title: Overcoming resistance to beta-lactamase inhibitors: comparing sulbactam to novel inhibitors against clavulanate resistant SHV enzymes with substitutions at Ambler position 244.
abstract: Amino acid changes at Ambler position R244 in class A TEM and SHV beta-lactamases confer resistance to ampicillin/clavulanate, a beta-lactam/beta-lactamase inhibitor combination used to treat serious infections. To gain a deeper understanding of this resistance phenotype, we investigated the activities of sulbactam and two novel penem beta-lactamase inhibitors with sp2 hybridized C3 carboxylates and bicyclic R1 side chains against a library of SHV beta-lactamase variants at the 244 position. Compared to SHV-1 expressed in Escherichia coli, all 19 R244 variants exhibited increased susceptibility to ampicillin/sulbactam, an important difference compared to ampicillin/clavulanate. Kinetic analyses of SHV-1 and three SHV R244 (-S, -Q, and -L) variants revealed the Ki for sulbactam was significantly elevated for the R244 variants, but the partition ratios, kcat/kinact, were markedly reduced (13 000 --> <or=500). A timed inactivation-mass spectrometry analysis of SHV inhibited by sulbactam showed that SHV-1 beta-lactamase was unmodified at 15 min. A parallel experiment with R244S demonstrated 70 and 88 +/- 3 Da fragments of sulbactam covalently attached to the beta-lactamase. We also observed that the Ki values of penems 1 and 2 were increased for R244 variants compared to those for SHV; however, these inhibitors effectively restored ampicillin susceptibility in vitro. Compared to that of sulbactam, the kcat/kinact values of penems for SHV-1 and R244S were low (<or=2), and unfragmented adducts of each penem were covalently attached to SHV-1 and R244S when studied using the timed inactivation-mass spectrometry method. The R244S mutation affects beta-lactamase inactivators differently, but resistance can be overcome by designing penem inhibitors with strategic chemical properties that improve affinity and impair turnover.
authors.affiliation: Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, USA.
authors.forename: Jodi M
authors.initials: JM
authors.lastname: Thomson
authors.forename: Anne M
authors.initials: AM
authors.lastname: Distler
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D019943:Amino Acid Substitution; D000667:Ampicillin / Q000737:chemistry / Q000494:pharmacology; D019818:Clavulanic Acid / Q000737:chemistry / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D013058:Mass Spectrometry; D015394:Molecular Structure; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D013407:Sulbactam / Q000737:chemistry / Q000494:pharmacology*; D018440:beta-Lactam Resistance / Q000235:genetics; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D000667:Ampicillin; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; D013407:Sulbactam