Abstract
β-Lactamases are the major reason β-lactam resistance is seen in Gram-negative bacteria. To combat this resistance mechanism, β-lactamase inhibitors are currently being developed. Presently, there are only three that are in clinical use (clavulanate, sulbactam and tazobactam). In order to address this important medical need, we explored a new inhibition strategy that takes advantage of a long-lived inhibitory trans-enamine intermediate. SA2-13 was previously synthesized and shown to have a lower k react than tazobactam. We investigated here the importance of the carboxyl linker length and composition by synthesizing three analogs of SA2-13 (PSR-4-157, PSR-4-155, and PSR-3-226). All SA2-13 analogs yielded higher turnover numbers and k react compared to SA2-13. We next demonstrated using protein crystallography that increasing the linker length by one carbon allowed for better capture of a trans-enamine intermediate; in contrast, this trans-enamine intermediate did not occur when the C2 linker length was decreased by one carbon. If the linker was altered by both shortening it and changing the carboxyl moiety into a neutral amide moiety, the stable trans-enamine intermediate in wt SHV-1 did not form; this intermediate could only be observed when a deacylation deficient E166A variant was studied. We subsequently studied SA2-13 against a relatively recently discovered inhibitor-resistant (IR) variant of SHV-1, SHV K234R. Despite the alteration in the mechanism of resistance due to the K→R change in this variant, SA2-13 was effective at inhibiting this IR enzyme and formed a trans-enamine inhibitory intermediate similar to the intermediate seen in the wt SHV-1 structure. Taken together, our data reveals that the C2 side chain linker length and composition profoundly affect the formation of the trans-enamine intermediate of penam sulfones. We also show that the design of SA2-13 derivatives offers promise against IR SHV β-lactamases that possess the K234R substitution.
Highlights
Bacteria harboring b-lactamase enzymes [E.C. 3.5.2.6] pose a significant threat to public health [1]
Inhibition was not observed for SHV-1 expressing cells by any of the inhibitors including tazobactam; this is likely a result of the high SHV-1 expression levels in the DH10B E. coli strain used
13 and its favorable inhibitory properties of wt SHV-1, we extended our analysis to perform a kinetic and crystallographic characterization of SA2-13-inhibited K234R SHV to aid in the understanding of this class A IR variant
Summary
Bacteria harboring b-lactamase enzymes [E.C. 3.5.2.6] pose a significant threat to public health [1]. These enzymes prevent blactam antibiotics from reaching their intended target, the penicillin binding proteins (PBPs). To overcome this resistance determinant, b-lactamase inhibitors were introduced into the medical pharmacopeia. There are only three blactamase inhibitors clinically available (sulbactam, tazobactam and clavulanic acid, Figure 1). These inhibitors, when administered concomitantly with b-lactams, can aid in the eradication of infection by restoring the potency of the partner ß-lactam. The inhibitors primarily inactivate class A blactamases [2,3]
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