Structure-Based Design Guides the Improved Efficacy of Deacylation Transition State Analogue Inhibitors of TEM-1 β-Lactamase^,

Abstract

Transition state analogue boronic acid inhibitors mimicking the structures and interactions of good penicillin substrates for the TEM-1 beta-lactamase of Escherchia coli were designed using graphic analyses based on the enzyme's 1.7 A crystallographic structure. The synthesis of two of these transition state analogues, (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1) and (1R)-1-acetamido-2-(3-carboxy-2-hydroxyphenyl)ethylboronic acid (2), is reported. Kinetic measurements show that, as designed, compounds 1 and 2 are highly effective deacylation transition state analogue inhibitors of TEM-1 beta-lactamase, with inhibition constants of 5.9 and 13 nM, respectively. These values identify them as among the most potent competitive inhibitors yet reported for a beta-lactamase. The best inhibitor of the current series was (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1, K(I) = 5.9 nM), which resembles most closely the best known substrate of TEM-1, benzylpenicillin (penicillin G). The high-resolution crystallographic structures of these two inhibitors covalently bound to TEM-1 are also described. In addition to verifying the design features, these two structures show interesting and unanticipated changes in the active site area, including strong hydrogen bond formation, water displacement, and rearrangement of side chains. The structures provide new insights into the further design of this potent class of beta-lactamase inhibitors.

Novel Insights into the Mode of Inhibition of Class A SHV-1 β-Lactamases Revealed by Boronic Acid Transition State Inhibitors

“…Considering the entropic relationships evident here, it would seem energetically favorable for the ceftazidime BATSI to occupy the deacylation water pocket, as it would release an ordered water molecule, thereby increasing entropy upon ceftazidime BATSI binding (18). Nevertheless, the K i values (Table 1) suggest that SHV-1 is an "outlier" in that it is not as readily inhibited by BATSIs as TEM-1 and AmpC.…”

“…Note that adopting this deacylation transition state conformation does not necessarily indicate weaker inhibition, as the chiral penicillin BATSI analogues, among the most potent BATSIs, are also observed to be in the deacylation transition state conformation (Fig. 4F) (18,24). Furthermore, the potent chiral cephalothin BATSI is observed to be in the deacylation transition state conformation (Fig.…”

“…Utilization of this ␤-lactamase carboxyl binding pocket by BATSIs has led to some of the most potent BATSIs (Fig. 4F) (18,24). Our design goal was in part successful for several reasons.…”

Targeting Antibiotic Resistance

“…The original inspiration for its design comes from a report published in 2000 by Ness and co‐workers in which compound 57 is described 182. This led to the idea of using cyclic boronates as inhibitors.…”

Boronic Acid Transition State Inhibitors Active against KPC and Other Class A β-Lactamases: Structure-Activity Relationships as a Guide to Inhibitor Design

“…In a parallel quest, BATSIs were studied and optimized for a variety of ␤-lactamase enzymes (7,(13)(14)(15)(16)(17)(18). Mechanistically, the boronic inhibitors act by binding to the active site of the enzyme, where they sterically resemble the quaternary transition state of the ␤-lactam hydrolysis reaction and occupy the active site with high affinity, leading to inhibition in a reversible competitive manner (9).…”