Pharmacomodulations of the benzoyl-thiosemicarbazide scaffold reveal antimicrobial agents targeting d-alanyl-d-alanine ligase in bacterio.

d-Alanyl-d-alanine ligase (Ddl) is a validated and attractive target among the bacterial enzymes involved in peptidoglycan biosynthesis. In the present work, we investigated the pharmacomodulations of the benzoylthiosemicarbazide scaffold to identify new Ddl inhibitors with antibacterial potency. Five novel series of thiosemicarbazide analogues, 1,2,4-thiotriazole-3-thiones, 1,3,4-thiadiazoles, phenylthiosemicarbazones, diacylthiosemicarbazides and thioureas were synthesized via straightforward procedures, then tested against Ddl and on susceptible or resistant bacterial strains. Among these, the thiosemicarbazone and thiotriazole were identified as the most promising scaffolds with Ddl inhibition potency in the micromolar range. Antimicrobial evaluation of salicylaldehyde-4(N)-(3,4-dichlorophenyl) thiosemicarbazone 33, one of the best compounds in our study, revealed interesting antimicrobial activities with values of 3.12-6.25 μM (1.06-2.12 μg/mL) against VRE strains and 12.5-25.0 μM (4.25-8.50 μg/mL) towards MRSA and VRSA strains. A detailed mechanistic study was conducted on the Ddl inhibitors 4-(3,4-dichlorophenyl)-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 20 and compound 33, and revealed a bactericidal effect at 5 × MIC concentration after 7 h and 24 h, respectively, and a bacteriostatic effect at 1 × MIC or 2 × MIC without any sign of bacterial membrane disruption at these lower concentrations. Finally, 20 and 33 were proved to target Ddl in bacterio via intracellular LC-MS dosage of d-Ala, l-Ala and d-Ala-d-Ala. Although, at this stage, our results indicate that other mechanisms might be involved to explain the antimicrobial potency of our compounds, their ability to inhibit the growth of strains resistant to usual antibiotics, as well as strains that express alternative ligases, sets the stage for the development of new antimicrobial agents potentially less sensitive to resistance mechanisms.