Decrypting orphan GPCR drug discovery via multitask learning.

The drug discovery of G protein-coupled receptors (GPCRs) superfamily using computational models is often limited by the availability of protein three-dimensional (3D) structures and chemicals with experimentally measured bioactivities. Orphan GPCRs without known ligands further complicate the process. To enable drug discovery for human orphan GPCRs, multitask models were proposed for predicting half maximal effective concentrations (EC50) of the pairs of chemicals and GPCRs. Protein multiple sequence alignment features, and physicochemical properties and fingerprints of chemicals were utilized to encode the protein and chemical information, respectively. The protein features enabled the transfer of data-rich GPCRs to orphan receptors and the transferability based on the similarity of protein features. The final model was trained using both agonist and antagonist data from 200 GPCRs and showed an excellent mean squared error (MSE) of 0.24 in the validation dataset. An independent test using the orphan dataset consisting of 16 receptors associated with less than 8 bioactivities showed a reasonably good MSE of 1.51 that can be further improved to 0.53 by considering the transferability based on protein features. The informative features were identified and mapped to corresponding 3D structures to gain insights into the mechanism of GPCR-ligand interactions across the GPCR family. The proposed method provides a novel perspective on learning ligand bioactivity within the diverse human GPCR superfamily and can potentially accelerate the discovery of therapeutic agents for orphan GPCRs.

Discovery of tetrasubstituted thiophenes as Cisd2 activators: A potential novel therapeutic option in nonalcoholic fatty liver disease.

Down-regulation of Cisd2 in the liver has been implicated in the development of nonalcoholic fatty liver disease (NAFLD) and increasing the level of Cisd2 is therefore a potential therapeutic approach to this group of diseases. Herein, we describe the design, synthesis, and biological evaluation of a series of Cisd2 activators, all thiophene analogs, based on a hit obtained using two-stage screening and prepared via either the Gewald reaction or by intramolecular aldol-type condensation of an N,S-acetal. Metabolic stability studies of the resulting potent Cisd2 activators suggest that thiophenes 4q and 6 are suitable for in vivo studies. The results from studies on 4q-treated and 6-treated Cisd2hKO-het mice, which carry a heterozygous hepatocyte-specific Cisd2 knockout, confirm that (1) there is a correlation between Cisd2 levels and NAFLD and (2) these compounds have the ability to prevent, without detectable toxicity, the development and progression of NAFLD.

A Convenient Diels-Alder Approach toward Potential Polyketide-like Antibiotics Using α-Activated α,ß-Unsaturated 4,4-Dimethyl-1-tetralones as Dienophiles.

Making use of a Diels-Alder approach based on various α,ß-unsaturated 2-carbomethoxy-4,4-dimethyl-1-tetralones as novel dienophiles, the corresponding polycyclic adducts could be efficiently synthesized in good to high yields (74~99%) in the presence of Lewis acid (e.g., SnCl4). Accordingly, a synthetically useful platform is established to provide a focused aromatic polyketide-like library for screening of potential natural and non-natural antimicrobial agents.

Design, synthesis, and anticancer evaluation of 1-benzo[1,3]dioxol-5-yl-3-N-fused heteroaryl indoles.

A series of 1-benzo[1,3]dioxol-5-yl-indoles bearing 3-N-fused heteroaryl moieties have been designed based on literature reports of the activity of indoles against various cancer cell lines, synthesized via a Pd-catalyzed C-N cross-coupling, and evaluated for their anticancer activity against prostate (LNCaP), pancreatic (MIA PaCa-2), and acute lymphoblastic leukemia (CCRF-CEM) cancer cell lines. A detailed structure-activity relationship study culminated in the identification of 3-N-benzo[1,2,5]oxadiazole 17 and 3-N-2-methylquinoline 20, whose IC50 values ranged from 328 to 644 nM against CCRF-CEM and MIA PaCa-2. Further mechanistic studies revealed that 20 caused cell cycle arrest at the S phase and induced apoptosis in CCRF-CEM cancer cells. These 1-benzo[1,3]dioxol-5-yl-3-N-fused heteroaryl indoles may serve as a template for further optimization to afford more active analogs and develop a comprehensive understanding of the structure-activity relationships of indole anticancer molecules.

Discovery of Potential Neuroprotective Agents against Paclitaxel-Induced Peripheral Neuropathy.

Chemotherapy-induced neurotoxicity is a common adverse effect of cancer treatment. No medication has been shown to be effective in the prevention or treatment of chemotherapy-induced neurotoxicity. Using minoxidil as an initial template for structural modifications in conjunction with an in vitro neurite outgrowth assay, an image-based high-content screening platform, and mouse behavior models, an effective neuroprotective agent CN016 was discovered. Our results showed that CN016 could inhibit paclitaxel-induced inflammatory responses and infiltration of immune cells into sensory neurons significantly. Thus, the suppression of proinflammatory factors elucidates, in part, the mechanism of action of CN016 on alleviating paclitaxel-induced peripheral neuropathy. Based on excellent efficacy in improving behavioral functions, high safety profiles (MTD > 500 mg/kg), and a large therapeutic window (MTD/MED > 50) in mice, CN016 might have great potential to become a peripherally neuroprotective agent to prevent neurotoxicity caused by chemotherapeutics as typified by paclitaxel.

Delayed intervention with a novel SGLT2 inhibitor NGI001 suppresses diet-induced metabolic dysfunction and non-alcoholic fatty liver disease in mice.

BACKGROUND AND PURPOSE: Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis, is closely related to metabolic diseases such as obesity and diabetes. Despite an accumulating number of studies, no pharmacotherapy that targets NAFLD has received general approval for clinical use. EXPERIMENTAL APPROACH: Inhibition of the sodium-glucose cotransporter 2 (SGLT2) is a promising approach to treat diabetes, obesity, and associated metabolic disorders. In this study, we investigated the effect of a novel SGLT2 inhibitor, NGI001, on NAFLD and obesity-associated metabolic symptoms in high-fat diet (HFD)-induced obese mice. KEY RESULTS: Delayed intervention with NGI001 protected against body weight gain, hyperglycaemia, hyperlipidaemia, and hyperinsulinaemia, compared with HFD alone. Adipocyte hypertrophy was prevented by administering NGI001. NGI001 inhibited impaired glucose metabolism and regulated the secretion of adipokines associated with insulin resistance. In addition, NGI001 supplementation suppressed hepatic lipid accumulation and inflammation but had little effect on kidney function. In-depth investigations showed that NGI001 ameliorated fat deposition and increased AMPK phosphorylation, resulting in phosphorylation of its major downstream target, acetyl-CoA carboxylase, in human hepatocyte HuS-E/2 cells. This cascade ultimately led to the down-regulation of downstream fatty acid synthesis-related molecules and the up-regulation of downstream ß oxidation-associated molecules. Surprisingly, NGI001 decreased gene and protein expression of SGLT1 and SGLT2 and glucose uptake in oleic acid-treated HuS-E/2 cells. CONCLUSION AND IMPLICATIONS: Our findings suggest the novel SGLT2 inhibitor, NGI001 has therapeutic potential to attenuate or delay the onset of diet-induced metabolic diseases and NAFLD.

Design, synthesis and anticancer evaluation of ß-carboline-1-one hydantoins.

Aim: Cancer is a major health burden and a leading cause of death worldwide. We sought to discover potential anticancer molecules with novel scaffold for further development of more active agents to address the issue. Methodology: A series of ß-carboline-1-one hydantoins were designed according to a conformational restriction strategy, synthesized via a one-pot Knoevenagel condensation-intramolecular cyclization, and tested in cytotoxicity assays. Results: The study culminated in the identification of 6b and 6c, both of which were found to potently inhibit breast and lung cancer cell lines. Of particular interest was 6c, which was 83 times more potent an inhibitor than 5-fluorouracil in inhibiting MCF-7. Conclusion: This work establishes ß-carboline-1-one hydantoin as a promising scaffold in the investigation of anticancer agents.

Synthesis and biological evaluation of N-glucosyl indole derivatives as sodium-dependent glucose co-transporter 2 inhibitors.

Sodium-dependent glucose co-transporter 2 (SGLT2) inhibition has been demonstrated to efficiently control hyperglycemia via an insulin secretion-independent pathway. The unique mode of action eliminates the risk of hypoglycemia and makes SGLT2 inhibitors an attractive option for the treatment of type 2 diabetes. In a continuation of our previous studies on SGLT2 inhibitors bearing different sugar moieties, sixteen new N-glucosyl indole derivatives were designed, synthesized, and evaluated for their inhibitory activity against hSGLT2. Of these sixteen, acethydrazide-containing N-glucosyl indole 9d was found to be the most potent SGLT2 inhibitor, and caused a significant elevation in urine glucose excretion in rats at 50 mg/kg, relative to the vehicle control.

Identification of an oxime-containing C-glucosylarene as a potential inhibitor of sodium-dependent glucose co-transporter 2.

Treatment of hyperglycemia with drugs that block renal glucose reabsorption via inhibition of sodium-dependent glucose cotransporter 2 (SGLT2) is a novel approach to diabetes management. In this study, twenty-seven aryl C-glycosides bearing a C=N/C-N linkage at the glucosyl C6 position were designed, synthesized and evaluated for their inhibitory activity against human SGLT2 (hSGLT2). Compounds with good hSGLT2 inhibition were further investigated to determine their selectivity over hSGLT1. Of these, five representative aryl C-glycosides were chosen for pharmacokinetic analysis. Oxime 2a was determined to have the most promising pharmacokinetic properties and was selected for in vivo glucosuria and plasma glucose level studies, which found it to exhibit comparable efficacy to dapagliflozin (1). Furthermore, 2a was not found to exhibit either significant cytotoxicity (CC50 > 50 µM) or human ether-a-go-go related gene (hERG) inhibition (2% inhibition at 10 µM). Taken together, these efforts culminated in the discovery of oxime 2a as a potential SGLT2 inhibitor.

Bromomethylthioindole Inspired Carbazole Hybrids as Promising Class of Anti-MRSA Agents.

Series of N-substituted carbazole analogues bearing an indole ring were synthesized as anti-methicillin-resistant Staphylococcus aureus (MRSA) agents from a molecular hybridization approach. The representative compound 19 showed an MIC = 1 µg/mL against a panel of MRSA clinical isolates as it possessed comparable in vitro activities to that of vancomycin. Moreover, compound 19 also exhibited MIC = 1 µg/mL activities against a recent identified Z172 MRSA strain (vancomycin-intermediate and daptomycin-nonsusceptible phenotype) and the vancomycin-resistant Enterococcus faecalis (VRE) strain. In a mouse model with lethal infection of MRSA (4N216), a 75% survival rate was observed after a single dose of compound 19 was intravenously administered at 20 mg/kg. In light of their equipotent activities against different MRSA isolates and VRE strain, the data underscore the importance of designed hybrid series for the development of new N-substituted carbazoles as potential anti-MRSA agents.

Mycotoxin Patulin Suppresses Innate Immune Responses by Mitochondrial Dysfunction and p62/Sequestosome-1-dependent Mitophagy.

Innate immune responses are important for pathogen elimination and adaptive immune response activation. However, excess inflammation may contribute to immunopathology and disease progression (e.g. inflammation-associated hepatocellular carcinoma). Immune modulation resulting from pattern recognition receptor-induced responses is a potential strategy for controlling immunopathology and related diseases. This study demonstrates that the mycotoxin patulin suppresses Toll-like receptor- and RIG-I/MAVS-dependent cytokine production through GSH depletion, mitochondrial dysfunction, the activation of p62-associated mitophagy, and p62-TRAF6 interaction. Blockade of autophagy restored the immunosuppressive activity of patulin, and pharmacological activation of p62-dependent mitophagy directly reduced RIG-I-like receptor-dependent inflammatory cytokine production. These results demonstrated that p62-dependent mitophagy has an immunosuppressive role to innate immune response and might serve as a potential immunomodulatory target for inflammation-associated diseases.

Total Synthesis of the Naturally Occurring Glycosylflavone Aciculatin.

The new flavone-glycoside aciculatin (1), from Chrysopogon aciculatus, has been shown to have cytotoxic, anti-inflammatory, and antiarthritis activity. Further biological studies have been limited because of the limited availability of 1 from natural sources. Herein the first total synthesis of 1 in an overall yield of 8.3% is described. The synthesis involved the regio- and stereoselective glycosylation-Fries-type O-to-C rearrangement to construct the C-aryl glycosidic linkage, followed by a Baker-Venkataraman rearrangement and cyclodehydration to form the flavone scaffold.

N-Indolylglycosides bearing modifications at the glucose C6-position as sodium-dependent glucose co-transporter 2 inhibitors.

Suppression of glucose reabsorption through the inhibition of sodium-dependent glucose co-transporter 2 (SGLT2) is a promising therapeutic approach for the treatment of type 2 diabetes. To investigate the effect of C6-substitution on inhibition of SGLT2 by N-indolylglucosides, a small library of 6-triazole, 6-amide, 6-urea, and 6-thiourea N-indolylglycosides were synthesized and tested. A detailed structure-activity relationship (SAR) study culminated in the identification of 6-amide derivatives 6a and 6o as potent SGLT2 inhibitors, which were further tested for inhibitory activity against SGLT1. The data obtained indicated that 6a and 6o are mildly to moderately selective for SGLT2 over SGLT1. Both compounds were also evaluated in a urinary glucose excretion test and pharmacokinetic study; 6a was found capable of inducing urinary glucose excretion in normal SD rats.

Development and application of a fluorescent glucose uptake assay for the high-throughput screening of non-glycoside SGLT2 inhibitors.

Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors are of current interest as a treatment for type 2 diabetes. Efforts have been made to discover phlorizin-related glycosides with good SGLT2 inhibitory activity. To increase structural diversity and better understand the role of non-glycoside SGLT2 inhibitors on glycemic control, we initiated a research program to identify non-glycoside hits from high-throughput screening. Here, we report the development of a novel, fluorogenic probe-based glucose uptake system based on a Cu(I)-catalyzed [3+2] cycloaddition. The safer processes and cheaper substances made the developed assay our first priority for large-scale primary screening as compared to the well-known [(14)C]-labeled α-methyl-D-glucopyranoside ([(14)C]-AMG) radioactive assay. This effort culminated in the identification of a benzimidazole, non-glycoside SGLT2 hit with an EC50 value of 0.62 µM by high-throughput screening of 41,000 compounds.

Discovery of selective inhibitors of Glutaminase-2, which inhibit mTORC1, activate autophagy and inhibit proliferation in cancer cells.

Glutaminase, which converts glutamine to glutamate, is involved in Warburg effect in cancer cells. Two human glutaminase genes have been identified, GLS (GLS1) and GLS2. Two alternative transcripts arise from each glutaminase gene: first, the kidney isoform (KGA) and glutaminase C (GAC) for GLS; and, second, the liver isoform (LGA) and glutaminase B (GAB) for GLS2. While GLS1 is considered as a cancer therapeutic target, the potential role of GLS2 in cancer remains unclear. Here, we discovered a series of alkyl benzoquinones that preferentially inhibit glutaminase B isoform (GAB, GLS2) rather than the kidney isoform of glutaminase (KGA, GLS1). We identified amino acid residues in an allosteric binding pocket responsible for the selectivity. Treatment with the alkyl benzoquinones decreased intracellular glutaminase activity and glutamate levels. GLS2 inhibition by either alkyl benzoquinones or GLS2 siRNA reduced carcinoma cell proliferation and anchorage-independent colony formation, and induced autophagy via AMPK mediated mTORC1 inhibition. Our findings demonstrate amino acid sequences for selective inhibition of glutaminase isozymes and validate GLS2 as a potential anti-cancer target.

Synthesis and biological evaluation of novel C-indolylxylosides as sodium-dependent glucose co-transporter 2 inhibitors.

Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors are the current focus on the indication for the management of hyperglycemia in diabetes. Here, a novel series of C-linked indolylxyloside-based inhibitors of SGLT2 has been discovered. Structure-activity relationship studies revealed that substituents at the 7-position of the indole moiety and a p-cyclopropylphenyl group in the distal position were necessary for optimum inhibitory activity. The pharmacokinetic study demonstrates that the most potent compound 1i is metabolically stable with a low clearance in rats. In further efficacy study, 1i is found to significantly lower blood glucose levels of streptozotocin (STZ)-induced diabetic rats.

Synthesis of heparin oligosaccharides and their interaction with eosinophil-derived neurotoxin.

A convenient route for the synthesis of heparin oligosaccharides involving regioselective protection of D-glucosamine and a concise preparation of rare L-ido sugars from diacetone α-D-glucose is described. Stereoselective coupling of a D-glucosamine-derived trichloroacetimidate with a 1,6-anhydro-ß-L-idopyranosyl 4-alcohol gave the desired α-linked disaccharide, which was used as repeating unit for dual chain elongation and termination. Stepwise assembly from the reducing to the non-reducing end with a D-glucosamine-derived monosaccharide as starting unit furnished the oligosaccharide skeletons having different chain lengths. A series of functional group transformations afforded the expected heparin oligosaccharides with 3, 5 and 7 sugar units. Interaction of these oligosaccharides with eosinophil-derived neurotoxin (EDN), a cationic ribonuclease and a mediator produced by human eosinophils, was further investigated. The results revealed that at 5 µg mL(-1), the heptasaccharide has sufficiently strong interference to block EDN binding to Beas-2B cells. The tri- and pentasaccharides have moderate inhibitory properties at 50 µg mL(-1) concentration, but no inhibition has been observed at 10 µg mL(-1). The IC(50) values of the tri-, penta- and heptasaccharides are 69.4, 47.2 and 0.225 µg mL(-1), respectively.

The first total synthesis of cytopiloyne, an anti-diabetic, polyacetylenic glucoside.

The first total synthesis of cytopiloyne 1, a novel bioactive polyacetylenic glucoside isolated from the extract of Bidens pilosa, is described. The structure of cytopiloyne was determined to be 2-ß-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne by using various spectroscopic methods, but the chirality of the polyyne moiety was unknown. Herein, the convergent synthesis of two diastereomers of cytopiloyne by starting from commercially available 4-(2-hydroxyethyl)-2,2-dimethyl-1,3-diozolane is described. The synthetic sequence involved two key steps: stereoselective glycosylation of the glucosyl trichloroacetimidate with 1-[(4-methoxybenzyl)oxy]hex-5-yn-2-ol to give the desired ß-glycoside and the construction of the glucosyl tetrayne skeleton by using a palladium/silver-catalyzed cross-coupling reaction to form the alkyne-alkyne bond, the first such use of this reaction. Comparison between the observed and published characterization data showed the 2R isomer to be the natural product cytopiloyne.

Development of sodium-dependent glucose co-transporter 2 inhibitors as potential anti-diabetic therapeutics.

The kidney plays an important role in the regulation of plasma glucose. It is estimated that greater than 99% of the renal glucose filtered by kidney glomerulus is resorbed by sodium-dependent glucose co-transporters (SGLTs), and that SGLT2 located in the proximal renal tubule achieves the most of this assignment. Studies of SGLT2 inhibitors indicate that raising renal glucose excretion by inhibiting SGLT2 helps effectively normalize the plasma glucose levels in individuals with type 2 diabetes mellitus (T2DM). This review discusses the discovery of SGLT2 inhibitors and the related structure-activity relationship (SAR) studies. The clinical trial data of dapagliflozin is also involved.

Discovery of novel N-ß-D-xylosylindole derivatives as sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors for the management of hyperglycemia in diabetes.

A novel series of N-linked ß-D-xylosides were synthesized and evaluated for inhibitory activity against sodium-dependent glucose cotransporter 2 (SGLT2) in a cell-based assay. Of these, the 4-chloro-3-(4-cyclopropylbenzyl)-1-(ß-D-xylopyranosyl)-1H-indole 19m was found to be the most potent inhibitor, with an EC(50) value similar to that of the natural SGLT2 inhibitor phlorizin. Further studies in Sprague-Dawley (SD) rats indicated that 19m significantly increased urine glucose excretion in a dose-dependent manner with oral administration. The antihyperglycemic effect of 19m was also observed in streptozotocin (STZ) induced diabetic SD rats. These results described here are a good starting point for further investigations into N-glycoside SGLT2 inhibitors.