Harmine is an ATP-competitive inhibitor for dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) - PubMed (original) (raw)

Harmine is an ATP-competitive inhibitor for dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A)

Tatyana Adayev et al. Arch Biochem Biophys. 2011.

Abstract

Harmine is a β-carboline alkaloid. The compound is a potent inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A), a kinase implicated in Down syndrome. In this study, we show that harmine functions as an ATP-competitive inhibitor against Dyrk1A. Our conclusion is supported by kinetic analysis of harmine inhibition as well as by the characterization of a Dyrk1A mutation conferring significant resistance to harmine. The mutation, V306A, is located next to the highly conserved D307 residue in kinases known to coordinate the phosphate groups of ATP through a Mg²+ ion. The V306A mutation offers harmine resistance by differentially altering Dyrk1A affinity for harmine and ATP. The V306A mutation causes no apparent alteration to Dyrk1A activity except for the reduction in ATP affinity. This deficiency could be fully compensated by supplying ATP with a concentration in the physiological range. Our results reveal that harmine inhibits Dyrk1A activity by interacting with residues in the ATP-binding pocket and displacing ATP. Our results also suggest that harmine will be a good lead compound for further designing of selective ATP-competitive Dyrk1A inhibitors through exploration of the ATP-binding pocket of Dyrk1A.

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Figures

Fig. 1

Fig. 1

Harmine derivatives, emodin, and TBB.

Fig. 2

Fig. 2. Inhibition of Dyrk1A by harmine

A. Competition of harmine against ATP. Harmine and ATP competition was performed with varied ATP (10-100 μM) and harmine (0 - 0.4 μM) at a constant dynatide 3 (24 μM) as described in the Materials and Methods. The initial rate (V) obtained from three independent trials was averaged and plotted as 1/V vs 1/[ATP]. KI was calculated from the secondary plot of apparent Km vs. [harmine]. B. Competition of harmine against dynatide 3. The reaction was performed with varied dynatide 3 (12-96 μM) and harmine (0 - 0.4 μM) at a constant ATP (50 μM) similarly as described above. The initial rate (V) obtained from three independent trials was averaged and was plotted as 1/V vs 1/[dynatide 3]. KI was calculated from the secondary plot of apparent Vmax vs. [harmine]. Harmine concentrations used in the reactions: (●) 0, (■) 0.1 μM, (◆) 0.2 μM, (▲) 0.4 μM.

Fig. 3

Fig. 3. Activity of the V306A mutant with high concentrations of ATP in vitro

Dynatide 3 concentration-dependent reactions were performed with 6-96 μM peptide in the presence of a constant ATP as described in Materials and Methods. Data (reaction rate) from three independent assays were averaged and used for plotting against [dynatide 3] followed by curve fitting using the Michaelis-Menten equation (v = Vmax*[dynatide 3]/(Km+[dynatide 3]). WT with 250 μM (□), 500 μM (not shown), and 1000 μM (○) ATP. The V306A mutant with 250 μM (■), 500 μM (◆), and 1000 μM (●) ATP.

Fig. 4

Fig. 4. Activity and harmine sensitivity of the V306A mutant in NIH 3T3 cells

A. Kinase activity. Dyrk1A activity in 3T3 cells was measured by using Gli 1-dependent transcription of luciferase as the reporter as described in Materials and Methods. The reaction was driven either by empty pCMV (vector), wild-type (WT) or mutant V306A (306) Dyrk1A. After correcting for the internal control, the luciferase output was normalized to the reactions promoted by empty pCMV(set as 1) before plotting. The data represent the average (± standard error) of four independent trials. The level of Dyrk1A expression (insert) in transfected cells was detected as described in Materials and Methods. A typical result was shown. B. Harmine sensitivity. WT and mutant V306A-driven luciferase synthesis was set up similarly as described above. After transfection, cells were incubated for 20 hours to allow Dyrk1A expression before treating with the indicated concentrations of harmine for 24-hour until luciferase determination. The luciferase activity at each harmine concentration was calculated as the percent of activity relative to each respective luciferase output for the no harmine control (set as 100%). The data represent the average (± standard error) of four independent trials. WT, open column and mutant V306A, solid column.

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