Design, synthesis and evaluation of D-alanyl-D-alanine ligase inhibitors : an attractive target for the development of antimicrobial agents

As the phenomenon of antibiotic resistance is dramatically increasing these days, the search for new therapeutic targets less vulnerable to resistance appears as a real need. The cell wall of bacteria and the enzymes involved in its synthesis are prime targets for many clinically important antibiotics, which inhibit the late stages of peptidoglycan biosynthetic pathway. However, the resistance phenomena have revealed the need to target other steps of peptidoglycan synthesis. D-alanyl-D-alanine ligase (Ddl) is of particular interest as it utilizes a substrate, D-alanine, which is specific for peptidoglycan biosynthesis and essential for bacterial growth. Our work focused on the development of Ddl inhibitors in order to target resistant strains of bacteria. The synthesis of benzoylthiosemicarbazides and their pharmacomodulations allowed identifying potent Ddl inhibitors with activities in the low micromolar range and good antimicrobial potencies, including against multidrug resistant strains. The in bacterio target was then determined by UPLC-MS dosage of intracellular peptidoglycan precursors levels in response to the three most promising derivatives of our study. An increase in D-Ala pools accompanied by a decrease in D-Ala-D-Ala were observed, thus confirming that these compounds exert, at least in part, their antibacterial activity through Ddl inhibition. This work thus highlights very promising tools for the development of novel antibacterial agents acting through an original mechanism of action.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 202

1-(2-Hydroxybenzoyl)-thiosemicarbazides are promising antimicrobial agents targeting d-alanine-d-alanine ligase in bacterio

The bacterial cell wall and the enzymes involved in peptidoglycan synthesis are privileged targets for the development of novel antibacterial agents. In this work, a series of 1-(2-hydroxybenzoyl)-thiosemicarbazides inhibitors of D-Ala-D-Ala ligase (Ddl) were designed and synthesized in order to target resistant strains of bacteria. Among these, the 4-(3,4-dichlorophenyl)-1-(2-hydroxybenzoyl)-3-thiosemicarbazide 29 was identified as a potent Ddl inhibitor with activity in the micromolar range. This compound, possessing strong antimicrobial activity including against multidrug resistant strains, was proven to act through a bactericidal mechanism and demonstrated very low cytotoxicity on THP-1 human monocytic cell line. Inhibition of Ddl activity by 29 was confirmed in bacterio using UPLC-MS/MS by demonstrating an increase in D-Ala intracellular pools accompanied by a commensurate decrease in D-Ala-D-Ala. Further structure-activity relationships (SARs) studies provided evidence that the hydroxyl substituent in the 2-position (R1) of the benzoylthiosemicarbazide scaffold is essential for the enzymatic inhibition. This work thus highlights the 1-(2-hydroxybenzoyl)-thiosemicarbazide motif as a very promising tool for the development of novel antibacterial compounds acting through an interesting mechanism of action and low cytotoxicity

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 to 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 5xMIC concentration after 7h and 24h, 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

CCDC 1980940: Experimental Crystal Structure Determination

Related Article: Alice Ameryckx, Lionel Pochet, Gang Wang, Esra Yildiz, Bouazza Es Saadi, Johan Wouters, Françoise Van Bambeke, Raphaël Frédérick|2020|Eur.J.Med.Chem.|200|112444|doi:10.1016/j.ejmech.2020.11244

CCDC 1980941: Experimental Crystal Structure Determination

Related Article: Alice Ameryckx, Lionel Pochet, Gang Wang, Esra Yildiz, Bouazza Es Saadi, Johan Wouters, Françoise Van Bambeke, Raphaël Frédérick|2020|Eur.J.Med.Chem.|200|112444|doi:10.1016/j.ejmech.2020.11244