Biology subjects

Veprintsev, D.

Publications and source records attributed to Veprintsev, D..

7 recordsLinked to original sources

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

Multiple triggers converge to preferential effector coupling in the CB2R through a complex allosteric communication network

G protein-coupled receptors are important therapeutic drug targets for a wide range of diseases. Their ability to preferentially engage specific signaling pathways over others can be exploited to design drugs that target only disease-associated pathways leading to an improved safety profile. However, the underlying molecular mechanisms for preferential pathway engagement are complex and remain largely elusive. To elucidate the multifaceted actions at the receptor level that lead to preferential coupling, we employ a combination of techniques. Our approach integrates systematic mutagenesis of the CB2R and comprehensive profiling of Gi2 and {beta}-arrestin1 engagements with computer simulations to track mutant-induced impacts on receptor dynamics. Most importantly, our research discloses multiple triggers on a complex allosteric communication network (ACN) that converge to preferential CB2R coupling by modulating evolutionary conserved motifs (e.g., CWxP, NPxxY, sodium binding site). Potent triggers for a preferential Gi2 response exhibit high levels of connectivity and are located in proximity to connections with high information transmission. Our insights highlight the complexity of GPCR signaling and can guide the rational design of drug candidates tailored to evoke specific functional responses that can enhance the precision and efficacy of therapeutic interventions.

cell biology↗

bioRxiv2024

Probing drug-target engagement of soluble Gαi1 protein using the SolThermoBRET thermal shift assay

IntroductionSensitive protein stability assays used during hit confirmation conventionally require high quantities of purified protein. Here, we describe a novel high-throughput 384-well BRET-based thermostability assay allowing for the ultrasensitive determination of Gi1 protein stability. Using this method we make use of the environmentally sensitive dye, SYPRO Red, which interacts with hydrophobic regions in the protein that become exposed upon denaturation. Assays are functional in crude cell lysates, without any requirement for protein purification. MethodsThe SolThermoBRET method measures resonance energy transfer between a thermally stabilised nano-luciferase (tsNLuc) genetically engineered to the N-terminus of Gi1 protein, and SYPRO Red, a fluorescent dye that binds to lipophilic residues exposed upon protein unfolding in response to thermal denaturation. Samples in 384-well PCR plate were subjected to a temperature gradient (30 - 60 {degrees}C) on a PCR block and incubated for 5 minutes. Isothermal SolThermoBRET at a fixed temperature 45{degrees}C allowed the measurement of IC50 curves to various ligands. Furimazine (10 M) was added to the samples and BRET read on the BMG Labtech PHERAstar FSX at room temperature. Melt curves were fit to a Boltzmann sigmoidal equation to obtain Tm values. ResultsLigand-induced stabilisation of Gi1 protein was demonstrated in non-purified samples. A range of Tm values were measured with increasing stabilisation observed for nucleotides/sides in the following order GDP<GTP<GppNHp<GTP{gamma}S. Correlation was observed between SolThermoBRET derived Tms determined in the presence of high ligand concentrations and IC50 determinations using Isothermal SolThermoBRET. ConclusionsSolThermoBRET represents a sensitive nanoscale method for the detection of changes in soluble protein thermostability. This method is attractive for hit conformation and the identification of novel ligands targeting soluble proteins.

biophysics↗

bioRxiv2024

Agonist efficacy at the B2AR is driven by agonist-induced differences in receptor affinity for the Gs protein, not ligand binding kinetics.

IntroductionThe {beta}2-adrenoceptor ({beta}2AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma, whereby {beta}2AR agonists relieve bronchoconstriction. The {beta}2AR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the {beta}2AR is not understood. We hypothesized that the kinetics of ligand binding and GPCR-G protein interactions could play a role in ligand efficacy. We characterised the molecular pharmacology of a range of {beta}2AR agonists and examined the correlation between ligand and mini-Gs binding kinetics and efficacy. MethodsWe used a Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) based competition ligand binding assay to measure the affinity and residence times of a range of {beta}2AR agonists binding to the human {beta}2AR. TR-FRET between Lumi4-Tb3+ on the N terminus of the {beta}2AR and fluorescent CA200693 (S)-propranolol-green was measured using a PHERAstar FSX. The ability of these {beta}2AR agonists to activate the heterotrimeric Gs protein was measured using the CASE Gs protein biosensor. This assay senses a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the G subunit and Venus acceptor on the G{psi}, on receptor activation, quantified using the operational model of agonism. NanoBRET was also used to measure interactions between DDM solubilised {beta}2AR-nLuc and purified Venus-mini-Gs. A large excess of unlabelled mini-Gs was used to dissociate the {beta}2AR-nLuc: Venus-mini-Gs complex. ResultsCharacterisation of the molecular pharmacology of seven {beta}2AR agonists showed a broad range of ligand binding affinities (pKi = 4.4 {+/-} 0.09 to 9.2 {+/-} 0.08) and kinetics parameters. There was no correlation between ligand residence times and their ability (log{tau} ) to activate the Gs protein (R2=0.26, p=0.29). However, there were statistically significant differences in the association rate (kon (fast)) (3.36{+/-}0.64x105 to 9.19{+/-} 0.42x105) and affinity (Kd) values of mini-Gs binding to the agonist-{beta}2AR complex (pKd =6.0 to 6.7). Both an increase in ligand driven mini-Gs kon(fast) rate and associated increase in mini-Gs pKd for the receptor, were moderately correlated with efficacy (log{tau} ) (R2 =0.58 and R2 =0.50 respectively). ConclusionsThese data support a model in which agonists of increased efficacy cause the {beta}2AR to adopt a conformation that is more likely to recruit G protein. Conversely, these data did not support a role for agonist binding kinetics in the molecular basis of efficacy.

pharmacology and toxicology↗

bioRxiv2021

Vasopressin V2 is a promiscuous G protein-coupled receptor that is biased by its peptide ligands

Activation of the G protein-coupled receptors by agonists may result in the activation of one or more G proteins, and in the recruitment of arrestins. The balance of activation of different pathways can be influenced by the ligand. Using BRET-based biosensors, we showed that the vasopressin V2 receptor activates or at least engages many different G proteins across all G protein subfamilies in response to its native agonist arginine vasopressin (AVP). This includes members of the Gi/o and G12/13 families that have not been previously reported. These signalling pathways are also activated by the synthetic peptide desmopressin and natural homologs of AVP, namely oxytocin and the non-mammalian hormone vasotocin. They demonstrated varying degrees of functional selectivity relative to AVP, as quantified using the operational model for quantifying ligand bias. Additionally, we modelled G protein activation as a Michaelis-Menten reaction. This approach provided a complementary way to quantify signalling bias, with an added benefit of clear separation of the effects of ligand affinity from the intrinsic activity of the receptor. These results showed that V2 receptor is not only promiscuous in its ability to engage several G proteins, but also that its signalling profile could be easily biased by small structural changes in the ligand.

pharmacology and toxicology↗

bioRxiv2020

Measurement of non-purified GPCR thermostability using the homogenous ThermoBRET assay

Measurements of membrane protein thermostability allows indirect detection of ligand binding. Current thermostability assays require protein purification or rely on pre-existing radiolabelled or fluorescent ligands, limiting their application to established target proteins. Alternative methods detect protein aggregation which requires sufficiently high level of protein expression. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptors (CB1 and CB2) and the {beta}2-adrenoceptor ({beta}2AR) as model systems. ThermoBRET reports receptor unfolding, does not need labelled ligands and can be used with non-purified proteins. It uses Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol-reactive fluorescent dye that binds cysteines exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc-fused GPCRs can be determined in non-purified detergent solubilised membrane preparations or solubilised whole cells, revealing differences in thermostability for different solubilising conditions and in the presence of stabilising ligands. We extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split-Nluc (Tm of 87 C vs 59 C). ThermoBRET allows determination of GPCR thermostability, which is useful for protein purification optimisation and as part of drug discovery screening strategies.

biochemistry↗

bioRxiv2017

A combinatorial approach for the synthesis of multi-phosphorylated peptides: new tool for studying phosphorylation patterns in proteins

Phosphorylation of proteins at multiple sites creates different phosphorylation patterns that are essential for their biological activity. For example, such patterns contribute to the redirection of signalling to alternative pathways. Multi-phosphorylated peptides are excellent tools to systematically study the impact of unique phosphorylation patterns on signalling, but their synthesis is extremely difficult. Here we present an efficient and general method for the synthesis of multi-phosphorylated peptides, using a combination of different tailor-made coupling conditions. The method was demonstrated for the synthesis of a library of Rhodopsin C terminal peptides with distinct phosphorylation patterns containing up to seven phosphorylated Ser (pSer) and Thr (pThr) residues in close proximity to one another. Our method can be used to synthesize peptides incorporating multiple phosphorylated amino acids at high efficiency. It does not require any special expertise and can be performed in any standard peptide laboratory. This approach opens the way for quantitative mechanistic studies of phosphorylation patterns and their biological roles.

biochemistry↗