Effects of site-directed mutagenesis of GLP-1 and glucagon receptors on signal transduction activated by dual and triple agonists.

The paradigm of one drug against multiple targets, known as unimolecular polypharmacology, offers the potential to improve efficacy while overcoming some adverse events associated with the treatment. This approach is best exemplified by targeting two or three class B1 G protein-coupled receptors, namely, glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulinotropic polypeptide receptor for treatment of type 2 diabetes and obesity. Some of the dual and triple agonists have already shown initial successes in clinical trials, although the molecular mechanisms underlying their multiplexed pharmacology remain elusive. In this study we employed structure-based site-directed mutagenesis together with pharmacological assays to compare agonist efficacy across two key signaling pathways, cAMP accumulation and ERK1/2 phosphorylation (pERK1/2). Three dual agonists (peptide 15, MEDI0382 and SAR425899) and one triple agonist (peptide 20) were evaluated at GLP-1R and GCGR, relative to the native peptidic ligands (GLP-1 and glucagon). Our results reveal the existence of residue networks crucial for unimolecular agonist-mediated receptor activation and their distinct signaling patterns, which might be useful to the rational design of biased drug leads.

Formyl peptide receptor 2 as a potential therapeutic target for inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a global health burden whose existing treatment is largely dependent on anti-inflammatory agents. Despite showing some therapeutic actions, their clinical efficacy and adverse events are unacceptable. Resolution as an active and orchestrated phase of inflammation involves improper inflammatory response with three key triggers, specialized pro-resolving mediators (SPMs), neutrophils and phagocyte efferocytosis. The formyl peptide receptor 2 (FPR2/ALX) is a human G protein-coupled receptor capable of binding SPMs and participates in the resolution process. This receptor has been implicated in several inflammatory diseases and its association with mouse model of IBD was established in some resolution-related studies. Here, we give an overview of three reported FPR2/ALX agonists highlighting their respective roles in pro-resolving strategies.

Molecular recognition of two endogenous hormones by the human parathyroid hormone receptor-1.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor-1 (PTH1R), a member of class B G protein- coupled receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.62 Å and 3.25 Å, respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders.

A high-throughput screening campaign against PFKFB3 identified potential inhibitors with novel scaffolds.

The growth of solid tumors depends on tumor vascularization and the endothelial cells (ECs) that line the lumen of blood vessels. ECs generate a large fraction of ATP through glycolysis, and elevation of their glycolytic activity is associated with angiogenic behavior in solid tumors. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) positively regulates glycolysis via fructose-2/6-bisphosphate, the product of its kinase activity. Partial inhibition of glycolysis in tumor ECs by targeting PFKFB3 normalizes the otherwise abnormal tumor vessels, thereby reducing metastasis and improving the outcome of chemotherapy. Although a limited number of tool compounds exist, orally available PFKFB3 inhibitors are unavailable. In this study we conducted a high-throughput screening campaign against the kinase activity of PFKFB3, involving 250,240 chemical compounds. A total of 507 initial hits showing >50% inhibition at 20 µM were identified, 66 of them plus 1 analog from a similarity search consistently displayed low IC50 values (<10 µM). In vitro experiments yielded 22 nontoxic hits that suppressed the tube formation of primary human umbilical vein ECs at 10 µM. Of them, 15 exhibited binding affinity to PFKFB3 in surface plasmon resonance assays, including 3 (WNN0403-E003, WNN1352-H007 and WNN1542-F004) that passed the pan-assay interference compounds screening without warning flags. This study provides potential leads to the development of new PFKFB3 inhibitors.

Signaling profiles in HEK 293T cells co-expressing GLP-1 and GIP receptors.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are regarded as 'incretins' working closely to regulate glucose homeostasis. Unimolecular dual and triple agonists of GLP-1R and GIPR have shown remarkable clinical benefits in treating type 2 diabetes. However, their pharmacological characterization is usually carried out in a single receptor-expressing system. In the present study we constructed a co-expression system of both GLP-1R and GIPR to study the signaling profiles elicited by mono, dual and triple agonists. We show that when the two receptors were co-expressed in HEK 293T cells with comparable receptor ratio to pancreatic cancer cells, GIP predominately induced cAMP accumulation while GLP-1 was biased towards ß-arrestin 2 recruitment. The presence of GIPR negatively impacted GLP-1R-mediated cAMP and ß-arrestin 2 responses. While sharing some common modulating features, dual agonists (peptide 19 and LY3298176) and a triple agonist displayed differentiated signaling profiles as well as negative impact on the heteromerization that may help interpret their superior clinical efficacies.

GLP-1 mimetics as a potential therapy for nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH), as a severe form of nonalcoholic fatty liver disease (NAFLD), is characterized by liver steatosis, inflammation, hepatocellular injury and different degrees of fibrosis. The pathogenesis of NASH is complex and multifactorial, obesity and type 2 diabetes mellitus (T2DM) have been implicated as major risk factors. Glucagon-like peptide-1 receptor (GLP-1R) is one of the most successful drug targets of T2DM and obesity, and its peptidic ligands have been proposed as potential therapeutic agents for NASH. In this article we provide an overview of the pathophysiology and management of NASH, with a special focus on the pharmacological effects and possible mechanisms of GLP-1 mimetics in treating NAFLD/NASH, including dual and triple agonists at GLP-1R, glucose-dependent insulinotropic polypeptide receptor or glucagon receptor.

[Baihe Yuzi Prescription improves asthenospermia by up-regulating the expression of CFTR in the sperm].

OBJECTIVE: To observe the therapeutic efficacy of Baihe Yuzi Prescription (BYP) in the treatment of clinical syndrome-free asthenospermia and its effects on semen parameters, sperm DNA fragmentation index (DFI) and the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in the sperm. METHODS: We randomly divided 112 patients with clinical syndrome-free asthenospermia into a control group (n = 55) and an experimental group (n = 57), the former treated orally with L-carnitine liquid combined with vitamin E capsules and the latter with BYP in addition, both for 3 months. After treatment, we obtained the total sperm count, sperm motility, percentages of progressively motile sperm (PMS) and morphologically normal sperm (MNS), sperm DFI and expression of CFTR in the sperm, and compared the above parameters between the two groups of patients before and after medication. RESULTS: The total effectiveness rate was significantly higher in the experimental group (82.46%) than in the control (65.45%) (P < 0.05). Compared with the baseline, the patients in the experimental group showed significant improvement after treatment in the total sperm count (��53.5��3.5�� vs ��86.5��3.9�� ��106, P < 0.05), sperm motility (��23.5��3.5��% vs ��38.8��3.7��%, P < 0.05), PMS (��20.1��3.2��% vs ��30.3��3.3��%, P < 0.05), MNS (��2.3��0.3��% vs ��3.9��0.4��%, P < 0.05), sperm DFI (��37.3��3.1��% vs ��25.2��3.4��%, P < 0.05) and the expression of CFTR (P < 0.05), and even better improvement than the controls in sperm motility (��23.8��3.7��% vs ��30.2��3.4��%, P < 0.05), PMS (��19.6��3.1��% vs ��25.3��2.9��%, P < 0.05), MNS (��2.4��0.4��% vs ��3.1��0.3��%, P < 0.05), and sperm DFI (��36.6��3.3��% vs ��30.3��3.1��%, P < 0.05). The total sperm count and the expression of CFTR, however, were not significantly improved in the control group after treatment (P > 0.05). CONCLUSION: Baihe Yuzi Prescription can increase sperm count and motility, improve sperm morphology and DFI in patients with clinical syndrome-free asthenospermia, which may be related to the up-regulated expression of CFTR in the sperm.

An in vitro Förster resonance energy transfer-based high-throughput screening assay identifies inhibitors of SUMOylation E2 Ubc9.

SUMOylation is one of the posttranslational modifications that mediate cellular activities such as transcription, DNA repair, and signal transduction and is involved in the cell cycle. However, only a limited number of small molecule inhibitors have been identified to study its role in cellular processes. Here, we report a Förster resonance energy transfer (FRET) high-throughput screening assay based on the interaction between E2 Ubc9 and E3 PIAS1. Of the 3200 compounds screened, 34 (1.1%) showed higher than 50% inhibition and 4 displayed dose-response inhibitory effects. By combining this method with a label-free surface plasmon resonance (SPR) assay, false positives were excluded leading to discovering WNN0605-F008 and WNN1062-D002 that bound to Ubc9 with KD values of 1.93 ± 0.62 and 5.24 ± 3.73 µM, respectively. We examined the effect of the two compounds on SUMO2-mediated SUMOylation of RanGAP1, only WNN0605-F008 significantly inhibited RanGAP1 SUMOylation, whereas WNN1062-D002 did not show any inhibition. These compounds, with novel chemical scaffolds, may serve as the initial material for developing new SUMOylation inhibitors.

High-throughput screening campaign identifies a small molecule agonist of the relaxin family peptide receptor 4.

Relaxin/insulin-like family peptide receptor 4 (RXFP4) is a class A G protein-coupled receptor (GPCR), and insulin-like peptide 5 (INSL5) is its endogenous ligand. Although the precise physiological role of INSL5/RXFP4 remains elusive, a number of studies have suggested it to be a potential therapeutic target for obesity and other metabolic disorders. Since selective agonists of RXFP4 are scarcely available and peptidic analogs of INSL5 are hard to make, we conducted a high-throughput screening campaign against 52,000 synthetic and natural compounds targeting RXFP4. Of the 109 initial hits discovered, only 3 compounds were confirmed in secondary screening, with JK0621-D008 displaying the best agonism at human RXFP4. Its S-configuration stereoisomer (JK1) was subsequently isolated and validated by a series of bioassays, demonstrating a consistent agonistic effect in cells overexpressing RXFP4. This scaffold may provide a valuable tool to further explore the biological functions of RXFP4.

Publisher Correction: Integrating yeast chemical genomics and mammalian cell pathway analysis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Integrating yeast chemical genomics and mammalian cell pathway analysis.

Chemical genomics has been applied extensively to evaluate small molecules that modulate biological processes in Saccharomyces cerevisiae. Here, we use yeast as a surrogate system for studying compounds that are active against metazoan targets. Large-scale chemical-genetic profiling of thousands of synthetic and natural compounds from the Chinese National Compound Library identified those with high-confidence bioprocess target predictions. To discover compounds that have the potential to function like therapeutic agents with known targets, we also analyzed a reference library of approved drugs. Previously uncharacterized compounds with chemical-genetic profiles resembling existing drugs that modulate autophagy and Wnt/ß-catenin signal transduction were further examined in mammalian cells, and new modulators with specific modes of action were validated. This analysis exploits yeast as a general platform for predicting compound bioactivity in mammalian cells.

Identification, structure modification, and characterization of potential small-molecule SGK3 inhibitors with novel scaffolds.

The serum and glucocorticoid-regulated kinase (SGK) family has been implicated in the regulation of many cellular processes downstream of the PI3K pathway. It plays a crucial role in PI3K-mediated tumorigenesis, making it a potential therapeutic target for cancer. SGK family consists of three isoforms (SGK1, SGK2, and SGK3), which have high sequence homology in the kinase domain and similar substrate specificity with the AKT family. In order to identify novel compounds capable of inhibiting SGK3 activity, a high-throughput screening campaign against 50,400 small molecules was conducted using a fluorescence-based kinase assay that has a Z' factor above 0.5. It identified 15 hits (including nitrogen-containing aromatic, flavone, hydrazone, and naphthalene derivatives) with IC50 values in the low micromolar to sub-micromolar range. Four compounds with a similar scaffold (i.e., a hydrazone core) were selected for structural modification and 18 derivatives were synthesized. Molecular modeling was then used to investigate the structure-activity relationship (SAR) and potential protein-ligand interactions. As a result, a series of SGK inhibitors that are active against both SGK1 and SGK3 were developed and important functional groups that control their inhibitory activity identified.

High-throughput screening campaigns against a PI3Kα isoform bearing the H1047R mutation identified potential inhibitors with novel scaffolds.

The phosphatidylinositol 3-kinase (PI3K) pathway is involved in many cellular functions including cell growth, metabolism, and transformation. Hyperactivation of this pathway contributes to tumorigenesis, therefore, PI3K is a major target for anticancer drug discovery. Since the PI3Kα isoform is implicated mostly in cancer, we conducted a high-throughput screening (HTS) campaign using a 3-step PI3K homogenous time-resolved fluorescence assay against this isoform bearing the H1047R mutation. A total of 288,000 synthetic and natural product-derived compounds were screened and of which, we identified 124 initial hits that were further selected by considering the predicted binding mode, relationship to known pan-assay interference compounds and previous descriptions as a lipid kinase inhibitor. A total of 24 compounds were then tested for concentration-dependent responses. These hit compounds provide novel scaffolds that can potentially be optimized to create novel PI3K inhibitors.

W2476 ameliorates ß-cell dysfunction and exerts therapeutic effects in mouse models of diabetes via modulation of the thioredoxin-interacting protein signaling pathway.

Recent evidence shows that high glucose levels recruit carbohydrate response element-binding protein, which binds the promoter of thioredoxin-interacting protein (txnip), thereby regulating its expression in ß-cells. Overexpression of txnip not only induces ß-cell apoptosis but also reduces insulin production. Thus, the discovery of compounds that either inhibit TXNIP activity or suppress its expression was the focus of the present study. INS-1E cells stably transfected with either a txnip proximal glucose response element connected to a luciferase reporter plasmid (BG73) or full-length txnip promoter connected to a luciferase reporter plasmid (CL108) were used in primary and secondary high-throughput screening campaigns, respectively. From 256 000 synthetic compounds, a small molecule compound, W2476 [9-((1-(4-acetyl-phenyloxy)-ethyl)-2-)adenine], was identified as a modulator of the TXNIP-regulated signaling pathway following the screening and characterized using a battery of bioassays. The preventive and therapeutic properties of W2476 were further examined in streptozotocin-induced diabetic and diet-induced obese mice. Treatment with W2476 (1, 5, and 15 µmol/L) dose-dependently inhibited high glucose-induced TXNIP expression at the mRNA and protein levels in INS-1E cells and rat pancreatic islets. Furthermore, W2476 treatment prevented INS-1E cells from apoptosis induced by chronic exposure of high glucose and enhanced insulin production in vitro. Oral administration of W2476 (200 mg·kg-1·d-1) rescued streptozotocin-induced diabetic mice by promoting ß-cell survival and enhancing insulin secretion. This therapeutic property of W2476 was further demonstrated by its ability to improve glucose homeostasis and insulin sensitivity in diet-induced obese mice. Thus, chemical intervention of the TXNIP-regulated signaling pathway might present a viable approach to manage diabetes.

Foxp3 promoter methylation impairs suppressive function of regulatory T cells in biliary atresia.

Biliary atresia (BA) is characterized by progressive inflammation of the biliary system leading to liver cirrhosis, necessitating liver transplantation in pediatric patients. Various cell types have been reported to participate in the proinflammatory response in rhesus rotavirus (RRV)-induced BA mouse models, including T helper (Th) 1, Th2, Th17, CD8+ T cells, and natural killer cells. The immune suppressive regulatory T (Treg) cells, on the contrary, were reported not to function properly. The underlying mechanism is largely unknown. Focusing on the impaired suppressive function of Treg, we found methylation status of CpG islands within the Foxp3 promoter region of Treg cells in BA patients and murine models were both increased. Moreover, by injecting 5-aza-2'-deoxycytidine (Aza) as DNA-methylation inhibitor to RRV-infected mice, BA phenotypes were alleviated. Furthermore, Treg cells isolated from "RRV+Aza"-injected mice had better suppressive function than Treg cells from mice injected with RRV only, both in vivo and ex vivo. Thus we concluded that aberrant increased methylation status of "Foxp3 promoter" in Treg cells leads to impaired Treg suppressive function, exacerbating inflammatory injury in BA.

Landmark studies on the glucagon subfamily of GPCRs: from small molecule modulators to a crystal structure.

The glucagon subfamily of class B G protein-coupled receptors (GPCRs) has been proposed to be a crucial drug target for the tretmaent of type 2 diabetes. The challenges associated with determining the crystal structures of class B GPCRs relate to their large amino termini and the lack of available small molecule ligands to stabilize the receptor proteins. Following our discovery of non-peptidic agonists for glucagon-like peptide-1 receptor (GLP-1R) that have therapeutic effects, we initiated collaborative efforts in structural biology and recently solved the three-dimensional (3D) structure of the human glucagon receptor (GCGR) 7-transmembrane domain, providing in-depth information about the underlying signaling mechanisms. In this review, some key milestones in this endeavor are highlighted, including discoveries of small molecule ligands, their roles in receptor crystallization, conformational changes in transmembrane domains (TMDs) upon activation and structure-activity relationship analyses.

Association of Ureaplasma urealyticum colonization with development of bronchopulmonary dysplasia: a systemic review and meta-analysis.

There is controversy regarding the roles of Ureaplasma urealyticum (U. urealyticum) colonization in the development of bronchopulmonary dysplasia (BPD). This study explored the association between U. urealyticum and bronchopulmonary dysplasia at 36 weeks post-menstrual age (BPD36). Studies published before December 31, 2013 were searched from Medline, Embase, Ovid, Web of Science, and Cochrane databases, with the terms "Ureaplasma urealyticum", "chronic lung disease", or "BPD36" used, and English language as a limit. The association between U. urealyticum colonization and BPD36 was analyzed with RevMan 4.2.10 software, using the odds ratio (OR) and relative risk (RR) for dichotomous variables. Out of the enrolled 81 studies, 11 investigated the BPD36 in total 1193 infants. Pooled studies showed no association between U. urealyticum colonization and subsequent development of BPD36, with the OR and RR being 1.03 (95% CI=0.78-1.37; P=0.84) and 1.01 (95% CI= 0.88-1.16, P=0.84), respectively. These findings indicated no association between U. urealyticum colonization and the development of BPD36.

[Expression of proto-oncogene Wip1 in breast cancer and its clinical significance].

OBJECTIVE: To investigate the expression of proto-oncogene Wip1 in breast cancer tissue and its clinical significance. METHODS: Through the uses of semi-RT-PCR, immunohistochemical technique and Western blot, the specimens from 70 patients of breast cancer and 20 normal controls were detected for Wip1 mRNA and protein expression. At the same time, the authors analyzed the relations between the expression of Wip1 in human breast cancer and different clinical pathologic parameters. RESULTS: RT-PCR: The values of gene expression of Wip1 mRNA in breast cancer tissue, pericancerous tissue and normal breast tissue were 0.715 +/- 0.087, 0.175 +/- 0.021 and 0.154 +/- 0.022 respectively. Thus the value of gene expression in breast cancer tissue was significantly higher than that in pericancerous tissue or normal breast tissue (P < 0.01). Immunohistochemistry: The high expression rates of Wip1 protein in breast cancer tissue, pericancerous tissue and normal breast tissue were 62.9% (44/70), 2.9% (2/70) and 0 (0/20) respectively and there was a significant difference among these three different tissues (P < 0.01). Western blot: The relative contents of Wip1 protein in breast cancer tissue, pericancerous tissue and normal breast tissue were 0.688 +/- 0.151, 0.251 +/- 0.043 and 0.234 +/- 0.044 respectively. The relative content of Wip1 protein in breast cancer tissue was significantly higher than that in pericancerous tissue or normal breast tissue (P < 0.01). The high expression of Wip1 protein was negatively correlated with the expression of p53, but it had nothing to do with tumor size, age, tumor staging, axillary lymph node metastasis and expressions of ER and PR. CONCLUSION: The high expression of Wip1 mRNA and its protein in breast cancer tissue may promote the growth of breast cancer. Wip1 may become a new target for therapy of breast cancer.

[The study of different operative style for abdominal aortic aneurysm during surrounding operation].

OBJECTIVE: To compare the the similarities and differences during the surrounding operation of endovascular repair (EVAR) and open surgical repair (OSR) for abdominal aortic aneurysm. METHODS: 112 patients with abdominal aortic aneurysms (AAA) were selected from 2004 to 2009: among them, 66 patients were treated with EVAR, 46 patients with OSR. Data of two groups were collected and analyzed during surrounding operation. RESULTS: Compared to OSR group, the mean blood lost, blood transfusion and intra-operative fluid in EVAR group were significantly less than OSR group (P < 0.05). The mean time of operation, observation period in ICU and being in hospital in EVAR group were shorter than OSR group (P < 0.05). But the cost of hospitalization in EVAR was far higher than that of OSR group (P < 0.05). In short term postoperative complications the OSR group was higher than the EVAR (P < 0.05), however, there was no statistically significant difference in death rate of the two groups during surrounding operation (P > 0.05). CONCLUSION: EVAR has the advantages of mild trauma, less blood loss, quicker recovery after operation, and less disturbance to internal environment. Especially, it is suitable for the patients who can not undergo open surgery repair, but its cost is still higher.