Inhibitor resistance in the KPC-2 beta-lactamase, a preeminent property of this class A beta-lactamase

Abstract

As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current beta-lactamase inhibitors.

FIG. 1.

Chemical structures of the classical β-lactamase inhibitors, the novel penem β-lactamase inhibitors, cefotaxime, and imipenem.

FIG. 2.

(A) UVD spectra for 25 μM penem 1, a mixture of 25 μM penem 1 and 0.25 μM KPC-2, and a mixture of 25 μM penem 1 and 50 mM NaOH after 50 s of incubation. (B) UVD spectra for a mixture of 25 μM penem 1 and 0.25 μM KPC-2 monitored over periods of 5, 25, and 50 s. (C) Penem 1 hydrolysis by KPC-2 monitored at A₂₉₀ using ratios of 125:1 (25 μM penem 1 and 200 nM KPC-2), 500:1 (25 μM penem 1 and 50 nM KPC-2), and 1,000:1 (25 μM penem 1 and 25 nM KPC-2). (D) Hydrolysis of NCF (100 μM) by 1.0 μM KPC-2 after inhibition by 1.0 mM penem 1 at a 1,000:1 inhibitor-to-enzyme ratio during 24 h of incubation at room temperature compared to hydrolysis by KPC-2 alone.

FIG. 3.

Superimposition of molecular representations of penem 1 (gray-orange) and penem 2 (gray-purple) within the KPC-2 active site. Atom colors: N (blue), O (red), S (green in penem 1, yellow in penem 2).