Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-carboxylic Acids as Inhibitors of Bacterial Serine Acetyltransferase in the Quest for Novel Potential Antibacterial Adjuvants

Abstract

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals; therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure–activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts.

Combatting antimicrobial resistance via the cysteine biosynthesis pathway in bacterial pathogens

“…These compounds mimic binding interactions with key active site residues similar to the natural inhibitor L-cysteine ( Figure 2 ). Recent studies have explored 2-aminothiazoles and 2-aminooxazole compounds as inhibitors of CysE from S. typhimurium ( St CysE) [ 55 , 56 ]. Since the crystal structure for St CysE (7E3Y) has been solved only recently [ 57 ], all virtual screening was carried out against both Ec CysE (1T3D) and H. influenzae CysE (1SSM) crystal structures, as there is strong conservation of active site residues with St CysE.…”

“…Further research into St CysE inhibition was conducted using an in-house library for further virtual screening of St CysE [ 55 , 57 ]. Using the same screening method as discussed previously the researchers identified seven compounds that reduced St CysE activity, with the most potent being the substituted 2-aminothiazole, compound 5, (5-{2-[(4-Methylphenyl)amino]-1,3-thiazol-4-yl}-1,2-oxazole-3-carboxylic acid) ( Figure 3 ), with an IC 50 of 110 µM, which displayed competitive inhibition relative to acetyl-CoA (K i = 64 μM).…”

“…Docking analysis showed compound 5 interacts with the CysE serine active site residues and acetyl-CoA binding pocket, where the carboxylic acid group interacts with the same residues as seen for inhibitor cysteine/substrate serine. The ‘L-shape’ of the inhibitor allows it to mimic acetyl-CoA, explaining the observation of competitive inhibition [ 55 ]. Structure–activity relationship analysis was undertaken through in vitro screening of compound 5 analogues.…”

“…Structure–activity relationship analysis was undertaken through in vitro screening of compound 5 analogues. Substitution of the 2-aminothiazole ring with a 2-aminooxazole was shown to increase affinity, and the presence of an ester, amide or carboxylic acid group connected to the isoxazole ring was shown to be essential for affinity [ 55 ]. Isoxazole-3-ester and isoxazole-3-carboxylic acid derivatives were further optimized through synthesis with different chemical groups connected to the oxazole ring [ 55 ].…”

A Competitive O-Acetylserine Sulfhydrylase Inhibitor Modulates the Formation of Cysteine Synthase Complex

“…In the last years, many efforts have been addressed to the development of potential inhibitors targeting cysteine pathway [32][33][34] and, in particular, OASS isoforms or SAT from several pathogen species [10,[35][36][37][38][39][40][41][42][43][44][45][46][47][48]. A drug discovery campaign carried out in our laboratories led to the identification of the synthetic compound UPAR415 ((1S,2S)-1-(4-methylbenzyl)-2-phenylcyclopropanecarboxylic acid, Figure 1a) as the most potent inhibitor developed so far of OASS-A from Salmonella Typhimurium (StOASS-A), with a nanomolar dissociation constant for the target [41,49].…”