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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., Significance: 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., (©2024 American Association for Cancer Research.)

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., (© 2024. Springer Nature Limited.)

In the present report, we describe the synthesis and structure-activity relationships of novel "four-arm" dihydropyrazoline compounds designed as peripherally restricted antagonists of cannabinoid-1 receptor (CB 1 R). A series of racemic 3,4-diarylpyrazolines were synthesized and evaluated initially in CB 1 receptor binding assays. The novel compounds, designed to limit brain penetrance and decreased lipophilicity, showed high affinity for CB 1 R and potent in vitro CB 1 R antagonist activities. Promising compounds with potent CB 1 R activity were evaluated in tissue distribution studies. Compounds 6a , 6f , and 7c showed limited brain penetrance attesting to its peripheral restriction. The 4 S -enantiomer of these compounds further showed a stereoselective affinity for the CB 1 receptor and behaved as inverse agonists. In vivo studies on food intake and body weight reduction in diet-induced obese (DIO) mice showed that these compounds could serve as potential leads for the development of selective CB 1 R antagonists with improved potency and peripheral restriction.

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.

Hermansky-Pudlak syndrome (HPS) is a rare genetic disorder which, in its most common and severe form, HPS-1, leads to fatal adult-onset pulmonary fibrosis (PF) with no effective treatment. We evaluated the role of the endocannabinoid/CB 1 R system and inducible nitric oxide synthase (iNOS) for dual-target therapeutic strategy using human bronchoalveolar lavage fluid (BALF), lung samples from patients with HPS and controls, HPS-PF patient-derived lung fibroblasts, and bleomycin-induced PF in pale ear mice (HPS1 ep/ep ). We found overexpression of CB 1 R and iNOS in fibrotic lungs of HPSPF patients and bleomycin-infused pale ear mice. The endocannabinoid anandamide was elevated in BALF and negatively correlated with pulmonary function parameters in HPSPF patients and pale ear mice with bleomycin-induced PF. Simultaneous targeting of CB 1 R and iNOS by MRI-1867 yielded greater antifibrotic efficacy than inhibiting either target alone by attenuating critical pathologic pathways. Moreover, MRI-1867 treatment abrogated bleomycin-induced increases in lung levels of the profibrotic interleukin-11 via iNOS inhibition and reversed mitochondrial dysfunction via CB 1 R inhibition. Dual inhibition of CB 1 R and iNOS is an effective antifibrotic strategy for HPSPF., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Seven-transmembrane receptors signal via G-protein- and β-arrestin-dependent pathways. We describe a peripheral CB 1 R antagonist (MRI-1891) highly biased toward inhibiting CB 1 R-induced β-arrestin-2 (βArr2) recruitment over G-protein activation. In obese wild-type and βArr2-knockout (KO) mice, MRI-1891 treatment reduces food intake and body weight without eliciting anxiety even at a high dose causing partial brain CB 1 R occupancy. By contrast, the unbiased global CB 1 R antagonist rimonabant elicits anxiety in both strains, indicating no βArr2 involvement. Interestingly, obesity-induced muscle insulin resistance is improved by MRI-1891 in wild-type but not in βArr2-KO mice. In C2C12 myoblasts, CB 1 R activation suppresses insulin-induced akt-2 phosphorylation, preventable by MRI-1891, βArr2 knockdown or overexpression of CB 1 R-interacting protein. MRI-1891, but not rimonabant, interacts with nonpolar residues on the N-terminal loop, including F108, and on transmembrane helix-1, including S123, a combination that facilitates βArr2 bias. Thus, CB 1 R promotes muscle insulin resistance via βArr2 signaling, selectively mitigated by a biased CB 1 R antagonist at reduced risk of central nervous system (CNS) side effects., Competing Interests: The authors declare the following competing financial interest(s): G.K., R.C., and M.R.I. are listed as co-inventors on two US Government patents covering (S)-MRI-1891. The remaining authors declare no competing interests., (Not subject to U.S. Copyright. Published 2021 by American Chemical Society.)

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 CB 2 , 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)benzo[d]thiazol-3(2H)-yl)ethyl acetate ligand (MRI-2646) acts as a partial agonist of CB 2 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 CB 2 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.

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.

ATP-binding cassette (ABC) transporters are evolutionarily conserved membrane proteins that pump a variety of endogenous substrates across cell membranes. Certain subfamilies are known to interact with pharmaceutical compounds, potentially influencing drug delivery and treatment efficacy. However, the role of drug resistance-associated ABC transporters has not been examined in idiopathic pulmonary fibrosis (IPF) or its animal model: the bleomycin (BLM)-induced murine model. Here, we investigate the expression of two ABC transporters, P-gp (permeability glycoprotein) and BCRP (breast cancer resistance protein), in human IPF lung tissue and two different BLM-induced mouse models of pulmonary fibrosis. We obtained human IPF specimens from patients during lung transplantation and administered BLM to male C57BL/6J mice either by oropharyngeal aspiration (1 U/kg) or subcutaneous osmotic infusion (100 U/kg over 7 d). We report that P-gp and BCRP expression in lungs of patients with IPF was comparable to controls. However, murine lungs expressed increased levels of P-gp and BCRP after oropharyngeal and subcutaneous BLM administration. We localized this upregulation to multiple pulmonary cell types, including alveolar fibroblasts, endothelial cells, and type 2 epithelial cells. Functionally, this effect reduced murine lung exposure to nintedanib, a U.S. Food and Drug Administration-approved IPF therapy known to be a P-gp substrate. The study reveals a discrepancy between IPF pathophysiology and the common animal model of lung fibrosis. BLM-induced drug efflux in the murine lungs may present an uncontrolled confounding variable in the preclinical study of IPF drug candidates, and these findings will facilitate disease model validation and enhance new drug discoveries that will ultimately improve patient outcomes.

Endocannabinoids acting on the cannabinoid-1 receptor (CB 1 R) or ghrelin acting on its receptor (GHS-R 1A ) both promote alcohol-seeking behavior, but an interaction between the two signaling systems has not been explored. Here, we report that the peripheral CB 1 R inverse agonist JD5037 reduces ethanol drinking in wild-type mice but not in mice lacking CB 1 R, ghrelin peptide or GHS-R 1A . 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 CB 1 R or GHS-R 1A blockade to reduce ethanol drinking. We conclude that blocking CB 1 R in ghrelin-producing cells reduces alcohol drinking by inhibiting the formation of active ghrelin and its signaling via gastric vagal afferents. Thus, peripheral CB 1 R blockade may have therapeutic potential in the treatment of alcoholism., (Published by Elsevier Inc.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cannabinoid-1 receptor (CB 1 R) antagonists/inverse agonists have great potential in the treatment of metabolic disorders like dyslipidemia, type 2 diabetes, and nonalcoholic steatohepatitis. Cannabinoid-1 receptor inverse agonists have also been reported to be effective in mitigating fibrotic disorders in murine models. Inducible nitric oxide synthase is another promising target implicated in fibrotic and inflammatory disorders. We have disclosed MRI-1867 as a potent and selective, peripherally acting dual-target inhibitor of the CB 1 R and inducible nitric oxide synthase (iNOS). Herein, we report the synthesis of [ 13 C 6 ]-MRI-1867 as a racemate from commercially available chlorobenzene- 13 C 6 as the starting, stable-isotope label reagent. The racemic [ 13 C 6 ]-MRI-1867 was further processed to the stable-isotope-labeled enantiopure compounds using chiral chromatography. Both racemic [ 13 C 6 ]-MRI-1867 and S- 13 C 6 -MRI-1867 will be used to quantitate unlabeled S-MRI-1867 during clinical drug metabolism and pharmacokinetics studies and will be used as a liquid chromatography-tandem mass spectrometry bioanalytical standard., (© 2018 John Wiley & Sons, Ltd.)

Aims: To determine the specific role of podocyte-expressed cannabinoid-1 receptor (CB 1 R) in the development of diabetic nephropathy (DN), relative to CB 1 R in other renal cell types., Material and Methods: We developed a mouse model with a podocyte-specific deletion of CB 1 R (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 CB 1 R activation., Results: High glucose exposure for 48 hours led to an increase in CB 1 R 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 CB 1 R on proximal tubular cells., Conclusions: Activation of CB 1 R in podocytes contributes to both glomerular and tubular dysfunction in type-1 DN, which highlights the therapeutic potential of peripheral CB 1 R blockade., (© 2017 John Wiley & Sons Ltd.)

Key Points: 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.

Objective: Obesity-induced accumulation of ectopic fat in the liver is thought to contribute to the development of insulin resistance, and increased activity of hepatic CB 1 R has been shown to promote both processes. However, lipid accumulation in liver can be experimentally dissociated from insulin resistance under certain conditions, suggesting the involvement of additional mechanisms. Obesity is also associated with pro-inflammatory changes which, in turn, can promote insulin resistance. Kupffer cells (KCs), the liver's resident macrophages, are the major source of pro-inflammatory cytokines in the liver, such as TNF-α, which has been shown to inhibit insulin signaling in multiple cell types, including hepatocytes. Here, we sought to identify the role of CB 1 R in KCs in obesity-induced hepatic insulin resistance., Methods: We used intravenously administered β-D-glucan-encapsulated siRNA to knock-down CB 1 R gene expression selectively in KCs., Results: We demonstrate that a robust knock-down of the expression of Cnr1, the gene encoding CB 1 R, results in improved glucose tolerance and insulin sensitivity in diet-induced obese mice, without affecting hepatic lipid content or body weight. Moreover, Cnr1 knock-down in KCs was associated with a shift from pro-inflammatory M1 to anti-inflammatory M2 cytokine profile and improved insulin signaling as reflected by increased insulin-induced Akt phosphorylation., Conclusion: These findings suggest that CB 1 R expressed in KCs plays a critical role in obesity-related hepatic insulin resistance via a pro-inflammatory mechanism., (Published by Elsevier GmbH.)

JD5037 (1) is a potent and selective, peripherally acting inverse agonist of the cannabinoid (CB 1 R) receptor. Peripheral CB 1 receptor antagonists/inverse agonists have great potential in the treatment of metabolic disorders like type 2 diabetes, obesity, and nonalcoholic steatohepatitis. We report the synthesis of octadeuterated [ 2 H 8 ]-JD5037 (S, S) (8) along with its (S, R) diastereomer (13) from commercially available L-valine-d 8 starting material. The [ 2 H 8 ]-JD5037 compound will be used to quantitate unlabeled JD5037 during clinical ADME studies and will be used as an LC-MS/MS bioanalytical standard., (Copyright © 2017 John Wiley & Sons, Ltd.)

Idiopathic pulmonary fibrosis (IPF) is a life-threatening disease without effective treatment, highlighting the need for identifying new targets and treatment modalities. The pathogenesis of IPF is complex, and engaging multiple targets simultaneously might improve therapeutic efficacy. To assess the role of the endocannabinoid/cannabinoid receptor 1 (endocannabinoid/CB1R) system in IPF and its interaction with inducible nitric oxide synthase (iNOS) as dual therapeutic targets, we analyzed lung fibrosis and the status of the endocannabinoid/CB1R system and iNOS in mice with bleomycin-induced pulmonary fibrosis (PF) and in lung tissue and bronchoalveolar lavage fluid (BALF) from patients with IPF, as well as controls. In addition, we investigated the antifibrotic efficacy in the mouse PF model of an orally bioavailable and peripherally restricted CB1R/iNOS hybrid inhibitor. We report that increased activity of the endocannabinoid/CB1R system parallels disease progression in the lungs of patients with idiopathic PF and in mice with bleomycin-induced PF and is associated with increased tissue levels of interferon regulatory factor-5. Furthermore, we demonstrate that simultaneous engagement of the secondary target iNOS by the hybrid CB1R/iNOS inhibitor has greater antifibrotic efficacy than inhibition of CB1R alone. This hybrid antagonist also arrests the progression of established fibrosis in mice, thus making it a viable candidate for future translational studies in IPF.

We report the design, synthesis, and structure-activity relationships of novel dual-target compounds with antagonist/inverse agonist activity at cannabinoid receptor type 1 (CB 1 R) and inhibitory effect on inducible nitric oxide synthase (iNOS). A series of 3,4-diarylpyrazolinecarboximidamides were synthesized and evaluated in CB 1 receptor (CB 1 R) binding assays and iNOS activity assays. The novel compounds, designed to have limited brain penetrance, elicited potent in vitro CB 1 R antagonist activities and iNOS inhibitory activities. Some key compounds displayed high CB 1 R binding affinities. Compound 7 demonstrated potent in vivo pharmacological activities such as reduction of food intake mediated by the antagonism of the CB 1 Rs and antifibrotic effect in the animal models of fibrosis mediated by iNOS inhibition and CB 1 R antagonism.

Each adult mammalian skeletal muscle has a unique complement of fast and slow myofibers, reflecting patterns established during development and reinforced via their innervation by fast and slow motor neurons. Existing data support a model of postnatal "matching" whereby predetermined myofiber type identity promotes pruning of inappropriate motor axons, but no molecular mechanism has yet been identified. We present evidence that fiber type-specific repulsive interactions inhibit innervation of slow myofibers by fast motor axons during both postnatal maturation of the neuromuscular junction and myofiber reinnervation after injury. The repulsive guidance ligand ephrin-A3 is expressed only on slow myofibers, whereas its candidate receptor, EphA8, localizes exclusively to fast motor endplates. Adult mice lacking ephrin-A3 have dramatically fewer slow myofibers in fast and mixed muscles, and misexpression of ephrin-A3 on fast myofibers followed by denervation/reinnervation promotes their respecification to a slow phenotype. We therefore conclude that Eph/ephrin interactions guide the fiber type specificity of neuromuscular interactions during development and adult life., (© 2015 Stark et al.)