Preclinical Drug Development of a Novel Antiviral Target in Rotavirus

Abstract

In 2021, rotavirus is the leading cause of severe gastroenteritis for children under the age of 5. While there is currently no cure for this virus, the use of medication is relied upon to alleviate pain from symptoms. A study conducted by the CDC in 2019 found that while only >1% of rotavirus infections under the age of 5 are fatal in high‐income countries, children from low‐income countries account for roughly 82% of all annual rotavirus‐related deaths. As a result, it is critical to developing new effective and affordable drugs to help improve accessibility in addition to decreasing the rates of infection. As the virally encoded ion channel (viroporin), non‐structural protein 4 (NSP4), is central to the replication and pathogenesis of rotavirus, it is a prime target for drug discovery studies. Similar viroporin proteins from HIV‐1 (Vpu) and Influenza A (M2) have been successfully targeted to treat their corresponding viral infections, but this method has not yet been tested for rotavirus infections. In this experiment, an E. coli‐based assay was developed to test known, antiviral ion channel inhibitors for their ability to bind and inhibit the channel activity of rotavirus NSP4. The subsequent data demonstrates that viroporin activity can be measured in a 96‐well format, rather than previous versions that employed 1‐2L cultures. As well, there is no perturbation of NSP4 protein expression or growth kinetics of the E.coli strains used in the assay. Ongoing experiments seek to test the anti‐viroporin activity of compounds representing a variety of core structures and mechanistic targets (Azelnidipine, Rimantadine hydrochloride, and 5‐(N,N‐Hexamethylene)amiloride). These compounds are being utilized to uncover the most effective molecular mechanism of NSP4 inhibition and to provide some biophysical information about the nature of inhibitor interactions with NSP4. These assays, along with drug binding kinetics being determined by thermofluor assay, will give us the first glimpse into NSP4 as a possible target for the treatment of rotavirus infections.