Inhibition of metallo-beta-lactamase by rational and combinatorial approaches



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Texas Tech University


The inhibition of the Bacillus cereus 5/B/6 metallo-P-lactamase, an enzyme that catalyzes the hydrolysis of neariy all P-lactam antibiotics, has been explored by rational and combinatorial approaches.

Inactivation of the metallo-P-lactamase activity by sodium 7- (dibromomethylene)-cephalosporanate and sodium 3-acetoxymethyl-7-(Ebromomethylidene)-3-cephen-4-carboxylate was time-dependent. The rate constants of inactivation (k.nact) were 0.20 and 0.25 min"', respectively. The values of Kmact were found to be 4.7 and 9.0 mM, respectively.

The penicillin derivatives sodium 6-(R- thiolmethyl) penicillate and sodium 6-(Sthiolmethyl) penicillate were reversible, noncompetitive inhibitors of the metallo-P lactamase. The IC50 (inhibitor concentration for 50 % inhibition), K, (dissociation constant for the enzyme-inhibitor complex), and K,' (dissociation constant for inhibitor from enzyme-substrate-inhibitor complex) values were 8.3, 4.2 and 11 |j.M, respectively for sodium 6-(R-thiolmethyl) penicillate and 18, 8.6 and 23 fiM, respectively for the Sisomer. Single-stranded DNA aptamers were developed using the SELEX (systematic evolution of ligands by exponential enrichment), combinatorial chemistry technology. A 61-mer containing a random 30-mer (comprising over lO'^ sequences) was synthesized. After twenty-one SELEX rounds, one sequence was found. This oligonucleotide synthesized. This 30-mer was a on competitive inhibitor of the metallo-P-lactamase with an IC50 of 1.2 nM, a K, of 0.92 nM and a K,' of 11 nM. Using the MFold program, a unique stem-loop secondary structure was predicted for a 10-nucleotide portion of the 30- mer. When this 10-mer was synthesized, it also was also found to be a noncompetitive inhibitor of the metallo-P-lactamase. The ICso was the same as that of the 30-mer; however, the K, (0.31 nM) and K,' (1.5 nM) were lower than the 30-mer. An 1 8- mer corresponding to the remainder of the 30-mer sequence did not show detectable inhibition. Both the 30-mer and 10-mer had no effect on the activity of the B. cereus 569/H/9 Plactamase 1 or bo\ ine carboxypeptidase A, indicating that the inhibition was specific for the metallo-P-lactamase. The rational drug design approach using chemical synthesis to produce new metallo-P-lactamase inhibitors was successful. The combinatorial chemistry approach (SELEX) was also successful and led to an entirely new class of highly efficient and specific metallo-P-lactamase inhibitors.



Beta lactam antibiotics, Penicillin, Organometallic chemistry