Résumé
Introduction et objectifsMetallo-β-lactamases (MBLs) are increasingly involved as a major mechanism of resistance to carbapenems in relevant opportunistic Gram-negative pathogens. Unfortunately, clinically efficient MBL inhibitors still represent an unmet medical need. We are developing compounds containing a 1,2,4-triazole-3-thione scaffold as an original zinc ligand and few promising series were already reported [1-4]. Here, a new series characterized by a diversely substituted amino acid moiety is presented. The primary objectives were to obtain compounds with a broader inhibition spectrum and higher synergistic activity in combination with meropenem in microbiological assays.Matériels et méthodes.More than 40 compounds were synthesized following known protocols. They were purified by HPLC and characterized by mass spectrometry (MS) and NMR. Their purity was above 95%. Their inhibitory potency was measured on recombinant clinically-relevant MBLs (VIM-1, -2, -4, NDM-1, IMP-1) in 96-wells plates. The inhibition mode of best compounds was assessed on VIM-2 and NDM-1 using equilibrium dialysis, native MS, isothermal calorimetry (ITC) and X-ray crystallography. The antibacterial synergistic activity of best compounds (32 g/mL) combined to meropenem was assessed on several clinical MBL-producing multidrug-resistant isolates (K. pneumoniae, E. coli, P. aeruginosa) using a broth microdilution method. Several experiments were perfomed to study the drug-like properties of selected compounds: cytotoxicity (HeLa cells), ADME profile, selectivity toward human metallo-enzymes, pharmacokinetic studies in rats. Finally, a preliminary in vivo evaluation was performed on a murine model of bacterial pneumonia.Résultats, discussion et conclusionSeveral compounds showed broad-spectrum activity with submicromolar to nanomolar inhibitory potencies against most relevant MBLs (i.e. VIMs and NDM-1). In addition, they fully restored the activity of meropenem against both VIM- and NDM-1-producing clinical isolates (e.g. up to 1000-fold reduction of meropenem MIC on a NDM-1-producing E. coli). The study of structure-activity relationships pointed the determining role of certain di-heteroaryl-substituted amino acid moieties in both NDM-1 inhibition and potentializing activity. Equilibrium dialysis, native MS, ITC and X-ray crystallography showed that the best compounds at least partly inhibited MBLs by zinc stripping. Finally, their low cytotoxicity, favourable ADME properties and selectivity against mammalian metallo-enzymes, and preliminary in vivo experiments further supported the promising potential of these compounds.