Binding Sites of Bicarbonate in Phosphoenolpyruvate Carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) is used in plant metabolism for fruit maturation or seed development as well as in the C4 and crassulacean acid metabolism (CAM) mechanisms in photosynthesis, where it is used for the capture of hydrated CO2 (bicarbonate). To find the yet unknown binding site of bicarbonate in this enzyme, we have first identified putative binding sites with nonequilibrium molecular dynamics simulations and then ranked these sites with alchemical free energy calculations with corrections of computational artifacts. Fourteen pockets where bicarbonate could bind were identified, with three having realistic binding free energies with differences with the experimental value below 1 kcal/mol. One of these pockets is found far from the active site at 14 Å and predicted to be an allosteric binding site. In the two other binding sites, bicarbonate is in direct interaction with the magnesium ion; neither sequence alignment nor the study of mutant K606N allowed to discriminate between these two pockets, and both are good candidates as the binding site of bicarbonate in phosphoenolpyruvate carboxylase.

Structural Optimization of Azacryptands for Targeting Three-Way DNA Junctions.

Transient melting of the duplex-DNA (B-DNA) during DNA transactions allows repeated sequences to fold into non-B-DNA structures, including DNA junctions and G-quadruplexes. These noncanonical structures can act as impediments to DNA polymerase progression along the duplex, thereby triggering DNA damage and ultimately jeopardizing genomic stability. Their stabilization by ad hoc ligands is currently being explored as a putative anticancer strategy since it might represent an efficient way to inflict toxic DNA damage specifically to rapidly dividing cancer cells. The relevance of this strategy is only emerging for three-way DNA junctions (TWJs) and, to date, no molecule has been recognized as a reference TWJ ligand, featuring both high affinity and selectivity. Herein, we characterize such reference ligands through a combination of in vitro techniques comprising affinity and selectivity assays (competitive FRET-melting and TWJ Screen assays), functional tests (qPCR and Taq stop assays) and structural analyses (molecular dynamics and NMR investigations). We identify novel azacryptands TrisNP-amphi and TrisNP-ana as the most promising ligands, interacting with TWJs with high affinity and selectivity. These ligands represent new molecular tools to investigate the cellular roles of TWJs and explore how they can be exploited in innovative anticancer therapies.

Are changes in antibiotic prophylaxis recommendations responsible for an increased risk of cefazolin allergy?

BACKGROUND: The first line of prevention of surgical site infection relies on the timely administration of antibiotic prophylaxis. First- and second-generation cephalosporins are the most recommended antibiotics in elective surgery. The incidence of cefazolin allergy has increased worldwide over the years. The sensitization mechanism of cefazolin is currently unknown, and data supporting cross-reactivity between penicillins and cephalosporins are lacking. Sensitization could occur through previous exposure either to cefazolin or to structurally related chemical agents. The objective of this study was to evaluate sensitization agents towards cefazolin. METHODS: The OpenBabel chemoinformatics toolbox was used to search for similarities between cefazolin and other molecules in an extensive drug database. Using the pholcodine-rocuronium similarity score as a threshold, we selected drugs with the most similar structure to that of cefazolin. Exposure to those drugs and cefazolin was assessed in a cohort of patients with skin test-proven cefazolin allergy at a specialized allergy centre via a self-administered anonymous questionnaire. RESULTS: Using the pholcodine-rocuronium similarity score as a threshold (score≥0.7), 42 molecules were found to be similar to cefazolin (all cephalosporins). Only 8 were marketed in France. None of the 14 cefazolin-allergic patients who answered the questionnaire (65% female, median age 56 years) reported exposure to any identified antibiotics. In contrast, 11 (78%) had at least one previous surgery requiring cefazolin before the index case. CONCLUSION: Direct previous cefazolin exposure was identified in 78% of cefazolin-allergic patients. Cefazolin started to take a central place in antibiotic prophylaxis after 2010, when cefamandole usage decreased drastically. Changes in antibiotic prophylaxis over the past 14 years in France could have been the turning point for the increased incidence of cefazolin allergy.