Substrate and Inhibitor Interactions of Class A b-Lactamases

Beta lactams are the most widely prescribed antibacterial drugs, and are of particular value against infections by Gram-negative bacteria, where few alternatives exist. In these organisms b-lactamase production is the main resistance mechanism. While these enzymes are many and varied, several have been structurally and biochemically characterised, making b-lactamases both relevant to human (and animal) health and suitable model systems for developing computational methods to study enzyme-catalysed reactions.

Carbapenems are the newest, most potent b-lactams and key antibiotics for severe bacterial infections. While most b-lactamases are inhibited by carbapenems, enzymes that efficiently hydrolyze these substrates (carbapenemases) are disseminating. The molecular basis for this expansion of activity remains obscure, and the activity of carbapenemases against other b-lactam classes is variable. We have previously (J Am Chem Soc 134 18275 (2012); Chem Comm, 50, 14736 (2014)) used structural and computational (molecular dynamics (MD) and quantum mechanical (QM)) approaches to study interaction of b-lactamases with carbapenems with the aim of both understanding the basis for this variation in activity and developing methodologies for predicting the activity of uncharacterised systems. We now extend this work to i) investigate, experimentally and in silico, interactions of carbapenemases and comparator b-lactamases with b-lactams of different classes, ii) study how carbapenemase activity against different b-lactams may be affected by point mutation, and iii) investigate b-lactamase interactions with candidate inhibitors.

Experimental work has encompassed crystallographic structure determination for both enzyme:ligand (b-lactam substrate and inhibitor) complexes and mutated enzyme variants; complemented with steady state kinetic measurements of their reactions with substrates and inhibitors. These data provide the first structural information revealing how KPC, the carbapenemase most relevant to human infections, interacts with different b-lactam classes, including carbapenems, and with mechanism-based inhibitors. These findings also indicate that KPC variants differing in their activity towards specific b-lactam substrates also vary in their susceptibility to inhibitors, identifying potential for evasion of inhibitor activity through accumulation of point mutations. The project also applied state-of-the-art serial femtosecond crystallographic (SFX) approaches in efforts to characterise short-lived species on the b-lactamase reaction pathway. b-lactamase complexes were studied computationally using MD and QM/molecular mechanics (QM/MM) approaches to simulate the deacylation stages of the reaction, extending our in silico characterisations of carbapenemases to deacylation of non-carbapenem substrates (cephalosporins). Our findings identify how combinations of specific interactions and structural/dynamic reorganisation on ligand binding control b-lactamase interactions with ligands and hence activity towards substrates and inhibitors.