title: Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ.
abstract: The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer's disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases.
authors.affiliation: 1] Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan [2] Laboratory of Functional Biology, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Sonamoto
authors.affiliation: Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Kii
authors.affiliation: Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Koike
authors.affiliation: Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
authors.forename: Yuto
authors.initials: Y
authors.lastname: Sumida
authors.affiliation: Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
authors.forename: Tomoe
authors.initials: T
authors.lastname: Kato-Sumida
authors.affiliation: Medical Research Support Center, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Okuno
authors.affiliation: Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Hosoya
authors.affiliation: Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Hagiwara
mesh_terms: D001572:Benzofurans / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D018833:Chaperonins / Q000235:genetics* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology; D053778:Thiazolidines / Q000494:pharmacology; D014230:Triazoles / Q000494:pharmacology; D000097603:Dyrk Kinases
chemical_list: D001572:Benzofurans; D018797:Cell Cycle Proteins; D018833:Chaperonins; D018841:HSP90 Heat-Shock Proteins; D008156:Luciferases; D018832:Molecular Chaperones; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D053778:Thiazolidines; D014230:Triazoles; D000097603:Dyrk Kinases; C000622094:5-((2,3-dihydrobenzofuran-5-yl)methylene)-2-iminothiazolidin-4-one; C498362:CDC37 protein, human; C533237:STA 9090