Advancing the development of a novel class of small molecules for treating pan-coronavirus infections

Abstract For the past decade, our laboratory has been studying the role of cellular kinases in intracellular trafficking of RNA viruses and as targets for broad-spectrum antivirals. Furthermore, we have provided a proof of concept for the potential feasibility of the host-targeted broad-spectrum antiviral approach by demonstrating that the inhibition of two cellular kinases, AAK1 and GAK, by novel or the approved anticancer drugs, sunitinib and erlotinib, protects mice from dengue and Ebola viruses with a high barrier to resistance. Since the therapeutic index (TI) of this drug combination is narrower for SARS-CoV-2 infection, here, we focus on an independent class of compounds, the isothiazolo[4,3-b]pyridine-based RMC-113 series, that emerged from our prior work, but does not inhibit AAK1 or GAK. We showed that RMC-113 and 25 related analogs have potent broad- spectrum antiviral activity with a high barrier to resistance. Excitingly, RMC-113 reduces SARS-CoV-2 titer to undetectable levels at non-toxic concentrations and binds PIKFYVE, a cell kinase that regulates endosomal trafficking. We hypothesize that RMC-113 analogs inhibit both multiple distinct steps in the SARS-CoV-2 life cycle and the inflammatory response to this virus, in part by targeting PIKFYVE, thereby offering attractive and safe candidate inhibitors to combat SARS-CoV-2, other pandemic coronaviruses and other emerging viruses. In Aim 1, we will use a multi-dimensional medicinal chemistry approach to optimize the TI and PK profile of lead RMC-113 analogs and define their in vitro therapeutic potential as broad anticoronavirus inhibitors. Aim 2 will determine the effect of prioritized analogs and apilimod, a repurposed drug candidate for COVID-19 that inhibits PIKFYVE, on viral replication, cytokine response and tissue injury in organoids derived from excised normal lung tissue supplemented with PBMCs from 20 human donors and in two rodent models. Aim 3 will generate ADME-toxicity and safety pharmacology datasets to select pre-IND candidates. In Aim 4, we will probe the mechanism of antiviral action of RMC-113. We will validate PIKFYVE as a candidate target and use an unbiased CRISPRi screen to identify RMC-113's target(s) and profile its chemical-genetic landscape. In parallel, we will design a clickable RMC-113 probe to confirm the molecular target via activity-based protein profiling and to monitor target engagement. Lastly, we will probe functional relevance and specific roles of PIKFYVE and other candidates emerging via these approaches in SARS-CoV-2 infection, and validate them as the molecular target(s) mediating the antiviral effect. The predicted immediate impact is that this project will provide insight into the therapeutic potential and MOA of apilimod, a repurposed drug candidate (beyond the reported effect on viral entry), and will establish a unique human lung organoid model for studying SARS-CoV- 2 pathogenesis and response to treatment under more natural conditions. In the longer term, successful completion of our study will deliver a drug-like small molecule candidate designed to protect against resurge of COVID-19 and to provide readiness for future outbreaks with coronaviruses and other emerging viruses.