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.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.

Clones encoding pro-phenol oxidase [pro-

Two isoforms of prophenoloxidase were isolated from pupae of Oregon-R strain of Drosophila melanogaster. The purification procedure included ammonium sulfate fractionation, Sephacryl S-200 gel chromatography, DEAE-cellulose, and hydroxylapatite column chromatography. The two isoforms, A1 and A3, could be separated by ammonium sulfate fractionation. The isoelectric points of A1 and A3 were determined to be pH 5.8 and 6.7, respectively. The molecular weights of the monomers of A1 and A3 were estimated by SDS-PAGE to be 78 and 77 kDa, respectively. The native states of A1 and A3 are considered to be homodimeric, as judged by gel-filtration chromatography.

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