Determining the binding kinetics and signalling effects of CB1 and CB2 receptor ligandsa closer look at residence time and functional selectivity

Resumen

The CB1 and CB2 receptors are the main GPCRs of the endocannabinoid system, and regulate many important physiological processed as pain, inflammation, or behaviour. Increasing interest exist to develop new drugs targeting these receptors, as they constitute potential drug targets for many diseases as Alzheimer disease, Huntington disease, chronic pain, or inflammation.During the last decades, the interest around the binding kinetics of drugs has hugely expanded, as it has been shown to be an important determinant of in vivo drug action in many cases. Moreover, studying the kinetic interactions of drug binding to its target can also provide valuable information in order to understand the efficacy at a molecular level.In this work, we generated a novel fluorescence-based assay that allows the determination of binding association and dissociation rates of cannabinoid compounds at CB1 and CB2 receptors. Using themethodology proposed by Motulsky and Mahan, we successfully determined the kinetic parameters of seven different cannabinoid compounds at both CB1 and CB2 receptors.We also studied the efficacy of the selected cannabinoid compounds using three different assays. On one hand, we determined the coupling to miniGsi and b-arrestin 2 using BRET biosensors. On the other hand, we successfully determine the internalization that cannabinoid compounds elicited using a technique based on diffusion-enhanced resonance energy transfer (DERET). Applying the operational model of agonism (OMA) proposed by Black and Leff, the bias exerted by the compounds at both CB1 and CB2 receptors was determined for the studied responses. Finally, we explore the relationship between the efficacy elicited at the different responses with the residence time of the cannabinoid agonists. The CB1 and CB2 receptors are the main GPCRs of the endocannabinoid system, and regulate many important physiological processed as pain, inflammation, or behaviour. Increasing interest exist to develop new drugs targeting these receptors, as they constitute potential drug targets for many diseases as Alzheimer disease, Huntington disease, chronic pain, or inflammation.During the last decades, the interest around the binding kinetics of drugs has hugely expanded, as it has been shown to be an important determinant of in vivo drug action in many cases. Moreover, studying the kinetic interactions of drug binding to its target can also provide valuable information in order to understand the efficacy at a molecular level.In this work, we generated a novel fluorescence-based assay that allows the determination of binding association and dissociation rates of cannabinoid compounds at CB1 and CB2 receptors. Using themethodology proposed by Motulsky and Mahan, we successfully determined the kinetic parameters of seven different cannabinoid compounds at both CB1 and CB2 receptors.We also studied the efficacy of the selected cannabinoid compounds using three different assays. On one hand, we determined the coupling to miniGsi and b-arrestin 2 using BRET biosensors. On the other hand, we successfully determine the internalization that cannabinoid compounds elicited using a technique based on diffusion-enhanced resonance energy transfer (DERET). Applying the operational model of agonism (OMA) proposed by Black and Leff, the bias exerted by the compounds at both CB1 and CB2 receptors was determined for the studied responses. Finally, we explore the relationship between the efficacy elicited at the different responses with the residence time of the cannabinoid agonists.