Harmine specifically inhibits protein kinase DYRK1A and interferes with neurite formation

Abstract

DYRK1A is a dual‐specificity protein kinase that autophosphorylates a conserved tyrosine residue in the activation loop but phosphorylates exogenous substrates only at serine or threonine residues. Tyrosine autophosphorylation of DYRKs is a one‐off event that takes place during translation and induces the activation of the kinase. Here we characterize the beta‐carboline alkaloid harmine as a potent and specific inhibitor of DYRK1A both in vitro and in cultured cells. Comparative in vitro assays of four kinases of the DYRK family showed that harmine inhibited substrate phosphorylation by DYRK1A more potently than it inhibited substrate phosphorylation by the closely related kinase DYRK1B [half maximal inhibitory concentrations (IC50) of 33 nm versus 166 nm, respectively] and by the more distant members of the family, DYRK2 and DYRK4 (1.9 μm and 80 μm, respectively). Much higher concentrations of harmine were required to suppress tyrosine autophosphorylation of the translational intermediate of DYRK1A in a bacterial in vitro translation system (IC50 = 1.9 μm). Importantly, harmine inhibited the phosphorylation of a specific substrate by DYRK1A in cultured cells with a potency similar to that observed in vitro (IC50 = 48 nm), without negative effects on the viability of the cells. Overexpression of the DYRK1A gene on chromosome 21 has been implicated in the altered neuronal development observed in Down syndrome. Here, we show that harmine interferes with neuritogenesis in cultured hippocampal neurons. In summary, our data show that harmine inhibits DYRK1A substrate phosphorylation more potently than it inhibits tyrosine autophosphorylation, and provide evidence for a role of DYRK1A in the regulation of neurite formation.

Mechanism of dual specificity kinase activity of DYRK1A

“…As we have previously found for harmine [22], all tested compounds inhibited the phosphorylation of T434 in SF3B1 much more potently than tyrosine autophosphorylation of DYRK1A (Fig. 4).…”

“…For a broad coverage of chemical scaffolds, we compiled a series of compounds comprising known DYRK1A inhibitors (harmine [22], TG003 [24,25], KH-CB19 [26], 4,5,6,7-tetrabromobenzotriazole (TBB) [27,28]) and unspecific kinase inhibitors (staurosporin, curcumin). TBB was analysed because this compound had been used to establish the differential inhibitor sensitivity of the translational intermediate and mature form of dDYRK2 [13].…”

“…S4). Inhibitors were dissolved in dimethylsulfoxide (DMSO) to create a 1 mM stock solution, except that harmine was dissolved in ethanol as described previously [22]. Further dilutions of the inhibitors were prepared in water.…”

A review on medicinal importance, pharmacological activity and bioanalytical aspects of beta-carboline alkaloid “Harmine”

“…Harmine is a potent inhibitor of DYRK1A, a kinase implicated in Down syndrome [30] . Harmine inhibits DYRK1A substrate phosphorylation more potently than it inhibits tyrosine autophosphorylation, providing an evidence for a role of DYRK1A in the regulation of neurite formation [31] . Harmine, an inhibitor of the forkhead box class O (FoxO) kinase DYRK1A, stimulated FoxO nuclear accumulation and DNA binding activity [32] .…”

Antitumor properties and toxicity effects of Peganum harmala L. (Zygophyllaceae)

“…In the past decades, more attention has been drawn on their anticancer potencies (Boeira, da Silva, Erdtmann, & Henriques, 2001;Go¨ckler et al, 2009;Adhami, Farsam, & Krenn, 2011). Recently, pharmacological and therapeutic effects of P. harmala and its main alkaloids have been reviewed (Moloudizargari, Mikaili, Aghajanshakeri, Asghari, & Shayegh, 2013).…”