Discovery of Bunyaviral Endonuclease Inhibitors for Anti Viral

Abstract Segmented negative-sense, single-stranded RNA viruses (sNSVs), which include bunyaviruses, are causative agents of human diseases. Rift Valley Fever Virus (RVFV), a mosquito-borne virus causing hemorrhagic fever in humans, is identified by the World Health Organization (WHO) as a priority disease due to its potential to cause outbreaks. The virus is a negative-strand, segmented RNA virus of the genus Phlebovirus in the family Bunyaviridae identified in 1931. Its epizootic/epidemic life cycle involves mosquitoes, livestock, and humans. It has caused 40 outbreaks since 1998, with the most recent one in Mauritania in 2022 with 47 confirmed cases and a case fatality rate of 49% (data from WHO). Even though the outbreaks occurred mainly in Africa countries where the virus is endemic, international travels resulted in RVFV diagnoses in Europe and Asia recently. RVFV has the potential to cause significant global health and economic impact. Unfortunately, there are no FDA- approved drugs or vaccines for the treatment of the RVFV infection. Therefore, there is an urgent medical need for potent therapeutics for RVFV. The overall goal of this project is to develop small molecule inhibitor of RVFV endonuclease as prophylactics and/or therapeutics for infections of RVFV and possibly other highly related bunyaviruses. The strategy is to address the unmet medical need by optimizing a small molecule SP3 series, represented by MBXC-4522, identified in the high throughput screen using a homogeneous FRET-based biochemical assay in Phase I. The compound inhibited the enzymatic activity of RVFV endonuclease by binding to and affecting the thermal stability of the vial protein. Analogs of MBXC-4522 showed structure-dependent activity in our limited preliminary SAR. A second series represented by apomorphine will be the backup series. Phase II of this project will be a continuation collaboration between Microbiotix, Inc., Dr. Gaya Amarasinghe from WUStL, and Dr. Amy Hartman from University of Pittsburgh. Analogs (≥200) will be designed and synthesized in the structural based chemical optimization in Aim 1 and evaluated in biochemical and cellular assays in Aim 2 using an assay funnel designed to prioritize compounds by potency, selectivity, and confirmed to act by specific non-toxic mechanisms of action in Aim 3. Crystallography (Aim 3) will also be employed to further optimize the series. Prioritized analogs will be formulated for in vivo studies. The maximum tolerated dose of the most promising lead compounds will be determined in mouse tolerability studies, and pharmacokinetic analyses will be used to further prioritize compounds and optimize dosing strategies. Inhibitors with promising exposure and tolerability in mice will be tested for efficacy in an established mouse model in Aim 4. The major milestone of this proposal is to identify in vivo-validated RVFV endonuclease inhibitors with sufficient safety profiles. The RVFV endonuclease inhibitors that meet the milestones (preclinical candidate and backup) will be developed further for use in therapies to treat RVFV infection.