Structure-activity relationship of inositol thiophosphate analogs as allosteric activators of Clostridioides difficile toxin B

Clostridioides difficile is a bacterium that causes life-threatening intestinal infections. Infection symptoms are mediated by a toxin secreted by the bacterium. Toxin pathogenesis is modulated by the intracellular molecule, inositol-hexakisphosphate (IP6). IP6 binds to a cysteine protease domain (CPD) on the toxin, inducing auto-proteolysis, which liberates a virulence factor in the cytosol. Here, we developped second-generation IP6 analogs designed to induce auto-proteolysis in the gut lumen, prior to toxin uptake, circumventing pathogenesis. We synthesized a panel of thiophosphate- and sulfate-containing IP6 analogs, and characterized their toxin binding affinity, auto-proteolysis induction as well as binding to physiological divalent cations. Our top candidate was soluble in physiological extracellular ion concentrations, unlike IP6. In addition, the IP6 analogs were more negatively charged than IP6, resulting in improved affinity and stabilization of the CPD, which enhanced toxin auto-proteolysis. Our data illustrate the optimization of IP6 with thiophosphate biomimetics which are more capable of inducing TcdB auto-proteolysis than the native ligand, warranting further studies in vivo.