Structural insights into ligand recognition and subtype selectivity of the human melanocortin-3 and melanocortin-5 receptors.

Members of the melanocortin receptor (MCR) family that recognize different melanocortin peptides mediate a broad spectrum of cellular processes including energy homeostasis, inflammation and skin pigmentation through five MCR subtypes (MC1R-MC5R). The structural basis of subtype selectivity of the endogenous agonist γ-MSH and non-selectivity of agonist α-MSH remains elusive, as the two agonists are highly similar with a conserved HFRW motif. Here, we report three cryo-electron microscopy structures of MC3R-Gs in complex with γ-MSH and MC5R-Gs in the presence of α-MSH or a potent synthetic agonist PG-901. The structures reveal that α-MSH and γ-MSH adopt a "U-shape" conformation, penetrate into the wide-open orthosteric pocket and form massive common contacts with MCRs via the HFRW motif. The C-terminus of γ-MSH occupies an MC3R-specific complementary binding groove likely conferring subtype selectivity, whereas that of α-MSH distances itself from the receptor with neglectable contacts. PG-901 achieves the same potency as α-MSH with a shorter length by rebalancing the recognition site and mimicking the intra-peptide salt bridge in α-MSH by cyclization. Solid density confirmed the calcium ion binding in MC3R and MC5R, and the distinct modulation effects of divalent ions were demonstrated. Our results provide insights into ligand recognition and subtype selectivity among MCRs, and expand the knowledge of signal transduction among MCR family members.

Correction to "A Novel G Protein-Biased and Subtype-Selective Agonist for a G Protein-Coupled Receptor Discovered from Screening Herbal Extracts".

[This corrects the article DOI: 10.1021/acscentsci.9b01125.].

A Novel G Protein-Biased and Subtype-Selective Agonist for a G Protein-Coupled Receptor Discovered from Screening Herbal Extracts.

Subtype selectivity and functional bias are vital in current drug discovery for G protein-coupled receptors (GPCRs) as selective and biased ligands are expected to yield drug leads with optimal on-target benefits and minimal side-effects. However, structure-based design and medicinal chemistry exploration remain challenging in part because of highly conserved binding pockets within subfamilies. Herein, we present an affinity mass spectrometry approach for screening herbal extracts to identify active ligands of a GPCR, the 5-HT2C receptor. Using this method, we discovered a naturally occurring aporphine 1857 that displayed strong selectivity for activating 5-HT2C without activating the 5-HT2A or 5-HT2B receptors. Remarkably, this novel ligand exhibited exclusive bias toward G protein signaling for which key residues were identified, and it showed comparable in vivo efficacy for food intake suppression and weight loss as the antiobesity drug, lorcaserin. Our study establishes an efficient approach to discovering novel GPCR ligands by exploring the largely untapped chemical space of natural products.

High-throughput screening against thioredoxin glutathione reductase identifies novel inhibitors with potential therapeutic value for schistosomiasis.

BACKGROUND: Schistosomiasis, a parasitic disease also known as bilharzia and snail fever, is caused by different species of flatworms, such as Schistosoma mansoni (S. mansoni). Thioredoxin glutathione reductase (TGR) from S. mansoni (SmTGR) is a well-characterized drug target for schistosomiasis, yet no anti-SmTGR compounds have reached clinical trials, suggesting that therapeutic development against schistosomiasis might benefit from additional scaffolds targeting this enzyme. METHODS: A high-throughput screening (HTS) assay in vitro against SmTGR was developed and applied to a diverse compound library. SmTGR activity was quantified with ThioGlo®, a reagent that fluoresces upon binding to the free sulfhydryl groups of the reaction product GSH (reduced glutathione). RESULTS: We implemented an HTS effort against 59,360 synthetic compounds. In the primary screening, initial hits (928 or 1.56 %) showing greater than 90 % inhibition on SmTGR activity at a final concentration of 10 µM for each compound were identified. Further tests were carried out to confirm the effects of these hits and to explore the concentration-dependent response characteristics. As a result, 74 of them (0.12 %) representing 17 chemical scaffolds were confirmed and showed a great concentration-dependent inhibitory trend against SmTGR, including structures previously shown to be lethal to schistosomal growth. Of these, two scaffolds displayed a limited structure-activity relationship. When tested in cultured larvae, 39 compounds had cidal activity in 48 h, and five of them killed larvae completely at 3.125 µM. Of these, three compounds also killed adult worms ex vivo at concentrations between 5 µM and 10 µM. CONCLUSION: These confirmed hits may serve as starting points for the development of novel therapeutics to combat schistosomiasis.

High-throughput screening of antagonists for the orphan G-protein coupled receptor GPR139.

AIM: To discover antagonists of the orphan G-protein coupled receptor GPR139 through high-throughput screening of a collection of diverse small molecules. METHODS: Calcium mobilization assays were used to identify initial hits and for subsequent confirmation studies. RESULTS: Five small molecule antagonists, representing 4 different scaffolds, were identified following high-throughput screening of 16 000 synthetic compounds. CONCLUSION: The findings provide important tools for further study of this orphan G-protein coupled receptor.

Supplementation of the maternal diet during pregnancy with chocolate and fructose interacts with the high-fat diet of the young to facilitate the onset of metabolic disorders in rat offspring.

Obesity and non-alcoholic fatty liver disease are the most common metabolic disorders in society today. Previously, we found that supplementing the maternal diet during pregnancy with chocolate and fructose has negative effects on the well-being of the offspring that were ameliorated if the offspring were fed a normal diet during postnatal life. In the present study, we investigated whether feeding offspring a high-fat diet would augment the maternal programming effects and whether extra protein supply can correct the low birth weight resulting from the chocolate-supplemented maternal diet. Pregnant Sprague-Dawley rats were divided into three groups and fed either standard chow (normal nutrition; NN), chocolate- and fructose-supplemented standard chow with casein sodium (overnutrition; ON) or the supplemented standard chow without casein sodium (malnutrition; MN) throughout pregnancy. Male offspring were weaned on either standard or high-fat chow. Dams in the MN group exhibited moderate weight gain, consumed 50% less protein (P < 0.001) but more carbohydrates during gestation and delivered pups with a 12% lower birth weight (P < 0.05) than pups in the NN group, results that are consistent with previous findings. When fed on a high-fat diet after birth, pups from dams in the MN group (MNHD) had 30% more body fat (P = 0.023) and liver triglyceride (TG) levels that were double (P < 0.01) those in offspring in the other groups, leading to fatty livers in these offspring at 14 weeks of age. Hepatic expression of the PPARα, ApoB100, MTTP, CPT1 and SREBP1c genes was significantly downregulated in the MNHD group (P < 0.05 for all), indicating changes in lipid metabolism. Although dams in the ON group exhibited marked gestational weight gain (P < 0.01), they gave birth to normal weight pups that only manifested mild increases in body fat and liver TG content (P < 0.05), without significant changes in the expression of most genes when fed with the high-fat diet. The results suggest that the extra protein supply in the form of casein sodium was able to correct some negative programming effects of the chocolate and fructose supplementation of the maternal diet, which, in conjunction with a high-fat diet in the offspring, may facilitate the onset of metabolic disorders, with impaired liver gene expression possibly a key contributor.

High-throughput screening for GPR119 modulators identifies a novel compound with anti-diabetic efficacy in db/db mice.

G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic ß cells and enteroendocrine cells. It is involved in glucose-stimulated insulin secretion and glucagon-like peptide-1 (GLP-1) release, thereby representing a promising target for the treatment of type 2 diabetes. Although a number of GPR119 agonists were developed, no positive allosteric modulator (PAM) to this receptor has been reported. Here we describe a high-throughput assay for screening GPR119 PAMs and agonists simultaneously. Following screening of a small molecule compound library containing 312,000 synthetic and natural product-derived samples, one potent GPR119 agonist with novel chemical structure, MW1219, was identified. Exposure of MIN6 and GLUTag cells to MW1219 enhanced glucose-stimulated insulin secretion and GLP-1 release; once-daily oral dosing of MW1219 for 6 weeks in diabetic db/db mice reduced hemoglobin A1c (HbA1c) and improved plasma glucose, insulin and GLP-1 levels; it also increased glucose tolerance. The results demonstrate that MW1219 is capable of effectively controlling blood glucose level and may have the potential to be developed as a new class of anti-diabetic agents.

Effects of a maternal diet supplemented with chocolate and fructose beverage during gestation and lactation on rat dams and their offspring.

1. Consumption of a high-fat and high-energy diet during pregnancy leads to a risk of long-term consequences on fetal development, as well as on the postnatal health of offspring. To investigate the effects of such a diet on fetal programming, we established a high-energy intake pregnant rat model using chocolate and fructose beverage as supplements to a normal chow diet. 2. Pregnant Sprague-Dawley rats were assigned to either chow (control) or a diet supplemented with chocolate and fructose beverage throughout gestation and lactation. The male F(1) pups received normal chow diet after weaning. Physiological or pathological changes in dams and pups (e.g. glucose and lipid metabolism) were evaluated. 3. The results showed that dams offered the high-fat (mainly from chocolate) and high-calorie diet during gestation consumed more energy and gained more weight than chow-fed dams. Over-consumption of chocolate reduced chow intake in dams, leading to low maternal protein supply. As a result, pups from these dams exhibited reduced birth weight that lasted until adulthood. The high-energy diet during lactation led to increased total body fat, as well as impaired liver function, in offspring; thus, the lactational diet is suggested to be a stronger determinant of offspring fat metabolism than gestational diet. 4. The results of the study suggest that over-supply of carbohydrates, such as chocolate and fructose, either during gestation or lactation has a negative impact on the well-being of offspring.

Identification of novel KCNQ4 openers by a high-throughput fluorescence-based thallium flux assay.

To develop a real-time thallium flux assay for high-throughput screening (HTS) of human KCNQ4 (Kv7.4) potassium channel openers, we used CHO-K1 cells stably expressing human KCNQ4 channel protein and a thallium-sensitive dye based on the permeability of thallium through potassium channels. The electrophysiological and pharmacological properties of the cell line expressing the KCNQ4 protein were found to be in agreement with that reported elsewhere. The EC(50) values of the positive control compound (retigabine) determined by the thallium and (86)rubidium flux assays were comparable to and consistent with those documented in the literature. Signal-to-background (S/B) ratio and Z factor of the thallium influx assay system were assessed to be 8.82 and 0.63, respectively. In a large-scale screening of 98,960 synthetic and natural compounds using the thallium influx assay, 76 compounds displayed consistent KCNQ4 activation, and of these 6 compounds demonstrated EC(50) values of less than 20 µmol/L and 2 demonstrated EC(50) values of less than 1 µmol/L. Taken together, the fluorescence-based thallium flux assay is a highly efficient, automatable, and robust tool to screen potential KCNQ4 openers. This approach may also be expanded to identify and evaluate potential modulators of other potassium channels.

Tetramethylpyrazine protects palmitate-induced oxidative damage and mitochondrial dysfunction in C2C12 myotubes.

AIMS: Tetramethylpyrazine (TMP), one of the active ingredients isolated from a Chinese herbal prescription, possesses protective effects against oxidative stress caused by high glucose in endothelial cells. In this study, the role of TMP in preventing muscle cells from palmitate-induced oxidative damage was investigated and the possible mechanisms of action elucidated. MAIN METHODS: Mitochondrial reactive oxygen species (ROS) were measured in C2C12 myotubes, a palmitate-induced oxidative stress cell model, with or without TMP. Both mitochondrial membrane potential (MMP) and oxygen consumption were assessed in conjunction with quantification of mitochondrial DNA and mitochondrial biogenesis-related factors, such as peroxisome proliferator-activated receptor-γ coactivator 1 α (PGC1α), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (Tfam), by real-time polymerase chain reaction. Expression of mitochondrial respiratory chain complex III as an index of mitochondrial function was evaluated by immunoblotting, and glucose transport into the C2C12 myotube examined by analyzing 2-deoxy-[(3)H]glucose uptake. KEY FINDINGS: TMP significantly alleviated palmitate-induced mitochondrial ROS production, mitigated mitochondrial dysfunction and increased D-loop mRNA expression as compared with the control. This was accompanied by a marked reversal of palmitate-induced down-regulation in the expression of mitochondrial biogenesis-related factors (PGC1α, NRF1 and Tfam) and decreased glucose uptake in C2C12 myotubes. As a result, cell respiration, as reflected by the elevated expression of mitochondrial respiratory chain complex III and oxygen consumption, was enhanced. SIGNIFICANCE: TMP is capable of protecting C2C12 myotubes against palmitate-induced oxidative damage and mitochondrial dysfunction, and improving glucose uptake in muscle cells partially through the up-regulation of mitochondrial biogenesis.

High-throughput screening for bioactive components from traditional Chinese medicine.

Throughout the centuries, traditional Chinese medicine has been a rich resource in the development of new drugs. Modern drug discovery, which relies increasingly on automated high throughput screening and quick hit-to-lead development, however, is confronted with the challenges of the chemical complexity associated with natural products. New technologies for biological screening as well as library building are in great demand in order to meet the requirements. Here we review the developments in these techniques under the perspective of their applicability in natural product drug discovery. Methods in library building, component characterizing, biological evaluation, and other screening methods including NMR and X-ray diffraction are discussed.

Microfluidic devices for characterizing the agonist of formyl peptide receptor in RBL-FPR cells.

The human formyl peptide receptor (FPR) plays an important role in inflammation and immunity. Finding of specific agonists and antagonists of FPR may provide potential therapeutic agents for FPR related disorders. The binding of agonist by FPR induces a cascade of G protein-mediated signaling events leading to neutrophil chemotaxis, intracellualr calcium mobilization, FPR ligand uptake and so on. This work proposed a microfluidic-based method to characterize FPR-related cellular events in response to small peptides, N-formyl-Met-Leu-Phe (fMLF), in rat basophilic leukemia cell line RBL-2H3 expressing human FPR (RBL-FPR). The results showed that fMLF triggered chemotaxis, calcium mobilization and FPR ligand uptake in RBL-FPR cells, indicating the potential role of FPR agonist. The chemotaxis index and the calcium mobilization intensity increased but the time course of calcium mobilization decreased, as the rising of fMLF concentration. The basic agreement between the microfluidic results and the previous studies demonstrated good feasibility of the microfluidic method for characterization of FPR agonist. Microfluidic technology displays significant advantages over traditional methods in terms of sample consumption and assay time. It also facilitates experimental process and real-time observation of cellular responses at single cell resolution.

Association between green tea intake and coronary artery disease in a Chinese population.

BACKGROUND: There is still conflicting evidence that green tea may protect against coronary atherosclerosis therefore the present study investigated the association between green tea consumption and arteriographically determined coronary atherosclerosis in a Chinese population. METHODS AND RESULTS: The study population consisted of 520 consecutive patients (379 men and 141 women) who underwent coronary arteriography for the first time. Patients were divided into 2 groups (Non-coronary artery disease [CAD] and CAD groups) according to the results of coronary arteriography. After adjusting the established and potential confounders, green tea consumption was associated with a reduced risk of CAD in male patients, with an adjusted odds ratio (OR) of 0.62 (95% confidence interval, 0.38-1.01) compared with those who did not drink green tea. Compared to non-tea drinkers, the adjusted ORs were 1.09 (0.61-1.96) in male patients consuming less than 125 g of dried green tea leaves per month, 0.36 (0.19-0.71) for 125-249 g per month and 0.36 (0.17-0.73) for > or =250 g per month, with a statistically significant test for trend (P<0.001). Similar dose-response relationships were also observed for frequency, duration, concentration and starting age of green tea drinking in male patients. In female patients, no inverse association was found between green tea consumption and CAD. CONCLUSIONS: Green tea consumption can protect against the development of coronary atherosclerosis in Chinese male patients.

[The clinical observation of bipolar transurethral plasma kinetic resection of prostate for high risk benign prostate hyperplasia].

OBJECTIVE: To evaluate therapeutic effect and reliability of bipolar transurethral plasma kinetic prostatectomy (TUPKP) for high risk level benign prostatic hyperplasia (BPH). METHODS: A total of 230 cases of high risk of BPH were treated with TUPKP. Among them, 132 cases with the residual urine of 40 to 420 ml had accepted long term but inefficient medical therapy, 98 cases were suffered with repeating acute urinary retention. One hundred and seventy-three cases with the functional capacity>4 MET were performed the standard transurethral resection of the prostate (TURP), the other 57 cases with the functional capacity<4 MET were accepted the minimally invasive TURP. Among them 12 cases complicated with bladder stones accepted Ho: YAG lithotripsy priory. The international prostate symptom score (IPSS), The maximal urinary flow rate (Qmax) and residual urine of the 2 groups before and after operation were analyzed. RESULTS: There was no transurethral resection syndrome occurred in both groups. After 3 to 12 months of follow-up postoperatively, the IPSS of the two groups were reduced from (21.9+/-5.7) and (23.7+/-5.0) to (4.4+/-2.3) and (5.5+/-2.4), residual urine were reduced from (61.8+/-18.4) ml and (103.9+/-77.3) ml to (13.0+/-6.2) ml and (15.8+/-6.1) ml, respectively. The Qmax was increased from (5.7+/-3.0) ml/s and (4.8+/-2.8) ml/s to (20.9+/-6.3) ml/s and (16.8+/-3.9) ml/s, there existed significant differences (P<0.01). However the IPSS, Qmax and residual urine of the standard group had progressed more obviously than the minimally invasive TURP group (P<0.05). CONCLUSIONS: It is safe and effective to use TUPKP for treating high risk patients of BPH with classic TURP and minimally invasive TURP according to different functional capacity. When the functional capacity is more than 4 MET, the standard procedures is preferred.

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators.

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newly developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.

Antioxidative effect of the herbal remedy Qin Huo Yi Hao and its active component tetramethylpyrazine on high glucose-treated endothelial cells.

AIMS: This study was designed to gain insights into the antioxidant mechanism of a Chinese herbal remedy, Qing Huo Yi Hao (QHYH), and its active components against oxidative stress induced by high glucose in endothelial cells. MAIN METHODS: Effects of QHYH on reactive oxygen species (ROS) production and nitric oxide (NO) generation were measured with the fluorescent markers H(2)DCF-DA and DAF-FM DA, respectively. Phosphorylation of Akt (protein kinase B)/eNOS (endothelial nitric oxide synthase) and uncoupling protein 2 (UCP2) expression were studied by Western blot techniques. Influences of QHYH and one of the active components (tetramethylpyrazine, TMP) on UCP2 expression were subsequently evaluated by quantitative real-time reverse transcription-polymerase chain reaction. Using RNA interference techniques, the involvement of UCP2 in high glucose-induced ROS production in mouse brain microvascular (bEnd.3) cells and its correlation with the antioxidant effect of QHYH were further assessed. KEY FINDINGS: Our results showed that QHYH could protect endothelial cells from high glucose-induced damages, such as ROS production, down-regulation of Akt/eNOS phosphorylation and reduction of NO generation. The protective properties of QHYH were partially attributed to UCP2 mRNA/protein expression, because silence of UCP2 gene by siRNAs (small interfering RNAs) abolished such effects. A total of 28 extracts and 11 active components isolated from QHYH were functionally analyzed. Of which, TMP displayed comparable antioxidant and endothelial protective effects as QHYH. SIGNIFICANCE: All of the data, taken together, point to some therapeutic potential of QHYH and TMP for vascular complications of diabetes.

[Effects of salvianolate on cardiomyocytes apoptosis and heart function in a swine model of acute myocardial infarction].

OBJECTIVE: To observe the effects of salvianolate on cardiomyocytes apoptosis and heart function in a swine model of acute myocardial infarction (AMI). METHODS: A total of 21 young swine were randomly divided into untreated group, low-dose salvianolate (LS) group and high-dose salvianolate (HS) group (7 in each group). AMI was induced by ligaturing left anterior descending coronary artery. After the operation, 400 or 200 mg salvianolate dissolved in 250 mL 5% glucose saline was administered by intravenous drip to swine in the HS group and the LS group respectively for 7 days. The swine in the untreated group were administered with 250 mL 5% glucose saline. The left ventricular ejection fraction (LVEF) and myocardial perfusion were measured by gated myocardial perfusion imaging at the end of the 4th week after operation. And myocardial apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Apoptosis index was calculated under an optical microscope. RESULTS: Myocardial apoptotic index in the edge of myocardial infarction was decreased in the HS group and the LS group, which in the HS group was lower than that in the untreated group (P<0.05). Radioactive defect regions were found by gated myocardial perfusion imaging in all the three groups, but those in the HS group and the LS group were less than those in the untreated group (P<0.05). And the levels of myocardial perfusion and LVEF in the HS group were significantly higher than those in the untreated group (P<0.05). CONCLUSION: Salvianolate administered by intravenous drip can inhibit the cardiomyocytes apoptosis and improve the function of heart after AMI in swine.

The role of tanshinone IIA in the treatment of obesity through peroxisome proliferator-activated receptor gamma antagonism.

Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor that coordinates carbohydrate and lipid metabolism, and is a therapeutic target for type 2 diabetes. Tanshinone IIA (Tan) is a lipophilic diterpene that is widely used to treat cardiovascular diseases in traditional Chinese medicine, and has recently been found to reduce body weight and lower blood lipids. However, its underlying mechanism of antiadipogenic effects remains unknown. Here, we report that Tan inhibits 3T3-L1 preadipocyte differentiation and transcriptional activities of full-length PPARgamma and PPARgamma ligand-binding domains. The effects of Tan are mediated through its property as a natural antagonist of PPARgamma (dissociation constant of an inhibitor value, 2.562 +/- 0.711 microm). Tan treatment reduced adipose mass and body weight, improved glucose tolerance, and lowered the low-density lipoprotein to high-density lipoprotein ratio without changing the food intake in a high-fat diet-induced obese animal model. Our results suggest that the combined properties of Tan in adipogenesis, glucose tolerance, lipogenesis, and cardiovascular protection are beneficial for treating diabetic patients with complex metabolic conditions, in which modulating a single target is often not sufficient to achieve the desired effect.

A new lignan glycoside from Peperomia duclouxii.

A new lignan glycoside was isolated from the n-BuOH extract of Peperomia duclouxii C. DC., and its structure was elucidated to be 2-(4''-hydroxy-3'', 5''-dimethoxybenzyl)-3-(5'-methoxy-3', 4'-methylenedioxybenzyl)butyrolactone-4''-O-beta-D-glucopyranoside (1) by spectral methods.