ORCID Profile
0000-0002-3583-5409
Current Organisation
University of Nottingham
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Publisher: American Chemical Society (ACS)
Date: 22-11-2019
DOI: 10.26434/CHEMRXIV.10283027.V1
Abstract: Cannabinoid type 2 receptor (CB 2 R) is a fundamental part of the endocannabinoid signaling system (eCB system), and is known to play an important role in tissue injury, inflammation, cancer and pain. In stark contrast to its significance, the underlying signaling mechanisms and tissue expression profiles are poorly understood. Due to its low expression in healthy tissue and lack of reliable chemical tools, CB 2 R visualization in live cells remains uncharted. Here we report the development of a drug derived toolbox of highly potent, CB 2 R-selective fluorescent probes based on reverse design. Extensive validation in several applications such as CB 2 R detection in flow cytometry and time-resolved imaging, and the development of a novel fluorescent-based TR-FRET assay to generate kinetic and equilibrium binding data demonstrate the high versatility of our toolbox. These probes are the first to preserve affinity and efficacy in both human and mouse CB 2 R, a crucial aspect for preclinical translatability, and to enable imaging of CB2R internalization in living cells using confocal microscopy.
Publisher: Cold Spring Harbor Laboratory
Date: 29-06-2020
DOI: 10.1101/2020.06.29.171512
Abstract: The β2-adrenoceptor (β2AR) is a well-established target in asthma and a prototypical GPCR for biophysical studies. Solubilisation of membrane proteins has classically involved the use of detergents. However, the detergent environment differs from the native membrane environment and often destabilises membrane proteins. Use of hiphilic copolymers is a promising strategy to solubilise membrane proteins within their native lipid environment in the complete absence of detergents. Here we show the isolation of the β 2 AR in the polymer Diisobutylene maleic acid (DIBMA). We demonstrate that β 2 AR remains functional in the DIBMA lipid particle (DIBMALP) and shows improved thermal stability compared to the n-Dodecyl-β-D-Maltopyranoside (DDM) detergent solubilised β 2 AR. This unique method of extracting β 2 AR offers significant advantages over previous methods routinely employed such as the introduction of thermostabilising mutations and the use of detergents, particularly for functional biophysical studies.
Publisher: Cold Spring Harbor Laboratory
Date: 06-08-2020
DOI: 10.1101/2020.08.05.237982
Abstract: 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 (CB 1 and CB 2 ) and the β 2 -adrenoceptor (β 2 AR) 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 (T m ) 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 ( T m of 87 ⁰C vs 59 ⁰C). ThermoBRET allows determination of GPCR thermostability, which is useful for protein purification optimisation and as part of drug discovery screening strategies.
Publisher: Cold Spring Harbor Laboratory
Date: 08-07-2020
DOI: 10.1101/2020.07.07.191957
Abstract: Sensitive protein stability assays for membrane proteins are crucial for developing purification protocols, for structural and biophysical characterisation and drug discovery. Here, we describe a novel high-throughput 384-well FRET-based thermostability methodology, ThermoFRET, allowing for the ultrasensitive determination of G protein coupled receptor (GPCR) stability. This method measures FRET between a terbium-cryptate labelled GPCR and BODIPY-FL-Cystine, a thiolreactive dye that reacts with cysteine residues exposed upon protein unfolding in response to thermal denaturation. ThermoFRET is functional in crude solubilised membrane preparations, without protein purification and can detect receptor stabilising ligands, making it ideally suited for orphan receptor screening.
Publisher: Proceedings of the National Academy of Sciences
Date: 15-05-2007
Abstract: Proteins are involved in various equilibria that play a major role in their activity or regulation. The design of molecules that shift such equilibria is of great therapeutic potential. This fact was demonstrated in the cases of allosteric inhibitors, which shift the equilibrium between active and inactive (R and T) states, and chemical chaperones, which shift folding equilibrium of proteins. Here, we expand these concepts and propose the shifting of oligomerization equilibrium of proteins as a general methodology for drug design. We present a strategy for inhibiting proteins by “shiftides”: ligands that specifically bind to an inactive oligomeric state of a disease-related protein and modulate its activity by shifting the oligomerization equilibrium of the protein toward it. We demonstrate the feasibility of our approach for the inhibition of the HIV-1 integrase (IN) protein by using peptides derived from its cellular-binding protein, LEDGF 75, which specifically inhibit IN activity by a noncompetitive mechanism. The peptides inhibit the DNA-binding of IN by shifting the IN oligomerization equilibrium from the active dimer toward the inactive tetramer, which is unable to catalyze the first integration step of 3′ end processing. The LEDGF 75-derived peptides inhibit the enzymatic activity of IN in vitro and consequently block HIV-1 replication in cells because of the lack of integration. These peptides are promising anti-HIV lead compounds that modulate oligomerization of IN via a previously uncharacterized mechanism, which bears advantages over the conventional interface dimerization inhibitors.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Dmitry Veprintsev.