Screening for small molecule competitors of the phosphorylated substrates ofβ-TrCP and their application for the induction of targeted protein degradation
Nobumoto Watanabe, PhD
Chemical Resource Development Research Unit, RIKEN Center for Sustainable Resource Science
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 IκBα, only when these serines are phosphorylated by the IκB 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.
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