The aim of this study was to perform homology modeling of the β-alanyl aminopeptidase sequence of Pseudomonas aeruginosa, and to use the model obtained in the design, synthesis and testing of inhibitors in order to evaluate the cellular role of this enzyme. A 3D model of β-alanyl aminopeptidase was derived and evaluated using Maestro (version 9.3, Schrödinger). Virtual database screening was then conducted in order to discover inhibitors which would be predicted to bind to the active site of the enzyme. Twenty-seven hit compounds with the highest docking scores were synthesized, purified and the in vitro antimicrobial activities for these synthesized agents, against both Gram-negative and Gram-positive strains were evaluated using both disc diffusion and micro-dilution assays. Under disc diffusion assay, N-(2-aminoethyl)benzenesulfonamide trifluoroacetate salt 1a, N-(2-aminoethyl)-4-methylbenzenesulfonamide trifluoroacetate salt 1b, N-(2-aminoethyl)-4-chlorophenylsulfonamide trifluoroacetate 1c, N-(2-aminoethyl)-4-nitrophenylsulfonamide trifluoroacetate 1d, N-(2-aminoethyl)-2-nitrophenylsulfonamide trifluoroacetate 1e, and 3-amino-N-{2-(2-nitrophenylsulfonamido)ethyl}propanamide trifluoroacetate 8e showed the greatest anti-pseudomonal activity. Based on disc diffusion assay, against Gram positive and Gram negative strains, 3-Amino-N-{2-(2-nitrophenylsulfonamido)ethyl}propanamide trifluoroacetate 8e had the greatest selectivity, followed by N-(2-aminoethyl)-4-chlorophenylsulfonamide trifluoroacetate 1c and N-(2-aminoethyl)-4-methylbenzenesulfonamide trifluoroacetate salt 1b. Additionally, compound N-(2-aminoethyl)-4-nitrophenylsulfonamide trifluoroacetate 1d showed the greatest anti-pseudomonal effeact, with an MIC of 78.125 µg/ml. This effect was not specific as it also had the greatest effect against B. subtilis and P. vulgaris, with an MIC of 156.25 µg/ml for both species. These sulfonamides (and their β-alanyl derivatives) represent new leads in the search for antimicrobial agents for the treatment of P. aeruginosa. Although compounds 8e, 1c, and 1b have selective effects upon PAO1, their anti-pseudomonal activities are not sufficient for the development of potent anti-pseudomonal agents, suggesting that β-alanyl aminopeptidase has no physiological role in the growth of Pseudomonas aeruginosa. The results obtained in this work do, however, support further work to enhance the activity of these non-classical sulphonamides, as well as proteomics and protein purification in order to isolate and study β-alanyl aminopeptidase crystallographicall
