Achieving ligand subtype selectivity within highly homologous subtypes of G-protein-coupled receptor (GPCR) is critical yet challenging for GPCR drug discovery, primarily due to the unclear mechanism underlying ligand subtype selectivity, which hampers the rational design of subtype-selective ligands. Herein, we disclose an unusual molecular mechanism of entropy-driven ligand recognition in cannabinoid (CB) receptor subtypes, revealed through atomic-level molecular dynamics simulations, cryoelectron microscopy structure, and mutagenesis experiments. This mechanism is attributed to the distinct conformational dynamics of the receptor\'s orthosteric pocket, leading to variations in ligand binding entropy and consequently, differential binding affinities, which culminate in specific ligand recognition. We experimentally validated this mechanism and leveraged it to design ligands with enhanced or ablated subtype selectivity. One such ligand demonstrated favorable pharmacokinetic properties and significant efficacy in rodent inflammatory analgesic models. More importantly, it is precisely due to the high subtype selectivity obtained based on this mechanism that this ligand does not show addictive properties in animal models. Our findings elucidate the unconventional role of entropy in CB receptor subtype selectivity and suggest a strategy for rational design of ligands to achieve entropy-driven subtype selectivity for many pharmaceutically important GPCRs.

Liver fibrosis is a condition characterized by aberrant proliferation of connective tissue in the liver resulting from diverse etiological factors. G protein-coupled receptor GPR55 has recently been identified as a regulator of liver diseases. Herein, we report the discovery of a cyclic peptide P1-1 that antagonizes GPR55 and suppresses collagen secretion in hepatic stellate cells. The alanine scanning and docking study was carried out to predict the binding mode and allowed for further structural optimization of peptide antagonists for GPR55. The subsequent in vivo study demonstrated that P1-1 ameliorates CCl4-induce and MCD-diet-induce acute liver inflammation and fibrosis. Further study indicates that P1-1 reduces reactive oxygen species (ROS) production, attenuates ER stress, and inhibits mitochondria-associated hepatocyte apoptosis. In this work, we provided the first successful example of antagonizing GPR55 for liver inflammation and fibrosis, which validates GPR55 as a promising target for the treatment of liver fibrosis and affords a high-potent GPR55 antagonist P1-1 as a potential therapeutic candidate.

Cannabis sativa has long been used for neurological and psychological healing. Recently, cannabidiol (CBD) extracted from cannabis sativa has gained prominence in the medical field due to its non-psychotropic therapeutic effects on the central and peripheral nervous systems. CBD, also acting as a potent antioxidant, displays diverse clinical properties such as anticancer, antiinflammatory, antidepressant, antioxidant, antiemetic, anxiolytic, antiepileptic, and antipsychotic effects. In this review, we summarized the structural activity relationship of CBD with different receptors by both experimental and computational techniques and investigated the mechanism of interaction between related receptors and CBD. The discovery of structural activity relationship between CBD and target receptors would provide a direction to optimize the scaffold of CBD and its derivatives, which would give potential medical applications on CBD-based therapies in various illnesses.

Cannabidiol (CBD) has antioxidant and anti-inflammatory activities. However, the anti-tumor effect of CBD on hepatocellular carcinoma (HCC) remains unclear. Here, we investigated whether CBD displays anti-tumorigenic effects in HCC cells and whether it could reduce tumorigenesis and metastases in vivo. First, this study treated HCC cells with different concentrations of CBD, followed by analyzing the changes in the proliferative, apoptotic, migratory and invasive abilities. The effects of CBD on the growth and metastasis of HCC cells in vivo were verified by tumorigenesis and metastasis assays. Subsequently, the target genes of CBD were predicted through the SwissTarget website and the genes differentially expressed in cells after CBD treatment were analyzed by microarray for intersection. The enrichment of the pathways after CBD treatment was analyzed by KEGG enrichment analysis, followed by western blot validation. Finally, rescue assays were used to validate the functions of genes as well as pathways in the growth and metastasis of HCC cells. A significant weakening of the ability of HCC cells to grow and metastasize in vitro and in vivo was observed upon CBD treatment. Mechanistically, CBD reduced GRP55 expression in HCC cells, along with increased TP53 expression and blocked MAPK signaling activation. In CBD-treated cells, the anti-tumor of HCC cells was restored after overexpression of GRP55 or deletion of TP53. CBD inhibits the MAPK signaling activation and increases the TP53 expression by downregulating GRP55 in HCC cells, thereby suppressing the growth and metastasis of HCC cells.

Icariin, a flavonoid glycoside, is extracted from Epimedium. This study aimed to investigate the vascular protective effects of icariin in type 1 diabetic rats by inhibiting high-mobility group box 1 (HMGB1)-related inflammation and exploring its potential mechanisms. The impact of icariin on vascular dysfunction was assessed in streptozotocin (STZ)-induced diabetic rats through vascular reactivity studies. Western blotting and immunofluorescence assays were performed to measure the expressions of target proteins. The release of HMGB1 and pro-inflammation cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The results revealed that icariin administration enhanced acetylcholine-induced vasodilation in the aortas of diabetic rats. It also notably reduced the release of pro-inflammatory cytokines, including interleukin-8 (IL-8), IL-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) in diabetic rats and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). The results also unveiled that the pro-inflammatory cytokines in the culture medium of HUVECs could be increased by rHMGB1. The increased release of HMGB1 and upregulated expressions of HMGB1-related inflammatory factors, including advanced glycation end products (RAGE), Toll-like receptor 4 (TLR4), and phosphorylated p65 (p-p65) in diabetic rats and HG-induced HUVECs, were remarkably suppressed by icariin. Notably, HMGB1 translocation from the nucleus to the cytoplasm in HUVECs under HG was inhibited by icariin. Meanwhile, icariin could activate G protein-coupled estrogen receptor (GPER) and sirt1. To explore the role of GPER and Sirt1 in the inhibitory effect of icariin on HMGB1 release and HMGB-induced inflammation, GPER inhibitor and Sirt1 inhibitor were used in this study. These inhibitors diminished the effects of icariin on HMGB1 release and HMGB1-induced inflammation. Specifically, the GPER inhibitor also negated the activation of Sirt1 by icariin. These findings suggest that icariin activates GPER and increases the expression of Sirt1, which in turn reduces HMGB1 translocation and release, thereby improving vascular endothelial function in type 1 diabetic rats by inhibiting inflammation.

The cannabinoid receptor 1 (CB1) is famous as the target of Δ9-tetrahydrocannabinol (THC), which is the active ingredient of marijuana. Suppression of CB1 is frequently suggested as a drug target or gene therapy for many conditions (e.g., obesity, Parkinson\'s disease). However, brain networks affected by CB1 remain elusive, and unanticipated psychological effects in a clinical trial had dire consequences. To better understand the whole brain effects of CB1 suppression we performed in vivo imaging on mice under complete knockout of the gene for CB1 (cnr1-/-) and also under the CB1 inverse agonist rimonabant. We examined white matter structural changes and brain function (network activity and directional uniformity) in cnr1-/- mice. In cnr1-/- mice, white matter (in both sexes) and functional directional uniformity (in male mice) were altered across the brain but network activity was largely unaltered. Conversely, under rimonabant, functional directional uniformity was not altered but network activity was altered in cortical regions, primarily in networks known to be altered by THC (e.g., neocortex, hippocampal formation). However, rimonabant did not alter many brain regions found in both our cnr1-/- results and previous behavioral studies of cnr1-/- mice (e.g., thalamus, infralimbic area). This suggests that chronic loss of cnr1 is substantially different from short-term suppression, subtly rewiring the brain but largely maintaining the network activity. Our results help explain why pathological mutations in CB1 (e.g., chronic pain) do not always provide insight into the side effects of CB1 suppression (e.g., clinical depression), and thus urge more preclinical studies for any drugs that suppress CB1.

Drug-induced cardiotoxicity has become one of the most common and detrimental health concerns, which causes significant loss to public health and drug resources. Cannabinoid receptors (CBRs) have recently achieved great attention for their vital roles in the regulation of heart health and disease, with mounting evidence linking CBRs with the pathogenesis and progression of drug-induced cardiotoxicity. This review aims to summarize fundamental characteristics of two well-documented CBRs (CB1R and CB2R) from aspects of molecular structure, signaling and their functions in cardiovascular physiology and pathophysiology. Moreover, we describe the roles of CB1R and CB2R in the occurrence of cardiotoxicity induced by common drugs such as antipsychotics, anti-cancer drugs, marijuana, and some emerging synthetic cannabinoids. We highlight the \'yin-yang\' relationship between CB1R and CB2R in drug-induced cardiotoxicity and propose future perspectives for CBR-based translational medicine toward cardiotoxicity curation and clinical monitoring.

N-(1,3-dimethylbutyl)-N\'-phenyl-p-phenylenediamine quinone (6PPD-Q) is a quinone derivative of a common tire additive 6PPD, whose occurrence has been widely reported both in the environment and human bodies including in adults, pregnant women and children. Yet, knowledge on the potential intestinal toxicity of 6PPD-Q in mammals at environmentally relevant dose remain unknown. In this study, the effects of 6PPD-Q on the intestines of adult ICR mice were evaluated by orally administering environmentally relevant dose or lower levels of 6PPD-Q (0.1, 1, 10, and 100 μg/kg) for 21 days. We found that 6PPD-Q disrupted the integrity of the intestinal barrier, mostly in the jejunum and ileum, but not in the duodenum or colon, in a dose-dependent manner. Moreover, intestinal inflammation manifested with elevated levels of TNF-α, IL-1, and IL-6 mostly observed in doses at 10 and 100 μg/kg. Using reverse target screening technology combining molecular dynamic simulation modeling we identified key cannabinoid receptors including CNR2 activation to be potentially mediating the intestinal inflammation induced by 6PPD-Q. In summary, this study provides novel insights into the toxic effects of emerging contaminant 6PPD-Q on mammalian intestines and that the chemical may be a cannabinoid receptor agonist to modulate inflammation.

The two subtypes of cannabinoid receptors (CBR), namely CB1R and CB2R, belong to the G protein-coupled receptor (GPCR) superfamily and are confirmed as potential therapeutic targets for a variety of diseases such as inflammation, neuropathic pain, and immune-related disorders. Since CB1R is mainly distributed in the central nervous system (CNS), it could produce severe psychiatric adverse reactions and addiction. In contrast, CB2R are predominantly distributed in the peripheral immune system with minimal CNS-related side effects. Therefore, more attention has been devoted to the discovery of CB2R ligands. In view of the favorable profile of CB2R, many high-binding affinity and selectivity CB2R ligands have been developed recently. This paper reviews recent research progress on CB2R ligands, including endogenous CB2R ligands, natural compounds, and novel small molecules, in order to provide a reference for subsequent CB2R ligand development.

Metal complexes exhibit a diverse range of coordination geometries, representing novel privileged scaffolds with convenient click types of preparation inaccessible for typical carbon-centered organic compounds. Herein, we explored the opportunity to identify biologically active organometallic complexes by reverse docking of a rigid, minimum-size octahedral organoruthenium scaffold against thousands of protein-binding pockets. Interestingly, cannabinoid receptor type 1 (CB1) was identified based on the docking scores and the degree of overlap between the docked organoruthenium scaffold and the hydrophobic scaffold of the cocrystallized ligand. Further structure-based optimization led to the discovery of organoruthenium complexes with nanomolar binding affinities and high selectivity toward CB2. Our work indicates that octahedral organoruthenium scaffolds may be advantageous for targeting the large and hydrophobic binding pockets and that the reverse docking approach may facilitate the discovery of novel privileged scaffolds, such as organometallic complexes, for exploring chemical space in lead discovery.