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Fluorescence detection of DNA and RNA G-quadruplexes (G4s) is a very efficient strategy to assess not only the existence and prevalence of cellular G4s but also their relevance as targets for therapeutic interventions. Among the fluorophores used to this end, turn-on probes are the most interesting since their fluorescence is triggered only upon interaction with their G4 targets, which ensures a high sensitivity and selectivity of detection. We reported on a series of twice-as-smart G4 probes, which are both smart G4 ligands (whose structure is reorganized upon interaction with G4s) and smart fluorescent probes (whose fluorescence is turned on upon interaction with G4s). The fine mechanistic details behind the excellent properties of the best prototype N-TASQ remain to be deciphered: to investigate this, we report here on the synthesis and studies of two analogues, Tz N-TASQ and Alk N-TASQ, and on a careful analysis of their G4-interacting properties, investigated both in vitro and in silico. Our results show that fine-tuning their constitutive structural elements allows for increasing the efficiency of both their 'off' (i. e., a conformation with a low fluorescence) and 'on' states (i. e., a conformation with a high fluorescence), which opens interesting ways for the design of more efficient fluorogenic G4 probes., (© 2024 Wiley-VCH GmbH.)

NMDA receptors (NMDARs) are glutamate-gated ion channels playing a central role in synaptic transmission and plasticity. NMDAR dysregulation is linked to various neuropsychiatric disorders. This is particularly true for GluN2B-containing NMDARs (GluN2B-NMDARs), which have major pro-cognitive, but also pro-excitotoxic roles, although their exact involvement in these processes remains debated. Traditional GluN2B-selective antagonists suffer from slow and irreversible effects, limiting their use in native tissues. We therefore developed OptoNAM-3, a photoswitchable negative allosteric modulator selective for GluN2B-NMDARs. OptoNAM-3 provided light-induced reversible inhibition of GluN2B-NMDAR activity with precise temporal control both in vitro and in vivo on the behavior of freely moving Xenopus tadpoles. When bound to GluN2B-NMDARs, OptoNAM-3 displayed remarkable red-shifting of its photoswitching properties allowing the use of blue light instead of UV light to turn-off its activity, which we attributed to geometric constraints imposed by the binding site onto the azobenzene moiety of the ligand. This study therefore highlights the importance of the binding site in shaping the photochemical properties of azobenzene-based photoswitches. In addition, by enabling selective, fast, and reversible photocontrol of native GluN2B-NMDARs with in vivo compatible photochemical properties (visible light), OptoNAM-3 should be a useful tool for the investigation of the GluN2B-NMDAR physiology in native tissues.

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., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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 CO 2 (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.

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., (Copyright © 2024 Société française d'anesthésie et de réanimation (Sfar). Published by Elsevier Masson SAS. All rights reserved.)

Interrogating G-quadruplex (G4) biology at its deepest roots in human cells relies on the design, synthesis, and use of ever smarter molecular tools. Here, we demonstrate the versatility of biomimetic G4 ligands referred to as TASQ (template assembled synthetic G-quartet) in which a biotin handle was incorporated for G4-focused chemical biology investigations. We have rethought the biotinylated TASQ design to make it readily chemically accessible via an efficient click-chemistry-based strategy. The resulting biotinylated, triazole-assembled TASQ, or BioTriazoTASQ, was thus shown to efficiently isolate both DNA and RNA G4s from solution by affinity purification protocols, for identification purposes. Its versatility was then further demonstrated by optical imaging that provided unique mechanistic insights into the actual strategic relevance of G4-targeting strategies, showing that ligand-stabilized G4 sites colocalize with and, thus, are responsible for DNA damage foci in human cells., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)