Inhibition of Nonessential Bacterial Targets: Discovery of a Novel Serine O-Acetyltransferase Inhibitor

Abstract

In ϒ-proteobacteria and Actinomycetales, cysteine biosynthetic enzymes are indispensable during persistence and become dispensable during growth or acute infection. The biosynthetic machinery required to convert inorganic sulfur into cysteine is absent in mammals; therefore, it is a suitable drug target. We searched for inhibitors of Salmonella serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of l-cysteine biosynthesis. The virtual screening of three ChemDiv focused libraries containing 91 243 compounds was performed to identify potential SAT inhibitors. Scaffold similarity and the analysis of the overall physicochemical properties allowed the selection of 73 compounds that were purchased and evaluated on the recombinant enzyme. Six compounds displaying an IC50 <100 μM were identified via an indirect assay using Ellman’s reagent and then tested on a Gram-negative model organism, with one of them being able to interfere with bacterial growth via SAT inhibition.

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

“…Since the primary structure of SAT is highly conserved among bacteria, [ 38 ] the virtual screening of our in-house library was performed using the available crystal structures of Ec SAT (pdb code: 1T3D) and Hi SAT (pdb code: 1SSM), setting the experimental parameters as previously reported by us [ 37 ]. After this preliminary analysis, a total of 7 compounds were identified and selected to be evaluated on the recombinant enzyme from S. typhimurium .…”

“…After this preliminary analysis, a total of 7 compounds were identified and selected to be evaluated on the recombinant enzyme from S. typhimurium . The enzyme used in these assays has a K m ,Ser = 1.07 ± 0.15 mM, a K m,AcCoA = 0.17 ± 0.04 mM, k cat = 3813 ± 169 min −1 [ 37 ]. The assay conditions for the initial screening were set to improve the sensitivity of the assay towards both competitive and uncompetitive inhibitors, i.e., the concentration of both substrates was equal to K m .…”

“…SAT from Salmonella enterica serovar Typhimurium was expressed as trxA fusion protein as previously described [ 37 ] and was more than 95% pure based on SDS-PAGE analysis. The enzyme activity was measured by a spectrophotometric indirect assay that exploits the signal at 412 nm generated by 5,5’-Dithiobis(2-nitrobenzoic acid (Ellmn’s reagent) reduction by the reaction product coenzyme A [ 37 ]. A total of 5% DMSO was present in all the assays and was proven not to perturb enzyme activity (data not shown).…”

“…From the structural point of view, SAT is a dimer of trimers, with an α-helical N-terminal domain and a carboxyl-terminal left-handed α-helix domain ending with a flexible, disordered C-terminal sequence that binds OASS-A at the active site and competitively inhibits it [ 35 , 36 ]. Our first attempt to deliver an SAT inhibitor relied on the virtual screening of a focused commercial library of chemical compounds [ 37 ]. This strategy was inspired by previous works where similar approaches were carried out obtaining encouraging results [ 27 , 33 ].…”

“…This strategy was inspired by previous works where similar approaches were carried out obtaining encouraging results [ 27 , 33 ]. In our first attempt, the virtual screening of three ChemDiv focused libraries containing 91,243 compounds was performed to identify six compounds displaying an IC 50 < 100 μM via an indirect assay using Ellman’s reagent [ 37 ]. In addition, one of the compounds was found to have measurable growth inhibitory effects against E. coli .…”