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

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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.

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R. Yu. Sidorov

Perm Federal Research Center, the Ural Branch of Russian Academy of Sciences; Perm State University

Author for correspondence.
Email: sidorov.r@iegm.ru
Russian Federation, Perm; Perm

A. G. Tkachenko

Perm Federal Research Center, the Ural Branch of Russian Academy of Sciences; Perm State University

Email: sidorov.r@iegm.ru
Russian Federation, Perm; Perm

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2. Fig. 1. Domain composition of RSH superfamily enzymes catalysing the synthesis and hydrolysis of alarmones (p)ppGpp. Bifunctional long RSHs, such as RelMsm from M. smegmatis and RelMtb from M. tuberculosis, include hydrolase (HYD) and synthetase (SYN) domains in the catalytic domain, as well as a C-terminal regulatory domain (REG). Monofunctional short RSHs contain either a hydrolase or synthetase domain. RelZ from M. smegmatis is an unusual small alarmonsynthetase because it also contains an HII RNase (HII) domain

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3. Fig. 2. Effect of DMNP inhibitor on the enzymatic kinetics of (p)ppGpp-synthetases of M. smegmatis. a - Chromatographic analysis of nucleotides in samples taken from the enzymatic reaction with RelNTD after: 1 - 0 h or 2 - 0.5 h (detection at 254 nM, 1 mM GTP, 4 mM ATP). b - Analysis of the enzymatic kinetics of the N-terminal domain of the RelMsm protein from M. smegmatis. The curves of reaction rate dependence (calculated per 1 μmol of protein) on the concentration of substrate GTP are given: 1 - in the absence of inhibitor addition; 2 - when 600 μM DMNP was added. c - Ds-Na-PAAG electrophoresis of purified RelNTD enzyme. d - Chromatographic analysis of nucleotides in samples taken from the enzymatic reaction with RelZ after 1 - 0 h or 2 - 0.5 h (detection at 254 nM, 1 mM GDP, 4 mM ATP). e - Enzymatic kinetics analysis of the small RelZ alarmonsynthetase from M. smegmatis. The curves of reaction rate dependence (calculated per 1 μmol of protein) on the substrate concentration GDP are shown: 1 - in the absence of inhibitor addition; 2 - when 600 μM DMNP was added. f - Ds-Na-PAAG electrophoresis of purified RelZ enzyme. g - Hill plot for the reaction with RelZ: 1 - in the absence of inhibitor addition; 2 - when 600 μM DMNP was added

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4. Fig. 3. pppGpp-Synthesising activity of the long RSH protein RelMtb from M. tuberculosis over a range of DMNP concentrations (0-600 μM) in 5% methanol. The reaction rate is estimated by the change in the concentration of the substrate GTP. MetOH- - reaction without methanol and DMNP addition

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5. Fig. 4. Spatial alignment and molecular docking results of three-dimensional structures of M. tuberculosis RelMtb (blue), M. smegmatis RelMsm (purple) and RelZ (yellow) target proteins predicted by AlphaFold. SYN, synthetase domain; HYD, hydrolase domain; CTD, regulatory C-terminal domain; TGS, TGS domain; HII, HII RNase domain. DMNP binding clusters (green): 1 - corresponding to the active site of the synthetase domain, 2 - located near the active site of the synthetase domain

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