Pyrimidinyl Biphenylureas Act as Allosteric Modulators to Activate Cannabinoid Receptor 1 and Initiate ß-Arrestin-Dependent Responses.

Cannabinoid receptor 1 (CB1) is a G-protein-coupled receptor that is abundant in the central nervous system. It binds several compounds in its orthosteric site, including the endocannabinoids, arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol, and the plant-derived Δ9-tetrahydrocannabinol, one of the main psychoactive components of marijuana. It primarily couples to Gi/o proteins to inhibit adenylate cyclase activity and typically induces downstream signaling that is Gi-dependent. Since this receptor is implicated in several maladies, such as obesity, pain, and neurodegenerative disorders, there is interest in developing therapeutics that selectively target this receptor. Allosteric modulators of CB1 offer one new approach that has tremendous therapeutic potential. Here, we reveal receptor- and cellular-level properties consistent with receptor activation by a series of pyrimidinyl biphenylureas (LDK1285, LDK1288, LDK1305, and PSNCBAM1), including promoting binding of the agonist CP55940 with positive cooperativity and inhibiting binding of the inverse agonist SR141716A with negative cooperativity, demonstrated via radioligand binding studies. Consistent with these findings, the allosteric modulators induced cellular internalization of the receptor and recruitment of ß-arrestin 2 in human embryonic kidney cell line 293 cells monitored with confocal and total internal reflective fluorescence microscopy, respectively. These allosteric modulators, however, caused G-protein-independent but ß-arrestin 1-dependent phosphorylation of the downstream kinases extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Src, shown by immunoblotting studies. These results are consistent with the involvement of ß-arrestin and suggest that these allosteric modulators induce biased signaling.

Extensive location bias of the GPCR-dependent translatome via site-selective activation of mTOR.

G protein-coupled receptors (GPCRs) modulate various physiological functions by re-wiring cellular gene expression in response to extracellular signals. Control of gene expression by GPCRs has been studied almost exclusively at the transcriptional level, neglecting an extensive amount of regulation that takes place translationally. Hence, little is known about the nature and mechanisms of gene-specific post-transcriptional regulation downstream of receptor activation. Here, we apply an unbiased multiomics approach to delineate an extensive translational regulatory program initiated by the prototypical beta2-adrenergic receptor (ß2-AR) and provide mechanistic insights into how these processes are orchestrated. Using ribosome profiling (Ribo-seq), we identify nearly 120 novel gene targets of adrenergic receptor activity which expression is exclusively regulated at the level of translation. We next show that all translational changes are induced selectively by endosomal ß2-ARs. We further report that this proceeds through activation of the mammalian target of rapamycin (mTOR) pathway. Specifically, within the set of translational GPCR targets we discover significant enrichment of genes with 5' terminal oligopyrimidine (TOP) motifs, a gene class classically known to be translationally regulated by mTOR. We then demonstrate that endosomal ß2-ARs are required for mTOR activation and subsequent mTOR-dependent TOP mRNA translation. Together, this comprehensive analysis of drug-induced translational regulation establishes a critical role for location-biased GPCR signaling in fine-tuning the cellular protein landscape.

Mechanistic insights on clearance and inhibition discordance between liver microsomes and hepatocytes when clearance in liver microsomes is higher than in hepatocytes.

Human liver microsomes (HLM) and human hepatocytes (HHEP) are two common in vitro systems used in metabolic stability and inhibition studies. The comparison between the assays using the two systems can provide mechanistic insights on the interplay of metabolism, passive permeability and transporters. This study investigated the critical factors impacting the unbound intrinsic clearance (CLint,u) and IC50 of CYP3A inhibition between HLM and HHEP. The HLM/HHEP CLint,u ratio and HHEP/HLM IC50 ratio are inversely correlated to passive permeability, but have no correlation with P-gp efflux ratio. Cofactor-supplemented permeabilized HHEP (MetMax™) collapses the IC50 differences between HHEP and HLM. P-gp inhibitor, encequidar, shows minimal impact on CLint,u and IC50 in HHEP. This is the first study that is able to separately investigate the effects of passive permeability and efflux transport. These data collectively show that passive permeability plays a critical role in metabolism and enzyme inhibition in HHEP, while P-gp efflux has a minor role. This may be due to low functional P-gp activity in suspension HHEP under the assay conditions. Low passive permeability may limit metabolism and enzyme inhibition in HHEP, leading to lower CLint,u and higher IC50 in HHEP compared to HLM. When liver microsomes give higher CLint,u than hepatocytes, microsomes are more predictive of in vivo clearance than hepatocytes.