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Recognition and resistance in TEM beta-lactamase.
Xiaojun Wang1, George Minasov, Jesús Blázquez
1Department of Pharmaceutical Chemistry, University of California San Francisco, Genentech Hall, San Francisco, California 94143, USA.
Biochemistry
|July 16, 2003
Summary
Developing substrate-like antimicrobials to combat resistance is key. While compound 10 strongly inhibited TEM-1 beta-lactamase, it was susceptible to the TEM-30 mutant, highlighting potential resistance liabilities in substrate-based drug design.
Area of Science:
- Biochemistry
- Microbiology
- Drug Discovery
Background:
- Antimicrobial resistance is a growing global health threat.
- Developing new antimicrobials that evade resistance mechanisms is crucial.
- Substrate-based drug design is a strategy to create inhibitors less prone to resistance.
Purpose of the Study:
- To test the hypothesis that inhibitors closely resembling substrates are more difficult for resistance enzymes to overcome.
- To evaluate transition-state analogues as inhibitors of TEM-1 beta-lactamase and its mutants.
- To understand the structural basis of inhibitor recognition and resistance.
Main Methods:
- Synthesis and testing of 10 transition-state analogues against TEM-1 beta-lactamase and four mutant variants (TEM-30, TEM-32, TEM-52, TEM-64).
- Determination of crystal structures of four inhibitors complexed with TEM-1.
- Assessment of an inhibitor's efficacy in reversing bacterial resistance to ampicillin in cell culture.
Main Results:
- The substrate-analogue inhibitor (compound 10) showed the highest potency against wild-type TEM-1 (K(i) = 64 nM).
- Compound 10 was highly susceptible to the TEM-30 mutant, with over a 100-fold decrease in inhibition.
- X-ray crystallography revealed structural differences between acylation and deacylation transition-state mimics and explained TEM-30's reduced affinity for compound 10.
- In cell culture, compound 10 reduced ampicillin minimum inhibitory concentrations by 4- to 64-fold.
Conclusions:
- While substrate-based design can yield potent inhibitors, resistance mutations can still emerge.
- The TEM-30 mutation (R244S) confers significant resistance to substrate-analogue inhibitors.
- Structural insights are vital for understanding inhibitor-enzyme interactions and predicting resistance liabilities.