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.

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.

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.

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.