Structural requirements for the stability of novel cephalosporins to AmpC β-lactamase based on 3D-structure

AmpC β-lactamase is one of the leading causes of Pseudomonas aeruginosa (P. aeruginosa) resistance to cephalosporins. FR259647 is a cephalosporin having a novel pyrazolium substituent at the 3-position and exhibits excellent activity (MIC = 1 μg/mL) against the AmpC β-lactamase overproducing P. aeruginosa FP1380 strain in comparison with the third-generation cephalosporins FK518 [Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21–24, 1990, Abs. 454; Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21–24, 1990, Abs. 455; Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21–24, 1990, Abs. 456; Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21–24, 1990, Abs. 457] (MIC = 16 μg/mL) and ceftazidime (CAZ) (MIC = 128 μg/mL). The stability of FR259647 and FK518 to AmpC β-lactamase was evaluated using MIC assays against both the P. aeruginosa PAO1 strain and a PAO1 mutant strain overproducing AmpC β-lactamase as a differential assay, which indicates that the main difference derives from their stability to AmpC β-lactamase. A structural analysis using computer simulations indicated that the difference in stability may be due to steric hindrance of the 3-position substituents causing differential affinity. This steric hindrance may disturb entry of the cephalosporins into the binding pocket. We predicted the possibility of inhibition of entry as a potential means of enhancing stability by conformational analysis. In order to validate this speculation, novel FR259647 derivatives 4–9 were designed, calculated, synthesized, and evaluated. As a result, we demonstrated that their probability of entry correlated with the MIC ratio of the mutant strain to the parent strain and supports the validity of our model.

We propose novel structural requirements that FR259647 derivatives with lower probability of entry into the binding pocket of AmpC β-lactamase are more stable to the enzyme.Figure optionsDownload as PowerPoint slide