Biology subjects

Janouskova-Randakova, A.

Publications and source records attributed to Janouskova-Randakova, A..

4 recordsLinked to original sources

bioRxiv2026

Steroid-based Tide Quencher 1 probes enable real-time mapping of novel non-canonical cholesterol sites on the M1 muscarinic receptor

Steroid-based fluorescent-quencher probes now enable real-time, residue-level mapping of previously inaccessible cholesterol-binding sites on G-protein-coupled receptors. We designed Tide Quencher 1 (TQ1) conjugated steroids that target two distinct peripheral sites on the M1 muscarinic receptor. One near the extracellular N-terminus and another adjacent to the intracellular C-terminus. Using pregnanolone glutamate as a versatile scaffold, we synthesised a library of probes varying in C-3 linker length ({gamma}-aminobutyric acid vs. L-glutamic acid) and C-3/C-5 stereochemistry (3/3{beta}/5/5{beta}). Fluorescence-quenching assays with CFP-tagged receptors revealed that TQ1 probes consistently outperformed Dabcyl, delivering up to 40 % quenching within minutes and sub-micromolar EC50 values. The most potent N-terminal probe (35-PRG-Glu-TQ1 (5)) achieved 300 nM potency, while the best C-terminal probe (35{beta}-PRG-Glu-TQ1 (3)) reached 1 {micro}M potency with rapid association. Molecular docking and MD simulations identified key residues (K20, Q24, W405 at the N-site; K57, Y62, W150 at the C-site) mediating binding, a prediction confirmed by alanine-scan mutagenesis that markedly reduced quenching at the N-terminus and only modestly affected the C-terminus. Competition experiments with non-quenching analogues further validated probe specificity. Crucially, the pregnane core proved essential; alternative steroid backbones failed to generate robust quenching. This fluorescence-quenching platform overcomes the limitations of traditional radioligand assays, providing kinetic insight, high-throughput compatibility, and the ability to dissect lipid-GPCR interactions in native membranes. The approach is readily extensible to other GPCR families, opening new avenues for structure-guided drug discovery targeting allosteric cholesterol sites.

pharmacology and toxicology↗

bioRxiv2026

Dualsteric and dual-acting modulation of muscarinic receptors by antagonist KH-5

Background and purposeMuscarinic acetylcholine receptors are key therapeutic targets, and ligands engaging both orthosteric and allosteric sites may offer improved selectivity and efficacy. The muscarinic antagonist KH-5 displays functional antagonistic potency exceeding its binding affinity, suggesting a non-classical mechanism of action. Here, we investigated whether KH-5 acts as a dualsteric antagonist and defined its mode of interaction with muscarinic receptors. Experimental approachFunctional responses at human M1 and M2 receptors expressed in CHO cells were assessed using inositol phosphate accumulation and [35S]GTP{gamma}S binding, respectively. Radioligand binding studies employed orthosteric antagonists and agonists in combination with KH-5 and classical allosteric modulators. Data were analysed using competitive, allosteric, and dualsteric binding and operational models. Molecular docking, molecular dynamics simulations, and site-directed mutagenesis were used to identify structural determinants of KH-5 binding. Key resultsKH-5 antagonised responses to multiple agonists in a saturable and probe-dependent manner consistent with an allosteric interaction. However, KH-5 did not decrease maximal response to agonists, contradicting simple allosteric antagonism. At M2 receptors, antagonism was largely competitive. Binding studies revealed transient enhancement of agonist binding at M1 receptors at nanomolar concentrations of KH-5, best described by a dualsteric binding model involving independent orthosteric and ectopic site interactions. KH-5 did not bind to the classical muscarinic allosteric site at the second extracellular loop but interacted with an extracellular vestibule site, supported by molecular modelling and mutation of key residues. Conclusions and implicationsAt M1 receptors, the most parsimonious model among those tested combines orthosteric competition with an ectopic/allosteric component. At M2 receptors, KH-5 behaves predominantly as an orthosteric antagonist under the present conditions, although a weak or probe-specific allosteric component cannot be excluded. SummaryO_ST_ABSWhat is already knownC_ST_ABSMuscarinic receptors are therapeutic targets with conserved orthosteric binding sites. Allosteric or dualsteric ligands may improve receptor-subtype selectivity. What this study addsKH-5 shows dualsteric, dual-acting modulation at M1 receptors. At M2 receptors, KH-5 behaves mainly as a competitive antagonist. Clinical significanceDualsteric muscarinic antagonists may enable subtype-selective anticholinergic drug development. KH-5 provides a framework for designing mixed orthosteric/allosteric ligands.

pharmacology and toxicology↗

bioRxiv2025

Antagonistic properties of 4-(hexyloxy)benzoate derivatives and their N-methyl ammonium salts at muscarinic acetylcholine receptors

Muscarinic acetylcholine receptors (mAChRs) are key regulators of diverse physiological processes and longstanding therapeutic targets. Building on the long-acting antagonist KH-5, we synthesised and evaluated a series of 4-(hexyloxy)benzoate derivatives and their quaternary N-methylated analogues to explore how structural modifications influence receptor affinity and the duration of functional antagonism. Our structure-activity analysis revealed that introducing a rigid azabicyclo[2.2.2]octan-1-ium group boosted binding affinity (up to 250-fold compared to parental compounds) yet reduced the half-life of functional antagonism. In contrast, analogues with moderate flexibility maintained high potency while preserving longer receptor residence time. Computational docking and molecular dynamics (MD) simulations demonstrated that stable hydrogen bonding with residue N6.52 and salt-bridge formation with D3.32 were critical for sustained ligand binding to the receptor, with MD-derived metrics outperforming docking energies in predicting biological activity. Crucially, a positively charged nitrogen and a 4-hexyloxy substituent are essential features for high-affinity binding and prolonged antagonism. Shortening the alkyl chain resulted in a marked loss of affinity and abolished sustained activity. These findings underscore the need to balance molecular rigidity with conformational flexibility and charge distribution in the design of long-residence mAChR antagonists, offering a framework for further development of mAChR-targeted long-acting antagonists. HighlightsO_LINew analogues show up to 250x higher affinity at muscarinic receptors C_LIO_LIN6.52 H-bonding during MD predicts compound binding better than docking energies C_LIO_LICharged nitrogen and 4-hexyloxy are key to high affinity and sustained action C_LIO_LIRigid azabicyclo groups boost potency but shorten antagonism duration C_LIO_LIFlexible analogues balance potency with longer receptor residence time better C_LI

pharmacology and toxicology↗

bioRxiv2024

Cholesterol differentially modulates the activity of opioid and muscarinic receptors via common non-canonical binding site

G protein-coupled receptors (GPCRs) are membrane proteins that represent the largest and most therapeutically targeted receptor class, accounting for 30% of currently marketed drugs. Two binding motifs for membrane cholesterol, the cholesterol recognition amino acid consensus (CRAC) domain and the cholesterol consensus motif (CCM), have been postulated. Using a simulation of the molecular dynamics of cholesterol association with the receptor, we predicted the binding of membrane cholesterol to non-canonical sites, distinct from CRAC and CCM, at muscarinic and opioid receptors. We identified a binding site common to muscarinic and opioid receptors at TM6, with arginine 6.35 as the major residue. Membrane cholesterol depletion mimics the effects of R6.35 mutations, confirming its role in receptor modulation. Targeting cholesterol-binding sites offers novel pharmacotherapeutic strategies, including tissue-specific sterol-based modulation. One sentence summaryThis study identifies a shared non-canonical cholesterol-binding site at TM6 in muscarinic and opioid receptors, with significant implications for drug development targeting tissue-specific GPCR modulation.

pharmacology and toxicology↗