Structure-based design guides the improved efficacy of deacylation transition state analogue inhibitors of TEM-1 beta-lactamase

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 enzym e's 1.7 Angstrom crystallographic structure. The synthesis of two of these transition state analogues, (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethlb oronic acid (1) and (1R)-1-acetamido2-(3-carboxy-2-hydroxyphenyl) ethylboro nic acid (2), is reported. Kinetic measurements show that, as designed, com pounds 1 and 2 are highly effective deacylation transition state analogue i nhibitors of TEM-1 beta-lactamase, with inhibition constants of 5.9 and 13 nM, respectively. These values identify them as among the most potent compe titive inhibitors yet reported for a beta-lactamase. The best inhibitor of the current series was (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboron ic acid (1, k(1) = 5.9 nM), which resembles most closely the best known sub strate of TEM-1, benzylpenicillin (penicillin G). The high-resolution cryst allographic structures of these two inhibitors covalently bound to TEM-1 ar e also described. In addition to verifying the design features, these two s tructures show interesting and unanticipated changes in the active site are a, including strong hydrogen bond formation, water displacement, and rearra ngement of side chains. The structures provide new insights into the furthe r design of this patent class of beta-lactamase inhibitors.