Dyrk1A (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A) is a serine/ threonine kinase essential for brain development and function, and its excessive activity is considered a pathogenic factor in Down syndrome. The development of potent, selective inhibitors of Dyrk1A would help to elucidate the molecular mechanisms of normal and diseased brains, and may provide a new lead compound for molecular-targeted drug discovery. Here, we report a novel Dyrk1A inhibitor, InDY, a benzothiazole derivative showing a potent ATPcompetitive inhibitory effect with IC 50 and K i values of 0.24 and 0.18 µm, respectively. X-ray crystallography of the Dyrk1A/InDY complex revealed the binding of InDY in the ATP pocket of the enzyme. InDY effectively reversed the aberrant tau-phosphorylation and rescued the repressed nFAT (nuclear factor of activated T cell) signalling induced by Dyrk1A overexpression. Importantly, proInDY, a prodrug of InDY, effectively recovered Xenopus embryos from head malformation induced by Dyrk1A overexpression, resulting in normally developed embryos and demonstrating the utility of proInDY in vivo.

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To identify novel vitamin D receptor (VDR) ligands that induce a novel architecture within the ligand-binding pocket (LBP), we have investigated eight 22-butyl-1alpha,24-dihydroxyvitamin D(3) derivatives (3-10), all having a butyl group as the branched alkyl side chain. We found that the 22S-butyl-20-epi-25,26,27-trinorvitamin D derivative 5 was a potent VDR agonist, whereas the corresponding compound 4 with the natural configuration at C(20) was a potent VDR antagonist. Analogues with the full vitamin D(3) side chain were less potent agonist, and whether they were agonists or antagonists depended on the 24-configuration. X-ray crystal structures demonstrated that the VDR-LBD accommodating the potent agonist 5 has an architecture wherein the lower side and the helix 11 side of the LBP is simply expanded relative to the canonical active-VDR situation; in contrast, the potent antagonist 4 induces an extra cavity to accommodate the branched moiety. This is the first report of a VDR antagonist that generates a new cavity to alter the canonical pocket structure of the ligand occupied VDR.

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Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors.

We investigate here the interaction between GroEL and two kinds of non-native alpha-lactalbumin. alpha-Lactalbumin is a Ca(2+)-binding protein which assumes a molten globule state in the absence of Ca2+ (apo-alpha-lactalbumin) at neutral pH. Our results, obtained by molecular-sieve chromatography and hydrogen-exchange measurements, show that apo-alpha-lactalbumin in this molten globule state is not bound to GroEL either in the absence or in the presence of KCl. On the other hand, we show by molecular-sieve chromatography that alpha-lactalbumin, in which the four disulphide bonds are fully reduced, is bound to GroEL when 50 mM KCl is present. The results demonstrate that the protein state recognized by GroEL is more unfolded and expanded than the typical molten globule state of alpha-lactalbumin.