bioRxiv - Pharmacology and Toxicology最新文献

{"title":"scRNA-seq identifies unique macrophage population in murine model of ozone induced asthma exacerbation","authors":"Jess L Ray, Joshua Walum, Daria Jelic, Ryelie Barnes, Ian D Bentley, Rodney J Britt, Joshua Englert, Megan N Ballinger","doi":"10.1101/2024.07.23.604740","DOIUrl":"https://doi.org/10.1101/2024.07.23.604740","url":null,"abstract":"Ozone (O3) inhalation triggers asthmatic airway hyperresponsiveness (AHR), but the mechanisms by which this occurs are unknown. Previously, we developed a murine model of dust mite, ragweed, and aspergillus (DRA)-induced allergic lung inflammation followed by O3 exposure for mechanistic investigation. The present study used single cell RNA-sequencing for unbiased profiling of immune cells within the lungs of mice exposed to DRA, O3, or DRA+O3, to identify the components of the immune cell niche that contribute to AHR. Alveolar macrophages (AMs) had the greatest number of differentially expressed genes following DRA+O3, most of which were unique to the 2-hit exposure. Following DRA+O3, AMs activated transcriptional pathways related to cholesterol biosynthesis, degradation of the extracellular matrix, endosomal TLR processing, and various cytokine signals. We also identified AM and monocyte subset populations that were unique to the DRA+O3 group. These unique AMs activated gene pathways related to inflammation, sphingolipid metabolism, and bronchial constriction. The unique monocyte population had a gene signature that suggested phospholipase activation and increased degradation of the extracellular matrix. Flow cytometry analysis of BAL immune cells showed recruited monocyte-derived AMs after DRA and DRA+O3, but not after O3 exposure alone. O3 alone increased BAL neutrophils but this response was attenuated in DRA+O3 mice. DRA-induced changes in the airspace immune cell profile were reflected in elevated BAL cytokine/chemokine levels following DRA+O3 compared to O3 alone. The present work highlights the role of monocytes and AMs in the response to O3 and suggests that the presence of distinct subpopulations following allergic inflammation may contribute to O3-induced AHR.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"354 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141781660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Suite of Biochemical and Cell-Based Assays for the Characterization of KRAS Inhibitors and Degraders","authors":"Medhanie Kidane, Rene M. Hoffman, Jennifer K. Wolfe-Demarco, Ting-Yu Huang, Chi-Ling Teng, Luis M. Gonzalez Lira, Jennifer Lin-Jones, Gabriel Pallares, Jane E. Lamerdin, Nicole B. Servant, Chun-Yao Lee, Chao-Tsung Yang, Jean A. Bernatchez","doi":"10.1101/2024.07.20.604418","DOIUrl":"https://doi.org/10.1101/2024.07.20.604418","url":null,"abstract":"KRAS is an important oncogenic driver which is mutated in numerous cancers. Recent advances in the selective targeting of KRAS mutants via small molecule inhibitors and targeted protein degraders have generated an increase in research activity in this area in recent years. As such, there is a need for new assay platforms to profile next generation inhibitors which improve on the potency and selectivity of existing drug candidates, while evading the emergence of resistance. Here, we describe the development of a new panel of biochemical and cell-based assays to evaluate the binding and function of known chemical entities targeting mutant KRAS. Our assay panels generated selectivity profiles and quantitative binding interaction dissociation constants for small molecules and degraders against wild type, G12C, G12D, and G12V KRAS, which were congruent with published data. These assays can be leveraged for additional mutants of interest beyond those described in this study, using both overexpressed cell-free systems and cell-based systems with endogenous protein levels.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141781661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Molecular Image-based Explainable Artificial intelligence Framework Identifies Novel Candidate Antibiotics","authors":"Kingsten Lin, Yuxin Yang, Feixiong Cheng","doi":"10.1101/2024.07.21.604494","DOIUrl":"https://doi.org/10.1101/2024.07.21.604494","url":null,"abstract":"The continued growth of antibiotic resistance and the slowing of antibiotic discovery poses a large challenge in fighting infectious diseases. Recent advances in Artificial intelligence (AI) technologies offer a time- and cost-effective solution for the rapid development of effective antibiotics. In this study, we presented an explainable AI framework from a pre-trained model using 10 million drug-like molecular images. Specifically, we created a fine-tuned ImageMol from experimental Staphylococcus aureus inhibition assays which contained 24,521 molecules consisting of 516 active compounds and 24,005 non-active compounds. Our optimized AI model achieved a strong AUROC of 0.926. The model was then used to predict the antibiotic activities from 10,247 FDA-approved, clinically investigational, or experimental molecules from the DrugBank database. After further filtering, 340 molecules were identified to have antibacterial behavior while simultaneously being dissimilar to known antibiotics. Finally, 76 candidates were identified as FDA-approved drugs for other applications. Thus, those candidates can be repurposed into needed novel antibiotics. We further illustrated explainable molecular images for top predicted candidate drugs via Gradient-weighted Class Activation Mapping (Grad-CAM) heatmap analysis. In summary, the presented molecular image-based AI model in drug discovery could be highly favorable due to its high performance, speed, and biological interpretation.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141781662","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Network-based investigation of petroleum hydrocarbons-induced ecotoxicological effects and their risk assessment","authors":"Ajaya Kumar Sahoo, Shreyes Rajan Madgaonkar, Nikhil Chivukula, Panneerselvam Karthikeyan, Kundhanathan Ramesh, Shambanagouda Rudragouda Marigoudar, Krishna Venkatarama Sharma, Areejit Samal","doi":"10.1101/2024.07.18.604159","DOIUrl":"https://doi.org/10.1101/2024.07.18.604159","url":null,"abstract":"Petroleum hydrocarbons (PHs) are compounds composed mostly of carbon and hydrogen, originating from crude oil and its derivatives. PHs are primarily released into the environment through the diffusion of oils, resulting from anthropogenic activities like transportation and offshore drilling, and accidental incidents such as oil spills. Once released, these PHs can persist in different ecosystems and cause long-term detrimental ecological impacts. While the hazards associated with such PH contaminations are often assessed by the concentrations of total petroleum hydrocarbons in the environment, studies focusing on the risks associated with individual PHs are limited. Here, we leveraged different network-based frameworks to explore and understand the adverse ecological effects associated with PH exposure. First, we systematically curated a list of 320 PHs from published reports. Next, we integrated biological endpoint data from toxicological databases, and constructed a stressor-centric adverse outcome pathway (AOP) network linking 75 PHs with 177 ecotoxicologically-relevant high confidence AOPs within AOP-Wiki. Further, we relied on stressor-species network constructions, based on reported toxicity concentrations and bioconcentration factors data for 80 PHs and 28 PHs, respectively, and found that crustaceans are documented to be affected by many of these PHs. Finally, we utilized the aquatic toxicity data within ECOTOX to construct species sensitivity distributions for polycyclic aromatic hydrocarbons (PAHs) prioritized by the US EPA, and derived their corresponding hazard concentrations (HC05) that protect 95% of species in the aquatic ecosystem. Overall, this study highlights the importance of using network-based approaches and risk assessment methods to understand the PH-induced toxicities effectively.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141781666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exploring the Mechanism of Gentiana in Treating Pancreatic Cancer Based on Network Pharmacology and Molecular Docking Techniques","authors":"Yuanyuan Qian, Zhaojunli Wang, Jiancheng Ji","doi":"10.1101/2024.07.18.604197","DOIUrl":"https://doi.org/10.1101/2024.07.18.604197","url":null,"abstract":"Objective: This study aims to investigate the mechanism of Gentiana in treating pancreatic cancer using network pharmacology and molecular docking techniques.\u0000Methods: Active compounds of Gentiana were screened from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. The 3D structures of the active compounds were downloaded from the PubChem database. Reverse docking was performed using the PharmMapper database to identify potential target proteins. Differential gene expression data related to colorectal cancer were obtained from the Gene Expression Omnibus (GEO) database, and differentially expressed genes were selected. Venn diagram analysis was employed to identify common genes between the protein targets and differentially expressed genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool. Molecular docking was performed using ChemDraw 20.0, AutoDock, and PyMOL.\u0000Results: A total of 72 common genes and 15 signaling pathways were identified from the reverse docking data of Gentiana and the pancreatic cancer dataset (GSE196009). Molecular docking results demonstrated favorable binding energies between the active compounds of Gentiana and proteins 1og5, 1pq2, 2bxr, 2bk3, 1u3w, 1wma, 1wuu, 1tdi, 1mlw, 1egc, 1s1p, 1f12, 1m51, 1kqu, 1ls6, 1ry0, 1nhx, and 1db4.\u0000Conclusion: Gentiana may exert its therapeutic effects on pancreatic cancer through a multi-component, multi-target, and multi-pathway mechanism.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"35 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141781664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Validation of an MPS based intestinal cell culture model for the evaluation of drug-induced toxicity","authors":"Stefanie Hoffmann, Philip Hewitt, Isabel Koscielski, Dorota Kurek, Wouter Strijker, Kinga Kosim","doi":"10.1101/2024.07.18.604106","DOIUrl":"https://doi.org/10.1101/2024.07.18.604106","url":null,"abstract":"The potential for drug-induced gastrointestinal (GI) toxicity is significant, since the GI tract is one of the first barriers which come in to contact with oral drugs. In pharmaceutical research, the complex behavior of human intestinal cells is traditionally investigated using 2D cultures, in which one cell type grows under static conditions. With the development of advanced microphysiological systems (MPS) more in vivo like conditions can be generated which increase the physiological nature and also the predictive validity of these models. Caco-2 cells are known for their capability to build tight junctions. These connections are responsible for the maintenance of intestinal homeostasis and can be used as a specific safety endpoint, by measuring the Trans Epithelial Electrical Resistance (TEER), for the investigation of drug-induced GI toxicity. Compared to a widely used Caco-2 cell 2D Transwell model, the advanced MPS model (Mimetas OrganoPlate) allows for the recapitulation of the enterocyte cell layer of the intestinal barrier as the Caco-2 cells grow in a tubular structure through which the medium continuously flows. The OrganoPlate intestinal model was qualified to be implemented as a routine test system for the early prediction of drug-induced GI toxicity based on the measurement of the tightness of the cell layer by measuring changes in the TEER. For this qualification 23 well known compounds as well as a positive, negative and solvent control were selected. The compounds were selected based on their known effect on the GI system (chemotherapeutics, tight junction disruptor, liver toxins, controls, NSAIDs and a mixed group of drugs). The TEER values were measured 4h and 24h after treatment. In parallel the cell viability was determined after 24h to be able to distinguish between an unspecific cytotoxic effect or direct tight junction damage. Overall, from the 23 tested compounds, 15 showed the expected outcome, i.e.,the compound led to a decrease of the TEER for the positive control compounds, or the TEER value remained stable after treatment with non-GI-toxic compounds. In summary, this MPS model allowed the recapitulation of the human intestinal GI barrier and will enable a faster and more robust assessment of drug-induced damage in the GI tract.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"115 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141781699","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification and characterization of thiamine analogues with antiplasmodial activity","authors":"Imam Fathoni, Terence CS Ho, Alex HY Chan, Finian J Leeper, Kai Matuschewski, Kevin J Saliba","doi":"10.1101/2024.07.18.604204","DOIUrl":"https://doi.org/10.1101/2024.07.18.604204","url":null,"abstract":"Thiamine is metabolized into thiamine pyrophosphate (TPP), an essential enzyme cofactor. Previous work has shown that oxythiamine, a thiamine analogue, is metabolized by thiamine pyrophosphokinase (TPK) into oxythiamine pyrophosphate (OxPP) within the malaria parasite Plasmodium falciparum, and then inhibits TPP-dependent enzymes, killing the parasite in vitro and in vivo. To identify a more potent antiplasmodial thiamine analogue, 11 commercially available compounds were tested against P. falciparum and P. knowlesi. Five active compounds were identified, but only N3-pyridyl thiamine (N3PT), a potent transketolase inhibitor and candidate anticancer lead compound, was found to suppress P. falciparum proliferation with an IC50 value 10-fold lower than that of oxythiamine. N3PT was active against P. knowlesi and was >17 times less toxic to human fibroblast, as compared to oxythiamine. Increasing the extracellular thiamine concentration reduced the antiplasmodial activity of N3PT, consistent with N3PT competing with thiamine/TPP. A transgenic P. falciparum line overexpressing TPK was found to be hypersensitized to N3PT. Docking studies showed an almost identical binding mode in TPK between thiamine and N3PT. Furthermore, we show that [3H]thiamine accumulation, resulting from a combination of transport and metabolism, in isolated parasites is reduced by N3PT. Treatment of P. berghei-infected mice with 200 mg/kg/day N3PT reduced their parasitemia, prolonged their time to malaria symptoms, and appeared to be non-toxic to mice. Collectively, our studies are consistent with N3PT competing with thiamine for TPK binding and inhibiting parasite proliferation by reducing TPP production, as well as being converted into a TPP antimetabolite that inhibits TPP-dependent enzymes.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745195","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Prebiotic Supplementation Modulates the Gut Microbiome for Improving Oral Antipsychotic Bioavailability","authors":"Kate Collins, Srinivas Kamath, Tahlia Meola, Anthony Wignall, Paul Joyce","doi":"10.1101/2024.07.17.604016","DOIUrl":"https://doi.org/10.1101/2024.07.17.604016","url":null,"abstract":"This study investigates the impact of gut microbiome composition on the bioavailability and biodistribution of lurasidone hydrochloride, an atypical antipsychotic used in treating mental health conditions. The research aims to uncover the relationship between gut metabolome, specifically short-chain fatty acids (SCFAs), and lurasidone absorption through modulation of intestinal pH. Male Sprague-Dawley rats underwent a 14-day microbiome depletion using antibiotics or prebiotics administered via drinking water, resulting in depleted, enriched, or normal (control) microbiomes. Following this treatment, lurasidone was orally administered to fasted rats, and blood samples were collected at specific intervals to correlate pharmacokinetic analysis with changes in microbiota and metabolome composition. Results revealed a significant 4.3-fold increase in lurasidone bioavailability following prebiotic administration, while antibiotic treatment decreased bioavailability. This effect is attributed to prebiotics increasing microbial diversity, which strongly correlates with SCFA production (R2 = 0.93). The subsequent lowering of intestinal pH created a more suitable environment for lurasidone solubility and absorption. This novel study establishes a clear relationship between gut microbiome composition and lurasidone bioavailability, with prebiotic administration positively impacting both gut microbial diversity and drug absorption. Thus, these findings suggest that gut microbiome manipulation may present an innovative approach to improving therapeutic outcomes for antipsychotic medications, potentially addressing poor response rates in some patients. Furthermore, this research highlights the importance of considering gut microbiome composition, in particular the use of SCFAs as a biomarker to produce predictive models of drug pharmacokinetics, especially for pH-dependent soluble drugs. In the context of drug efficacy these gut microbiome-based predictive pharmacokinetic models will pave the path for personalised medicine approaches for mental health treatment.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"49 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effect of Voltage-Gated K+ Channel Inhibition by 4-aminopyridine in Spinal Cord Injury Recovery in Zebrafish","authors":"Payge Hoffman, Karen Mruk","doi":"10.1101/2024.07.15.603582","DOIUrl":"https://doi.org/10.1101/2024.07.15.603582","url":null,"abstract":"Spinal cord injury (SCI) affects between 250,000 to 500,000 individuals annually. After the initial injury, a delayed secondary cascade of cellular responses occurs causing progressive degeneration and permanent disability. One part of this secondary process is disturbance of ionic homeostasis. The K+ channel blocker, 4-aminopyridine (4-AP), is used clinically to alleviate symptoms of multiple sclerosis (MS). Several ongoing studies are being conducted to explore additional areas where 4-AP may have an effect, including stroke, traumatic brain injury, and nervous system recovery after SCI. The goal of our study was to determine whether 4-AP affects recovery from SCI in zebrafish (Danio rerio). Using the transgenic line Tg(gfap:EGFP), we created a spinal transection and tracked swim recovery. We found that constant treatment with 10 μM 4-AP increases swimming distance 40%. Live imaging demonstrated that treatment with 4-AP increases radial glial cells bridging at the site of injury in the presence of 4-AP. We conclude that 10 μM 4-AP is pro-regenerative after SCI.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"35 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A universal cannabinoid CB1 and CB2 receptor TR-FRET kinetic ligand binding assay.","authors":"Leire Borrega-Roman, Bradley L. Hoare, Miroslav Kosar, Roman C. Sarott, Kacper J. Patej, Jara Bouma, Morgan Scott-Dennis, Eline J. Koers, Thais Gazzi, Leonard Mach, Sergio Barrondo, Joan Salles, Wolfgang Guba, Eric Kusznir, Marc Nazare, Arne C. Rufer, Uwe Grether, Laura H. Heitman, Erick M. Carreira, David A. Sykes, Dmitry B. Veprintsev","doi":"10.1101/2024.07.16.603654","DOIUrl":"https://doi.org/10.1101/2024.07.16.603654","url":null,"abstract":"INTRODUCTION: The 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).\u0000METHODS: We designed and synthesized D77, a novel universal tracer based on the lower affinity non-selective naturally occurring psychoactive cannabinoid, delta8-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. RESULTS: D77 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. DISCUSSION: The 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 strong","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}