Ureidopenicillins Are Potent Inhibitors of Penicillin-Binding Protein 2 from Multidrug-Resistant Neisseria gonorrhoeae H041.

Effective treatment of gonorrhea is threatened by the increasing prevalence of Neisseria gonorrhoeae strains resistant to the extended-spectrum cephalosporins (ESCs). Recently, we demonstrated the promise of the third-generation cephalosporin cefoperazone as an antigonococcal agent due to its rapid second-order rate of acylation against penicillin-binding protein 2 (PBP2) from the ESC-resistant strain H041 and robust antimicrobial activity against H041. Noting the presence of a ureido moiety in cefoperazone, we evaluated a subset of structurally similar ureido ß-lactams, including piperacillin, azlocillin, and mezlocillin, for activity against PBP2 from H041 using biochemical and structural analyses. We found that the ureidopenicillin piperacillin has a second-order rate of acylation against PBP2 that is 12-fold higher than cefoperazone and 85-fold higher than ceftriaxone and a lower MIC against H041 than ceftriaxone. Surprisingly, the affinity of ureidopenicillins for PBP2 is minimal, indicating that their inhibitory potency is due to a higher rate of the acylation step of the reaction compared to cephalosporins. Enhanced acylation results from the combination of a penam scaffold with a 2,3-dioxopiperazine-containing R1 group. Crystal structures show that the ureido ß-lactams overcome the effects of resistance mutations present in PBP2 from H041 by eliciting conformational changes that are hindered when PBP2 interacts with the weaker inhibitor ceftriaxone. Overall, our results support the potential of piperacillin as a treatment for gonorrhea and provide a framework for the future design of ß-lactams with improved activity against ESC-resistant N. gonorrhoeae.

Effect of Ca(2+) on Aß40 fibrillation is characteristically different.

Alzheimer's disease (AD) is the only one among top ten diseases in USA that cannot be cured, prevented or slowed down. At molecular level the mechanism of onset has been closely associated with mis-folding of Aß40 and Aß42 and is well supported by the genetic data for AD. Extensive research efforts have led to identification of factors and metal ions that could manipulate Aß equilibrium, especially Ca(2+). Previously, we reported selectively acceleration of Aß42 fibril formation by Ca(2+)in vitro within physiological concentrations (BBA (2009) 1794:1536). Aß40 on the other hand did not appear to be significantly affected by Ca(2+) addition. In an effort to understand the distinctive behavior of Aß40, we monitored changes of Aß40 aggregation by intrinsic tyrosine fluorescence and CD and took different approaches for data processing. Our analysis of CD data indicates a complex effect induced by the addition of 2mM Ca(2+) resulting in an increase in the rate of transformation from monomer to ß-sheet rich fibrilar or intermediate species formation in Aß40. Surprisingly, the kinetics observed by intrinsic fluorescence studies in this article and ThT, SEC or EM studies in our previous report were not able to unravel the existence of this effect in Aß40.