Active-Site Druggability of Carbapenemases and Broad-Spectrum Inhibitor Discovery

Abstract

Serine and metallo-carbapenemases are a serious health concern due to their capability to hydrolyze nearly all β-lactam antibiotics. However, the molecular basis for their unique broad-spectrum substrate profile is poorly understood, particularly for serine carbapenemases, such as KPC-2. Using substrates and newly identified small molecules, we compared the ligand binding properties of KPC-2 with the noncarbapenemase CTX-M-14, both of which are Class A βlactamases with highly similar active sites. Notably, compared to CTX-M-14, KPC-2 was more potently inhibited by hydrolyzed β-lactam products (product inhibition), as well as by a series of novel tetrazole-based inhibitors selected from molecular docking against CTX-M-14. Together with complex crystal structures, these data suggest that the KPC-2 active site has an enhanced ability to form favorable interactions with substrates and small molecule ligands due to its increased hydrophobicity and flexibility. Such properties are even more pronounced in metallo-carbapenemases, such as NDM-1, which was also inhibited by some of the novel tetrazole compounds, including one displaying comparable low μM affinities against both KPC-2 and NDM-1. Our results suggest that carbapenemase activity confers an evolutionary advantage on producers via a broad β-lactam substrate scope but also a mechanistic Achilles' heel that can be exploited for new inhibitor discovery. The complex structures demonstrate, for the first time, how noncovalent inhibitors can be engineered to simultaneously target both serine and metallo-carbapenemases. Despite the relatively modest activity of the current compounds, these studies also demonstrate that hydrolyzed products and tetrazole-based chemotypes can provide valuable starting points for broad-spectrum inhibitor discovery against carbapenemases.

Discovery of dual-activity small-molecule ligands of Pseudomonas aeruginosa LpxA and LpxD using SPR and X-ray crystallography

“…We included these compounds as positive results because their binding kinetics were distinct from non-binders, and because, as described below, crystal structures suggested that they were likely binders. To further exclude the possibility of non-specific protein binding caused by small molecule aggregation 33,34 , we analyzed compounds 1 and 2 against two unrelated enzymes, CTX-M-14 and KPC-2 Class A β-lactamase 35,36 , in a nitrocefin-hydrolysis assay using the SPR buffer. The compounds showed no inhibition of CTX-M-14 or KPC-2 even when tested at 3 mM concentrations, suggesting that the binding to LpxA and LpxD was specific.…”

“…CTX-M-14 and KPC-2 were purified as previously described 35,36,44 . The hydrolytic activity of CTX-M-14 and KPC-2 was determined using the β-lactamase substrate nitrocefin in a PBS-P+ buffer containing 200 mM phosphate buffer pH 7.4, 27 mM potassium chloride and 1.37 M sodium chloride, 0.5% (v/v) surfactant P20 supplemented with 2% DMSO.…”

A Lysine-Targeted Affinity Label for Serine-β-Lactamase Also Covalently Modifies New Delhi Metallo-β-lactamase-1 (NDM-1)

“…Previous design efforts to develop wide-spectrum β-lactamase inhibitors have also relied on the shared function of these divergent enzymes to bind β-lactams, but have not previously targeted Lys for covalent modification. [42][43][44][45][46][47][48][49] This particular Lys residue is conserved in most other B1-subclass metallo-β-lactamases (e.g. NDM, IMP, CcrA, BcII, SPM), but not in VIMs, which have a more distant Arg residue that serves an analogous function.…”

4-Amino-1,2,4-triazole-3-thione as a Promising Scaffold for the Inhibition of Serine and Metallo-β-Lactamases

“…Conversely, in KPC-2, like in all SBLs, the process involves the catalytic Ser70, which performs a nucleophilic attack on the β-lactam core. The β−lactam substrate is further stabilized in the active site by an extensive network of interactions [22][23][24]. The first inhibitor active against both SBLs and MBLs classes has, only recently, reached clinical trials [19,20].…”