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  3. Temperature vaulting: A method for screening of slow- and tight-binding inhibitors that selectively target kinases in their non-native state

Temperature vaulting: A method for screening of slow- and tight-binding inhibitors that selectively target kinases in their non-native state

  • Eur J Med Chem. 2025 Oct 5:295:117789. doi: 10.1016/j.ejmech.2025.117789.
Sora Suzuki 1,  Koji Umezawa 2,  Gaku Furuie 1,  Masaki Kikuchi 3,  Daichi G M Nakamura 4,  Nanae Fukahori 1,  Ninako Kimura 1,  Masato Yamakawa 1,  Takashi Niwa 5,  Takashi Umehara 6,  Takamitsu Hosoya 7,  Isao Kii 8
Affiliations

Affiliations

  • 1 Laboratory for Drug Target Research, Department of Agriculture, Graduate School of Science and Technology, Shinshu University, 8304 Minami-Minowa, Kami-Ina, Nagano, 399-4598, Japan.
  • 2 Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, 8304 Minami-Minowa, Kami-Ina, Nagano, 399-4598, Japan.
  • 3 Department of Structural Biology, Medical Research Laboratory, Institute of Integrated Research, Institute of Science Tokyo, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan; Laboratory for Epigenetics Drug Discovery, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.
  • 4 Laboratory for Chemical Biology, RIKEN Center for Biosystems Dynamics Research, 6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan.
  • 5 Laboratory for Chemical Biology, RIKEN Center for Biosystems Dynamics Research, 6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan; Chemical Bioscience Team, Laboratory for Biomaterials and Bioengineering, Institute of Integrated Research, Institute of Science Tokyo, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-0062, Japan; Laboratory for Molecular Transformation Chemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
  • 6 Laboratory for Epigenetics Drug Discovery, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan; College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga, 525-8577, Japan.
  • 7 Laboratory for Chemical Biology, RIKEN Center for Biosystems Dynamics Research, 6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan; Chemical Bioscience Team, Laboratory for Biomaterials and Bioengineering, Institute of Integrated Research, Institute of Science Tokyo, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-0062, Japan.
  • 8 Laboratory for Drug Target Research, Department of Agriculture, Graduate School of Science and Technology, Shinshu University, 8304 Minami-Minowa, Kami-Ina, Nagano, 399-4598, Japan; Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, 8304 Minami-Minowa, Kami-Ina, Nagano, 399-4598, Japan. Electronic address: ikii@shinshu-u.ac.jp.
PMID: 40412300 DOI: 10.1016/j.ejmech.2025.117789
Abstract

A polypeptide folds into its protein tertiary structure in the native state through a folding intermediate in the non-native state. The transition between these states is thermodynamically driven. A folding intermediate of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) autophosphorylates intramolecularly, whereas DYRK1A in the native state no longer catalyzes this reaction. The alteration in substrate specificity suggests a conformational transition of DYRK1A during its folding process. Consistent with this hypothesis, we identified FINDY (1), which inhibits the intramolecular autophosphorylation but not the intermolecular phosphorylation, suggesting that DYRK1A in the non-native state possesses an alternative inhibitor-binding site. Meanwhile, it remains an issue that the methods for approaching the alternative binding site require an intricate assay tailored to the individual target. Here we show a method, designated as "temperature vaulting," for inhibitor screening that targets the non-native state. Transient heating of recombinant DYRK1A protein drove the reversible transition between the native state and the non-native state targeted by FINDY (1). At physiological temperature, FINDY (1) slowly bound to the DYRK1A protein. These results indicate that transient heating accelerates the slow-binding process by assisting the protein to overcome the high-energy barrier leading to the target non-native state. The energy barrier also slowed down the dissociation, resulting in tight binding between DYRK1A and FINDY (1). Structure-activity relationship revealed that both the methoxy group and the alkyne moiety underlie the selectivity of FINDY (1) toward DYRK1A in the non-native state. Furthermore, this study suggests that the dissociation rate underlies the inhibition selectivity of FINDY (1) between DYRK1A and its family kinase DYRK1B. This method could leverage conventional assays to identify slow- and tight-binding inhibitors.

Keywords

DYRK1A; DYRK1B; Folding; Kinetics; Residence time; SAR; Thermodynamic equilibrium.

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