Identification of small molecules targeting homoserine acetyl transferase from Mycobacterium tuberculosis and Staphylococcus aureus

Abstract

There is an urgent need to validate new drug targets and identify small molecules that possess activity against both drug-resistant and drug-sensitive bacteria. The enzymes belonging to amino acid biosynthesis have been shown to be essential for growth in vitro, in vivo and have not been exploited much for the development of anti-tubercular agents. Here, we have identified small molecule inhibitors targeting homoserine acetyl transferase (HSAT, MetX, Rv3341) from M. tuberculosis. MetX catalyses the first committed step in L-methionine and S-adenosyl methionine biosynthesis resulting in the formation of O-acetyl-homoserine. Using CRISPRi approach, we demonstrate that conditional repression of metX resulted in inhibition of M. tuberculosis growth in vitro. We have determined steady state kinetic parameters for the acetylation of L-homoserine by Rv3341. We show that the recombinant enzyme followed Michaelis–Menten kinetics and utilizes both acetyl-CoA and propionyl-CoA as acyl-donors. High-throughput screening of a 2443 compound library resulted in identification of small molecule inhibitors against MetX enzyme from M. tuberculosis. The identified lead compounds inhibited Rv3341 enzymatic activity in a dose dependent manner and were also active against HSAT homolog from S. aureus. Molecular docking of the identified primary hits predicted residues that are essential for their binding in HSAT homologs from M. tuberculosis and S. aureus. ThermoFluor assay demonstrated direct binding of the identified primary hits with HSAT proteins. Few of the identified small molecules were able to inhibit growth of M. tuberculosis and S. aureus in liquid cultures. Taken together, our findings validated HSAT as an attractive target for development of new broad-spectrum anti-bacterial agents that should be effective against drug-resistant bacteria.

Amino Acid Biosynthesis Inhibitors in Tuberculosis Drug Discovery

“…NSC369066 was the most potent, with killing levels comparable to INH, which were partially restored after Met supplementation. Unfortunately, this first-in-class MetX inhibitor suffers from high cytotoxicity (5 µM) and multitarget killing activity, and further effort is required to improve its antimycobacterial activity and its SI before considering it as a promising candidate for further hit-to-lead optimization [ 112 ].…”

TRAF6 triggers Mycobacterium-infected host autophagy through Rab7 ubiquitination

“…Mycobacterium tuberculosis (Mtb), the pathogenic bacterium responsible for tuberculosis (TB), typically infiltrates the respiratory tract and results in chronic lung lesions [ 1 ]. An effective host response against Mtb necessitates the concerted actions of innate and adaptive immune cells, including macrophages, dendritic cells (DCs), neutrophils, and T cells [ 2 , 3 ]. Macrophages are both the primary host cells and the initial defense against Mtb infection [ 4 – 6 ].…”