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.

Selective inhibition of the kinase DYRK1A by targeting its folding process

“…Our recent study indicated that mutations affecting autophosphorylation strengthen the interaction between DYRK1A and the kinase-specific co-chaperone CDC37 (ref. 36 ). CDC37 assists in the folding of target kinases in cooperation with HSP90 (refs 37 , 38 ).…”

“…To investigate whether the mutation of Ser97 affects the thermodynamic stability of DYRK1A, we examined the interaction between the S97A mutant and CDC37 fused with luciferase nanoKAZ, as described in our recent study 36 , with some modifications (see the Methods for details). The S97A, Y321F, Y319F/Y321F and K188R mutations significantly strengthened the interaction of DYRK1A with CDC37-nanoKAZ ( Fig.…”

DYRK1B mutations associated with metabolic syndrome impair the chaperone-dependent maturation of the kinase domain

“…Therefore, CDC37 fused to luciferase can be used as a thermodynamic sensor for kinase structures 11, 15 . Here we show that the pathogenic DYRK1B variants interact more strongly with CDC37 than wild DYRK1B.…”

Retinoid derivative Tp80 exhibits anti‐hepatitis C virus activity through restoration of GI‐GPx expression

“…In our past efforts with chemical library screening, we have developed anti‐viral compounds targeting hepatitis B virus (HBV), human herpesviruses, human immunodeficiency virus‐1 (HIV‐1), and dengue virus . In this study, we applied our screening system for HCV in combination with Rep‐Feo reporter replicon system, and identified retinoid derivative Tp80 as a potent inhibitor of HCV replication.…”

“…RT‐PCR was performed, as described previously . Briefly, total RNA was extracted from 1.0 × 10 5 of cells with Sepasol RNA‐I (Nacalai Tesque) and treated with RQ DNase I (Promega, Fitchburg, WI).…”