Biology subjects

Drewry, D.

Publications and source records attributed to Drewry, D..

4 recordsLinked to original sources

bioRxiv2018

New tools for carbohydrate sulphation analysis: Heparan Sulphate 2-O-sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors

Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2-O-sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3-phosphoadenosine 5-phosphosulphate) to the 2-O position of -L-iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.\n\nSUMMARY STATEMENTWe report that HS2ST, which is a PAPS-dependent glycan sulphotransferase, can be assayed using a variety of novel biochemical procedures, including a non-radioactive enzyme-based assay that detects glycan substrate sulphation in real time. HS2ST activity can be inhibited by different classes of compounds, including known protein kinase inhibitors, suggesting new approaches to evaluate the roles of HS2ST-dependent sulphation with small molecules in cells.

biochemistry↗

bioRxiv2018

New tools for evaluating protein tyrosine sulphation: Tyrosyl Protein Sulphotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

Protein tyrosine sulphation is a post-translational modification (PTM) best known for regulating extracellular protein-protein interactions. Tyrosine sulphation is catalysed by two Golgi-resident enzymes termed Tyrosyl Protein Sulpho Transferases (TPSTs) 1 and 2, which transfer sulphate from the co-factor PAPS (3-phosphoadenosine 5-phosphosulphate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulphation assays has hampered the development of chemical biology approaches for the identification of small molecule inhibitors of tyrosine sulphation. In this paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulphation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and 2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set (PKIS), we identified oxindole-based inhibitors of the Ser/Thr kinase RAF as low micromolar inhibitors of TPST1/2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulphotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulphation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.\n\nSUMMARY STATEMENTWe develop new assays to quantify tyrosine sulphation by the human tyrosine sulphotransferases TPST1 and 2. TPST1 and 2 catalytic activities are inhibited by protein kinase inhibitors, suggesting new starting points to synthesise (or repurpose) small molecule compounds to evaluate biological TPST using chemical biology.

biochemistry↗

bioRxiv2018

AAK1 inhibits WNT signaling by promoting clathrin-mediated endocytosis of LRP6

{beta}-catenin-dependent WNT signal transduction governs normal development and adult tissue homeostasis. Inappropriate pathway activity mediates a vast array of human diseases, including bone density disorders, neurodegeneration and cancer. Although several WNT-directed therapeutics are in clinical trials, new targets, compounds and strategies are needed. We performed a gain-of-function screen of the human kinome to identify new druggable regulators of {beta}-catenin-dependent transcription. We found that over-expression of the AP2 Associated Kinase 1 (AAK1) strongly inhibited WNT signaling. Reciprocally, silencing of AAK1 expression or pharmacological inhibition of AAK1 kinase activity using a new, selective and potent small molecule inhibitor activated WNT signaling. This small molecule is a cell active dual AAK1/BMP2K inhibitor that represents the best available tool to study AAK1-dependent signaling pathways. We report that AAK1 and the WNT co-receptor LRP6 physically co-complex and that AAK1 promotes clathrin-mediated endocytosis of LRP6. Collectively, our data support a WNT-induced negative feedback loop mediated by AAK1-driven, clathrin-mediated endocytosis of LRP6.\n\nSummary StatementA gain-of-function screen of the human kinome revealed AAK1 as a negative regulator of WNT signaling. We show that AAK1 promotes clathrin-mediated endocytosis of LRP6, resulting in downregulation of WNT signaling. We use a new selective and potent AAK1/BMP2K small molecule probe to validate our findings.

cell biology↗

bioRxiv2017

Repurposed kinase inhibitors and β-lactams as a novel therapy for antibiotic resistant bacteria

Antibiotic resistant bacteria are an increasing global problem, and pathogenic actinomycetes and firmicutes are particularly challenging obstacles. These pathogens share several eukaryotic-like kinases that present antibiotic development opportunities. We used computational modelling to identify human kinase inhibitors that could be repurposed towards bacteria as part of a novel combination therapy. The computational model suggested a family of inhibitors, the imidazopyridine aminofurazans (IPAs), bind PknB with high affinity. We found that these inhibitors biochemically inhibit PknB, with potency roughly following the predicted models. A novel x-ray structure confirmed that the inhibitors bind as predicted and made favorable protein contacts with the target. These inhibitors also have antimicrobial activity towards Mycobacteria and Nocardia, and normally ineffective {beta}-lactams can potentiate IPAs to more efficiently inhibit growth of these pathogens. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can synergize with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.

microbiology↗