New ß-phospholactam as a carbapenem transition state analog: Synthesis of a broad-spectrum inhibitor of metallo-ß-lactamases.

In an effort to test whether a transition state analog is an inhibitor of the metallo-ß-lactamases, a phospholactam analog of carbapenem has been synthesized and characterized. The phospholactam 1 proved to be a weak, time-dependent inhibitor of IMP-1 (70%), CcrA (70%), L1 (70%), NDM-1 (53%), and Bla2 (94%) at an inhibitor concentration of 100µM. The phospholactam 1 activated ImiS and BcII at the same concentration. Docking studies were used to explain binding and to offer suggestions for modifications to the phospholactam scaffold to improve binding affinities.

Binding of 2-(Triazolylthio)acetamides to Metallo-ß-lactamase CcrA Determined with NMR.

Metallo-ß-lactamase (MBL)-producing bacteria resistant to ß-lactam antibiotics are a serious threat to human health. Despite great efforts and important progress in the discovery of MBL inhibitors (MBLIs), there is none in clinical use. Herein, inhibitor complexes of the MBL CcrA were investigated by NMR spectroscopy to provide perspectives on the further development of 2-(triazolylthio)acetamide-type MBLIs. By using the NMR-based chemical shift perturbation (CSP) and direction of CSP methodologies together with molecular docking, the spatial orientation of three compounds in the CcrA active site was investigated (4-6). Inhibitor 6 showed the best binding affinity (K d ≈ 2.3 ± 0.3 µM), followed by 4 (K d = 11 ± 11 µM) and 5 (K d = 34 ± 43 µM), as determined from the experimental NMR data. Based on the acquired knowledge, analogues of other MBLIs (1-3) were designed and evaluated in silico with the purpose of examining a strategy for promoting their interactions with the catalytic zinc ions.

The structure of the metallo-ß-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor.

The increasing number of pathogens expressing metallo-ß-lactamases (MBLs), and in this way achieving resistance to ß-lactam antibiotics, is a significant threat to global public health. A promising strategy to treat such resistant pathogens is the co-administration of MBL inhibitors together with ß-lactam antibiotics. However, an MBL inhibitor suitable for clinical use has not yet been identified. Verona integron-encoded metallo-ß-lactamase 2 (VIM-2) is a widespread MBL with a broad substrate spectrum and hence is an interesting drug target for the treatment of ß-lactam-resistant infections. In this study, three triazolylthioacetamides were tested as inhibitors of VIM-2. One of the tested compounds showed clear inhibition of VIM-2, with an IC50 of 20 µM. The crystal structure of the inhibitor in complex with VIM-2 was obtained by DMSO-free co-crystallization and was solved at a resolution of 1.50 Å. To our knowledge, this is the first structure of a triazolylthioacetamide inhibitor in complex with an MBL. Analysis of the structure shows that the inhibitor binds to the two zinc ions in the active site of VIM-2 and revealed detailed information on the interactions involved. Furthermore, the crystal structure showed that binding of the inhibitor induced a conformational change of the conserved residue Trp87.

Azolylthioacetamide: A Highly Promising Scaffold for the Development of Metallo-ß-lactamase Inhibitors.

A new scaffold, azolylthioacetamide, was constructed and assayed against metallo-ß-lactamases (MßLs). The obtained molecules specifically inhibited MßL ImiS, and 1c was found to be the most potent inhibitor, with a K i = 1.2 µM using imipenem as substrate. Structure-activity relationships reveal that the aromatic carboxyl improves inhibitory activity of the inhibitors, but the aliphatic carboxyl does not. Compounds 1c-d and 1h-i showed the best antibacterial activities against E. coli BL21(DE3) cells producing CcrA or ImiS, resulting in 32- and 8-fold reduction in MIC values, respectively; 1c and 1f-j resulted in a reduction in MIC against P. aeruginosa. Docking studies revealed that 1a, 1c, and 1d fit tightly into the substrate binding site of CphA as a proxy for ImiS with the aromatic carboxylate forming interactions with Lys224, the Zn(II) ion, the backbone of Asn233, and hydrophobic portions of the inhibitors aligning with hydrophobic patches of the protein surface.