title: Energetic, structural, and antimicrobial analyses of beta-lactam side chain recognition by beta-lactamases.
abstract: BACKGROUND
  Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these beta-lactams, most often through bacterial expression of beta-lactamases, threatens public health. To understand how beta-lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because beta-lactams form covalent adducts with beta-lactamases. This has complicated functional analyses and inhibitor design.
  
  
  RESULTS
  To investigate the contribution to interaction energy of the key amide (R1) side chain of beta-lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well as four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine beta-lactamases. Therefore, binding energies can be calculated directly from K(i) values. The K(i) values measured span four orders of magnitude against the Group I beta-lactamase AmpC and three orders of magnitude against the Group II beta-lactamase TEM-1. The acylglycineboronic acids have K(i) values as low as 20 nM against AmpC and as low as 390 nM against TEM-1. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of beta-lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of beta-lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to beta-lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray crystallography to 1.90 A and 1.75 A resolution; these structures suggest interactions that are important to the affinity of the inhibitors.
  
  
  CONCLUSIONS
  Acylglycineboronic acids allow us to begin to dissect interaction energies between beta-lactam side chains and beta-lactamases. Surprisingly, there is little correlation between the affinity contributed by R1 side chains and their occurrence in beta-lactam inhibitors or beta-lactam substrates of serine beta-lactamases. Nevertheless, presented in acylglycineboronic acids, these side chains can lead to inhibitors with high affinities and specificities. The structures of their complexes with AmpC give a molecular context to their affinities and may guide the design of anti-resistance compounds in this series.
authors.affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Chicago, IL 60611, USA.
authors.forename: E
authors.initials: E
authors.lastname: Caselli
authors.forename: R A
authors.initials: RA
authors.lastname: Powers
authors.forename: L C
authors.initials: LC
authors.lastname: Blasczcak
authors.forename: C Y
authors.initials: CY
authors.lastname: Wu
authors.forename: F
authors.initials: F
authors.lastname: Prati
authors.forename: B K
authors.initials: BK
authors.lastname: Shoichet
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D016972:Enterobacter cloacae / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D055672:Static Electricity; D013816:Thermodynamics; D018440:beta-Lactam Resistance; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*; D047090:beta-Lactams
chemical_list: D000900:Anti-Bacterial Agents; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D001618:beta-Lactamases