Biology subjects

Ayub, H.

Publications and source records attributed to Ayub, H..

4 recordsLinked to original sources

bioRxiv2026

PCO371 intracellular agonism at the parathyroid hormone 1 receptor produces pan-activation of signalling partners

Small-molecule agonists of class B1 G-protein-coupled receptors (GPCRs) remain rare because these receptors typically require large extracellular peptide ligands for activation. PCO371 is a notable exception: an intracellular non-peptide agonist, originally developed for osteoporosis treatment, that activates parathyroid hormone 1 receptor (PTH1R) from the cytoplasmic face of the receptor and is considered Gs-protein biased. In this study, we compared the functional, pharmacological and structural properties of PCO371 with the canonical extracellular peptide agonist PTH 1-34 at the PTH1R to define the molecular mechanisms underlying PCO371's unusual signalling profile. By manipulating receptor reserve, we demonstrate that PCO371 is a partial agonist relative to PTH 1-34, contrary to previous observations. Across Gs, Gi3, Gq, cAMP, and {beta}-arrestin-2 pathways, operational model analysis showed that PCO371 is non-biased, engaging the same transducers as PTH1-34 but with ~1000-fold lower potency. Mechanistic modelling using the Cubic Ternary Complex Activation model revealed that reduced intrinsic efficacy is sufficient to reproduce PCO371's distinct monotonic signalling kinetics, providing a quantitative explanation for its partial agonism. PCO371 binding is {beta}-arrestin-compatible but only drives measurable {beta}-arrestin-2 recruitment when PTH1R is highly expressed. Molecular modelling suggests that PC0371 stable binding needs an intact receptor-G protein complex. Overall, these insights define the mechanistic basis of intracellular agonism at a class B1 GPCR and provide a framework for designing next-generation small-molecule modulators that exploit this emerging pharmacological space.

pharmacology and toxicology↗

bioRxiv2026

BzAM terpolymer series with a step-wise systematic change in hydrophobicity, provides a nuanced platform for functional analysis of GPCRs in native-like nanodiscs

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Sulfo-DIBMA encapsulation uniquely preserves signalling-competent active states of the class B1 GPCRs, calcitonin gene-related peptide and parathyroid hormone 1 receptors, in native-like nanodiscs

Class B1 G-protein-coupled receptors (GPCRs), such as the calcitonin gene-related peptide (CGRP) receptor and parathyroid hormone 1 (PTH1) receptor, require native lipid interactions to maintain signalling-competent conformations. However, conventional detergents disrupt these environments. Amphipathic copolymers offer a detergent-free alternative, yet the field still lacks a clear understanding of which polymer architectures best preserve active-state GPCR pharmacology, limiting their broader translational utility. Here, we examine how distinct copolymer chemistries influence the functional integrity of class B1 GPCRs by comparing SMA 2000, DIBMA-12, and the electroneutral sulfo-DIBMA. Using NanoLuciferase bioluminescence resonance energy transfer (NanoBRET) ligand-binding, competition, and mini-G-protein recruitment assays on nanodisc-encapsulated receptors, we show that all three copolymers maintain high-affinity extracellular ligand binding but differ markedly in their ability to preserve intracellular signalling. Despite lower receptor extraction efficiency, only sulfo-DIBMA support mini-Gs engagement at the CGRP receptor and enable G-protein-dependent allosteric modulation at the PTH1 receptor, including conserved ligand affinity and prolonged residence time. These data reveal that polymer charge and backbone chemistry, rather than extraction yield, determine whether native-like nanodiscs retain the conformational landscape required for active-state signalling. Controlling non-specific ligand binding to the copolymer is a key requirement for a successful assay. Our findings identify sulfo-DIBMALP as a particularly superior environment for preserving native signalling behaviour in class B1 GPCRs, highlighting copolymer chemistry as an important determinant in detergent-free membrane protein studies. HIGHLIGHTSO_LISulfo-DIBMA encapsulated nanodiscs preserve active-state conformation of human calcitonin gene-related peptide receptor and parathyroid hormone 1 receptor. C_LIO_LIAll three copolymers (SMA 2000, DIBMA-12 and sulfo-DIBMA) preserve extracellular ligand binding but only sulfo-DIBMA preserves intracellular functional competence, including mini-Gs recruitment and G-protein-dependent allosteric modulation. C_LIO_LICopolymer chemistry, particularly the electroneutral, aliphatic nature of sulfo-DIBMA, may influence the preservation of signalling-competent states in two class B1 GPCRs by minimising charge-driven perturbations during solubilisation. C_LIO_LISulfo-DIBMALP provides a novel platform for studying dynamic membrane proteins with potential to provide mechanistic insights and facilitate drug discovery programmes in the future. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/724797v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1c19452org.highwire.dtl.DTLVardef@1f09464org.highwire.dtl.DTLVardef@19e582forg.highwire.dtl.DTLVardef@af85e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

AER-270 and TGN-020 are not aquaporin-4 water channel blockers

Aquaporin-4 (AQP4) is the most abundant water channel protein in the brain. It controls water homeostasis, facilitates glymphatic function and is a drug target for brain edema following injury or stroke. Dysregulation of brain water homeostasis affects millions of people every year leading to death, disability and cognitive decline, for which no medicines are available. Two compounds, AER-270 and TGN-020, are sold as AQP4 inhibitors and a prodrug of AER-270 is currently in a phase I human trial. However, the direct effect of these compounds on AQP4 function has not been unequivocally demonstrated. Our data across multiple cellular and molecular assay systems demonstrate, unexpectedly, that AER-270 and TGN-020 do not inhibit AQP4. Although we observed an apparent inhibitory effect of AER-270 and TGN-020 in the Xenopus laevis oocyte assay, there was no effect in assays using reconstituted recombinant AQP4 or mammalian cells expressing exogenous or endogenous AQP4. We identify alternative mechanisms of action for both molecules that may explain previously reported in vivo results that were interpreted in the context of AQP4 inhibition. Overall, we conclude that AER-270 and TGN-020 should not be used to investigate the AQP4-dependence of biological processes in the brain.

biophysics↗