title: Kinetic and Structural Characterization of the Interaction of 6-Methylidene Penem 2 with the β-Lactamase from Mycobacterium tuberculosis.
abstract: Mycobacterium tuberculosis is intrinsically resistant to most β-lactam antibiotics because of the constitutive expression of the blaC-encoded β-lactamase. This enzyme has extremely high activity against penicillins and cephalosporins, but weaker activity against carbapenems. The enzyme can be inhibited by clavulanate, avibactam, and boronic acids. In this study, we investigated the ability of 6-methylidene β-lactams to inhibit BlaC. One such compound, penem 2, inhibited BlaC more than 70 times more efficiently than clavulanate. The compound forms a covalent complex with BlaC as shown by mass spectrometry. Crystallization of the complex revealed that the bound inhibitor was covalently attached via the Ser70 active site residue and that the covalently, acylated form of the inhibitor had undergone additional chemistry yielding a 4,7-thiazepine ring in place of the β-lactam and a thiazapyroline ring generated as a result of β-lactam ring opening. The stereochemistry of the product of the 7-endo-trig cyclization was the opposite of that observed previously for class A and D β-lactamases. Addition of penem 2 greatly synergized the antibacterial properties of both ampicillin and meropenem against a growing culture of M. tuberculosis. Strikingly, penem 2 alone showed significant growth inhibition, suggesting that in addition to its capability of efficiently inhibiting BlaC, it also inhibited the peptidoglycan cross-linking transpeptidases.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology, Roorkee , Roorkee, Uttarakhand 247667, India.
authors.forename: Saugata
authors.initials: S
authors.lastname: Hazra
authors.affiliation: Tufts University School of Medicine , 800 Washington Street, #257, Boston, Massachusetts 02111, United States.
authors.forename: Sebastian G
authors.initials: SG
authors.lastname: Kurz
authors.affiliation: Early Discovery, Infectious Diseases Antibacterial & Antifungal, Merck Research Laboratories , Kenilworth, New Jersey 07033, United States.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Wolff
authors.forename: Liem
authors.initials: L
authors.lastname: Nguyen
authors.affiliation: Department of Medicine, Louis Stokes Cleveland Veterans Affairs Medical Center , Cleveland, Ohio 44106, United States.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: John S
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D000667:Ampicillin / Q000737:chemistry / Q000494:pharmacology; D000995:Antitubercular Agents / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry*; D002511:Cephalosporins / Q000737:chemistry* / Q000494:pharmacology; D004357:Drug Synergism; D007700:Kinetics; D000077731:Meropenem; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D013845:Thienamycins / Q000737:chemistry / Q000494:pharmacology; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D002511:Cephalosporins; D013845:Thienamycins; D000667:Ampicillin; D001618:beta-Lactamases; D000077731:Meropenem