ABSTRACT 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 agonist, originally developed for osteoporosis treatment, that activates parathyroid hormone 1 receptor (PTH1R) from the cytoplasmic face of the receptor. In this study, we compared the functional, pharmacological and structural properties of PCO371 with the canonical extracellular peptide PTH1-34 at the PTH1R to define the mechanism underlying PCO371’s unusual signalling profile. Functionally, PCO371 exhibited markedly lower functional affinity and a strong dependence on receptor reserve, achieving full agonism only at high receptor expression, whereas PTH1-34 maintained robust signalling under receptor depletion. Across Gαs, Gαi3, Gαq(R183Q), cAMP, and β-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. Our findings establish PCO371 as a non-biased but globally less potent agonist, compared to PTH1-34, whose signalling efficacy depends on receptor reserve and G-protein engagement. PCO371 binding is β-arrestin-compatible but only drives measurable β-arrestin-2 recruitment when PTH1R is highly expressed. 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. HIGHLIGHTS PCO371 requires high PTH1R expression to achieve full agonism. PCO371 activates Gás, Gái and Gáq families but is significantly less potent than PTH1-34. Operational modelling shows no detectable signalling bias for PCO371; reduced signalling is global rather than pathway selective. PCO371-bound PTH1R structure is compatible with β-arrestin engagement, where PCO371 elicits a measurable β-arrestin-2 response only under high receptor expression. Molecular dynamic simulations reveal that PCO371 becomes stably bound only within a preassembled PTH1R-G-protein complex. PCO371 stabilises a distinct rearrangement in PTH1R:Gαs/Gαq/Gαi3 TM6 and the TM1/TM7 bundle and forms G-protein subtype specific α5 helix interactions. Establish a mechanistic basis for intracellular agonism, informing future design of therapeutically relevant modulators. GRAPHICAL ABSTRACT

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‑Gαs 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.

ABSTRACT 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. HIGHLIGHTS Stepwise-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. All BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. CGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. The 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. GRAPHICAL ABSTRACT

Vasopressin (AVP) and oxytocin (OT) receptors (nomenclature as recommended by NC-IUPHAR [98]) are activated by the endogenous cyclic nonapeptides vasopressin and oxytocin. These peptides are derived from precursors which also produce neurophysins (neurophysin I for oxytocin; neurophysin II for vasopressin). Vasopressin and oxytocin differ at only 2 amino acids (positions 3 and 8). There are metabolites of these neuropeptides that may be biologically active [71].

Membrane proteins (MPs) are critical to cellular processes and serve as essential therapeutic targets. However, their isolation and characterization are often impeded by traditional detergent-based methods, which can compromise their native states, and retention of their native lipid environment. Amphiphilic polymers have emerged as effective alternatives, enabling the formation of nanoscale discs that preserve MPs' structural and functional integrity. We introduce a novel series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers with tunable amphiphilicity, synthesized through controlled polymerization. Designed to mimic and improve upon industry-standard poly(styrene-co-maleic acid), these well-defined terpolymers offer enhanced control over molecular weight and distribution, allowing for systematic evaluation of polymer properties and their effect on membrane solubilization. The BzAM series effectively solubilized membranes and demonstrated a direct correlation between polymer hydrophobicity and solubilization efficiency of bacterial ABC transporter, Sav1866. This research highlights the importance of rational polymer design in MP research and provides a foundation for future developments.

Doxorubicin (Doxo)-associated cardio-and vasotoxicity has been recognised as a serious complication of cancer chemotherapy. The purpose of this novel paper was to determine the effect of Doxo on G-protein coupled receptor (GPCR)-mediated vasocontraction located on vascular smooth muscle cells. Rat left anterior descending artery segments were incubated for 24 h with 0.5 µM Doxo. The vasocontractile responses by activation of endothelin receptor type A (ET A ) and type B (ET B ), serotonin receptor 1B (5-HT 1B ) and thromboxane A2 prostanoid receptor (TP) were investigated by a sensitive myography using specific agonists, while the specificity of the GPCR agonists was verified by applying selective antagonists (i.e. ET A and ET B agonist = 10 − 14 -10 − 7.5 M endothelin-1 (ET-1); ET A antagonist = 10 µM BQ123; ET B agonists = 10 − 14 -10 − 7.5 M sarafotoxin 6c (S6c) and ET-1; ET B antagonist = 0.1 µM BQ788; 5-HT 1B agonist = 10 − 12 -10 − 5.5 M 5-carboxamidotryptamine (5-CT); 5-HT 1B antagonist = 1 µM GR55562; TP agonist = 10 − 12 -10 − 6.5 M U46619; TP antagonist = 1 µM Seratrodast). Our results show that 0.5 µM Doxo incubation of LAD segments leads to an increased VSMC vasocontraction through the ET B , 5-HT 1B and TP GPCRs, with a 2.2-fold increase in ET B -mediated vasocontraction at 10 − 10.5 M S6c, a 2.0-fold increase in 5-HT 1B -mediated vasocontraction at 10 − 5.5 M 5-CT, and a 1.3-fold increase in TP-mediated vasocontraction at 10 − 6.5 M U46619. Further studies unravelling the involvement of intracellular GPCR signalling pathways will broaden our understanding of the Doxo-induced vasotoxicity, and thus pave the way to mitigate the adverse effects by potential implementation of adjunct therapy options.

G-protein-coupled receptors (GPCRs) are the largest family of membrane proteins, regulate a plethora of physiological responses and are the therapeutic target for 30–40% of clinically-prescribed drugs. They are integral membrane proteins deeply embedded in the plasma membrane where they activate intracellular signalling via coupling to G-proteins and β-arrestin. GPCRs are in intimate association with the bilayer lipids and that lipid environment regulates the signalling functions of GPCRs. This complex lipid ‘landscape’ is both heterogeneous and dynamic. GPCR function is modulated by bulk membrane properties including membrane fluidity, microdomains, curvature, thickness and asymmetry but GPCRs are also regulated by specific lipid:GPCR binding, including cholesterol and anionic lipids. Understanding the molecular mechanisms whereby GPCR signalling is regulated by lipids is a very active area of research currently. A major advance in membrane protein research in recent years was the application of styrene maleic acid (SMA) copolymers. These spontaneously generate SMA lipid particles (SMALPs) encapsulating membrane protein in a nano-scale disc of cell membrane, thereby removing the historical need for detergent and preserving lipid:GPCR interaction. The focus of this review is how GPCR-SMALPs are increasing our understanding of GPCR structure and function at the molecular level. Furthermore, an increasing number of ‘second generation’ SMA-like copolymers have been reported recently. These are reviewed from the context of increasing our understanding of GPCR molecular mechanisms. Moreover, their potential as a novel platform for downstream biophysical and structural analyses is assessed and looking ahead, the translational application of SMA-like copolymers to GPCR drug discovery programmes in the future is considered.

Hepatorenal syndrome (HRS) is a life-threatening complication of end-stage liver disease first reported over a century ago, but its management still poses an unmet challenge. A therapeutic agent found to stabilize the condition is a short cyclic peptide, vasopressin analogue, terlipressin (TP). While TP is commonly prescribed for HRS patients in most parts of the world, it was only recently approved for use in the United States. TP exhibits short circulation half-lives and adverse side effects associated with the dose required. Herein, we present a 1,18-octadecanedioic acid (ODDA) conjugate of the cyclic peptide (ODDA-TP), which enables noncovalent binding to serum albumin via native fatty acid binding modes. ODDA-TP is demonstrated to outperform TP alone in studies including in vitro cellular receptor activation, stability in plasma, pharmacokinetics, and performance in vivo in rats. Specifically, ODDA-TP had an elimination half-life 20 times that of TP alone while exhibiting a superior safety profile.

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 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. ### Competing Interest Statement P.K., R.M.B. and M.M.S. are founders and shareholders in Estuar Pharmaceuticals. L.U. has been offered vesting shares in Estuar Pharmaceuticals.

Vasopressin (AVP) and oxytocin (OT) receptors (nomenclature as recommended by NC-IUPHAR [94]) are activated by the endogenous cyclic nonapeptides vasopressin and oxytocin. These peptides are derived from precursors which also produce neurophysins (neurophysin I for oxytocin; neurophysin II for vasopressin). Vasopressin and oxytocin differ at only 2 amino acids (positions 3 and 8). There are metabolites of these neuropeptides that may be biologically active [69].

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2) are related peptides, which are potent vasodilators. The receptors for these peptides are unusual, comprising a G-protein-coupled receptor (GPCR) with receptor activity-modifying proteins (RAMPs). The GPCR, the calcitonin receptor-like receptor (CLR), forms a CGRP receptor with RAMP1, (the CGRPR), whereas CLR with RAMP2 or RAMP3 forms two subtypes of adrenomedullin receptor (AM1 and AM2 receptors). CGRP is also a potent agonist at the AMY1(a) receptor, a heterodimer between the calcitonin receptor (CTR) and RAMP1. This article covers the pharmacology of these receptors, their structure, distribution, and potential involvement in disease.

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N ',N '-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a 'toolkit' of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.

The fundamental importance of membrane proteins in cellular processes has driven a marked increase in the use of membrane mimetic approaches for studying and exploiting these proteins. Nano-encapsulation strategies which preserve the native lipid bilayer environment are particularly attractive. Consequently, the use of poly(styrene co-maleic acid) (SMA) has been widely adopted to solubilise proteins directly from cell membranes by spontaneously forming "SMA Lipid Particles" (SMALPs). G-protein-coupled receptors (GPCRs) are ubiquitous "chemical switches", are central to cell signalling throughout the evolutionary tree, form the largest family of membrane proteins in humans and are a major drug discovery target. GPCR-SMALPs that retain binding capability would be a versatile platform for a wide range of down-stream applications. Here, using the adenosine A2A receptor (A2AR) as an archetypical GPCR, we show for the first time the utility of fluorescence correlation spectroscopy (FCS) to characterise the binding capability of GPCRs following nano-encapsulation. Unbound fluorescent ligand CA200645 exhibited a monophasic autocorrelation curve (dwell time, τD = 68 ± 2 μs; diffusion coefficient, D = 287 ± 15 μm2 s-1). In the presence of A2AR-SMALP, bound ligand was also evident (τD = 625 ± 23 μs; D = 30 ± 4 μm2 s-1). Using a non-receptor control (ZipA-SMALP) plus competition binding confirmed that this slower component represented binding to the encapsulated A2AR. Consequently, the combination of GPCR-SMALP and FCS is an effective platform for the quantitative real-time characterisation of nano-encapsulated receptors, with single molecule sensitivity, that will have widespread utility for future exploitation of GPCR-SMALPs in general.

The adenosine 2A receptor (A(2A)R), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A(2A)R-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A(2A)R-SMALP encapsulated native lipids. The fluorescence spectrum of the A(2A)R-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A(2A)R-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp246(6.48) in TM6 and Trp268(7.33) at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 231(6.33)) to report on the dynamic cytoplasmic face of the A(2A)R. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A(2A)R and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.