FINDY(1)

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.

The kinase DYRK1A phosphorylates substrate proteins that are involved in the progression of many diseases. DYRK1A also phosphorylates its own residues on key elements intramolecularly to activate and stabilize itself during the folding process. Once the folding process of DYRK1A has completed, it can no longer catalyzes the intramolecular reaction, suggesting that a transitional intermediate state that catalyzes the autophosphorylation exists. In the previous study, we identified a small molecule, designated as FINDY, that selectively inhibits the folding intermediate of DYRK1A. Although evidence has suggested that FINDY targets the ATP-binding pocket of DYRK1A, it remains elusive as to whether the DYRK1A kinase domain could be purified as a complex with FINDY. In this study, we successfully expressed and purified the kinase domain of DYRK1A in complex with FINDY. The DYRK1A kinase domain was expressed as a fusion protein with a hexahistidine tag and ZZ-domain (His-ZZ-DYRK1A) at 6 °C by using a cold shock induction system in Escherichia coli cells. The cells were incubated with FINDY. The cell pellets were gently extracted on ice and subjected to immobilized-metal affinity chromatography. The amount of FINDY in the elution fraction was measured by UV absorbance specific for FINDY. The eluate contained FINDY with the ratio of FINDY to DYRK1A protein being 0.15 in quadruplicate experiments. Thus, this study demonstrates the direct interaction between the DYRK1A kinase domain and FINDY, paving the way for structural determination of the complex.