Biology subjects

Koers, E. J.

Publications and source records attributed to Koers, E. J..

7 recordsLinked to original sources

bioRxiv2024

A universal cannabinoid CB1 and CB2 receptor TR-FRET kinetic ligand binding assay.

INTRODUCTIONThe kinetics of ligand binding to G protein-coupled receptors (GPCRs) is an important determining factor in the preclinical evaluation of a molecule. Therefore, efforts should be made to measure this property as part of any drug development plan. The original assays used to assess ligand binding kinetics were developed using radioligands. However, these types of assays are very labor-intensive, limiting their application to the later phases of the drug discovery process. Recently, fluorescence-based ligand binding assays have been developed for multiple GPCRs, demonstrating their superiority through a homogeneous format and continuous data acquisition capabilities. The overriding aim of this study was to develop a fluorescence-based homogeneous ligand binding assay to profile the kinetics of compounds binding to human cannabinoid type 1 and 2 receptors (CB1R and CB2R). METHODSWe designed and synthesized D77, a novel universal tracer based on the lower affinity non-selective naturally occurring psychoactive cannabinoid, {Delta}8-THC. Using the TR-FRET (time-resolved Forster resonance energy transfer) technique to develop an assay to study the kinetics of ligand binding to CB1R and CB2R at physiological temperature. To establish a CB1R construct suitable for this assay, it was necessary to truncate the first 90 amino acids of the flexible CB1R N-terminal domain, in order to reduce the FRET distance between the terbium cryptate (donor) and the fluorescent ligand (acceptor), while the full length CB2R construct remained functional due to its shorter N-terminus. We then used the Motulsky-Mahan competition binding model to study the binding kinetics of non-fluorescent ligands. RESULTSD77 tracer displayed affinity for the truncated human CB1R (CB1R91-472) and full length CB2R (CB2R1-360) in the nanomolar range, and competitive binding behavior with orthosteric ligands. Crucially, D77 displayed fast dissociation kinetics from both CB1R and CB2R, comparable to those of the most rapidly dissociating reference compounds tested. This unique property of D77 proved pivotal to accurately determining the on- and off-rates of the fastest dissociating compounds. Using D77, we successfully determined the kinetic binding properties of a series of CB1R and CB2R agonists and antagonists at 37{degrees}C, including rimonabant, which was marketed for the treatment of obesity but later withdrawn due to serious neurological side effects. DISCUSSIONThe kon values of molecules binding CB1R showed a difference of three orders of magnitude from the slowest associating compound, HU308 to the most rapid, rimonabant. Interestingly, we found a strong correlation between kon and affinity for compounds binding to CB1R, suggesting that the association rate is the main parameter determining the affinity of compounds binding to CB1R. For compounds binding to CB2R, both kon and koff parameters contributed as affinity determinants. However, in contrast to CB1R, a stronger correlation was found between the dissociation constant rate parameter and the affinity of these molecules, suggesting that a combination of kon and koff dictates the overall affinity of compounds binding to CB2R. Ultimately, exploring the kinetic parameters of potential cannabinoid drug candidates could help future drug development programs targeting these receptors.

pharmacology and toxicology↗

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↗

bioRxiv2023

Molecular recognition of an aversive odorant by the murine trace amine-associated receptor TAAR7f

There are two main families of G protein-coupled receptors that detect odours in humans, the odorant receptors (ORs) and the trace amine-associated receptors (TAARs). Their amino acid sequences are distinct, with the TAARs being most similar to the aminergic receptors such as those activated by adrenaline, serotonin and histamine. To elucidate the structural determinants of ligand recognition by TAARs, we have determined the cryo-EM structure of a murine receptor, mTAAR7f, coupled to the heterotrimeric G protein Gs and bound to the odorant N,N-dimethylcyclohexylamine (DMCH) to an overall resolution of 2.9 [A]. DMCH is bound in a hydrophobic orthosteric binding site primarily through van der Waals interactions and a strong charge-charge interaction between the tertiary amine of the ligand and an aspartic acid residue. This site is distinct and non-overlapping with the binding site for the odorant propionate in the odorant receptor OR51E2. The structure, in combination with mutagenesis data and molecular dynamics simulations suggests that the activation of the receptor follows a similar pathway to that of the {beta}-adrenoceptors, with the significant difference that DMCH interacts directly with one of the main activation microswitch residues.

molecular biology↗

bioRxiv2021

Individual cells traffic the Vasopressin 2 Receptor to their cell surface with different success

G protein coupled receptors (GPCRs) translate the actions of hormones into intracellular signalling events. Mutations in GPCRs can prevent their correct expression and trafficking to the cell surface and cause disease. We use single cell measurements in HEK293 cells to show that the balance between endoplasmic reticulum (ER) and cell surface localisation of the Vasopressin 2 receptor (V2R) varies significantly from cell to cell. We find that mutations in the V2R affect the proportion of cells able to send this GPCR to the cell surface but do not prevent all cells in the population from correctly trafficking the mutant receptors. These findings reveal that the ability of cells to correctly traffic V2R to the cell surface depends not only on the expressed V2R mutant but also on the individual cell environment. Significance statementMissense mutations in the Vasopressin 2 Receptor (V2R) cause Nephrogenic Diabetes Insipidus. Some of these mutations prevent correct expression and trafficking of V2R to the cell surface resulting in a loss-of-function. We show -using single cell measurements-that the balance between endoplasmic reticulum and cell surface localisation of the V2R varies significantly from cell to cell, independent from its expression level. Mutations affect the proportion of cells able to send V2R to the cell surface but do not prevent all cells in the population from correctly trafficking the mutant receptors. Hence, the ability of cells to correctly traffic V2R to the cell surface depends not only on expressed V2R mutant but also on the cell environment.

molecular biology↗

bioRxiv2021

Direct observation of Hsp90-induced compaction in a protein chain

The chaperone Hsp90 is well known to undergo important conformational changes, which depend on nucleotide, co-chaperones, substrate interactions and post-translational modifications. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally, yet central to its function. Here, using optical tweezers and luciferase and glucocorticoid receptor substrates, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable kinases and receptor clients, regulates their activity, and how Hsp90 cooperates with Hsp70.

biophysics↗

bioRxiv2021

Prediction of ligand-receptor pharmacological activities using a combined docking and machine learning approach

G protein coupled receptors (GPCRs) are valuable therapeutic targets for many diseases. A central question of GPCR drug discovery is to understand what determines the agonism or antagonism of ligands which bind them. Ligands exert their action via the interactions in the ligand binding pocket. We hypothesised that there is a common set of receptor interactions made by ligands of diverse structures that mediate their action and that among a large dataset of different ligands, the functionally important interactions will be over-represented. We computationally docked ~2700 known {beta}2AR ligands to multiple {beta}2AR structures, generating ca 75,000 docking poses and predicted all atomic interactions between the receptor and the ligand. We used machine learning (ML) techniques to identify specific interactions that correlate with the agonist or antagonist activity of these ligands. The interpretation of ML analysis in human understandable form allowed us to construct an exquisitely detailed structure-activity relationship that identifies small changes to the ligands that invert their activity and thus helps to guide the drug discovery process. This approach can be readily applied to any drug target.

bioinformatics↗

bioRxiv2020

Polypeptide collapse modulation and folding stimulation by GroEL-ES

Unfolded proteins ubiquitously collapse into a compact yet dynamic state1,2. While this compaction is pivotal to protein folding3, aggregation4,5, intrinsic disorder6, and phase separation7, its role in protein quality control mechanisms remains obscure8. Collapse has been characterized mainly for polypeptides that are free in solution, in terms of kinetics, chain expansion, and effect on folding9,10. Yet, theory suggests that the solvent-mediated forces driving collapse can be altered near hydrophobic and charged surfaces, which are observed for many proteins including GroEL-ES11,12. Notably, while GroEL-ES is the archetypal protein-folding chaperone, its folding mechanism remains unresolved13,14. GroEL-ES is proposed to sterically confine polypeptides within its closed chamber15, unfold misfolded states16,17, or promote folding indirectly by suppressing aggregation18,19. Here, using integrated protein manipulation and imaging, we show that GroEL-ES can strengthen the collapse of polypeptide substrates, and hence stimulate folding directly. Strikingly, attractive forces pull substrate chains into the open GroEL cavity -unclosed by GroES-, and hence trigger a gradual compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL, and is aided by GroES binding to the cavity rim, and by the amphiphilic C-terminal tails at the cavity bottom. Peptides corresponding to these C-termini alone are sufficient to strengthen the collapse. The results show a mechanism that allows folding to be stimulated: by strengthening the collapse, residues are brought together that must contact to fold. The notion that one protein can modulate the collapse of another may be generally important in protein conformation and coacervation control, for systems ranging from the GroEL-ES homologue TRiC/CCT20, to the oncogenic c-Myc/Max complex21, and the nuclear pore transporter transportin22.

biophysics↗