The Mechanism of Mycobacterial (p)ppGpp Synthetase Inhibition by Synthetic Erogorgiaene Analog

Abstract

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism cessation, growth rate control, virulence, bacterial persistence, and biofilm formation. The RelA/SpoT homologs superfamily proteins are responsible for (p)ppGpp alarmone synthesis, including long bifunctional RSH proteins and small alarmone synthetases. This study employs enzyme kinetics and dose-dependent inhibition methods to investigate the specific mechanism of action of DMNP involving RelMsm and RelZ proteins, which are (p)ppGpp synthetases in Mycolicibacterium smegmatis belonging to both types, as well as RelMtb protein from Mycobacterium tuberculosis. The compound DMNP has demonstrated its capability to inhibit the activity of the RelMtb protein. According to enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor targeting the RelMsm and RelZ proteins. Molecular docking analysis allowed to localize the DMNP binding site in proximity to the (p)ppGpp synthetase domain active site. This study advances the development of alarmone synthetase inhibitor class of compounds, which includes relacin and its derivatives, alongside the investigated compound DMNP – a synthetic analog of the marine coral metabolite erogorgiaene. Unlike the conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining the conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may potentially address prevailing challenges associated with antimicrobial resistance and bacterial persistence.