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

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Roman Y. Sidorov^1,2,a* and Alexander G. Tkachenko^1,2

¹Institute of Ecology and Genetics of Microorganisms, Perm Federal Research Center, Ural Branch of Russian Academy of Sciences, 614000 Perm, Russia

²Perm State University, 614990 Perm, Russia

Received June 29, 2023; Revised January 9, 2024; Accepted January 9, 2024

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The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases Rel_Msm and RelZ from Mycolicibacterium smegmatis and Rel_Mtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of Rel_Mtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of Rel_Msm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP – a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.

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KEY WORDS: (p)ppGpp, alarmones, mycobacteria, RelA/SpoT homologs, small alarmone synthetases, alarmone synthetase inhibitors

DOI: 10.1134/S0006297924030027

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