Biology subjects

Rufer, A. C.

Publications and source records attributed to Rufer, A. C..

3 recordsLinked to original sources

bioRxiv2024

CellEKT: A robust chemical proteomics workflow to profile cellular target engagement of kinase inhibitors

The human genome encodes 518 protein kinases that are pivotal for drug discovery in various therapeutic areas such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, making it difficult to develop selective inhibitors. To characterize and prioritize kinase-inhibiting drug candidates, efficient methods are desired to determine target engagement across the cellular kinome. In this study, we present CellEKT (Cellular Endogenous Kinase Targeting), an optimized and robust chemical proteomics platform for investigating cellular target engagement of endogenously expressed kinases using the sulfonyl fluoride-based probe XO44 and two new probes ALX005 and ALX011. The optimized workflow enabled the determination of the kinome interaction landscape of covalent and non-covalent drugs across over 300 kinases, expressed as half maximum inhibitory concentration (IC50), which were validated using distinct platforms like phosphoproteomics and NanoBRET. With CellEKT, target engagement profiles were linked to their substrate space. CellEKT has the ability to decrypt drug actions and to guide the discovery and development of drugs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/616061v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@5dd891org.highwire.dtl.DTLVardef@1353379org.highwire.dtl.DTLVardef@1c67382org.highwire.dtl.DTLVardef@1c964eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

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↗

bioRxiv2020

Diverse chemotypes drive biased signaling by cannabinoid receptors

Cannabinoid CB1 and CB2 receptors are members of the G protein-coupled receptor family, which is the largest class of membrane proteins in the human genome. As part of the endocannabinoid system, they have many regulatory functions in the human body. Their malfunction therefore triggers a diverse set of undesired conditions, such as pain, neuropathy, nephropathy, pruritus, osteoporosis, cachexia and Alzheimers disease. Although drugs targeting the system exist, the molecular and functional mechanisms involved are still poorly understood, preventing the development of better therapeutics with fewer undesired effects. One path toward the development of better and safer medicines targeting cannabinoid receptors relies on the ability of some compounds to activate a subset of pathways engaged by the receptor while sparing or even inhibiting the others, a phenomenon known as biased signaling. To take advantage of this phenomenon for drug development, a better profiling of the pathways engaged by the receptors is required. Using a BRET-based signaling detection platform, we systematically analyzed the primary signaling cascades activated by CB1 and CB2 receptors, including 9 G protein and 2 {beta}-arrestin subtypes. Given that biased signaling is driven by ligand-specific distinct active conformations of the receptor, establishing a link between the signaling profiles elicited by different drugs and their chemotypes may help designing compounds that selectively activate beneficial pathways while avoiding those leading to undesired effects. We screened a selection of 35 structurally diverse ligands, including endocannabinoids, phytocannabinoids and synthetic compounds structurally similar or significantly different from natural cannabinoids. Our data show that biased signaling is a prominent feature of the cannabinoid receptor system and that, as predicted, ligands with different chemotypes have distinct signaling profiles. The study therefore allows for better understanding of cannabinoid receptors signaling and provides the information about tool compounds that can now be used to link signaling pathways to biological outcomes, aiding the design of improved therapeutics.

pharmacology and toxicology↗