Search Results (282)

γ-Aminobutyric acid type A receptors (GABA_ARs) are pentameric ligand-gated ion channels mediating fast inhibitory neurotransmission in the mammalian brain. Although recent structural and kinetic studies have advanced understandings regarding their activation mechanisms, the molecular determinants coupling agonist binding to channel gating remain unclear. We investigated the contribution of the β₂E153 residue, located on loop B of the extracellular domain, to the activation of α₁β₂γ₂ GABA_ARs. Macroscopic and single-channel patch clamp recordings were used to characterize two β₂E153-mutants: charge reversal (β₂E153K) and hydrophobic substitution (β₂E153A). Both substitutions disrupted normal receptor kinetics, with β₂E153K selectively accelerating deactivation and β₂E153A affecting both deactivation and desensitization. Single-channel analysis showed that β₂E153A reduced open probability and mean open times, consistent with altered gating transitions inferred from kinetic modeling. Structural inspection suggested that β₂E153 forms electrostatic interactions with β₂K196 and β₂R207 to stabilize loop C and maintain the agonist-bound conformation. The disruption of this interaction likely destabilizes loop C, leading to weakened agonist binding and modified gating. Overall, our results identify β₂E153 as a key element in the long-range allosteric network linking the binding site to the channel gate in GABA_ARs. Full article

Magnesium ions regulate synaptic and nonsynaptic neuronal excitability from intracellular (Mg²⁺_i) and extracellular (Mg²⁺_o) domains, modulating voltage- and ligand-gated ion channels. K+ inward rectifier (Kir) channel inward rectification arises from Mg²⁺_i blocking the pore and outward K⁺ current, while Mg²⁺_o targets external sites. Mg²⁺_i causes voltage-dependent Ca²⁺ voltage-gated (Ca_V) and Na⁺ voltage-gated (Na_V) channel block while phosphorylation modulates channel activity. Mg²⁺_o elicits direct voltage-dependent Ca_V channel block, and screens surface charge, and in Na_V channels reduces conduction and may cause depolarization by quantum tunneling across closed channels. Mg²⁺_i is an allosteric large conductance Ca²⁺-activated K⁺ (BK) channel activator, binding to low-affinity sites to alter Ca²⁺ and voltage sensitivity but reduces small conductance Ca²⁺-activated K⁺ (SK) channels’ outward K⁺ current and induces inward rectification. N-Methyl-D-aspartate receptor (NMDAR) channels are inhibited by Mg²⁺_i binding within the pore, while Mg²⁺_o stabilizes excitability through voltage-dependent block, Mg²⁺_o forms Mg-ATP complex modifying purinergic P2X receptor (P2XR) channel affinity and gating and directly blocks the pore. Mg²⁺_o reduces gamma-aminobutyric acid type A receptor (GABA_AR) channel Cl⁻ current amplitude and augments susceptibility to blockers. Mg²⁺_o and Mg²⁺_i block nicotinic acetylcholine receptor (nAChR) channels through voltage-dependent pore binding and surface charge screening, impeding current flow and altering gating. Full article

Marrubium vulgare L. is a medicinal plant widely used in traditional medicine, with emerging evidence of anticancer potential. This study investigated its bioactive compounds as inhibitors of Heat Shock Protein 90 alpha (Hsp90α), a molecular chaperone essential for oncogenic protein stability. Organic and aqueous extracts were profiled using high-performance liquid chromatography–mass spectrometry (HPLC–MS), revealing a diverse phytochemical composition. Identified compounds were screened against the full-length crystal structure of Hsp90α using a structure-based computational workflow that included extra-precision and domain-specific molecular docking, molecular dynamics (MD) simulations, and MM/GBSA binding free energy calculations. Pharmacokinetic and toxicity profiles were evaluated through ADMET predictions. This study elucidated the chemical composition of the plant and identified two hit compounds: Forsythoside B bound preferentially to the middle domain, potentially interfering with client protein interactions, and chlorogenic acid targeted the C-terminal domain, which regulates dimerization and allosteric activity. Both ligands displayed stable protein–ligand interactions during MD and favorable ADMET properties. These findings provide the first integrated chemical and computational prediction framework, suggesting that some M. vulgare metabolites may interact with Hsp90, highlighting its potential as a source of novel anticancer scaffolds and laying the groundwork for experimental validation and drug development. Full article

The cannabinoid receptor type 2 (CB₂) is increasingly recognized as a crucial regulator of neuroimmune balance in the brain. In addition to its well-established role in immunity, the CB₂ receptor has been identified in specific populations of neurons and glial cells throughout various brain regions, and its expression is dynamically increased during inflammatory and neuropathological conditions, positioning it as a potential non-psychoactive target for modifying neurological diseases. The expression of the CB₂ gene (CNR2) is finely tuned by epigenetic processes, including promoter CpG methylation, histone modifications, and non-coding RNAs, which regulate receptor availability and signaling preferences in response to stress, inflammation, and environmental factors. CB₂ signaling interacts with TRP channels (such as TRPV1), nuclear receptors (PPARγ), and orphan G Protein-Coupled Receptors (GPCRs, including GPR55 and GPR18) within the endocannabinoidome (eCBome), influencing microglial characteristics, cytokine production, and synaptic activity. We review how these interconnected mechanisms affect neurodegenerative and neuropsychiatric disorders, underscore the species- and cell-type-specificities that pose challenges for translation, and explore emerging strategies, including selective agonists, positive allosteric modulators, and biased ligands, that leverage the signaling adaptability of the CB₂ receptor while reducing central effects mediated by the CB₁ receptor. This focus on the neuro-centric perspective repositions the CB₂ receptor as an epigenetically informed, context-dependent hub within the eCBome, making it a promising candidate for precision therapies in conditions featuring neuroinflammation. Full article

Blind docking predicts binding interactions between two molecular entities without prior knowledge of the binding site. This approach is essential because it explores the entire surface of the receptor to identify potential interaction sites. Blind docking widely works for both protein–protein and ligand–protein interaction studies. In protein–protein blind docking, the method aims to predict the correct orientation and interface of two proteins forming a complex. Protein blind docking is particularly valuable in studying transient interactions, protein–protein recognition, signaling pathways, tentative and significant biomolecular assemblies where structural data is limited. Ligand–protein blind docking discovers potential binding pockets across the entire protein surface. It is frequently applied in early-stage drug discovery, especially for novel or poorly characterized targets. The method helps identify allosteric sites or novel binding regions that are not evident from known structures. Overall, blind docking provides a versatile and powerful tool for studying molecular interactions, enabling discovery even in the absence of detailed structural information. In this scenario, we reported a timeline of attempts to improve this kind of computational approach with ML and hybrid approaches to obtain more reliable predictions. We dedicate two main sections to protein–protein and protein-ligand blind docking, presenting the reliability and caveats for each approach and outlining potential future directions. Full article

The inactivation of thrombin by antithrombin is highly enhanced by the presence of heparin chains forming “bridges” between the two proteins. X-ray structures for such ternary complexes have been published, but the molecular background of the lower efficiency of smaller heparinoids on thrombin inhibition remains poorly understood. Antithrombin-resistant prothrombin mutants (mutations affecting Arg596 in prothrombin) have been reported that cause severe thrombophilia. Our aim was to study the interactions in the antithrombin–thrombin Michaelis complex both in the presence and the absence of a heparinoid chain and in the presence of pentasaccharide by using molecular dynamics. We also intended to study the complexes of thrombin mutants as well as a known alternative antithrombin conformation at the “hinge” region built using docking. The binding between the proteins was investigated by Gaussian Accelerated Molecular Dynamics (GaMD). We compared the contribution of several amino acids at the binding “exosites” between AT and the wild type and mutant thrombins and between systems containing or not containing a heparinoid. In the docking-based simulations, several of the analyzed amino acid pairs no longer contributed to the interaction, suggesting that the open “hinge” conformation has limited biological relevance. We could identify multiple conformational types using clustering, revealing high flexibility in mutants and systems without heparinoid, probably indicating lower stability. We were also able to detect the allosteric effects of the ligands on the bound thrombin. In summary, we were able to obtain conformations using GaMD that can explain the better protein–protein interactions in the ternary complexes and the impaired AT binding of the thrombin Arg596 mutants at an atomic level. Full article

Gamma-aminobutyric acid type A receptors (GABA_ARs) are pentameric ligand-gated ion channels essential for inhibitory neurotransmission in the central nervous system. Subtype-specific expression patterns of GABA_AR subunits underlie their diverse roles in regulating anxiety, motor function, and sedation. While non-selective GABA_AR positive allosteric modulators (PAMs), such as benzodiazepines, are clinically effective anxiolytic drugs, their non-selective activity across α1/2/3/5 subunit-containing GABA_ARs leads to sedation, cognitive impairment, and risk of dependence. To address this, we evaluated ENX-102, a novel GABA_AR PAM, which exhibits selectivity for α2/3/5 subunits. In rodents, ENX-102 demonstrated dose-dependent anxiolytic-like activity following acute and sub-chronic administration, without sedation. ENX-102 exhibited a dose-dependent quantitative electroencephalography (qEEG) spectral signature in rodents that was distinct from that of benzodiazepines. In a double-blind, placebo-controlled, multiple-ascending dose study in healthy human volunteers, ENX-102 was evaluated using the NeuroCart, a CNS test battery including saccadic peak velocity (SPV), adaptive tracking, pupillometry, body sway, the Bond and Lader Visual Analog Scale (VAS), the Visual Verbal Learning Task (VVLT), and qEEG. ENX-102 produced reductions in SPV that were indicative of central target engagement, with minimal effects on alertness and motor coordination, which is consistent with subtype-selective GABA_AR targeting. Notably, qEEG revealed increased β-band power and decreased δ- and θ-band activity, which were distinct from the spectral profile of non-selective PAMs, supporting translational alignment with preclinical findings. Across dose levels, ENX-102 was well tolerated and exhibited favorable pharmacokinetics. These results support further clinical development of ENX-102 as a next-generation GABA_AR subtype-selective anxiolytic drug. Full article

TGR5 has emerged as a promising therapeutic target for obesity and metabolic disorders due to its regulatory roles in energy expenditure, glucose homeostasis, thermogenesis, and gut hormone secretion. This review summarizes the structural mechanisms of TGR5 activation, focusing on orthosteric and allosteric ligand interactions, toggle switch dynamics, and G protein coupling based on cryo-EM and docking-based models. A wide range of bioactive natural compounds including oleanolic acid, curcumin, betulinic acid, ursolic acid, quinovic acid, obacunone, nomilin, and 5β-scymnol are examined for their ability to modulate TGR5 signaling and elicit favorable metabolic effects. Molecular docking simulations using CB-Dock2 and PDB ID 7BW0 revealed key interactions within the orthosteric pocket, supporting their mechanistic potential as TGR5 agonists. Emerging strategies in TGR5-directed drug development are also discussed, including gut-restricted agonism to minimize gallbladder-related side effects, biased and allosteric modulation to fine-tune signaling specificity, and AI-guided optimization of natural product scaffolds. These integrated insights provide a structural and pharmacological framework for the rational design of safe and effective TGR5-targeted therapeutics. Full article

Obesity is a complex, multifactorial disease and a growing global health challenge associated with type 2 diabetes, cardiovascular disorders, cancer, and reduced quality of life. The existing pharmacological therapies are characterized by their limited number and efficacy, and safety concerns further restrict their utilization. This review synthesizes extensive knowledge regarding the role of the endocannabinoid system (ECS) in the pathogenesis of obesity, as well as its potential as a therapeutic target. A thorough evaluation of preclinical and clinical data concerning endocannabinoid ligands, cannabinoid receptors (CB1, CB2), their genetic variants, and pharmacological interventions targeting the ECS was conducted. Literature data suggests that the overactivation of the ECS may play a role in the pathophysiology of excessive food intake, dysregulated energy balance, adiposity, and metabolic disturbances. The pharmacological modulation of ECS components, by means of CB1 receptor antagonists/inverse agonists, CB2 receptor agonists, enzyme inhibitors, and hybrid or allosteric ligands, has demonstrated promising anti-obesity effects in animal models. However, the translation of these findings into clinical practice remains challenging due to safety concerns, particularly neuropsychiatric adverse events. The development of novel strategies, including peripherally restricted compounds, hybrid dual-target agents, dietary modulation of endocannabinoid tone, and non-pharmacological interventions, promises to advance the field of obesity management. Full article

The proposed physiological roles of bile acids have expanded beyond the digestion of fats to encompass cell signaling via the activation of a variety of nuclear and plasma membrane receptors in multiple organ systems. The current in silico study was inspired by previous observations from our group and others that bile acids interact functionally with cardiac, pulmonary, and gastrointestinal muscarinic receptors and more recent work demonstrating allosteric binding of cholesterol, the parent molecule for bile acid synthesis, to M₁ muscarinic receptors (M₁R). Here, we computationally tested the hypothesis that bile acids can allosterically bind to M₁R and thereby modulate receptor activation. Utilizing de novo site identification by the ligand competitive saturation (SILCS) method, putative novel allosteric binding sites of bile acid targeting M₁R were identified. Molecular dynamics simulations were used to uncover the molecular details of the activation mechanism of M₁R due to agonist binding along with allosteric modulation of bile acids on M₁R activation. Allosteric binding of bile acids and their glycine and taurine conjugates to M₁R negatively impacts the activation process, findings consistent with recent reports that M₁R expression and activation inhibit colon cancer cell proliferation. Thus, bile acids may augment colon cancer risk by inhibiting the tumor suppressor actions of M₁R. When validated experimentally, these findings are anticipated to shed light on our understanding of how bile acids in the membrane microenvironment can allosterically modulate the function of M₁R and possibly other G protein-coupled receptors. Full article

Rhodopsin, the prototypical Class A G protein-coupled receptor (GPCR) and visual pigment of rod photoreceptors, has long served as a structural and mechanistic model for GPCR biology. Mutations in rhodopsin are the leading cause of autosomal dominant retinitis pigmentosa (adRP), making this receptor a critical therapeutic target. In this review, we summarize the chemical, structural, and biophysical features of small-molecule modulators of this receptor, spanning both classical retinoid analogs and emerging non-retinoid scaffolds. These ligands reveal recurrent binding modes within the orthosteric chromophore pocket as well as peripheral allosteric and bitopic sites, where they mediate folding, rescue trafficking, photocycle modulation, and mutant stabilization. We organize ligand performance into a three-tier framework linking binding affinity, cellular rescue potency, and stability gains. Chemotypes in tier 2, which show sub-micromolar to low-micromolar activity with broad mutant coverage, emerge as promising candidates for optimization into next-generation scaffolds. Across scaffolds, a recurring minimal pharmacophore is evident by a contiguous hydrophobic π-surface anchored in the β-ionone region, coupled with a strategically oriented polar handle that modulates the Lys296/Glu113 microenvironment, offering tractable design vectors for non-retinoid chemotypes. Beyond the chromophore binding pocket, we highlight opportunities to exploit extracellular loop epitopes, cytoplasmic microswitch clefts, dimer/membrane interfaces, and ion co-binding sites to engineer safer, state-biased control with fewer photochemical liabilities. By integrating rhodopsin photobiophysics with environment-aware, multi-state medicinal chemistry, and by addressing current translational challenges in drug delivery, this review outlines a rational framework for advancing rhodopsin-targeted therapeutics toward clinically credible interventions for RP and related retinal degenerations. Full article

The mammalian target of rapamycin pathway, mTOR, is a crucial signaling pathway that regulates cell growth, proliferation, metabolism, and survival. Due to its dysregulation it is involved in several ailments such as cancer or age-related diseases. The discovery of mTOR and the understanding of its biological functions were greatly facilitated by the use of rapamycin, an antibiotic of natural origin, which allosterically inhibits mTORC1, effectively blocking its function. In this entirely computational study, we investigated mTOR’s interaction with seven ligands: two clinically established inhibitors (everolimus and rapamycin) and five lignin-derived oligomers, a renewable natural polyphenol recently used for the drug delivery of everolimus. The seven complexes were analyzed through all-atom molecular dynamics simulations in explicit solvent using a high-performance computing platform. Trajectory analyses revealed stable interactions between mTOR and all ligands, with lignin-derived compounds showing comparable or enhanced binding stability relative to reference drugs. To evaluate the stability of the molecular complex and the behavior of the ligand over time, we analyzed key parameters including root mean square deviation, root mean square fluctuation, number of hydrogen bonds, binding free energy, and conformational dynamics assessed through principal component analysis. Our results suggest that lignin fragments are a promising, sustainable scaffold for developing novel mTOR inhibitors. Full article

Background/Objectives: Endozepines known as the endogenous ligands of benzodiazepine-binding sites, include the diazepam binding inhibitor (DBI) and its processing products, the triakontatetraneuropeptide (TTN) and the octadecaneuropeptide (ODN). Despite indisputable evidence of the binding of ODN on GABA_AR-BZ-binding sites, their action on this receptor lacks compelling electrophysiological observations, with some studies reporting that ODN acts as a negative allosteric modulator (NAM) of GABA_AR while others suggest the opposite (positive allosteric modulation, PAM effect). All these studies were carried out in vitro with various neuronal cell types. To further elucidate the role of ODN in neuronal excitability, we tested its effect in vivo in the cerebral cortex of the anesthetized mouse. Methods: Spontaneous neuronal spikes were recorded by means of an extracellular pipette, in the vicinity of which ODN was micro-infused, either at a high dose (10⁻⁵ M) or low dose (10⁻¹¹ M). Results: ODN at a high dose induced a significant increase in neuronal spiking. This effect could be antagonized by the GABA_AR-BZ-binding site blocker flumazenil. In sharp contrast, at low concentrations, ODN reduced neuronal spiking with a magnitude similar to GABA itself. Interestingly, this decrease in neuronal activity by low dose of ODN was not flumazenil-dependent, suggesting that this effect is mediated by another receptor. Finally, we show that astrocytes in culture, known to be stimulated by picomolar doses of ODN via a GPCR, increased their export of GABA when stimulated by low dose of ODN. Conclusion: Our results confirm the versatility of ODN in the control of GABA transmission, but suggest that its PAM-like effect is, at least in part, mediated via an astrocytic non-GABA_AR ODN receptor release of GABA. Full article

Almost every cell of a multicellular organism is in contact with the extracellular matrix (ECM), which provides the shape and mechanic stability of tissue, organs and the entire body. At the molecular level, cells contact the ECM via integrins. Integrins are transmembrane cell adhesion molecules that connect the ECM to the cytoskeleton, which they bind with their extracellular and intracellular domains. Cysteine residues are abundant in both integrin subunits α and β. If pairwise oxidized into disulfide bridges, they stabilize the folding and molecular structure of the integrin. However, despite the oxidative environment of the extracellular space, not all pairs of cysteines in the extracellular integrin domains are permanently engaged in disulfide bridges. Rather, the reversible and temporary linkage of cystine bridges of these cysteine pairs by oxidation or their reductive cleavage can cause major conformational changes within the integrin, thereby changing ligand binding affinity and altering cellular functions such as adhesion and migration. During recent years, several oxidoreductases and thiol isomerases have been characterized which target such allosteric disulfide bridges. This outlines much better, albeit not comprehensively, the role that such thiol switches play in the redox regulation of integrins. The platelet integrin αIIbβ3 is the best examined example so far. Mostly referring to this integrin, this review will provide insights into the thiol switch-based redox regulation of integrins and the known effects of their allosteric disulfide bridges on conformational changes and cell functions, as well as on the machinery of redox-modifying enzymes that contribute to the redox regulation of cell contacts with the ECM. Full article

Arginine kinase is a key phosphagen kinase in invertebrates that facilitates rapid ATP regeneration by reversibly transferring phosphate groups between phosphoarginine and ADP. Structural studies have shown that the enzyme adopts distinct conformations in its ligand-free and ligand-bound states, known as the “open” and “closed” forms, respectively. These conformational changes are crucial for catalytic activity, enabling precise positioning of active-site residues and loop closure during phosphoryl transfer. Transition-state analog complexes have provided additional insights by mimicking intermediate states of catalysis, supporting the functional relevance of the open/closed structural model. Furthermore, studies across multiple species reveal how monomeric and dimeric forms of arginine kinase contribute to its allosteric regulation and substrate specificity. Beyond its metabolic role, arginine kinase is also recognized as a major allergen in crustaceans. Its structural uniqueness and absence in vertebrates make it a promising candidate for selective drug targeting. By integrating crystallographic data with functional context, this review highlights conserved features and species-specific variations of arginine kinase that may inform the design of inhibitors. Such molecules have the potential to serve both as antiparasitic agents and as novel therapeutics to manage crustacean-related allergic responses in humans. Full article