Design, Synthesis, and Evaluation of Site-Specific Hsp90 Inhibitors

The heat shock protein 90 (Hsp90) family of molecular chaperones is responsible for the conformational maturation of nascent polypeptides and refolding of denatured proteins. Hsp90 is known to play an important role in the regulation of cell signaling, survival and proliferation, transforming it into a promising target for the treatment of several diseases, including cancer. Proteins associated with all six hallmarks of cancer, as described by Weinberg, are Hsp90 clients. Since these proteins depend upon Hsp90, its inhibition has the potential to simultaneously disrupt all six and halt the malignant progression using a single small molecule. Hsp90 is abundantly expressed in the cell and accounts for 1-2% of the total cellular protein, making itone of the most prevalent proteins in eukaryotic cells. The Hsp90 isoforms are responsible for the conformational maintenance of greater than 150 proteins. There are four distinct isoforms, including Hsp90á, Hsp90â, GRP94 and TRAP1. Hsp90á and â are located in the cytoplasm; Hsp90á is the major inducible form, while Hsp90â is the constitutively active form. In contrast, GRP94 is found in the endoplasmic reticulum and TRAP1 is located in the mitochondrial matrix. Hsp90 exists as a homodimer consisting of an N- and C-terminal domain, connected by a middle domain. The Hsp90 N-terminus contains an ATP binding site, responsible for the ATPase activity of the chaperone associated with folding of clients. Small molecules that preferentially bind to this nucleotide binding site inhibit the ability of Hsp90 to properly fold polypeptides, ultimately tagging them for degradation through the ubiquitin-proteasome pathway. Natural product inhibitors of the N-terminal ATP binding site include the ansamycin antibiotic geldanamycin (GDA) and the macrocyclic lactone radicicol (RDC). Blagg and co-workers have previously reported the chimeric N-terminal inhibitor radamide, which combines the resorcinol portion of RDC and the quinone portion of GDA through a flexible linker. Radamide manifests a slightly greater binding affinity for GRP94 (Kd = 0.52 ìM) versus cytosolic Hsp90 (Kd = 0.87 ìM). This phenomenon is elucidated by examining the unique binding conformations adopted by radamide when crystal structures of were obtained using each isoform. When bound to Hsp90, radamide exhibits a linear conformation, while when bound to GRP94 the quinone portion is bent towards a pocket that is not accessible in Hsp90á or â. By employing conformational constraint, it was proposed that the quinone portion of radamide could exhibit the bent conformation seen in the natural products and result in increased affinity for Hsp90 as well as selective binding to the cytosolic isoforms. In contrast to radamide, a recent paper by Gasiewicz and co-workers examined the effects of flavones and (-)-Epigallocatechin-3-Gallate (EGCG), the major polyphenolic catechin found in green tea, on the aryl hydrocarbon receptor (AHR). Due to its diverse medicinal properties, EGCG has been proposed as a potential treatment for several diseases. The study by Gasiewicz revealed that EGCG functions through a different mechanism of action than the known flavone antagonists, interacting with the Hsp90 C-terminus rather than the AHR. It was confirmed that EGCG binds specifically to the C-terminal nucleotide binding site, either within or proximal to the site where the natural product novobiocin is thought to bind. Previous studies have determined that EGCG manifests an IC50 of ~150 ìM against MCF-7 cells. Upon examination of an overlay of EGCG and novobiocin, the structural similarities of the coumarin and the catechin cores become apparent. It was proposed that design of molecules based on the structure-activity relationships established for novobiocin may result in Hsp90-specific inhibitors based upon the EGCG scaffold

WNT activates the AAK1 kinase to promote clathrin-mediated endocytosis of LRP6 and establish a negative feedback loop

beta-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity2617993FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/50724-5; 2016/17469-0M.B.M. acknowledges support from the NIH (RO1-CA187799 and U24-DK116204-01). M.J.A. received financial support from NIH T32 Predoctoral Training Grants in Pharmacology (T32-GM007040-43 and T32-GM007040-42), an Initiative for Maximizing Student Diversity Grant (R25-GM055336-16), and the NIH National Cancer Institute (NCI) NRSA Predoctoral Fellowship to Promote Diversity in Health-Related Research (F31CA228289). M.P.W. received support from the Lymphoma Research Foundation (337444) and the NIH (T32-CA009156-35). Y.N. was supported by grants-in-aid from the Japan Society for the Promotion of Science (JSPS) (15KK0356 and 16K11493). T.T. was supported by the Howard Hughes Medical Institute Gilliam Fellowship for Advanced Study. M.V.G. was supported by Cancer Research UK (grants C7379/A15291 and C7379/A24639 to Mariann Bienz). The UNC Flow Cytometry Core Facility is supported in part by Cancer Center Core Support Grant P30 CA016086 to the UNC Lineberger Comprehensive Cancer Center, and research reported in this publication was supported by the Center for AIDS Research (award number 5P30AI050410), and the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The Structural Genomics Consortium (SGC) is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, the Eshelman Institute for Innovation, Genome Canada, the Innovative Medicines Initiative (European Union [EU]/European Federation of Pharmaceutical Industries and Associations [EFPIA]) (ULTRA-DD grant no. 115766), Janssen, Merck & Company, Merck KGaA, Novartis Pharma AG, the Ontario Ministry of Economic Development and Innovation, Pfizer, the São Paulo Research Foundation (FAPESP) (2013/50724-5), Takeda, and the Wellcome Trust (106169/ZZ14/Z). R.R.R. received financial support from FAPESP (2016/17469-0). We would also like to thank Claire Strain-Damerell and Pavel Savitsky for cloning various mutants of AAK1 and BMP2K proteins that were used in the crystallization trials. Additionally, we thank Dr. Sean Conner for providing the AAK1 plasmids, Dr. Stephane Angers for kindly providing the HEK293T DVL TKO cells, and Dr. Mariann Bienz for providing comments and feedback. We would like to thank members of the Major laboratory for their feedback and expertise regarding experimental design and project directio

CD4陽性T細胞に発現するA20(tnfaip3) はTh2型アレルギー性気道炎症を抑制する

研究科: 千葉大学大学院医学薬学府（先端医学薬学専攻）学位記番号: 千大院医薬博甲第医1412号博士（医学）千葉大学 = Chiba Universit

Novel Antitubercular 6‑Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure–Activity Relationship and Target Identification Studies

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against <i>Mycobacterium tuberculosis</i> (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome <i>bc</i>₁ complex, disruption of cell–wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylamino­pyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure–activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties