DESIGN, SYNTHESIS, AND EVALUATION OF A POTENT MECHANISM-BASED INHIBITOR FOR THE TEM BETA-LACTAMASE WITH IMPLICATIONS FOR THE ENZYME MECHANISM

The design, synthesis, and evaluation of 6 alpha-(hydroxymethyl)penicillanic acid (4) as a new mechanism-based inhibitor for the class A TEM-1 beta-lactamase is described. The design of this compound was aided by computer modeling, using the high-resolution crystal structure for the TEM-1 beta-lactamase. The molecule is believed to displace the hydrolytic water molecule (Wat-712) with its hydroxymethyl function. This interaction would impart longevity to the acyl-enzyme intermediate, accounting for the onset of inhibition, a process that results in an 11-h period for recovery of 90% of activity for the inhibited enzymes. This molecule inhibited the TEM-1 beta-lactamase rapidly, for which the kinetic parameters were evaluate. The molecule showed a partition ratio (i.e., k(cat)/k(inact)) of 28+/-2, a value which is lower than the corresponding parameter for all of the clinically used class A beta-lactamase inactivators. The design concepts outlined for 6 alpha-(hydroxymethyl)penicillanic acid as an inhibitor support the mechanistic roles proposed for Glu-166 and Wat-712 in the deacylation step for turnover chemistry by class A beta-lactamases. Furthermore, the principles that we disclose herein for the design of compound 4 as an inhibitor for beta-lactamases should be of general interest for designing specific inhibitors for any hydrolytic enzyme for which high-resolution crystal structure is available.