Investigational Studies on a Hit Compound Cyclopropane–Carboxylic Acid Derivative Targeting O-Acetylserine Sulfhydrylase as a Colistin Adjuvant

Abstract

Antibacterial adjuvants are of great significance, since they allow the therapeutic dose of conventional antibiotics to be lowered and reduce the insurgence of antibiotic resistance. Herein, we report that an O-acetylserine sulfhydrylase (OASS) inhibitor can be used as a colistin adjuvant to treat infections caused by Gram-positive and Gram-negative pathogens. A compound that binds OASS with a nM dissociation constant was tested as an adjuvant of colistin against six critical pathogens responsible for infections spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, Klebisiella pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus pseudintermedius. The compound showed promising synergistic or additive activities against all of them. Knockout experiments confirmed the intracellular target engagement supporting the proposed mechanism of action. Moreover, compound toxicity was evaluated by means of its hemolytic activity against sheep defibrinated blood cells, showing a good safety profile. The 3D structure of the compound in complex with OASS was determined at 1.2 Å resolution by macromolecular crystallography, providing for the first time structural insights about the nature of the interaction between the enzyme and this class of competitive inhibitors. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants and the structural basis for further structure−activity relationship studies.

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

“…Microbiologic assays demonstrated a synergic and/or additive action of the compound with colistin, a last-line polycationic antibiotic, in several bacterial strains prone to the insurgence of antibiotic resistance. Target engagement experiments on S. Typhimurium cultures and cytotoxicity assays on eukaryotic cells, moreover, determined the inhibitor to be specific and nontoxic [49]. These data support the viability of the pursued approach and represent a good starting point for the development of a new class of antimicrobial compounds, targeting the enzymes of the sulfur reductive assimilation pathway.…”

“…UPAR415, identified after several rounds of modification and selection of substituted cyclopropanes, is inspired to the C-terminal isoleucine of SAT, an extremely well-conserved residue responsible for CS anchorage [24,50]; because of its structure, this compound might destabilize the CS complex by competing with SAT tails for the occupation of OASS-A active site. Very recently, the pose of UPAR415 bound to StOASS-A was confirmed through crystallization studies (Figure 1, Protein Data Bank (PDB) id 6z4n, [49]). With this in mind, we decided to extend the mechanistic characterization of UPAR415 to a broader biochemical context, involving the engagement of OASS-A in the CS complex.…”

“…The structure of StOASS-A in complex with UPAR415 has been recently solved. The molecule accommodates in the enzyme active site and occupies, through its carboxylate moiety, the anchoring subsite of the C-terminal tail of SAT (Figure 1b, [16,49]). As demonstrated by the superimposition with the structure of H. influenzae OASS-A (HiOASS-A) bound to the SAT C-terminal decapeptide (PDB id 1y7l, [16]) or pentapeptides [36], the pose of UPAR415 mimics that of the C-terminal isoleucine residue.…”

“…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]. Microbiologic assays demonstrated a synergic and/or additive action of the compound with colistin, a last-line polycationic antibiotic, in several bacterial strains prone to the insurgence of antibiotic resistance.…”

“…(a) UPAR415 ((1S,2S)-1-(4-methylbenzyl)-2-phenylcyclopropanecarboxylic acid)[41]. (b) superimposition of an StOASS-A monomer (light cyan) in complex with UPAR415 (magenta, PDB id 6z4n,[49]) and an OASS-A monomer from H. influenzae (light pink) in complex with the NLNI tetrapeptide (violet, PDB id 1y7l,[16]), representing the C-terminal sequence of SAT. UPAR415 is inspired to the C-terminal isoleucine residue, which is key for SAT binding to OASS-A and is conserved in the SAT sequences of S. Typhimurium, E. coli, and H. influenzae.…”