A specific peptide inhibitor of the class B metallo-beta-lactamase L-1 from Stenotrophomonas maltophilia identified using phage display.
OBJECTIVE
In Gram-negative bacteria, resistance to beta-lactam antibiotics and to known inhibitors mediated by metallo-beta-lactamases is a major concern and a serious threat to public health. Since no clinically useful inhibitors are available against class B metallo-beta-lactamases, the aim of the study was to identify peptides as inhibitors.


METHODS
The L-1 metalloenzyme from Stenotrophomonas maltophilia was cloned, over-expressed, purified to homogeneity and used in screening of peptide libraries by phage display with a selective and competitive biopanning assay. This was based upon the high affinity of L-1 for cefoxitin and its slow hydrolysis.


RESULTS
From six peptides, the consensus sequence Cys-Val-His-Ser-Pro-Asn-Arg-Glu-Cys was identified as a promising inhibitor of L-1 hydrolytic activity. This peptide showed a mixed inhibition of L-1 with a K(i competitive) of 16 +/- 4 microM and a K(i uncompetitive) of 9 +/- 1 microM. The same peptide was prepared without flanking Cys residues and demonstrated no detectable inhibition of L-1 hydrolytic activity with nitrocefin as a substrate. These data confirmed the importance of the peptide conformation for the inhibition of L-1 hydrolytic activity. Further analysis revealed rescue by Zn2+ ions. The mixed inhibition indicated peptide binding near the active site of L-1 and blocking of zinc atoms for optimal conformation in the pocket of the active site.


CONCLUSIONS
This is the first report of a peptide inhibitor for Class B metallo-beta-lactamases. It will be used as a lead to identify more potent small molecule inhibitors via peptidomimetics.
Sanschagrin
François
F
Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4.
Levesque
Roger C
RC
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019151:Peptide Library; D020539:Sequence Analysis, Protein / Q000379:methods; D020615:Stenotrophomonas maltophilia / Q000201:enzymology*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D019151:Peptide Library; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; C496277:metallo-beta-lactamase L1, Stenotrophomonas maltophilia