dependent A3G degradation, ii) inhibition of pseudotyped HIV replication and iii) promoted an increased incorporation of A3G in virions. Here we propose an innovative drug discovery critical path to optimize a novel anti-HIV lead compound from C5 in anticipation of phase II SBIR pharmacokinetic analysis, in vivo efficacy testing, and IND-enabling studies. The Phase I SBIR Aims will focus on structure-activity relationship (SAR) medicinal chemistry of the C5 scaffold for hit-to-lead optimization of anti-HIV compound whose antiviral activity is mechanistically due to its function as a Vif antagonist that protects A3G from Vif-dependent degradation.

Public Health Relevance Statement:
PROJECT NARRATIVE HIV-1 Viral Infectivity Factor (Vif) is a viral accessory protein expressed late during infection that is essential for viral replication. Vif is required because it mediates degradation of the host defense factor APOBEC3G (A3G) that otherwise will inhibit viral replication by catalyzed HIV genomic hypermutation. Binding of Vif with A3G is essential for Vif-dependent degradation of A3G. Using a high-throughput screen that selectively quantifies Vif binding to A3G, we identified and validated a compound that blocks the Vif protein-protein interaction with A3G, increases A3G virion incorporation and reduces HIV replication. We propose hit-to-lead optimization of a novel anti-HIV drug candidate in this phase I SBIR by medicinal chemistry guided by a drug discovery critical pathway comprising primary, secondary, orthogonal, and ADMET validation assays.

Project Terms:
Address; Affect; Agonist; analog; Anti-HIV Agents; Anti-Retroviral Agents; anti-viral efficacy; Antiviral Agents; APOCEC3G gene; base; Binding; Biological Assay; Cell Membrane Permeability; Cells; Chemicals; Clinical; Complementary DNA; Complex; Critical Pathways; Custom; cytotoxicity; Degradation Pathway; design; Development; Dose; drug candidate; drug development; drug discovery; Drug Kinetics; Drug Screening; Drug Targeting; efficacy testing; elongin C; Fluorescence Resonance Energy Transfer; Genomics; high throughput screening; HIV; HIV Budding; HIV Genome; HIV Protease; HIV-1; Host Defense; In Vitro; in vivo; indexing; Infection; inhibitor/antagonist; innovation; Integrase; Lead; lead optimization; Mediating; Molecular Probes; multicatalytic endopeptidase complex; Mutagenesis; mutant; Mutation; novel; particle; Peripheral Blood Mononuclear Cell; Pharmaceutical Chemistry; Pharmaceutical Preparations; pharmacophore; Phase; phase 1 study; phase 2 study; Polyubiquitination; Positioning Attribute; pre-clinical; preclinical development; prevent; Property; protein protein interaction; Proteins; receptor; Recovery; recruit; Reporter; Reporting; Research; Reverse Transcription; scaffold; screening; Selection Criteria; Signal Transduction; Small Business Innovation Research Grant; small molecule; small molecule libraries; Source; Structure-Activity Relationship; Testing; Therapeutic; ubiquitin-protein ligase; Validation; Viral; Viral Genome; Viral Proteins; Virion; Virus Inhibitors; Virus Replication

The HIV host restriction factor APOBEC3G (A3G) can inhibit HIV by inducing catastrophicmutations in the viral genome. The HIV Vif protein protects HIV by binding to A3G and directingit to the proteasomal degradation pathway. The significance of the proposed SBIR phase Iresearch is that Vif remains a new antiviral target whose clinical potential has yet to be fullyexplored. Despite much academic research on Vif, only a limited effort has gone toward identifyingsmall molecule antagonists of Vif. OyaGen, Inc. is the only commercial entity currently pursuingthe identification of chemical scaffolds that specifically interact with Vif in meaningful ways fordrug development. An inherent limitation of prior anti-Vif drug discovery efforts has been the useof primary drug screens in which ‘hits’ may be due to more than one mechanisms of action. Wedesigned a live cell quenched FRET (FqRET) reporter assay that enables selection of membrane-permeable antagonists of protein-protein interactions between Vif and A3G using a Vif mutantthat retains wild type binding to A3G but no longer binds to Elongin C and no longer facilitatesA3G degradation. From a screen of a 110K small molecule library of drug-like compounds weidentified 165 prioritized his for further analysis. Although a few compounds were dose-dependentantagonists of Vif binding of A3G, one compound, hereafter referred to as C5, was non-cytotoxicand displayed dose-dependent: i) inhibition of Vif-dependent A3G degradation, ii) inhibition ofpseudotyped HIV replication and iii) promoted an increased incorporation of A3G in virions. Herewe propose an innovative drug discovery critical path to optimize a novel anti-HIV lead compoundfrom C5 in anticipation of phase II SBIR pharmacokinetic analysis, in vivo efficacy testing, andIND-enabling studies. The Phase I SBIR Aims will focus on structure-activity relationship (SAR)medicinal chemistry of the C5 scaffold for hit-to-lead optimization of anti-HIV compound whoseantiviral activity is mechanistically due to its function as a Vif antagonist that protects A3G fromVif-dependent degradation.

Public Health Relevance Statement:
PROJECT NARRATIVE HIV-1 Viral Infectivity Factor (Vif) is a viral accessory protein expressed late during infection that is essential for viral replication. Vif is required because it mediates degradation of the host defense factor APOBEC3G (A3G) that otherwise will inhibit viral replication by catalyzed HIV genomic hypermutation. Binding of Vif with A3G is essential for Vif-dependent degradation of A3G. Using a high-throughput screen that selectively quantifies Vif binding to A3G, we identified and validated a compound that blocks the Vif protein-protein interaction with A3G, increases A3G virion incorporation and reduces HIV replication. We propose hit-to-lead optimization of a novel anti-HIV drug candidate in this phase I SBIR by medicinal chemistry guided by a drug discovery critical pathway comprising primary, secondary, orthogonal, and ADMET validation assays.

Project Terms:
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