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

Total Synthesis of the Naturally Occurring Glycosylflavone Aciculatin

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

Decrypting orphan GPCR drug discovery via multitask learning

Abstract 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

High Throughput Mass Spectrometry Based Enzymatic Assays for Biofuels Development

Mass spectrometry's ability to efficiently generate intact biomolecular ions in the gas phase has led to a wide range of biological applications and is recently being applied for global metabolite profiling (`metabolomics?) primarily though liquid chromatography coupled to electrospray mass spectrometry. However the complexity and relatively low throughput of this approach has limited application for high throughput enzymatic assays. To overcome this, we have developed the Nanostructure-Initiator Mass Spectrometry enzymatic (Nimzyme1) assay where enzyme substrates are immobilized on the mass spectrometry surface using fluorous phase interactions. This `soft? immobilization allows efficient desorption/ionization while also allowing surface washing to reduce signal suppression from complex biological samples as a result of the preferential retention of the tagged products and reactants. We have also shown that Nimzyme can detect multiple and competing enzymatic activities and screen for optimal pH, temperature, and enzyme inhibition from crude cell lysates and a hot springs microbial community. This approach is being implemented at the DOE Joint BioEnergy Institute for high throughput functional characterization of both enzyme libraries and environmental samples. Specifically, we are constructing a complete set of glucose polysaccharides (cellobiose to cellihexose) for screening glucohydralase and glucotransferase activities and a p-coumaryl alcohol substrate for characterization of laccase activity. Together these assays will help to identify and optimize the conversion of lignocellulose into biofuels

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

Series of <i>N</i>-substituted carbazole analogues bearing an indole ring were synthesized as anti-methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) agents from a molecular hybridization approach. The representative compound <b>19</b> showed an MIC = 1 μg/mL against a panel of MRSA clinical isolates as it possessed comparable <i>in vitro</i> activities to that of vancomycin. Moreover, compound <b>19</b> also exhibited MIC = 1 μg/mL activities against a recent identified Z172 MRSA strain (vancomycin-intermediate and daptomycin-nonsusceptible phenotype) and the vancomycin-resistant <i>Enterococcus faecalis</i> (VRE) strain. In a mouse model with lethal infection of MRSA (4N216), a 75% survival rate was observed after a single dose of compound <b>19</b> 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 <i>N</i>-substituted carbazoles as potential anti-MRSA agents