Specific Agonist Research Articles

Structural basis and molecular mechanism of biased GPBAR signaling in regulating NSCLC cell growth via YAP activity

The G protein-coupled bile acid receptor (GPBAR) is the membrane receptor for bile acids and a driving force of the liver-bile acid-microbiota-organ axis to regulate metabolism and other pathophysiological processes. Although GPBAR is an important therapeutic target for a spectrum of metabolic and neurodegenerative diseases, its activation has also been found to be linked to carcinogenesis, leading to potential side effects. Here, via functional screening, we found that two specific GPBAR agonists, R399 and INT-777, demonstrated strikingly different regulatory effects on the growth and apoptosis of non-small cell lung cancer (NSCLC) cells both invitro and invivo. Further mechanistic investigation showed that R399-induced GPBAR activation displayed an obvious bias for β-arrestin 1 signaling, thus promoting YAP signaling activation to stimulate cell proliferation. Conversely, INT-777 preferentially activated GPBAR-Gs signaling, thus inactivating YAP to inhibit cell proliferation and induce apoptosis. Phosphorylation of GPBAR by GRK2 at S310/S321/S323/S324 sites contributed to R399-induced GPBAR-β-arrestin 1 association. The cryoelectron microscopy (cryo-EM) structure of the R399-bound GPBAR-Gs complex enabled us to identify key interaction residues and pivotal conformational changes in GPBAR responsible for the arrestin signaling bias and cancer cell proliferation. In summary, we demonstrate that different agonists can regulate distinct functions of cell growth and apoptosis through biased GPBAR signaling and control of YAP activity in a NSCLC cell model. The delineated mechanism and structural basis may facilitate the rational design of GPBAR-targeting drugs with both metabolic and anticancer benefits.

SJPYT-195: A Designed Nuclear Receptor Degrader That Functions as a Molecular Glue Degrader of GSPT1.

We previously reported a specific inverse agonist (SPA70) of the nuclear receptor pregnane X receptor (PXR). However, derivatization of SPA70 yielded only agonists and neutral antagonists, suggesting that inverse agonism of PXR is difficult to achieve. Therefore, we sought to design proteolysis targeting chimeras (PROTACs) aimed at inducing PXR degradation. Conjugation of a SPA70 derivative to ligands of the E3 substrate receptor cereblon (CRBN) resulted in one molecule, SJPYT-195, that reduced PXR protein level in an optimized degradation assay described here. Further analysis revealed that SJPYT-195 was a molecular glue degrader of the translation termination factor GSPT1 and that GSPT1 degradation resulted in subsequent reduction of PXR protein. GSPT1 has recently gained interest as an anticancer target, and our results give new insights into chemical determinants of drug-induced GSPT1 degradation. Additionally, we have developed assays and cell models for PXR degrader discovery that can be applied to additional protein targets.

Feeding desensitizes A1 adenosine receptors in adipose through FOXO1-mediated transcriptional regulation

ObjectiveAdipose tissue is a critical regulator of energy balance that must rapidly shift its metabolism between fasting and feeding to maintain homeostasis. Adenosine has been characterized as an important regulator of adipocyte metabolism primarily through its actions on A1 adenosine receptors (A1R). We sought to understand the role A1R plays specifically in adipocytes during fasting and feeding to regulate glucose and lipid metabolism. MethodsWe used Adora1 floxed mice with an inducible, adiponectin-Cre to generate FAdora1−/− mice, where F designates a fat-specific deletion of A1R. We used these FAdora1−/− mice along with specific agonists and antagonists of A1R to investigate changes in adenosine signaling within adipocytes between the fasted and fed state. ResultsWe found that the adipose tissue response to adenosine is not static, but changes dynamically according to nutrient conditions through the insulin-Akt-FOXO1 axis. We show that under fasted conditions, FAdora1−/− mice had impairments in the suppression of lipolysis by insulin on normal chow and impaired glucose tolerance on high-fat diet. FAdora1−/− mice also exhibited a higher lipolytic response to isoproterenol than WT controls when fasted, however this difference was lost after a 4-hour refeeding period. We demonstrate that FOXO1 binds to the A1R promoter, and refeeding leads to a rapid downregulation of A1R transcript and desensitization of adipocytes to A1R agonism. Obesity also desensitizes adipocyte A1R, and this is accompanied by a disruption of cyclical changes in A1R transcription between fasting and refeeding. ConclusionsWe propose that FOXO1 drives high A1R expression under fasted conditions to limit excess lipolysis during stress and augment insulin action upon feeding. Subsequent downregulation of A1R under fed conditions leads to desensitization of these receptors in adipose tissue. This regulation of A1R may facilitate reentrance into the catabolic state upon fasting.

The Role of Dexmedetomidine in Tumor-Progressive Factors in the Perioperative Period and Cancer Recurrence: A Narrative Review.

Dexmedetomidine, a specific α2 adrenergic receptor agonist, is highly frequently used in the perioperatively for its favorable pharmacology, such as mitigating postoperative cognitive dysfunction. Increasing attention has been recently focused on the effect of whether dexmedetomidine influences cancer recurrence, which urges the discussion of the role of dexmedetomidine in tumor-progressive factors. The pharmacologic characteristics of dexmedetomidine, the tumor-progressive factors in the perioperative period, and the relationships between dexmedetomidine and tumor-progressive factors were described in this review. Available evidence suggests that dexmedetomidine could reduce the degree of immune function suppression, such as keeping the number of CD3+ cells, NK cells, CD4+/CD8+ ratio, and Th1/Th2 ratio stable and decreasing the level of proinflammatory cytokine (interleukin 6 and tumor necrosis factor-alpha) during cancer operations. However, dexmedetomidine exhibits different roles in cell biological behavior depending on cancer cell types. The conclusions on whether dexmedetomidine would influence cancer recurrence could not be currently drawn for the lack of strong clinical evidence. Therefore, this is still a new area that needs further exploration.

Long-term treatment with the mPXR agonist PCN promotes hepatomegaly and lipid accumulation without hepatocyte proliferation in mice.

Pregnane X receptor (PXR) is highly expressed in the liver and plays a pivotal role in xenobiotic and endobiotic metabolism. We previously reported that PXR activation by its specific mouse agonist pregnenolone 16α-carbonitrile (PCN) significantly induces liver enlargement and lipid accumulation. However, the effect of long-term PCN treatment on PXR and mouse liver is still unknown. This study aimed to explore the influence of long-term administration of PCN on mouse liver and hepatic lipid homeostasis. Male C57BL/6 mice were injected intraperitoneally with PCN (100 mg/kgonce aweek) for 42 weeks. Serum and liver samples were collected for biochemical and histological analysis. PXR activation was investigated by Western blot. Ultra-high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (UHPLC-ESI-HRMS)-based lipidomics analysis was performed to explore the change in different lipid categories. The results showed that long-term treatment with PCN significantly promoted hepatomegaly without hepatocyte proliferation and enlargement. Long-term treatment with PCN did not upregulate PXR target proteins in mice, and there was no significant upregulation of CYP3A11, CYP2B10, UGT1A1, MRP2, or MRP4. Lipidomics analysis showed obvious hepatic lipid accumulation in the PCN-treated mice, and the most significant change was found in triglycerides (TGs). Additionally, long-term treatment with PCN had no risk for carcinogenesis. These findings demonstrated that long-term PCN treatment induces hepatomegaly and lipid accumulation without hepatocyte proliferation or enlargement.

Blockage of PD-L1 by FERMT3-mediated Wnt/β-catenin signalling regulates chemoresistance and immune evasion of colorectal cancer cells.

Colorectal cancer (CRC) constitutes a major public health problem because of the high rate of morbidity and mortality. Chemotherapy and immunotherapy are the major and promising strategies for cancer patients including CRC; nevertheless, chemoresistance and immune escape limit the final efficacy of the above approaches. FERMT3 has proven to exert a critical role in the immune system and has contradictive effects on cancer progression. In this study, bioinformatics database analysis and clinical specimen detection both corroborated the downregulation of FERMT3 in CRC tissues and cells. Of interest, overexpression of FERMT3 suppressed CRC cell invasion and sensitized cells to 5-fluorouracil (5-FU) by reducing cell viability and increasing cell apoptosis and caspase 3 activity. Noticeably, FERMT3 upregulation enhanced natural killer (NK) cells activation by increasing secretions of interferon γ (IFN-γ) and tumour necrosis factor α (TNF-α) when NK cells were co-cultured with CRC cells. Importantly, upregulation of FERMT3 promoted NK cell-mediated killing of CRC cells. Mechanically, FERMT3 inhibited the aberrant activation of Wnt/β-catenin signalling and the subsequent programmed death-ligand 1 (PD-L1) expression in CRC cells. Moreover, knockdown of PD-L1 suppressed CRC cell invasion, 5-FU resistance and NK cells-mediated tumour killing. Additionally, reactivating the Wnt/β-catenin signalling with a specific WNT agonist CAS 853220-52-7 overturned the efficacy of FERMT3 overexpression against CRC cell invasion, 5-FU chemoresistance and cell susceptibility to NK cell-mediated cytotoxicity. Therefore, the current findings substantiate that FERMT3 elevation may attenuate CRC cell chemoresistance and NK cell-mediated immune response to tumour cells by inhibiting Wnt/β-catenin-PD-L1 signalling. Therefore, FERMT3 elevation may be a promising therapeutic approach to overcome chemoresistance and immune evasion in CRC.

Characterization of Endothelium-Dependent Relaxation in the Saphenous Artery and Its Caudal Branches in Young and Old Adult Sprague Dawley Rats.

Ageing is associated with reduced endothelium-derived nitric oxide (NO) production in the femoral artery of Sprague Dawley (SD) rats. In the current study, we examined endothelium-dependent relaxation (EDR) in the saphenous artery and its caudal branches. We used acetylcholine and the Proteinase-Activated receptor-2 (PAR2)-specific agonist (2fLIGRLO) with nitroarginine methylester (L-NAME) to assess EDR in two groups of male SD rats (age in weeks: young, 10–12; old, 27–29). Acetylcholine and 2fLIGRLO were potent NO-dependent relaxant agents in all arteries. For all arteries, EDR by acetylcholine decreased significantly in old compared to young SD rats. Interestingly, PAR2-induced EDR of proximal saphenous artery segments and caudal branches decreased significantly in old compared to young, but did not differ for the in-between middle and distal ends of the saphenous artery. L-NAME treatment increased subsequent contractions of proximal and middle segments of saphenous arteries by phenylephrine and U46619 in young, but not in old, SD rats. We conclude the SD saphenous artery and caudal branches exhibit regional characteristics that differ in response to specific EDR agonists, endothelial NO synthase inhibitor, and changes to endothelium function with increased age, which are, in part, attributed to decreased sensitivity of vascular smooth muscle to the gaseous transmitter NO.

Group II Metabotropic Glutamate Receptors Reduce Apoptosis and Regulate BDNF and GDNF Levels in Hypoxic-Ischemic Injury in Neonatal Rats.

Birth asphyxia causes brain injury in neonates, but a fully successful treatment has yet to be developed. This study aimed to investigate the effect of group II mGlu receptors activation after experimental birth asphyxia (hypoxia-ischemia) on the expression of factors involved in apoptosis and neuroprotective neurotrophins. Hypoxia-ischemia (HI) on 7-day-old rats was used as an experimental model. The effects of intraperitoneal application of mGluR2 agonist LY379268 (5 mg/kg) and the specific mGluR3 agonist NAAG (5 mg/kg) (1 h or 6 h after HI) on apoptotic processes and initiation of the neuroprotective mechanism were investigated. LY379268 and NAAG applied shortly after HI prevented brain damage and significantly decreased pro-apoptotic Bax and HtrA2/Omi expression, increasing expression of anti-apoptotic Bcl-2. NAAG or LY379268 applied at both times also decreased HIF-1α formation. HI caused a significant decrease in BDNF concentration, which was restored after LY379268 or NAAG administration. HI-induced increase in GDNF concentration was decreased after administration of LY379268 or NAAG. Our results show that activation of mGluR2/3 receptors shortly after HI prevents brain damage by the inhibition of excessive glutamate release and apoptotic damage decrease. mGluR2 and mGluR3 agonists produced comparable results, indicating that both receptors may be a potential target for early treatment in neonatal HI.

Roles of EP Receptors in the Regulation of Fluid Balance and Blood Pressure.

Prostaglandin E2 (PGE2) is an important prostanoid expressing throughout the kidney and cardiovascular system. Despite the diverse effects on fluid metabolism and blood pressure, PGE2 is implicated in sustaining volume and hemodynamics homeostasis. PGE2 works through four distinct E-prostanoid (EP) receptors which are G protein-coupled receptors. To date, pharmacological specific antagonists and agonists of all four subtypes of EP receptors and genetic targeting knockout mice for each subtype have helped in uncoupling the diverse functions of PGE2 and discriminating the respective characteristics of each receptor. In this review, we summarized the functions of individual EP receptor subtypes in the renal and blood vessels and the molecular mechanism of PGE2-induced fluid metabolism and blood pressure homeostasis.

Activation of transient receptor potential vanilloid 4 is involved in pressure overload-induced cardiac hypertrophy.

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is expressed in hearts and implicated in cardiac remodeling and dysfunction. However, the effects of TRPV4 on pressure overload-induced cardiac hypertrophy remain unclear. In this study, we found that TRPV4 expression was significantly increased in mouse hypertrophic hearts, human failing hearts, and neurohormone-induced hypertrophic cardiomyocytes. Deletion of TRPV4 attenuated transverse aortic constriction (TAC)-induced cardiac hypertrophy, cardiac dysfunction, fibrosis, inflammation, and the activation of NFκB - NOD - like receptor pyrin domain-containing protein 3 (NLRP3) in mice. Furthermore, the TRPV4 antagonist GSK2193874 (GSK3874) inhibited cardiac remodeling and dysfunction induced by TAC. In vitro, pretreatment with GSK3874 reduced the neurohormone-induced cardiomyocyte hypertrophy and intracellular Ca2+ concentration elevation. The specific TRPV4 agonist GSK1016790A (GSK790A) triggered Ca2+ influx and evoked the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). But these effects were abolished by removing extracellular Ca2+ or GSK3874. More importantly, TAC or neurohormone stimulation-induced CaMKII phosphorylation was significantly blocked by TRPV4 inhibition. Finally, we show that CaMKII inhibition significantly prevented the phosphorylation of NFκB induced by GSK790A. Our results suggest that TRPV4 activation contributes to pressure overload-induced cardiac hypertrophy and dysfunction. This effect is associated with upregulated Ca2+/CaMKII mediated activation of NFκB-NLRP3. Thus, TRPV4 may represent a potential therapeutic drug target for cardiac hypertrophy and dysfunction after pressure overload.

Abstract 1852: G protein-coupled estrogen receptor-1 agonist LNS8801 induces mitotic arrest and cell death in uveal melanoma cells

Abstract Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults. Nearly 50% of the patients develop hepatic metastasis, and when this occurs the 1-year survival rate is 12 months. While the primary tumor can be treated with radiotherapy and surgery, current treatments for metastatic disease have shown limited clinical activity in UM patients and there is an urgent need for new effective therapies. Recent reports have shown that male patients have more metastases than female patients, and the time from the diagnosis until development of metastases was shorter, suggesting a potential role of female hormones in disease progression. The G-protein-coupled estrogen receptor-1 (GPER) has distinct functions from those of the classic estrogen receptors ERα/β and its activation with a specific agonist has tumor-suppressive roles in several cancers. Here, we demonstrate that the novel clinical GPER agonist, LNS8801 (Linnaeus Therapeutics), has potent anticancer activities in UM cells including inhibition of cell migration and cell proliferation. LNS8801 treatment induced a G2/M cell cycle arrest due to disruption of tubulin polymerization and defective mitotic spindle formation. There was a time-dependent increase of mitotic proteins, such as phosphorylated aurora-A, aurora-B and histone-3, while c-Myc and phospho-Rb were downregulated. The drug treatment also induced significant cell death, as detected by caspase 3/7 activation and PARP cleavage. Finally, LNS8801 inhibited tumor growth in a UM xenograft model in vivo. In conclusion, our studies suggest that GPER agonists may be a novel treatment for uveal melanoma, and clinical trials with the GPER agonist LNS8801 are warranted and currently underway. Citation Format: Grazia Ambrosini, Christopher A. Natale, Gary K. Schwartz. G protein-coupled estrogen receptor-1 agonist LNS8801 induces mitotic arrest and cell death in uveal melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1852.

Abstract 3568: Robust and potent dendritic cells for cancer immunotherapy

Abstract Background: Dendritic cells (DCs) are fundamental to adaptive immunity by detecting, engulfing and presenting foreign or changed antigenic peptides to lymphocytes that thereafter initiate antigen-specific immune attacks. DC-based immunotherapy against cancer has been promising for a long time, but has not yet met expectations. Cancer cell heterogeneity and numerous immune evasion mechanisms represent challenges. According to textbook renderings, DCs may induce either tolerance or immune activation depending upon the nature of the encountered antigen and the status of the microenvironment. Recently, we have discovered a more complicated scenario according to which monocyte-derived DCs (moDCs) exhibit dual pro- and anti-inflammatory features, suggesting that selected conditions that enhance pro-inflammatory features and counteract tolerogenic features could generate more robust and potent DCs for cancer therapy. Materials and Methods: Healthy blood donor buffy coats were used for the isolation of monocytes and conventional CD141+ and CD1c+ natural circulating DCs. IL-4 and GM-CSF were used to differentiate monocytes into immature moDCs. Toll-like receptor agonists were used to induce DC maturation, and specific agonists and inhibitors were tested in different DC variants. Flow cytometry, ELISA determination of cytokine secretion, HPLC-based quantitation of kynurenine secretion, genome-wide gene expression analyses and functional assays, such as antigen uptake, migration and the mixed leukocyte reaction were used for evaluation of treated DCs. Results: Conventional DCs were more susceptible to apoptosis, and dependent upon culture conditions, than moDCs. Inhibition of β-catenin signaling stimulated several pro-inflammatory features, e.g. increased IL-12 and decreased IL-10 secretion, with reciprocal effects caused by β-catenin stimulation. Maturation of moDCs was accompanied by very strong induction of indoleamine 2, 3-dioxygenase 1 (IDO1) transcription and paralleled by increased kynurenine secretion, but this was partially counteracted by β-catenin stimulation. Conclusions: Preparation of more potent DCs for cancer therapy needs to take into account DC longevity and conflicting activation patterns of pro- and anti-inflammatory abilities. Citation Format: Waqas Azeem, Seyed Mohammad Lellahi, Yaping Hua, Anne Margrete Øyan, Benjamin Gabriel, Karl-Henning Kalland. Robust and potent dendritic cells for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3568.

Abstract 1856: Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206

Abstract ONC201 (originally discovered as TRAIL-Inducing Compound #10 or TIC10) and analogue ONC206 have been found to induce an integrated stress response with suggested primary targets and mechanisms involving agonist activity against mitochondrial ClpP and antagonism of dopamine receptors D2/3 (DRD2/3). We hypothesized that dopamine, the agonist of DRD2, may compete with ONC201 or ONC206 for DRD2/3 and impair the anti-cancer effect of ONC201 or ONC206. We therefore pre-treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, lung cancer, GBM, and DIPG with dopamine for 12 hours, 24 hours or 48 hours, respectively, followed by treatment of ONC201 or ONC206 for 72hours. Cell viability was determined by the CellTiter Glo assay. Single treatment with dopamine did not increase cell viability. We observed that 48 hours of pre-treatment with dopamine at 50 μM, 100 μM or 200 μM impaired the cell viability suppression effect of ONC201 at 4 μM and 8 μM, while ONC206 at 0.5 μM, 1 μM and 2 μM led to similar protection for loss of cell viability in pancreatic cancer cells. The impairment of the anti-cancer effect by dopamine was also observed in colorectal cancer cells with 48 hours pre-treatment with dopamine at 50 μM, 100 μM, 200 μM or 400 μM and treatment with ONC201 at 4 μM and 8 μM, or ONC206 at 0.5 μM, 1 μM, or 2 μM. In addition, we treated pancreatic cancer and colorectal cancer cells with ONC212 after 48 hours pre-treatment with dopamine and observed a similar impairment of the anti-cancer effect, even though ONC212 is not a DRD2 antagonist. We pre-treated pancreatic cancer and colorectal cancer cells with specific DRD2 agonist, sumanirole, for 48 hours followed by treatment of ONC201, ONC206, or ONC212 for 72 hours, and no impairment was seen. We pre-treated multiple cancer cell lines with 2 μM or 10 μM dopamine for one week followed by ONC201, ONC206, or ONC212 treatment and performed colony assays. Pre-treatment with dopamine at 10 μM impaired the anti-cancer effect of ONC201 or ONC206 in pancreatic cancer. Pre-treatment with dopamine at 2 μM or 10 μM impaired the anti-tumor effect of ONC201 in GBM cells. By contrast to the cell viability result, no impairment of ONC212 effect from dopamine was seen in pancreatic cancer cells. Western blotting was conducted to investigate whether dopamine pre-treatment impacted signaling pathways reported to be affected by ONC201. Dopamine pre-treatment did not impact changes induced by ONC201 in ATF4, CHOP, DR5, ClpX, clpP, pERK, pAkt and pS6. We are pursuing whether the protective effect of dopamine is due to the agonism of DRD2 or alternative pro-survival mechanisms. Citation Format: Yiqun Zhang, Wafik S. El-Deiry. Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1856.

Improving Genomic Selection for Heat Tolerance in Dairy Cattle: Current Opportunities and Future Directions.

Heat tolerance is the ability of an animal to maintain production and reproduction levels under hot and humid conditions and is now a trait of economic relevance in dairy systems worldwide because of an escalating warming climate. The Australian dairy population is one of the excellent study models for enhancing our understanding of the biology of heat tolerance because they are predominantly kept outdoors on pastures where they experience direct effects of weather elements (e.g., solar radiation). In this article, we focus on evidence from recent studies in Australia that leveraged large a dataset [∼40,000 animals with phenotypes and 15 million whole-genome sequence variants] to elucidate the genetic basis of thermal stress as a critical part of the strategy to breed cattle adapted to warmer environments. Genotype-by-environment interaction (i.e., G × E) due to temperature and humidity variation is increasing, meaning animals are becoming less adapted (i.e., more sensitive) to changing environments. There are opportunities to reverse this trend and accelerate adaptation to warming climate by 1) selecting robust or heat-resilient animals and 2) including resilience indicators in breeding goals. Candidate causal variants related to the nervous system and metabolic functions are relevant for heat tolerance and, therefore, key for improving this trait. This could include adding these variants in the custom SNP panels used for routine genomic evaluations or as the basis to design specific agonist or antagonist compounds for lowering core body temperature under heat stress conditions. Indeed, it was encouraging to see that adding prioritized functionally relevant variants into the 50k SNP panel (i.e., the industry panel used for genomic evaluation in Australia) increased the prediction accuracy of heat tolerance by up to 10% units. This gain in accuracy is critical because genetic improvement has a linear relationship with prediction accuracy. Overall, while this article used data mainly from Australia, this could benefit other countries that aim to develop breeding values for heat tolerance, considering that the warming climate is becoming a topical issue worldwide.

Deubiquitinase OTUD3 regulates metabolism homeostasis in response to nutritional stresses

The ovarian-tumor-domain-containing deubiquitinases (OTUDs) block ubiquitin-dependent protein degradation and are involved in diverse signaling pathways. We discovered a rare OTUD3 c.863G>A mutation in a family with an early age of onset of diabetes. This mutation reduces the stability and catalytic activity of OTUD3. We next constructed an experiment with Otud3-/- mice and found that they developed worse obesity, dyslipidemia, and insulin resistance than wild-type mice when challenged with a high-fat diet (HFD). We further found that glucose and fatty acids stimulate CREB-binding-protein-dependent OTUD3 acetylation, promoting its nuclear translocation, where OTUD3 regulates various genes involved in glucose and lipid metabolism and oxidative phosphorylation by stabilizing peroxisome-proliferator-activated receptor delta (PPARδ). Moreover, targeting PPARδ using a specific agonist can partially rescue the phenotype of HFD-fed Otud3-/- mice. We propose that OTUD3 is an important regulator of energy metabolism and that the OTUD3 c.863G>A is associated with obesity and a higher risk of diabetes.

Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion.

TMEM16F, a Ca2+-activated phospholipid scramblase (CaPLSase), is critical for placental trophoblast syncytialization, HIV infection, and SARS-CoV2-mediated syncytialization, however, how TMEM16F is activated during cell fusion is unclear. Here, using trophoblasts as a model for cell fusion, we demonstrate that Ca2+ influx through the Ca2+ permeable transient receptor potential vanilloid channel TRPV4 is critical for TMEM16F activation and plays a role in subsequent human trophoblast fusion. GSK1016790A, a TRPV4 specific agonist, robustly activates TMEM16F in trophoblasts. We also show that TRPV4 and TMEM16F are functionally coupled within Ca2+ microdomains in a human trophoblast cell line using patch-clamp electrophysiology. Pharmacological inhibition or gene silencing of TRPV4 hinders TMEM16F activation and subsequent trophoblast syncytialization. Our study uncovers the functional expression of TRPV4 and one of the physiological activation mechanisms of TMEM16F in human trophoblasts, thus providing us with novel strategies to regulate CaPLSase activity as a critical checkpoint of physiologically and disease-relevant cell fusion events.

Discovery of novel Thieno[2,3-d]imidazole derivatives as agonists of human STING for antitumor immunotherapy using systemic administration

The activation of stimulator of interferon genes (STING) signaling pathways plays an important role in the innate immune response. Although several STING agonists have been developed recently, the majority of clinical CDN STING agonists are administered by intratumoral (IT) injection. Therefore, there remains a need to develop diverse non-CDN small-molecule STING agonists with systemic administration. Herein, by using a scaffold hopping strategy, we designed a series of thieno [2,3-d]imidazole derivatives as novel STING agonists. Further structure-activity relationship study and optimization led to the discovery of compound 45 as a highly potent human STING agonist with an EC50 value of 1.2 nM. Compound 45 was found to bind to multiple human STING isoforms and accordingly activated the downstream TBK1/IRF3 and NF-κB signaling pathways in the reporter cells bearing with different STING isoforms. The activation on STING signaling pathway was abolished in the STING knock-out cells, indicating that it is a specific STING agonist. Compound 45 significantly inhibited the tumor growth in allograft 4T1 and CT26 tumor models by systemic administration, and more significantly, 45 was able to induce tumor regression in CT26 tumor model without inducing weight loss, suggesting that compound 45 is a highly promising candidate worthy for further development.

The Nonpeptide Agonist MK-5046 Functions As an Allosteric Agonist for the Bombesin Receptor Subtype-3.

Allosteric ligands of various G-protein-coupled receptors are being increasingly described and are providing important advances in the development of ligands with novel selectivity and efficacy. These unusual properties allow expanded opportunities for pharmacologic studies and treatment. Unfortunately, no allosteric ligands are yet described for the bombesin receptor family (BnRs), which are proposed to be involved in numerous physiologic/pathophysiological processes in both the central nervous system and peripheral tissues. In this study, we investigate the possibility that the bombesin receptor subtype-3 (BRS-3) specific nonpeptide receptor agonist MK-5046 [(2S)-1,1,1-trifluoro-2-[4-(1H-pyrazol-1-yl)phenyl]-3-(4-[[1-(trifluoromethyl)cyclopropyl]methyl]-1H-imidazol-2-yl)propan-2-ol] functions as a BRS-3 allosteric receptor ligand. We find that in BRS-3 cells, MK-5046 only partially inhibits iodine-125 radionuclide (125I)-Bantag-1 [Boc-Phe-His-4-amino-5-cyclohexyl-2,4,5-trideoxypentonyl-Leu-(3-dimethylamino) benzylamide N-methylammonium trifluoroacetate] binding and that both peptide-1 (a universal BnR-agonist) and MK-5046 activate phospholipase C; however, the specific BRS-3 peptide antagonist Bantag-1 inhibits the action of peptide-1 competitively, whereas for MK-5046 the inhibition is noncompetitive and yields a curvilinear Schild plot. Furthermore, MK-5046 shows other allosteric behaviors, including slowing dissociation of the BRS-3 receptor ligand 125I-Bantag-1, dose-inhibition curves being markedly affected by increasing ligand concentration, and MK-5046 leftward shifting the peptide-1 agonist dose-response curve. Lastly, receptor chimeric studies and site-directed mutagenesis provide evidence that MK-5046 and Bantag-1 have different binding sites determining their receptor high affinity/selectivity. These results provide evidence that MK-5046 is functioning as an allosteric agonist at the BRS-3 receptor, which is the first allosteric ligand described for this family of receptors. SIGNIFICANCE STATEMENT: G-protein-coupled receptor allosteric ligands providing higher selectivity, selective efficacy, and safety that cannot be obtained using usual orthosteric receptor-based strategies are being increasingly described, resulting in enhanced usefulness in exploring receptor function and in treatment. No allosteric ligands exist for any of the mammalian bombesin receptor (BnR) family. Here we provide evidence for the first such example of a BnR allosteric ligand by showing that MK-5046, a nonpeptide agonist for bombesin receptor subtype-3, is functioning as an allosteric agonist.

Mitochondrial Calcium Disorder Affects Early Embryonic Development in Mice through Regulating the ERK/MAPK Pathway.

The homeostasis of mitochondrial calcium ([Ca2+]mt) in oocytes plays a critical role in maintaining normal reproductive cellular progress such as meiosis. However, little is known about the association between [Ca2+]mt homeostasis and early embryonic development. Two in vitro mouse MII oocyte models were established by using a specific agonist or inhibitor targeting mitochondrial calcium uniporters (MCU) to upregulate or downregulate [Ca2+]mt concentrations. The imbalance of [Ca2+]mt in MII oocytes causes mitochondrial dysfunction and morphological abnormity, leading to an abnormal spindle/chromosome structure. Oocytes in drug-treated groups are less likely to develop into blastocyst during in vitro culture. Abnormal [Ca2+]mt concentrations in oocytes hindered epigenetic modification and regulated mitogen-activated protein kinase (MAPK) signaling that is associated with gene expression. We also found that MAPK/ERK signaling is regulating DNA methylation in MII oocytes to modulate epigenetic modification. These data provide a new insight into the protective role of [Ca2+]mt homeostasis in early embryonic development and also demonstrate a new mechanism of MAPK signaling regulated by [Ca2+]mt that influences epigenetic modification.

A Critical Role of δ-Opioid Receptor in Anti-microglial Activation Under Stress

Microglia are involved in the regulation of cerebral homeostasis and pathogen confrontation. There is, however, evidence showing that excessive microglia activation is implicated in various age-related cerebral diseases. On the other hand, microglia may experience complex changes of polarization in pathological insults, i.e., from a proinflammatory M1 to an anti-inflammatory M2 phenotype, which differentially contribute to the exacerbation or alleviation of cellular injury. Remolding the phenotype of microglia or inhibiting the excessive activation of microglia seems to be a promising approach against neurodegenerative pathologies. Since δ-opioid receptor (DOR) activation exhibits a strong protective capacity against various neuronal injuries, especially the hypoxic/ischemic injury, we asked if the DOR-induced neuroprotection is associated with its effect on microglia. We explored this fundamental issue by using pharmacological and genetic approaches in the BV2 cell line, a general type of microglial cells. The results showed that DOR expression significantly increased in the activated microglial M2 phenotype, but slightly decreased in the microglial M1 phenotype. Hypoxia induced dual polarizations of BV2 cells with an increase in DOR expression. Administration of a specific DOR agonist, UFP-512, largely inhibited lipopolysaccharide (LPS) or hypoxia-induced microglial M1 activation and inflammatory activity with high concentrations of UFP-512 being effective to reverse the interleukin-4 (IL4)-induced microglial activation. Consistent with these observations, inhibiting DOR or knocking-down DOR promoted the excessive activation of BV2 cells in both M1 and M2 directions, while DOR overexpression did the opposite. Furthermore, the PC12 cells exposed to the conditioned medium of BV2 cells treated by UFP-512 grew better than those treated directly with UFP-512 under LPS or hypoxic insults. DOR inhibitor naltrindole could block all the effects of DOR activation. The medium from the BV2 cells with DOR knock-down decreased the viability of PC12 cell, while the medium from the BV2 cells with DOR overexpression largely attenuated LPS or hypoxic injury in the PC12 cells. These first data suggest a close linkage between DOR expression/function and microglial polarization and a critical role of DOR in negative controlling microglial activation. Our work provides a novel clue for new protective strategies against neurodegenerative pathophysiology through DOR-mediated regulation of microglia.