Synthesis and Antimicrobial Evaluation of Amixicile-Based Inhibitors of the Pyruvate-Ferredoxin Oxidoreductases of Anaerobic Bacteria and Epsilonproteobacteria

Amixicile is a promising derivative of nitazoxanide (an antiparasitic therapeutic) developed to treat systemic infections caused by anaerobic bacteria, anaerobic parasites, and members of theEpsilonproteobacteria (Campylobacter andHelicobacter ). Amixicile selectively inhibits pyruvate-ferredoxin oxidoreductase (PFOR) and related enzymes by inhibiting the function of the vitamin B1 cofactor (thiamine pyrophosphate) by a novel mechanism. Here, we interrogate the amixicile scaffold, guided by docking simulations, direct PFOR inhibition assays, and MIC tests againstClostridium difficile ,Campylobacter jejuni , andHelicobacter pylori . Docking simulations revealed that the nitro group present in nitazoxanide interacts with the protonated N4′-aminopyrimidine of thiamine pyrophosphate (TPP). Theortho -propylamine on the benzene ring formed an electrostatic interaction with an aspartic acid moiety (B456) of PFOR that correlated with improved PFOR-inhibitory activity and potency by MIC tests. Aryl substitution with electron-withdrawing groups and substitutions of the propylamine with other alkyl amines or nitrogen-containing heterocycles both improved PFOR inhibition and, in many cases, biological activity againstC. difficile . Docking simulation results correlate well with mechanistic enzymology and nuclear magnetic resonance (NMR) studies that show members of this class of antimicrobials to be specific inhibitors of vitamin B1 function by proton abstraction, which is both novel and likely to limit mutation-based drug resistance.