Development of novel antivirals against mpox (monkeypox) virus

A. Project Summary / Abstract The ongoing monkeypox (mpox) outbreak, with a total of more than 87,000 cases reported in over 100 non- endemic countries, highlights the pandemic potential of poxviruses. Due to its zoonotic nature with broad hosts and unidentified natural reservoirs of the causative agent, mpox virus (MPXV), future spillovers and outbreaks are expected even if the current outbreak is contained. Although typically manifesting in milder symptoms than smallpox, MPXV infections can still cause significant morbidity and mortality (up to 10% for clade I) that need to be therapeutically mitigated. The clinical efficacy of FDA-approved smallpox antiviral drugs is unclear or unpromising for treating MPXV, necessitating dedicated and expanded efforts in developing MPXV antivirals. The goal of this grant application is to develop novel antivirals for treating MPXV. Prior research from our team has developed and validated an efficient and robust reporter assay using the vaccinia virus (VACV), and identified and characterized three high quality and chemically distinct antiviral hits with strong potencies (EC50 = 0.14‒2.1 M; plaque reduction at 10 M 1,400‒160,000-fold), no cytotoxicity (CC50 > 250 M) and largely favorable ADME properties. The immediate focus of this grant is to further develop these three hit series into pre-clinical candidates using a highly integrated, multi-disciplinary approach combining medicinal chemistry, virology, ADME / pharmacokinetic (PK), toxicity, animal efficacy and proteomics. Three Specific Aims are proposed: 1) optimization and expansion of the identified hits. Iterative structure-activity relationship (SAR) and structure-property relationship (SPR) studies will be carried out via extensive analog synthesis, antiviral assay and ADME profiling to obtain optimized leads. Hit expansion will also be conducted by screening the high quality synthetic compounds in PI's lab as well as preselected commercial compounds; Aim 2) in vivo PK, toxicity and efficacy studies, where the acute and sub-acute toxicity and PK studies in mice will guide compound selection for efficacy studies in mice; Aim 3) determine the antiviral mechanism of action (MOA). The molecular targets and antiviral MOA of optimized leads will be determined using a few different methods: a) identify the viral replication stage(s) targeted by the leads; b) select resistant mutants by passaging virus in cultured cells in the presence of individual lead compound, and sequence viral genomes to identify affected genes; The compound will then be tested in recombinant viruses harboring the selected mutations; c) design and synthesize chemical probes to label, pull down, and identify the target protein(s); d) test metal chelating analogs in resolvase biochemical assays. Overall, 2-3 antiviral leads with strong potency, favorable PK profiles, minimal toxicities, and established MOA will be developed, setting the stage for further pre-clinical development of MPXV drugs that will also contribute substantially to countering other orthopoxviruses.