Nobumoto Watanabe
Screening for small molecule competitors of the phosphorylated substrates ofβ-TrCP and their application for the induction of targeted protein degradation
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Nobumoto Watanabe, PhDRIKEN-Max Planck Joint Research Division for Systems Chemical Biology, Bioprobe Application Research Unit, RIKEN Center for Sustainable Resource Science |
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Research summary
β-TrCP is a F-box protein, the substrate recognition subunit in the SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligase complex. F-box proteins frequently recognize their substrates not only via primary amino acid sequence motifs but also as a result of phosphorylation. The interaction between β-TrCP and its substrates is one of the most extensively studied examples of the phosphorylation dependent substrate recognition mechanism of F-box proteins. β-TrCP recognizes a DSGXXS motif in the N-terminal region of IkBa, only when these serines are phosphorylated by the IkB kinase, IKK. β-TrCP also recognizes other substrates, including β-catenin, Cdc25A, human immunodeficiency virus Vpu protein, Emi1 and others, in a phosphorylation-dependent manner.
Previously, we have identified human Wee1A, a protein tyrosine kinase that phosphorylates and keeps Cdk1 inactive at G2 phase of the cell cycle before cells are ready for mitosis, as one of the substrates for β-TrCP. When cells are ready for mitosis, Cdk1 phosphorylates 123th Serine of Wee1A and this phosphorylated serine (pS123) triggers the phosphorylation cascades for the Wee1A ubiquitination. The pS123 not only directly interacts with basic residues in the WD40 repeat domain of β-TrCP, but also primes phosphorylation by two independent protein kinases, Polo like kinase 1 (Plk1) and CK2, to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation creates a Polo box domain (PBD) binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 can phosphorylate S121, but only if S123 is phosphorylated first, thereby generating the second β-TrCP binding site surrounding S121.
We also developed a high-throughput screening system to identify inhibitors of PBD-dependent binding and screened a chemical library. We isolated a benzotropolone-containing natural compound derived from nutgalls (purpurogallin (PPG)) that inhibited PBD-dependent binding in vitro and in vivo. PPG not only delayed the onset of mitosis, but also pro-longed the progression of mitosis in HeLa cells.
Using this system, we are going to isolate small molecule competitors of the phosphorylated substrates ofβ-TrCP in this project. When such small molecules are identified, we will generate a synthetic molecule by binding it to a substrate recognition ligand for a target protein. We expect such molecule will induce the degradation of the target protein throughβ-TrCP dependent ubiquitination.
Publications
- Subedi A, Muroi M, Futamura Y, Kawamura T, Aono H, Nishi M, Ryo A, *Watanabe N, *Osada H.
A novel inhibitor of tumorspheres revealed activation of serine biosynthetic pathway upon mitochondrial inhibition.
FEBS Letters 593, 763-776 (2019)
PMID: 30874300 - Förster T, Shang E, Shimizu K, Sanada E, Schölermann B, Hübecker M, Hahne G, Pascual Lopez Alberca M, Janning P, Watanabe N, Sievers S, Giordanetto F, Shimizu T, Ziegler S, Osada H *Waldmann, H.
2-Sulfonylpyrimidines target the kinesin HSET via cysteine alkylation.
Eur. J. Org. Chem. 2019, 5486-5496 (2019) - Kwong MMY, Lee JW, Samian MR, Watanabe N, Osada H, Ong EBB.
Comparison of microplate- and bottle-based methods to age yeast for chronological life span assays.
J. Microbiol. Methods. 167, 105743 (2019)
PMID: 31629019 - Yang N, Liu S, Qin T, Liu X, Watanabe N, Mayo KH, *Li J, *Li X.
SUMO3 Modification by PIAS1 Modulates Androgen Receptor Cellular Distribution and Stability.
Cell Commun. Signal. 17, 153 (2019)
PMID: 31752909 - Suvarna K, Honda K, Muroi M, Kondoh Y, Osada H, *Watanabe N.
Measurement of ATPase activity of valosin-containing protein/p97.
Bio-protocol 10, e3516 (2020) - Suvarna K, Honda K, Muroi M, Kondoh Y, *Watanabe N, *Osada H
Identification of target protein for bio-active small molecule using photo-cross linked beads and MALDI-TOF mass spectrometry.
Bio-protocol 10, e3517 (2020) - Wang L, Liu S, Xu J, Watanabe N, Di J, Wei W, Mayo K.H, Li J, Li X.
A traditional Chinese Medicine, YXQN, Reduces Amyloid-induced Cytotoxicity by Inhibiting Aβ42 Aggregation and Fibril Formation.
Curr. Pharm. Des. 26, 780-789 (2020)
PMID: 32031066 - Wang L, Liu S, Xu J, Watanabe N, Mayo KH, Li J, Li X.
Emodin inhibits aggregation of amyloid-β peptide 1-42 and improves cognitive deficits in Alzheimer's disease transgenic mice.
J. Neurochem. AOP (2020)
PMID: 32799401 - Kawamura T, Futamura Y, Shang E, Muroi M, Janning P, Ueno M, Wilke J, Takeda S, Kondoh Y, Ziegler S, Watanabe N, Waldmann H, Osada H.
Discovery of small-molecule modulator of heterotrimeric Gi-Protein by integrated phenotypic profiling and chemical proteomics.
Biosci. Biotech. Biochem. 84, 2484-2490 (2020)
PMID: 32867616 - Pan Y, Zhao Y, Kuang R, Liu H, Sun D, Mao T, Jiang K, Yang X, Watanabe N, Mayo KH, Lin Q, Li J.
Injectable hydrogel-loaded nano-hydroxyapatite that improves bone regeneration and alveolar ridge promotion.
Mater. Sci. Eng. ,C 116, 111158 (2020)
PMID: 32806272 - Wilke J, Kawamura T, Xu H, Brause A, Friese A, Metz M, Schepmann D, Wünsch B, Artacho-Cordón A, Nieto FR, Watanabe N, Osada H, Ziegler S, *Waldmann H.
Discovery of a σ1 receptor antagonist by combination of unbiased cell painting and thermal proteome profiling.
Cell Chem. Biol. 28, 1-7 (2021)
PMID: 33567254
Former Publications
- Ooi L-C, *Watanabe N, Futamura Y, Sulaiman SF, Darah I, *Osada H.
Identification of small molecule inhibitors of p27Kip1 ubiquitination by high-throughput screening.
Cancer Sci. 104, 1461-1467 (2013)
PMID: 23910095 - Suvarna K, Honda K, Kondoh Y, Osada H, *Watanabe N.
Identification of a small molecule ligand of β-arrestin1 as an inhibitor of stromal fibroblast cell migration accelerated by cancer cells.
Cancer Med. 7, 883-893 (2018)
PMID: 29380537 - Suvarna K, Honda K, Muroi M, Kondoh Y, Osada H, *Watanabe N.
A small-molecule ligand of valosin-containing protein/p97 inhibits cancer cell-accelerated fibroblast migration.
J. Biol. Chem. 294, 2988-2996 (2019)
PMID: 30610116 - *Watanabe N, Osada H.
Small molecules that target phosphorylation dependent protein-protein interaction.
Bioorg. Med. Chem. 24, 3264-3254 (2016)
PMID: 27017542