Peripherally Restricted CB1 Receptor Inverse Agonist JD5037 Treatment Exacerbates Liver Injury in MDR2-Deficient Mice.

Previous research highlighted the involvement of the cannabinoid CB1 receptor in regulating the physiology of hepatocytes and hepatic stellate cells. The inhibition of the CB1 receptor via peripherally restricted CB1 receptor inverse agonist JD5037 has shown promise in inhibiting liver fibrosis in mice treated with CCl4. However, its efficacy in phospholipid transporter-deficiency-induced liver fibrosis remains uncertain. In this study, we investigated the effectiveness of JD5037 in Mdr2-/- mice. Mdr2 (Abcb4) is a mouse ortholog of the human MDR3 (ABCB4) gene encoding for the canalicular phospholipid transporter. Genetic disruption of the Mdr2 gene in mice causes a complete absence of phosphatidylcholine from bile, leading to liver injury and fibrosis. Mdr2-/- mice develop spontaneous fibrosis during growth. JD5037 was orally administered to the mice for four weeks starting at eight weeks of age. Liver fibrosis, bile acid levels, inflammation, and injury were assessed. Additionally, JD5037 was administered to three-week-old mice to evaluate its preventive effects on fibrosis development. Our findings corroborate previous observations regarding global CB1 receptor inverse agonists. Four weeks of JD5037 treatment in eight-week-old Mdr2-/- mice with established fibrosis led to reduced body weight gains. However, contrary to expectations, JD5037 significantly exacerbated liver injury, evidenced by elevated serum ALT and ALP levels and exacerbated liver histology. Notably, JD5037-treated Mdr2-/- mice exhibited significantly heightened serum bile acid levels. Furthermore, JD5037 treatment intensified liver fibrosis, increased fibrogenic gene expression, stimulated ductular reaction, and upregulated hepatic proinflammatory cytokines. Importantly, JD5037 failed to prevent liver fibrosis formation in three-week-old Mdr2-/- mice. In summary, our study reveals the exacerbating effect of JD5037 on liver fibrosis in genetically MDR2-deficient mice. These findings underscore the need for caution in the use of peripherally restricted CB1R inverse agonists for liver fibrosis treatment, particularly in cases of dysfunctional hepatic phospholipid transporter.

Cannabidiol is a behavioral modulator in BTBR mouse model of idiopathic autism.

Introduction: The prevalence of Autism Spectrum Disorder (ASD) has drastically risen over the last two decades and is currently estimated to affect 1 in 36 children in the U.S., according to the center for disease control (CDC). This heterogenous neurodevelopmental disorder is characterized by impaired social interactions, communication deficits, and repetitive behaviors plus restricted interest. Autistic individuals also commonly present with a myriad of comorbidities, such as attention deficit hyperactivity disorder, anxiety, and seizures. To date, a pharmacological intervention for the treatment of core autistic symptoms has not been identified. Cannabidiol (CBD), the major nonpsychoactive constituent of Cannabis sativa, is suggested to have multiple therapeutic applications, but its effect(s) on idiopathic autism is unknown. We hypothesized that CBD will effectively attenuate the autism-like behaviors and autism-associated comorbid behaviors in BTBR T+Itpr3tf/J (BTBR) mice, an established mouse model of idiopathic ASD. Methods: Male BTBR mice were injected intraperitoneally with either vehicle, 20 mg/kg CBD or 50 mg/kg CBD daily for two weeks beginning at postnatal day 21 ± 3. On the final treatment day, a battery of behavioral assays were used to evaluate the effects of CBD on the BTBR mice, as compared to age-matched, vehicle-treated C57BL/6 J mice. Results: High dose (50 mg/kg) CBD treatment attenuated the elevated repetitive self-grooming behavior and hyperlocomotion in BTBR mice. The social deficits exhibited by the control BTBR mice were rescued by the 20 mg/kg CBD treatment. Discussion: Our data indicate that different doses for CBD are needed for treating specific ASD-like behaviors. Together, our results suggest that CBD may be an effective drug to ameliorate repetitive/restricted behaviors, social deficits, and autism-associated hyperactivity.

Effect of N-oleoyl dopamine on myofibroblast trans-differentiation of retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells contribute to several clinical conditions resulting in retinal fibrotic scars. Myofibroblast trans-differentiation of RPE cells is a critical step in the process of retinal fibrosis. In this study, we investigated the effects of N-oleoyl dopamine (OLDA), a newer endocannabinoid with a structure distinct from classic endocannabinoids, on TGF-ß2-induced myofibroblast trans-differentiation of porcine RPE cells. Using an in vitro collagen matrix contraction assay, OLDA was found to inhibit TGF-ß2 induced contraction of collagen matrices by porcine RPE cells. This effect was concentration-dependent, with significant inhibition of contraction observed at 3 µM and 10 µM. OLDA did not affect the proliferation of porcine RPE cells. Immunocytochemistry showed that at 3 µM, OLDA decreased incorporation of α-SMA in the stress fibers of TGF-ß2-treated RPE cells. In addition, western blot analysis showed that 3 µM OLDA significantly downregulated TGF-ß2-induced α-SMA protein expression. Taken together these results demonstrate that OLDA inhibits TGF-ß induced myofibroblast trans-differentiation of RPE cells. It has been established that classic endocannabinoid such as anandamide, by activating the CB1 cannabinoid receptor, promote fibrosis in multiple organ systems. In contrast, this study demonstrates that OLDA, an endocannabinoid with a chemical structure distinct from classic endocannabinoids, inhibits myofibroblast trans-differentiation, an important step in fibrosis. Unlike classic endocannabinoids, OLDA has weak affinity for the CB1 receptor. Instead, OLDA acts on non-classic cannabinoid receptors such as GPR119, GPR6, and TRPV1. Therefore, our study indicates that the newer endocannabinoid OLDA and its non-classic cannabinoid receptors could potentially be novel therapeutic targets for treating ocular diseases involving retinal fibrosis and fibrotic pathologies in other organ systems.

LC-MS/MS quantitation of non-psychotropic cannabinoid cannabidiol in aqueous humor.

Cannabidiol (CBD) is the most abundant non-psychotropic phytocannabinoid isolated from Cannabis sativa. To support preclinical studies of ocular pharmacology of CBD, a bioanalytical method was developed and validated for quantification of CBD in aqueous humor using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Aqueous humor samples were subjected to protein precipitation by acetonitrile, followed by chromatographic separation using reversed phase LC on a Raptor ARC-18 column with mobile phase A: 0.1 % (v/v) formic acid in water (B) 0.1 % formic acid in acetonitrile (B) as eluents. Detection was carried out with a triple quadrupole mass spectrometer with electrospray ionization operated in positive ion mode. Stable-isotope labeled CBD (CBD-d3) was used as internal standard. The total run time was 8 min. Quantification was accomplished within the validated concentration range of 0.5-500 ng/mL for CBD using a 5 µL sample. The lower limit of quantitation was 0.5 ng/mL. Inter- and intra-day precision is 4.737-7.620 % and 3.426-5.830 %, respectively. Inter- and intra-day accuracy ranged between 99.01 % and 100.2 % and 99.85-101.4 % respectively. The extraction recoveries were found to be 66.06 ± 5.146 %. The established method was successfully applied to investigate ocular pharmacokinetics of CBD in mice. Following intraperitoneal (i.p.) administration of 50 mg/kg CBD, its concentration reaches a Cmax of 71.55 ± 36.64 ng/mL in aqueous humor, with a Tmax of 2 h and a half-life of 1.046 h. The AUC was 183.4 ± 49.17 ng * h/mL. The development and validation of this LC-MS/MS method is an important step toward the goal of assessing the aqueous humor concentrations of CBD and correlating the concentrations of this phytocannabinoid with its ocular pharmacologic effects.

The Effects of Cannabidiol on Aqueous Humor Outflow and Trabecular Meshwork Cell Signaling.

Intraocular pressure (IOP) is regulated primarily through aqueous humor production by ciliary body and drainage through uveoscleral and trabecular meshwork (TM) tissues. The goal of this study was to measure the effect of non-psychotropic cannabidiol (CBD) on aqueous humor outflow through TM and assess the effect of CBD on the TM cell signaling pathways that are important for regulating outflow. Perfused porcine eye anterior segment explants were used to investigate the effects of CBD on aqueous humor outflow. Cultured porcine TM cells were used to study the effects of CBD on TM cell contractility, myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation, and RhoA activation. In the anterior segment perfusion experiments, aqueous humor outflow was increased significantly within 1 h after adding 1 µM CBD and the effect was sustained over the 5 h of measurement. Treatment of TM cells with 1 µM CBD significantly decreased TM cell-mediated collagen contraction, inhibited phosphorylation of MLC and MYPT1, and reduced RhoA activation. Our data demonstrate, for the first time, that as a potential therapeutic agent for lowering intraocular pressure, CBD can enhance aqueous humor outflow and modify TM cell signaling.

Opposing roles of ZEB1 in the cytoplasm and nucleus control cytoskeletal assembly and YAP1 activity.

Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and is initiated by mesenchyme-driving transcription factors and actin cytoskeletal assembly. We show a cytoplasmic-to-nuclear transport gradient of the EMT transcription factor Zeb1 toward sites of invasion in lung adenocarcinoma (LUAD), driven by the EMT inducer Tgfb, which is expressed in M2 polarized macrophages. We show that Zeb1 binds free actin monomers and RhoA in the cytoplasm to inhibit actin polymerization, blocking cell migration and Yap1 nuclear transport. Tgfb causes turnover of the scaffold protein Rassf1a, which targets RhoA. Release of this RhoA inhibition in response to Tgfb overcomes Zeb1's block of cytoskeleton assembly and frees it for nuclear transport. A ZEB1 nuclear transport signature highlights EMT progression, identifies dedifferentiated invasive/metastatic human LUADs, and predicts survival. Blocking Zeb1 nuclear transport with a small molecule identified in this study inhibits cytoskeleton assembly, cell migration, Yap1 nuclear transport, EMT, and precancerous-to-malignant transition.

Reparative effects of chronic intermittent hypobaric hypoxia pre­treatment on intervertebral disc degeneration in rats.

Previous studies have indicated that chronic intermittent hypobaric hypoxia (CIHH) preconditioning can inhibit TNF­α and other related inflammatory cytokines and exerts protective effect on intervertebral disc degeneration disease (IDD) in rats; however, the mechanism is still unclear. The present study aimed to explore the repair mechanisms of CIHH on IDD in rats. In the experiment, 48 adult Sprague­Dawley rats were selected and randomly divided into an experimental group (CIHH­IDD), a degenerative group (IDD) and a control group (CON). The CIHH­IDD group of rats (n=16) were treated with CIHH (simulated 3000 m altitude, 5 h per day, 28 days; PO2=108.8 mmHg) before disc degeneration surgery. The IDD group of rats (n=16) underwent tail­vertebral intervertebral disc surgery to establish a model of intervertebral disc degeneration. The CON group of rats (n=16) did not receive any treatments. After surgery, the disc height index was calculated using X­ray analysis of rat tail vertebrae, the degeneration process was observed and repair was evaluated by chemically staining degenerative intervertebral disc tissue slices. The expression levels of basic fibroblast growth factor (bFGF), TGFß1, Collagen I and Collagen II were measured in the intervertebral disc tissue using western blotting; while the expression levels of bFGF, TGFß1 and hypoxia­inducible factor 1­α (HIF­1α) were measured in rat serum using ELISA. The results demonstrated that: i) The degree of intervertebral disc height degeneration in CIHH­IDD rats was significantly lower compared with that in IDD rats (P<0.05); ii) the expression levels of bFGF, TGFß1 and HIF­1α were higher in CIHH­IDD rat serum compared with those in IDD rat serum (P<0.05); iii) optical microscopy revealed that the degree of disc degeneration was relatively mild in CIHH­IDD rats; and iv) the protein expression levels of bFGF, TGFß1 and collagen II were increased in CIHH­IDD rat intervertebral disc tissues compared with those of IDD rats, while the overexpression of collagen I protein was inhibited. Overall, after CIHH pre­treatment, the expression levels of bFGF and TGFß1 were up­regulated, which play notable roles in repairing degenerative intervertebral discs in rats.

Treatment of posterolateral tibial plateau fractures with a rotational support plate and special pressurizer: technical note and retrospective case series.

BACKGROUND: In tibial plateau fractures, the posterolateral segment of the tibia plateau is frequently affected and challenging to treat. Although there are many surgical approaches and fixation methods for the treatment of these fractures, all of these methods have limitations. We designed a new rotational support plate (RSP) and a special pressurizer that can fix the fracture directly via the anterolateral approach. This method is advantageous because it leads to little trauma, involves a simple operation, and has a reliable fixation effect. This study details the technique of treating these fractures with the RSP and special pressurizer and provides the outcomes. METHODS: From May 2016 to January 2019, the data of 12 patients with posterolateral tibial plateau fractures treated with the RSP and special pressurizer in our hospital were retrospectively analyzed. Postoperative rehabilitation was advised, knee X-rays were taken at follow-ups, and fracture healing, complications, and knee range of motion were assessed. The Hospital for Special Surgery (HSS) knee score and Knee Injury and Osteoarthritis Outcome Score (KOOS) were used to evaluate knee function at the last follow-up. RESULTS: The average follow-up time of all patients was 16.5 months (range, 12-25 months). The average bony union time was 3.2 months (range, 3-4.5 months). At the last follow-up, the average knee range of motion was 138° (range, 107-145°). The average HSS score was 91 (range, 64-98). The average KOOS Symptoms score was 90 (range, 75-96). The average KOOS Pain score was 91 (range, 72-97). The average KOOS ADL score was 91 (range, 74-97). The average KOOS sport/recreation score was 83 (range, 70-90). The average KOOS QOL score was 88 (range, 69-93). Skin necrosis, incision infections, and fixation failure did not occur during the follow-up period. CONCLUSIONS: With our newly designed RSP and special pressurizer, posterolateral tibial plateau fractures can be easily and effectively reduced and fixed through the anterolateral approach, which serves as a novel treatment for posterolateral tibial plateau fractures.

Cannabidiol Signaling in the Eye and Its Potential as an Ocular Therapeutic Agent.

Cannabidiol (CBD), the major non-intoxicating constituent of Cannabis sativa, has gained recent attention due to its putative therapeutic uses for a wide variety of diseases. CBD was discovered in the 1940s and its structure fully characterized in the 1960s. However, for many years most research efforts related to cannabis derived chemicals have focused on D9-tetrahydrocannabinol (THC). In contrast to THC, the lack of intoxicating psychoactivity associated with CBD highlights the potential of this cannabinoid for clinical drug development. This review details in vitro and in vivo studies of CBD related to the eye, the therapeutic potential of cannabidiol for various ocular conditions, and molecular targets and mechanisms for CBD-induced ocular effects. In addition, challenges of CBD applications for clinical ocular therapeutics and future directions are discussed.

Studies of involvement of G-protein coupled receptor-3 in cannabidiol effects on inflammatory responses of mouse primary astrocytes and microglia.

Cannabidiol (CBD) exhibits anti-inflammatory and neuroprotective properties and is suggested to be effective in the pre-clinical and clinical treatment of illnesses of the central nervous system (CNS). Two major types of CNS glial cells, astrocytes and microglia, play critical roles in the development and pathogenesis of CNS diseases. However, the mechanisms by which CBD plays an anti-inflammatory and neuroprotective role for these glial cells have not been fully elucidated. In this study, we examined the effects of CBD on the inflammatory response of mouse primary astrocytes and microglia. We also investigated whether the effect of CBD on cytokine release is mediated by the G protein coupled receptor 3 (GPR3), which was recently identified as a novel receptor for CBD. Our results showed that CBD inhibited inflammatory responses of astrocytes and microglia stimulated with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand in vitro and in vivo. In addition, CBD reduced the phosphorylation of STAT3 and NF-κB signaling pathways in LPS-stimulated astrocytes. However, the inhibitory effect of CBD on pro-inflammatory cytokine production was independent of GPR3 expression in both types of glial cells. Thus, although CBD is effective in ameliorating the activation of astrocytes and microglia, its mechanism of action still requires further study. Our data support the concept that CBD may have therapeutic potential for neurological disorders that involve neuroinflammation.

Involvement of dopamine receptor in the actions of non-psychoactive phytocannabinoids.

Nematode Caenorhabditis elegans (C. elegans) exhibited a vigorous swimming behavior in liquid medium. Addition of dopamine inhibited the swimming behavior, causing paralysis in 65% of wild-type nematodes. Interestingly, phytocannabinoids cannabidiol (CBD) or cannabidivarin (CBDV), caused paralysis in 40% of the animals. Knockout of DOP-3, the dopamine D2-like receptor critical for locomotor behavior, eliminated the paralysis induced by dopamine, CBD, and CBDV. In contrast, both CBD and CBDV caused paralysis in animals lacking CAT-2, an enzyme necessary for dopamine synthesis. Co-administration of dopamine with either CBD or CBDV caused paralysis similar to that of either phytocannabinoid treatment alone. These data support the notion that CBD and CBDV act as functional partial agonists on dopamine D2-like receptors in vivo. The discovery that dopamine receptor is involved in the actions of phytocannabinoids moves a significant step toward our understanding of the mechanisms for medical uses of cannabis in the treatment of neurological and psychiatric disorders.

Discovery of endogenous inverse agonists for G protein-coupled receptor 6.

The orphan G protein-coupled receptor 6 (GPR6) is highly expressed in the striatum and has been linked to multiple striatal pathologies. The identification of endogenous ligands and their mechanisms of action at GPR6 will help to elucidate the physiological and pathological roles of the receptor. In the current study, we tested the concentration-dependent effects of a variety of endocannabinoid-like N-acylamides on GPR6 signaling. Here, we demonstrate for the first time that N-arachidonoyl dopamine, N-docosahexaenoyl dopamine, N-oleoyl dopamine and N-palmitoyl dopamine exert inverse agonism at GPR6. This effect was concentration-dependent, with potencies in the micromolar range, and functionally selective for ß-arrestin2 recruitment. Structure-activity relationship studies demonstrate that both the N-acyl side chain and the dopamine head group are important for these ligands to act on GPR6. Our discovery of these N-acyl dopamines as endogenous inverse agonists for GPR6 moves us one step further in understanding the roles GPR6 play in neurodegenerative and neuropsychiatric disorders related to striatal dysfunction.

GPR3, GPR6, and GPR12 as novel molecular targets: their biological functions and interaction with cannabidiol.

The G protein-coupled receptors 3, 6, and 12 (GPR3, GPR6, and GPR12) comprise a family of closely related orphan receptors with no confirmed endogenous ligands. These receptors are constitutively active and capable of signaling through G protein-mediated and non-G protein-mediated mechanisms. These orphan receptors have previously been reported to play important roles in many normal physiological functions and to be involved in a variety of pathological conditions. Although they are orphans, GPR3, GPR6, and GPR12 are phylogenetically most closely related to the cannabinoid receptors. Using ß-arrestin2 recruitment and cAMP accumulation assays, we recently found that the nonpsychoactive phytocannabinoid cannabidiol (CBD) is an inverse agonist for GPR3, GPR6, and GPR12. This discovery highlights these orphan receptors as potential new molecular targets for CBD, provides novel mechanisms of action, and suggests new therapeutic uses of CBD for illnesses such as Alzheimer's disease, Parkinson's disease, cancer, and infertility. Furthermore, identification of CBD as a new inverse agonist for GPR3, GPR6, and GPR12 provides the initial chemical scaffolds upon which potent and efficacious agents acting on these receptors can be developed, with the goal of developing chemical tools for studying these orphan receptors and ultimately new therapeutic agents.

Novel inverse agonists for the orphan G protein-coupled receptor 6.

The orphan G protein-coupled receptor 6 (GPR6) displays unique promise as a therapeutic target for the treatment of neuropsychiatric disorders due to its high expression in the striatopallidal neurons of the basal ganglia. GPR6, along with closely related orphan receptors GPR3 and GPR12, are phylogenetically related to CB1 and CB2 cannabinoid receptors. In the current study, we performed concentration-response studies on the effects of three different classes of cannabinoids: endogenous, phyto-, and synthetic, on both GPR6-mediated cAMP accumulation and ß-arrestin2 recruitment. In addition, structure-activity relationship studies were conducted on cannabidiol (CBD), a recently discovered inverse agonist for GPR6. We have identified four additional cannabinoids, cannabidavarin (CBDV), WIN55212-2, SR141716A and SR144528, that exert inverse agonism on GPR6. Furthermore, we have discovered that these cannabinoids exhibit functional selectivity toward the ß-arrestin2 recruitment pathway. These novel, functionally selective inverse agonists for GPR6 can be used as research tools and potentially developed into therapeutic agents.

Cannabidiol, a novel inverse agonist for GPR12.

GPR12 is a constitutively active, Gs protein-coupled receptor that currently has no confirmed endogenous ligands. GPR12 may be involved in physiological processes such as maintenance of oocyte meiotic arrest and brain development, as well as pathological conditions such as metastatic cancer. In this study, the potential effects of various classes of cannabinoids on GPR12 were tested using a cAMP accumulation assay. Our data demonstrate that cannabidiol (CBD), a major non-psychoactive phytocannabinoid, acted as an inverse agonist to inhibit cAMP accumulation stimulated by the constitutively active GPR12. Thus, GPR12 is a novel molecular target for CBD. The structure-activity relationship studies of CBD indicate that both the free hydroxyl and the pentyl side chain are crucial for the effects of CBD on GPR12. Furthermore, studies using cholera toxin, which blocks Gs protein and pertussis toxin, which blocks Gi protein, revealed that Gs, but not Gi is involved in the inverse agonism of CBD on GPR12. CBD is a promising novel therapeutic agent for cancer, and GPR12 has been shown to alter viscoelasticity of metastatic cancer cells. Since we have demonstrated that CBD is an inverse agonist for GPR12, this provides novel mechanism of action for CBD, and an initial chemical scaffold upon which highly potent and efficacious agents acting on GPR12 may be developed with the ultimate goal of blocking cancer metastasis.

Mass Spectrometry Analysis of Human CB2 Cannabinoid Receptor and Its Associated Proteins.

Studying the assemblies and dynamics of the complex formed by cannabinoid receptors and their associated proteins is important for understanding the molecular basis for the functions of these receptors. In the absence of the crystal structures of these macromolecular complexes, mass spectrometry is a sensitive technique that can be used to study cannabinoid receptors and their associated proteins. In this chapter, three aspects of the work are presented: (1) mass spectrometry analysis of the primary sequence of the human CB2 cannabinoid receptor after affinity chromatography purification of the receptor proteins, (2) functional proteomic analysis of CB2-associated proteins coimmunoprecipitated with CB2, and (3) chemical cross-linking and mass spectrometry analysis of CB2-G protein complex. The mass spectrometry approaches, in combination with mutagenesis and molecular modeling techniques, have been successfully used for studying the CB2-G protein complex. Similar approaches can also be applied for studying other G protein-coupled receptors in general.

GPR3 and GPR6, novel molecular targets for cannabidiol.

GPR3 and GPR6 are members of a family of constitutively active, Gs protein-coupled receptors. Previously, it has been reported that GPR3 is involved in Alzheimer's disease whereas GPR6 plays potential roles in Parkinson's disease. GPR3 and GPR6 are considered orphan receptors because there are no confirmed endogenous agonists for them. However, GPR3 and GPR6 are phylogenetically related to the cannabinoid receptors. In this study, the activities of endocannabinoids and phytocannabinoids were tested on GPR3 and GPR6 using a ß-arrestin2 recruitment assay. Among the variety of cannabinoids tested, cannabidiol (CBD), the major non-psychoactive component of marijuana, significantly reduced ß-arrestin2 recruitment to both GPR3 and GPR6. In addition, the inhibitory effects of CBD on ß-arrestin2 recruitment were concentration-dependent for both GPR3 and GPR6, with a higher potency for GPR6. These data show that CBD acts as an inverse agonist at both GPR3 and GPR6 receptors. These results demonstrate for the first time that both GPR3 and GPR6 are novel molecular targets for CBD. Our discovery that CBD acts as a novel inverse agonist on both GPR3 and GPR6 indicates that some of the potential therapeutic effects of CBD (e.g. treatment of Alzheimer's disease and Parkinson's disease) may be mediated through these important receptors.

A categorical structure-activity relationship analysis of GPR119 ligands.

The categorical structure-activity relationship (cat-SAR) expert system has been successfully used in the analysis of chemical compounds that cause toxicity. Herein we describe the use of this fragment-based approach to model ligands for the G protein-coupled receptor 119 (GPR119). Using compounds that are known GPR119 agonists and compounds that we have confirmed experimentally that are not GPR119 agonists, four distinct cat-SAR models were developed. Using a leave-one-out validation routine, the best GPR119 model had an overall concordance of 99%, a sensitivity of 99%, and a specificity of 100%. Our findings from the in-depth fragment analysis of several known GPR119 agonists were consistent with previously reported GPR119 structure-activity relationship (SAR) analyses. Overall, while our results indicate that we have developed a highly predictive cat-SAR model that can be potentially used to rapidly screen for prospective GPR119 ligands, the applicability domain must be taken into consideration. Moreover, our study demonstrates for the first time that the cat-SAR expert system can be used to model G protein-coupled receptor ligands, many of which are important therapeutic agents.

Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4ß6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1ß1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the ß2-AR*·Gαsß1γ2 complex crystal structure, the Gαi1ß1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1ß1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins.

Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119.

The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor.