The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, ...and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257’s unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.
Display omitted
•Crystal structure of human CB2 in complex with antagonist AM10257 is determined•A high degree of conformational similarity with the agonist-bound CB1 is uncovered•The yin-yang relationship of CB2 and CB1 will facilitate the design of selective drugs
The structure of the human cannabinoid receptor CB2 reveals how small molecules affect CB2 differently than CB1 and point to principles that could inform rational and selective drug design.
Coding variants in apolipoprotein L1 (APOL1), termed G1 and G2, can explain most excess kidney disease risk in African Americans; however, the molecular pathways of APOL1-induced kidney dysfunction ...remain poorly understood. Here, we report that expression of G2 APOL1 in the podocytes of Nphs1rtTA/TRE-G2APOL1 (G2APOL1) mice leads to early activation of the cytosolic nucleotide sensor, stimulator of interferon genes (STING), and the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. STING and NLRP3 expression was increased in podocytes from patients with high-risk APOL1 genotypes, and expression of APOL1 correlated with caspase-1 and gasdermin D (GSDMD) levels. To demonstrate the role of NLRP3 and STING in APOL1-associated kidney disease, we generated transgenic mice with the G2 APOL1 risk variant and genetic deletion of Nlrp3 (G2APOL1/Nlrp3 KO), Gsdmd (G2APOL1/Gsdmd KO), and STING (G2APOL1/STING KO). Knockout mice displayed marked reduction in albuminuria, azotemia, and kidney fibrosis compared with G2APOL1 mice. To evaluate the therapeutic potential of targeting NLRP3, GSDMD, and STING, we treated mice with MCC950, disulfiram, and C176, potent and selective inhibitors of NLRP3, GSDMD, and STING, respectively. G2APOL1 mice treated with MCC950, disulfiram, and C176 showed lower albuminuria and improved kidney function even when inhibitor treatment was initiated after the development of albuminuria.
The study of cancer cell metabolism has traditionally focused on glycolysis and glutaminolysis. However, lipidomic technologies have matured considerably over the last decade and broadened our ...understanding of how lipid metabolism is relevant to cancer biology 1-3. Studies now suggest that the reprogramming of cellular lipid metabolism contributes directly to malignant transformation and progression 4, 5. For example, de novo lipid synthesis can supply proliferating tumor cells with phospholipid components that comprise the plasma and organelle membranes of new daughter cells 6, 7. Moreover, the upregulation of mitochondrial β-oxidation can support tumor cell energetics and redox homeostasis 8, while lipid-derived messengers can regulate major signaling pathways or coordinate immunosuppressive mechanisms 9-11. Lipid metabolism has, therefore, become implicated in a variety of oncogenic processes, including metastatic colonization, drug resistance, and cell differentiation 10, 12-16. However, whether we can safely and effectively modulate the underlying mechanisms of lipid metabolism for cancer therapy is still an open question.
Kidney cancer is the seventh leading cause of cancer in the world, and its incidence is on the rise. Renal cell carcinoma (RCC) is the most common form and is a heterogeneous disease comprising three ...major subtypes that vary in their histology, clinical course and driver mutations. These subtypes include clear cell RCC, papillary RCC and chromophobe RCC. Molecular analyses of hereditary and sporadic forms of RCC have revealed that this complex and deadly disease is characterized by metabolic pathway alterations in cancer cells that lead to deregulated oxygen and nutrient sensing, as well as impaired tricarboxylic acid cycle activity. These metabolic changes facilitate tumour growth and survival. Specifically, studies of the metabolic features of RCC have led to the discovery of oncometabolites - fumarate and succinate - that can promote tumorigenesis, moonlighting functions of enzymes, and substrate auxotrophy owing to the disruption of pathways that enable the production of arginine and cholesterol. These metabolic alterations within RCC can be exploited to identify new therapeutic targets and interventions, in combination with novel approaches that minimize the systemic toxicity of metabolic inhibitors and reduce the risk of drug resistance owing to metabolic plasticity.
Endocannabinoids acting on the cannabinoid-1 receptor (CB1R) or ghrelin acting on its receptor (GHS-R1A) both promote alcohol-seeking behavior, but an interaction between the two signaling systems ...has not been explored. Here, we report that the peripheral CB1R inverse agonist JD5037 reduces ethanol drinking in wild-type mice but not in mice lacking CB1R, ghrelin peptide or GHS-R1A. JD5037 treatment of alcohol-drinking mice inhibits the formation of biologically active octanoyl-ghrelin without affecting its inactive precursor desacyl-ghrelin. In ghrelin-producing stomach cells, JD5037 reduced the level of the substrate octanoyl-carnitine generated from palmitoyl-carnitine by increasing fatty acid β-oxidation. Blocking gastric vagal afferents abrogated the ability of either CB1R or GHS-R1A blockade to reduce ethanol drinking. We conclude that blocking CB1R in ghrelin-producing cells reduces alcohol drinking by inhibiting the formation of active ghrelin and its signaling via gastric vagal afferents. Thus, peripheral CB1R blockade may have therapeutic potential in the treatment of alcoholism.
Display omitted
•CB1R and ghrelin work together to promote alcohol intake via a gut-brain axis•Alcohol intake is inhibited by CB1R blockade, but only when ghrelin signaling is intact•Inhibition of peripheral CB1R reduces plasma levels of biologically active ghrelin•CB1R blockade promotes the degradation of the substrate needed to activate ghrelin
Endocannabinoids and ghrelin are both implicated in promoting alcohol intake. Godlewski et al. show that cannabinoid-1 receptor (CB1R) signaling in ghrelin-producing stomach cells modulates the formation of biologically active octanoyl-ghrelin which then acts via gastric vagal afferents to promote alcohol consumption. They further show that peripherally-restricted pharmacological inhibition of CB1R reduces ethanol drinking in mice.
The study of cancer cell metabolism has traditionally focused on glycolysis and glutaminolysis. However, lipidomic technologies have matured considerably over the last decade and broadened our ...understanding of how lipid metabolism is relevant to cancer biology 1–3. Studies now suggest that the reprogramming of cellular lipid metabolism contributes directly to malignant transformation and progression 4, 5. For example, de novo lipid synthesis can supply proliferating tumor cells with phospholipid components that comprise the plasma and organelle membranes of new daughter cells 6, 7. Moreover, the upregulation of mitochondrial β-oxidation can support tumor cell energetics and redox homeostasis 8, while lipid-derived messengers can regulate major signaling pathways or coordinate immunosuppressive mechanisms 9–11. Lipid metabolism has therefore become implicated in a variety of oncogenic processes, including metastatic colonization, drug resistance, and cell differentiation 10, 12–16. However, whether we can safely and effectively modulate the underlying mechanisms for cancer therapy is still an open question.
Signaling through integral membrane G protein-coupled receptors (GPCRs) is influenced by lipid composition of cell membranes. By using novel high affinity ligands of human cannabinoid receptor CB2, ...we demonstrate that cholesterol increases basal activation levels of the receptor and alters the pharmacological categorization of these ligands. Our results revealed that (2-(6-chloro-2-((2,2,3,3-tetramethylcyclopropane-1-carbonyl)imino)benzodthiazol-3(2H)-yl)ethyl acetate ligand (MRI-2646) acts as a partial agonist of CB2 in membranes devoid of cholesterol and as a neutral antagonist or a partial inverse agonist in cholesterol-containing membranes. The differential effects of a specific ligand on activation of CB2 in different types of membranes may have implications for screening of drug candidates in a search of modulators of GPCR activity. MD simulation suggests that cholesterol exerts an allosteric effect on the intracellular regions of the receptor that interact with the G-protein complex thereby altering the recruitment of G protein.
We report the design, synthesis, and structure–activity relationships of novel dual-target compounds with antagonist/inverse agonist activity at cannabinoid receptor type 1 (CB1R) and inhibitory ...effect on inducible nitric oxide synthase (iNOS). A series of 3,4-diarylpyrazolinecarboximidamides were synthesized and evaluated in CB1 receptor (CB1R) binding assays and iNOS activity assays. The novel compounds, designed to have limited brain penetrance, elicited potent in vitro CB1R antagonist activities and iNOS inhibitory activities. Some key compounds displayed high CB1R binding affinities. Compound 7 demonstrated potent in vivo pharmacological activities such as reduction of food intake mediated by the antagonism of the CB1Rs and antifibrotic effect in the animal models of fibrosis mediated by iNOS inhibition and CB1R antagonism.
Clear cell renal cell carcinoma (ccRCC) incidence has risen steadily over the last decade. Elevated lipid uptake and storage is required for ccRCC cell viability. As stored cholesterol is the most ...abundant component in ccRCC intracellular lipid droplets, it may also play an important role in ccRCC cellular homeostasis. In support of this hypothesis, ccRCC cells acquire exogenous cholesterol through the high-density lipoprotein receptor SCARB1, inhibition or suppression of which induces apoptosis. Here, we showed that elevated expression of 3 beta-hydroxy steroid dehydrogenase type 7 (HSD3B7), which metabolizes cholesterol-derived oxysterols in the bile acid biosynthetic pathway, is also essential for ccRCC cell survival. Development of an HSD3B7 enzymatic assay and screening for small-molecule inhibitors uncovered the compound celastrol as a potent HSD3B7 inhibitor with low micromolar activity. Repressing HSD3B7 expression genetically or treating ccRCC cells with celastrol resulted in toxic oxysterol accumulation, impaired proliferation, and increased apoptosis in vitro and in vivo. These data demonstrate that bile acid synthesis regulates cholesterol homeostasis in ccRCC and identifies HSD3B7 as a plausible therapeutic target.
The bile acid biosynthetic enzyme HSD3B7 is essential for ccRCC cell survival and can be targeted to induce accumulation of cholesterol-derived oxysterols and apoptotic cell death.
Aims
To determine the specific role of podocyte‐expressed cannabinoid‐1 receptor (CB1R) in the development of diabetic nephropathy (DN), relative to CB1R in other renal cell types.
Material and ...methods
We developed a mouse model with a podocyte‐specific deletion of CB1R (pCB1Rko) and challenged this model with streptozotocin (STZ)‐induced type‐1 DN. We also assessed the podocyte response to high glucose in vitro and its effects on CB1R activation.
Results
High glucose exposure for 48 hours led to an increase in CB1R gene expression (CNR1) and endocannabinoid production in cultured human podocytes. This was associated with podocyte injury, reflected by decreased podocin and nephrin expression. These changes could be prevented by Cnr1‐silencing, thus identifying CB1R as a key player in podocyte injury. After 12 weeks of chronic hyperglycaemia, STZ‐treated pCB1Rko mice showed elevated blood glucose similar to that of their wild‐type littermates. However, they displayed less albuminuria and less podocyte loss than STZ‐treated wild‐type mice. Unexpectedly, pCB1Rko mice also have milder tubular dysfunction, fibrosis and reduction of cortical microcirculation compared to wild‐type controls, which is mediated, in part, by podocyte‐derived endocannabinoids acting via CB1R on proximal tubular cells.
Conclusions
Activation of CB1R in podocytes contributes to both glomerular and tubular dysfunction in type‐1 DN, which highlights the therapeutic potential of peripheral CB1R blockade.
PDF
