[165.2C166.7 C], lit. VIM-1 was first identified in Italy a few years later, in 1997, in a strain. This was followed, soon after, by the isolation of an allelic variant (VIM-2) in France. VIM-1 possesses the broadest range of substrate hydrolysis and can degrade nearly all configuration, which possibly provides the aromatic group with the proper orientation to form closer – interactions with Tyr67. In order to better investigate the binding pose and the binding path of the most promising candidate, 2b, in VIM-1, we performed 15 Molecular Dynamics (MD) docking replicas (20 ns each). Even though the MD docking shed light on the dynamic path the ligand experiences before binding VIM-1, no relevant information was added to the rigid docking results: the width/openness of the binding cavity offered straightforward access to 2b, and hydrophobic interactions with Tyr67 and His240 were principally responsible for stabilising the ligand in the binding site. The MD docking in VIM-1 therefore almost identically reflected the interactions and observations that Ispinesib (SB-715992) had already been reported in rigid docking studies, and hence corroborate the reliability of the static analyses in MBLs. The most probable pose assumed by the ligands in IMP-1 binding site largely resembled the one in VIM-1. Again, the zinc ions are coordinated by the thiolate and by the triazole nitrogen, and the rest of the ligand forms – interactions with Trp28, which replaces Tyr67 in VIM-1 (Figure 1eCh). The best inhibition was obtained for compound 2g, which forms an almost perfect contact with Trp28. 2.3.2. KPC-2 Given the absence of the zinc ions, the compounds were modelled in the thione form when docked in the KPC-2 binding site. In accordance with the architecture that this binding cavity presents, the docking returned a ligand orientation in which the triazole moiety always sinks into the active site, while the substituents introduced at position 4 points towards the opening of the binding site, which is delimited by Trp105 (Figure 1iCl). Compound 1d, the least active, only forms a – interaction with Trp105. On the other hand, compound 1f loses this contact with Trp105, but H-bonds to Asn132 through the triazole nitrogens at positions 1 and 2, and to Thr235 and Thr237 by means of a benzodioxole oxygen. Compounds 2b and 2g also show similar inhibition activity and a similar binding mode. They both form good – interactions with Thr235; with that of 2g, possibly being stronger because of the larger aromatic system. Compound 2b also H-bonds Asn132, similarly to 1f. In general, the similarities of the poses satisfactorily explain the comparable inhibition activity of the four compounds. The H-bonds formed by some of them with the residues lining the pocket open the way for the optimization of these derivatives to provide them with substituents able to interact more extensively within KPC-2 active site. Indeed, while the hydrophobic requirements of the binding site are well met by the compound aromatic regions, the number of polar interactions should be increased to further improve the binding affinity. In particular, compounds could be functionalized to contact Arg220 via a stronger electrostatic interaction. Furthermore, polar substituents could be attached to the aromatic portion to better reach the residues lining the oxyanion hole, that is Thr235 and Thr237. 2.4. Determination of Minimum Inhibitory Concentration (MIC) against Clinical Strains To investigate the ability of the compounds to reach the periplasmic space, where BLs are secreted and Ispinesib (SB-715992) concentrated in Gram-negative bacteria, and to synergically protect -lactam antibiotics from BLs hydrolysis, the minimum inhibitory concentration (MIC) values were determined against clinical strains overexpressing BLs targets of our studies (Table S2). Unfortunately, the obtained Ispinesib (SB-715992) MIC showed no synergistic effect for none of the tested compounds, except for 1a, 1c, 2c and 2g which very slightly potentiated meropenem (MEM) activity. It is important to consider that, in clinical strains, multiple mechanisms of resistance are co-present and employed by bacteria, thus complicating the interpretation of the obtained results. At the same time the necessity to validate designed compounds against their clinical bacterial targets is of critical importance for the further effective selection of candidates for hit-to-lead optimization. 3. Conclusions A library of 14 derivatives has been designed, synthesized and tested, both, in vitro and in biological tests against MBLs and SBLs. The most active compounds showed weak, but cross-class, inhibitory activity against all targeted BLs, confirming the possibility to introduce broad-spectrum activity on recurrent moieties targeting only one class. Starting from a scaffold that is widely recognised to bind to the MBLs active site, extended affinity against SBLs was achieved, although only at micromolar potency. Molecular.E.G. first identified in Italy a few years later, in 1997, in a strain. This was followed, soon after, by the isolation of an allelic variant (VIM-2) in France. VIM-1 possesses the broadest range of substrate hydrolysis and can degrade nearly all configuration, which possibly provides the aromatic group with the proper orientation to form closer – interactions with Tyr67. In order to better investigate the binding pose and the binding path of the most promising candidate, 2b, in VIM-1, we performed 15 Molecular Dynamics (MD) docking replicas (20 ns each). Even though the MD docking shed light on the dynamic path the ligand experiences before binding VIM-1, no relevant information was added to the rigid docking results: the width/openness of the binding cavity offered straightforward access to 2b, and hydrophobic interactions with Tyr67 and His240 were principally responsible for stabilising the ligand in the binding site. The MD docking in VIM-1 therefore almost identically reflected the interactions and observations that had already been reported in rigid docking studies, and hence corroborate the reliability of the static analyses in MBLs. The most probable pose assumed by the ligands in IMP-1 binding site largely resembled the one in VIM-1. Again, the zinc ions are coordinated by the thiolate and by the triazole nitrogen, and the rest of the ligand forms – interactions with Trp28, which replaces Tyr67 in VIM-1 (Figure 1eCh). The best inhibition was obtained for compound 2g, which forms an almost perfect contact with Trp28. 2.3.2. KPC-2 Given the absence of the zinc ions, the compounds were modelled in the thione form when docked in the KPC-2 binding site. In accordance with the architecture that this binding cavity presents, the docking returned a ligand orientation in which the triazole moiety always sinks into the active site, while the substituents introduced at position 4 points towards the opening of the binding site, which is delimited by Trp105 (Figure 1iCl). Compound 1d, the least active, only forms a – interaction with Trp105. On the other hand, compound 1f loses this contact with Trp105, but H-bonds to Asn132 through the triazole nitrogens at positions 1 and 2, and to Thr235 and Thr237 by means of Ispinesib (SB-715992) a benzodioxole oxygen. Compounds 2b and 2g also show similar inhibition activity and a similar binding mode. They both form good – interactions with Thr235; with that of 2g, possibly being stronger because of the larger aromatic system. Compound 2b also H-bonds Asn132, similarly to 1f. In general, the similarities of the poses satisfactorily explain the comparable inhibition activity of the four compounds. The H-bonds formed by some of them with the residues lining the pocket open the way for the optimization of these derivatives to provide them with substituents able to interact more extensively within KPC-2 active site. Indeed, while the hydrophobic requirements of the binding site are well met by the compound aromatic regions, the number of polar interactions should be increased to CD38 further improve the binding affinity. In particular, compounds could be functionalized to contact Arg220 via a stronger electrostatic interaction. Furthermore, polar substituents could be attached to the aromatic portion to better reach the residues lining the oxyanion hole, that is Thr235 and Thr237. 2.4. Determination of Minimum Inhibitory Concentration (MIC) against Clinical Strains To investigate the ability of the compounds to reach the periplasmic space, where BLs are secreted and concentrated in Gram-negative bacteria, and to synergically protect -lactam antibiotics from BLs hydrolysis, the minimum inhibitory concentration (MIC) values were determined against clinical strains overexpressing BLs targets.