Optimizing lipid RVn monophosphate prodrugs to maximize RVn-triphosphate delivery

PROJECT SUMMARY/ABSTRACT Emerging RNA viruses, especially respiratory viruses, are among the leading threats to global health. With few treatments currently available, there is an urgent and ongoing need for the development of safe, effective oral antivirals. The objective of this application is to optimize an innovative lipid prodrug delivery strategy for remdesivir nucleoside monophosphate (RVn-MP), and additional broad-spectrum nucleoside antivirals, to achieve 1) excellent oral bioavailability, 2) efficient intracellular activation across tissue types, and 3) bypass of liver metabolism to enhance tissue delivery. The central hypothesis is that specific modifications to the lipid prodrug scaffold can improve in vivo antiviral efficacy by enhancing prodrug metabolism to the active metabolite and augmenting tissue delivery. The rationale for this project is that a better understanding of how lipid prodrug modifications increase antiviral activity will allow for the rational design and development of novel broad- spectrum oral antivirals for the treatment of clinically important RNA viruses. Strategy: Aim 1 will identify the mechanisms that determine prodrug antiviral potency in vitro to maximize antiviral activity. Quantitation of lipid RVn-MP prodrugs and their metabolites in cell culture using mass spectrometry will determine how scaffold modifications alter uptake, metabolism, and antiviral activity. Genetic knockout studies will identify the specific phospholipase C (PLC) enzyme/s that are necessary for lipid RVn-MP prodrug metabolism across cell types. Finally, PLC enzyme kinetic studies will identify scaffold modifications that maximize metabolism and antiviral activity in vitro. These data will inform lipid prodrug scaffold design that optimizes lipid RVn-MP potency. Aim 2 will determine how lipid prodrug modifications control distribution to maximize tissue delivery. First-pass removal of oral drugs by the liver is a common problem in drug development. We will evaluate how oral lipid nucleoside prodrugs can partition into chylomicrons, move through lymphatics to the thoracic duct, and thereby avoid first-pass liver metabolism while increasing lung delivery. Structure-activity relationship studies using a library of lipid RVn prodrugs will identify scaffold modifications that increase intestinal lymphatic trafficking and improve serum pharmacokinetics and tissue distribution. Scaffolds that maximize in vitro antiviral activity (Aim 1) and in vivo lung delivery (Aim 2) will be selected to rationally design new lipid RVn- MP prodrugs and novel lipid prodrugs containing nucleosides with broad spectrum activity against RNA viruses. New compounds will be evaluated for increased metabolism and antiviral activity in vitro, tissue delivery in vivo, and efficacy against pathogenic coronaviruses and dengue in mice. Collectively, this proposal will optimize the antiviral efficacy of oral lipid RVn-MP prodrugs for the treatment of many clinically important RNA viruses. Additionally, a better understanding of how to maximize the efficacy of lipid nucleoside prodrug design may be the key to unlocking a whole new generation of broad-spectrum antivirals.