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Biophysical and Structural Studies of Chelator Fragment‐Library Candidates as Inhibitors of Metallo‐β‐lactamases
Author(s) -
Williams Cameron,
Tierney David L,
Crowder Michael W,
Cohen Seth,
Fast Walter,
Page Richard C
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.613.1
Subject(s) - antibiotics , bacteria , antibiotic resistance , microbiology and biotechnology , enzyme , biology , chemistry , biochemistry , genetics
Antibiotic resistance is a serious worldwide health problem. In the 2013 Antibiotic Resistance Threat Report, the CDC estimates that over 2 million people suffer from antibiotic‐resistant infections every year in the U.S. and 23,000 of these individuals die as a result. Metallo‐β‐lactamases are a class of enzymes that catalyze the hydrolysis of almost all β‐lactam antibiotics and, as a result, confer dangerous antibiotic resistance to the bacteria expressing them. Gram‐negative bacteria possessing metallo‐β‐lactamases are of utmost concern because these enzymes also break down carbapenems, a class of antibiotics commonly used as a last resort to treat antibiotic‐resistant bacterial infections. Enterobacteriaceae with carbapenemase activity are becoming increasingly widespread. This is a major problem because these gram‐negative bacteria of the intestinal flora are easily spread between humans and commonly found in clinical settings. Metallo‐β‐lactamase inhibitors are of particular interest because they could potentially restore the efficacy of current generation β‐lactam antibiotics. A library of chelator fragments has been developed to inhibit metallo‐β‐lactamases by taking advantage of the active site Zn 2+ ions critical for proper enzymatic function. Our in vitro activity assays have identified several potent candidates from the library for further study. VIM‐2, a variant of Verona integron‐encoded metallo‐β‐lactamase (VIM), was overexpressed and purified for the purpose of studying these chelator fragments. We utilized differential scanning fluorimetry to demonstrate that high concentrations of chelators destabilize VIM‐2 by removing Zn 2+ cofactors. Co‐crystallization and crystal soaks of VIM‐2 with several chelators was attempted in order to gain insight into the structure of the protein‐ligand complex but x‐ray diffraction data suggests that the chelators are not present in the crystal structure. Future work will involve binding affinity studies by isothermal titration calorimetry (ITC) and structural studies with advanced crystallographic techniques. Additional studies will be employed to examine the importance of a loop near the active site of all metallo‐β‐lactamases. Overall, these studies will contribute to the development of more effective chelators. Support or Funding Information This work was supported by NIH grant R01 GM111926.