Suchergebnisse

This journal is © The Royal Society of Chemistry 2020. ; The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation. Molecular dynamic simulations also showed this interaction for two ligand/BMX complexes. Kinome selectivity profiling showed that the most potent compound is the strongest binder, displays intracellular target engagement in BMX-transfected cells with two-digit nanomolar inhibitory potency, and leads to BMX degradation PC3 in cells. The new inhibitors displayed anti-proliferative effects in androgen-receptor positive prostate cancer cells that where further increased when combined with known inhibitors of related signaling pathways, such as PI3K, AKT and Androgen Receptor. We expect these findings to guide development of new selective BMX therapeutic approaches. ; Funded under the Royal Society (URF to G. J. L. B., URF\R\180019), FCT Portugal (iFCT to G. J. L. B., IF/00624/2015, Postdoctoral Fellowship SFRH/BPD/95253/2013 to J. D. S., project PTDC/MED-QUI/28764/2017 to J. D. S., 02/SAICT/2017 grant 28333 to T. R., doctoral studentship SFRH/BD/143583/2019 to B. B. S., and DL 57/2016/CP1451/CT0025 to J. D. S.). This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreements No. 807281 and 702428. The authors acknowledge the Wellcome Trust (104633/Z/14/Z, to D. W. and C. V. R.), Ministerio de Economía y Competitividad (project RTI2018-099592-B-C21 to F. C.), the Royal G. and Mae H. Westaway Family Memorial Fund (A. N. K.), National Science Foundation Graduate Research Fellowship (Grant No ...

Funding: We thank Sabine Baxter for assistance with hybridoma and cell culture at the University of Pennsylvania Perelman School of Medicine Cell Center Services Facility. We acknowledge the use of cryo-EM instruments at Pfizer Inc. The cryo-EM work was supported by a grant from the National Institute of Health (R01GM103899 and R01GM129357 to V.Y.M.-B.). The TRPV2-flag-RFP construct cloned into the pEGFP-N2 vector was kindly provided by Prof. Kojima (Gunma University, Japan). The Developmental Therapeutics Program is acknowledged for screening PL against the NCI-60 cancer cell line panel. We are grateful to Carlos Custódia, Eunice Paisana, and Dr. Joao Barata for their assistance with cell-line modulation ̃ and the GBM tumor model, and to Dr. Vikki Cantrill for her assistance with the editing of this manuscript. Figure design by Claudia Flandoli (draw.science). The Histology and Comparative Pathology Laboratory of the Instituto de Medicina Molecular is acknowledged for technical support. We also thank FCT Portugal (FCT Investigator IF/00624/2015 to G.J.L.B., IF/01693/2014 to V.A.M. and IF/00595/2014 to N.L.B.-M., CEECIND/00436/2018 to N.L.B.-M., PhD studentship SFRH/BD/143583/2019 to B.B.S., PhD studentship PD/BD/128289/2017 to E.P.L., Postdoctoral fellowship SFRH/BPD/1187731/2016 to M.C.M.), Agencia Estatal Investigación of Spain (AEI, RTI-2018-099592-B-C21 to F.C.), EMBO (Installation Grant 3057 to N.L.B.-M.), and the Medical Research Council (MR/N010051/1 to P.F.). This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement 630731, 675007, 807281, and 743640. G.J.L.B. is a Royal Society University Research Fellow (URF\R\180019). ; The use of computational tools to identify biological targets of natural products with anticancer properties and unknown modes of action is gaining momentum. We employed self-organizing maps to deconvolute the phenotypic effects of piperlongumine (PL) and establish a link to modulation of the human transient receptor potential vanilloid 2 (hTRPV2) channel. The structure of the PL-bound full-length rat TRPV2 channel was determined by cryo-EM. PL binds to a transient allosteric pocket responsible for a new mode of anticancer activity against glioblastoma (GBM) in which hTRPV2 is overexpressed. Calcium imaging experiments revealed the importance of Arg539 and Thr522 residues on the antagonistic effect of PL and calcium influx modulation of the TRPV2 channel. Downregulation of hTRPV2 reduces sensitivity to PL and decreases ROS production. Analysis of GBM patient samples associates hTRPV2 overexpression with tumor grade, disease progression, and poor prognosis. Extensive tumor abrogation and long term survival was achieved in two murine models of orthotopic GBM by formulating PL in an implantable scaffold/hydrogel for sustained local therapy. Furthermore, in primary tumor samples derived from GBM patients, we observed a selective reduction of malignant cells in response to PL ex vivo. Our results establish a broadly applicable strategy, leveraging data-motivated research hypotheses for the discovery of novel means tackling cancer. ; publishersversion ; published