# Development of a high-throughput screening platform for C. difficile toxin synthesis inhibitors unveils meclizine as an antivirulence agent

## TL;DR

A new screening method identified meclizine, an antihistamine, as a potential treatment for C. difficile infection by reducing toxin production.

## Contribution

A high-throughput screening platform was developed to identify compounds that inhibit C. difficile toxin synthesis without affecting bacterial growth.

## Key findings

- Meclizine reduced TcdA and TcdB protein levels and cytopathic effects in C. difficile cultures.
- Meclizine altered central carbon metabolism, leading to intracellular accumulation of glucose and pyruvate.
- The screening platform successfully identified meclizine as a toxin synthesis inhibitor.

## Abstract

Clostridioides difficile, a leading cause of hospital-acquired diarrhea, exerts its virulence through two co-regulated toxins, TcdA and TcdB. Despite their pivotal roles, the discovery of inhibitors targeting their biosynthesis is underexplored. Here, we present a high-throughput screening (HTS) platform designed to identify toxin synthesis inhibitors (TSIs) that minimally impact bacterial growth. The primary screen utilized a C. difficile reporter strain expressing secreted Nano-luciferase (secNluc) under the tcdA promoter, whereby inhibition of secNluc production indicates toxin biosynthesis inhibition. Screening the Prestwick Chemical Library at 10 and 100 µM identified several compounds that reduced secNluc activity. Through counter-screening, we eliminated compounds that caused spectral interference. Orthogonal dose-response assays assessing the effectiveness of inhibiting toxin production without affecting growth identified meclizine, an antihistamine, as the primary antivirulence candidate. Meclizine was confirmed as a TSI by showing that it reduced TcdA and TcdB protein levels, the cytopathic potential of cultures, and tcdA and tcdB transcription as determined by ELISA, cell-rounding assays, and RT-qPCR, respectively. Meclizine significantly altered central carbon metabolism in C. difficile, upregulating carbohydrate transport systems and the conversion of lactate to pyruvate, while downregulating glycolytic genes. These changes were associated with intracellular accumulation of glucose and pyruvate, metabolites known to negatively impact toxin production. Taken together, our findings underscore the utility of the above HTS platform to identify anti-C. difficile TSIs, which can serve as molecular and cellular probes, as well as chemical starting points for developing novel therapeutics for C. difficile infection.