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Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABA A ) receptors to dampen neuronal activity in the brain 1 – 5 . However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABA A receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABA A receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABA A receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain. Cryo-electron microscopy structures of GABA A receptors bound to intravenous anaesthetics and benzodiazepines reveal both common and distinct transmembrane binding sites, and show that the mechanisms of action of anaesthetics partially overlap with those of benzodiazepines.

Pheromone-binding proteins (PBPs) are specific odorant-binding proteins that can specifically recognize insect pheromones. Through transcriptional analysis of the antennae of adult Endoclita signifer, EsigPBP3 was discovered and identified, and EsigPBP3 was found to be highly expressed in the antennae of male moths. Based on the binding characteristics and ability of EsigPBP3, we can find the key ligands and binding site to consider as a target to control the key wood bore E. signifier. In this study, the fluorescence competitive binding assays (FCBA) showed that EsigPBP3 had a high binding affinity for seven key eucalyptus volatiles. Molecular docking analysis revealed that EsigPBP3 had the strongest binding affinity for the sexual pheromone component, (3E,7E)-4,7,11-trimethyl-1,3,7,10-dodecatetraene. Furthermore, same as the result of FCBA, the EsigPBP3 exhibited high binding affinities to key eucalyptus volatiles, eucalyptol, α-terpinene, (E)-beta-ocimene, (−)-β-pinene, and (−)-α-pinene, and PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 are key sites. In summary, EsigPBP3 exhibits high binding affinity to male pheromones and key volatile compounds and the crucial binding sites PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 can act as targets in the recognition of E. signifier pheromones.

Removal of protein-bound uremic toxins (PBUTs) during conventional dialysis is insufficient. PBUTs are associated with comorbidities and mortality in dialysis patients. Albumin is the primary carrier for PBUTs and only a small free fraction of PBUTs are dialyzable. In the past, we proposed a novel method where a binding competitor is infused upstream of a dialyzer into an extracorporeal circuit. The competitor competes with PBUTs for their binding sites on albumin and increases the free PBUT fraction. Essentially, binding competitor-augmented hemodialysis is a reactive membrane separation technique and is a paradigm shift from conventional dialysis therapies. The proposed method has been tested in silico, ex vivo, and in vivo, and has proven to be very effective in all scenarios. In an ex vivo study and a proof-of-concept clinical study with 18 patients, ibuprofen was used as a binding competitor; however, chronic ibuprofen infusion may affect residual kidney function. Binding competition with free fatty acids significantly improved PBUT removal in pre-clinical rat models. Based on in silico analysis, tryptophan can also be used as a binding competitor; importantly, fatty acids or tryptophan may have salutary effects in HD patients. More chemoinformatics research, pre-clinical, and clinical studies are required to identify ideal binding competitors before routine clinical use.

The pathophysiological mechanisms underlying the constellation of symptoms that characterize COVID-19 are only incompletely understood. In an effort to fill these gaps, a “nicotinic hypothesis,” which posits that nicotinic acetylcholine receptors (AChRs) act as additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptors, has recently been put forth. A key feature of the proposal (with potential clinical ramifications) is the suggested competition between the virus’ spike protein and small-molecule cholinergic ligands for the receptor’s orthosteric binding sites. This notion is reminiscent of the well-established role of the muscle AChR during rabies virus infection. To address this hypothesis directly, we performed equilibriumtype ligand-binding competition assays using the homomeric human α7-AChR (expressed on intact cells) as the receptor, and radio-labeled α-bungarotoxin (α-BgTx) as the orthosteric-site competing ligand.We tested different SARS-CoV-2 spike protein peptides, the S1 domain, and the entire S1–S2 ectodomain, and found that none of them appreciably outcompete [125I]-α-BgTx in a specific manner. Furthermore, patchclamp recordings showed no clear effect of the S1 domain on α7-AChR–mediated currents. We conclude that the binding of the SARS-CoV-2 spike protein to the human α7-AChR’s orthosteric sites—and thus, its competition with ACh, choline, or nicotine—is unlikely to be a relevant aspect of this complex disease.

Quercetin is a flavonol found in edible plants and possesses a significant anticancer activity. This study explored the mechanism by which quercetin prevented liver cancer via inducing apoptosis in HepG2 cells. Quercetin induced cell proliferation and apoptosis through inhibiting YY1 and facilitating p53 expression and subsequently increasing the Bax/Bcl-2 ratio. The results revealed that YY1 knockdown promoted apoptosis, whilst YY1 overexpression suppressed apoptosis via direct physical interaction between YY1 and p53 to regulate the p53 signaling pathway. Molecular docking using native and mutant YY1 proteins showed that quercetin could interact directly with YY1, and the binding of quercetin to YY1 significantly decreased the docking energy of YY1 with p53 protein. The interactions between quercetin and YY1 protein included direct binding and non-bonded indirect interactions, as confirmed by cellular thermal shift assay, UV-Vis absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. It was likely that quercetin directly bound to YY1 protein to compete with p53 for the binding sites of YY1 to disrupt the YY1-p53 interaction, thereby promoting p53 activation. This study provides insights into the mechanism underlying quercetin’s anticancer action and supports the development of quercetin as an anticancer therapeutic agent.

In this work, we report on the in vitro and in vivo pharmacological properties of LP1 analogs to complete the series of structural modifications aimed to generate compounds with improved analgesia. To do that, the phenyl ring in the N-substituent of our lead compound LP1 was replaced by an electron-rich or electron-deficient ring and linked through a propanamide or butyramide spacer at the basic nitrogen of the (−)-cis-N-normetazocine skeleton. In radioligand binding assays, compounds 3 and 7 were found to display nanomolar binding affinity for the μ opioid receptor (MOR) (Ki = 5.96 ± 0.08 nM and 1.49 ± 0.24 nM, respectively). In the mouse vas deferens (MVD) assay, compound 3 showed an antagonist effect against DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin), a highly selective MOR prototype agonist, whereas compound 7 produced naloxone reversible effect at MOR. Moreover, compound 7, as potent as LP1 and DAMGO at MOR, was able to reduce thermal and inflammatory pain assessed by the mouse tail-flick test and rat paw pressure thresholds (PPTs) measured by a Randall–Selitto test.

The tomato leafminer (Tuta absoluta), a globally invasive pest, poses a major economic threat to tomato production. Although chemical control remains the primary management method, sustainable alternatives are urgently needed. Sex pheromone communication is critical for moth courtship and mating, with pheromone-binding proteins (PBPs) playing a key role in this process. In this study, we identified a PBP gene, TabsPBP2, from the T. absoluta transcriptome. Real-time quantitative PCR (RT-qPCR) revealed that TabsPBP2 is highly expressed in the antennae, with a strong male-biased expression pattern. Ligand-binding assays demonstrated that TabsPBP2 has the highest affinity for the sex pheromones (3E, 8Z, 11Z)-tetradecatrienyl acetate (TDTA) and (3E, 8Z)-tetradecadienyl acetate (TDDA). It also demonstrated a moderate-to-strong binding affinity to several tomato volatiles, including 2-carene, myrcene, α-pinene, cis-3-hexen-l-ol, methyl salicylate, sabinene, and α-terpinene. Molecular docking suggested that hydrophobic interactions predominantly stabilize the TabsPBP2–ligand complexes, with PHE118, PHE12, LEU90, LEU68, and ALA73 identified as key interacting residues. Electroantennogram (EAG) and Y-tube olfactometer assays confirmed that TDTA and TDDA act as strong attractants for male T. absoluta. This study enhances our understanding of the pheromone recognition in T. absoluta and provides a foundation for developing novel, pheromone-based pest control strategies.

The split-green fluorescent protein (GFP) reassembly assay is a well-established approach to study protein–protein interactions (PPIs). In this assay, when two interacting proteins X and Y, respectively fused to residues 1–157 and to residues 158–237 of GFP, are co-expressed in E. coli, the two GFP halves are brought to sufficient proximity to reassociate and fold to recreate the functional GFP. At constant protein expression level, the intensity of fluorescence produced by the bacteria is proportional to the binding affinity of X to Y. We hypothesized that adding a third partner (Z) endowed with an affinity for either X or Y would lead to an in vivo competition assay. We report here the different steps of the set-up of this competition assay, and define the experimental conditions required to obtained reliable results. Results show that this competition assay is a potentially interesting tool for screening libraries of binding inhibitors, Z being either a protein or a chemical reagent.

Uranyl–protein interactions participate in uranyl trafficking or toxicity to cells. In addition to their qualitative identification, thermodynamic data are needed to predict predominant mechanisms that they mediate in vivo. We previously showed that uranyl can substitute calcium at the canonical EF-hand binding motif of calmodulin (CaM) site I. Here, we investigate thermodynamic properties of uranyl interaction with site II and with the whole CaM N-terminal domain by spectrofluorimetry and ITC. Site II has an affinity for uranyl about 10 times lower than site I. Uranyl binding at site I is exothermic with a large enthalpic contribution, while for site II, the enthalpic contribution to the Gibbs free energy of binding is about 10 times lower than the entropic term. For the N–terminal domain, macroscopic binding constants for uranyl are two to three orders of magnitude higher than for calcium. A positive cooperative process driven by entropy increases the second uranyl-binding event as compared with the first one, with ΔΔG = −2.0 ± 0.4 kJ mol−1, vs. ΔΔG = −6.1 ± 0.1 kJ mol−1 for calcium. Site I phosphorylation largely increases both site I and site II affinity for uranyl and uranyl-binding cooperativity. Combining site I phosphorylation and site II Thr7Trp mutation leads to picomolar dissociation constants Kd1 = 1.7 ± 0.3 pM and Kd2 = 196 ± 21 pM at pH 7. A structural model obtained by MD simulations suggests a structural role of site I phosphorylation in the affinity modulation.

A dye-release method for investigating the effect of a competitive lipid environment on the activity of two membrane-disrupting antimicrobial peptides (AMP), maculatin 1.1 and aurein 1.2, is presented. The results support the general conclusion that AMP have greater affinity for negatively charged membranes, for example bacterial membranes, than for the neutral membrane surface found in eukaryotic cells, but only within a competitive lipid environment. Indeed, in a single-model membrane environment, both peptides were more potent against neutral vesicles than against charged vesicles. The approach was also used to investigate the effect of pre-incubating the peptides in a neutral lipid environment then introducing charged lipid vesicles. Maculatin was shown to migrate from the neutral lipid bilayers, where pores had already formed, to the charged membrane bilayers. This result was also observed for charged-to-charged bilayers but, interestingly, not for neutral-to-neutral lipid interfaces. Aurein was able to migrate from either lipid environment, indicating weaker binding to lipid membranes, and a different molecular mechanism for lysis of lipid bilayers. Competitive lipid environments could be used to assess other critical conditions that modulate the activity of membrane peptides or proteins.