2. Academic Validation
  3. Development of a novel AAK1 inhibitor via Kinobeads-based screening

Development of a novel AAK1 inhibitor via Kinobeads-based screening

  • Sci Rep. 2024 Mar 20;14(1):6723. doi: 10.1038/s41598-024-57051-9.
Akari Yoshida # 1,  Satomi Ohtsuka # 1,  Fumiya Matsumoto 2,  Tomoyuki Miyagawa 2,  Rei Okino 2,  Yumeya Ikeda 1,  Natsume Tada 1,  Akira Gotoh 1,  Masaki Magari 1,  Naoya Hatano 1,  Ryo Morishita 3,  Ayano Satoh 4,  Yukinari Sunatsuki 5,  Ulf J Nilsson 6,  Teruhiko Ishikawa 7,  Hiroshi Tokumitsu 8
Affiliations

Affiliations

  • 1 Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama, 700-8530, Japan.
  • 2 Department of Science Education, Graduate School of Education, Okayama University, Okayama, 700-8530, Japan.
  • 3 CellFree Sciences Co. Ltd, Matsuyama, 790-8577, Japan.
  • 4 Organelle Systems Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama, 700-8530, Japan.
  • 5 Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
  • 6 Department of Chemistry, Lund University, Box 124, 221 00, Lund, Sweden.
  • 7 Department of Science Education, Graduate School of Education, Okayama University, Okayama, 700-8530, Japan. teruhiko@cc.okayama-u.ac.jp.
  • 8 Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama, 700-8530, Japan. tokumit@okayama-u.ac.jp.
  • # Contributed equally.
PMID: 38509168 DOI: 10.1038/s41598-024-57051-9
Abstract

A chemical proteomics approach using CA2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKKα/1 and β/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 µM), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 Inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 µM) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 µM), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 µM TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 Inhibitor.

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