Selectivity Profiling and Biological Activity of Novel β-Carbolines as Potent and Selective DYRK1 Kinase Inhibitors

Abstract

DYRK1A is a pleiotropic protein kinase with diverse functions in cellular regulation, including cell cycle control, neuronal differentiation, and synaptic transmission. Enhanced activity and overexpression of DYRK1A have been linked to altered brain development and function in Down syndrome and neurodegenerative diseases such as Alzheimer's disease. The β-carboline alkaloid harmine is a high affinity inhibitor of DYRK1A but suffers from the drawback of inhibiting monoamine oxidase A (MAO-A) with even higher potency. Here we characterized a series of novel harmine analogs with minimal or absent MAO-A inhibitory activity. We identified several inhibitors with submicromolar potencies for DYRK1A and selectivity for DYRK1A and DYRK1B over the related kinases DYRK2 and HIPK2. An optimized inhibitor, AnnH75, inhibited CLK1, CLK4, and haspin/GSG2 as the only off-targets in a panel of 300 protein kinases. In cellular assays, AnnH75 dose-dependently reduced the phosphorylation of three known DYRK1A substrates (SF3B1, SEPT4, and tau) without negative effects on cell viability. AnnH75 inhibited the cotranslational tyrosine autophosphorylation of DYRK1A and threonine phosphorylation of an exogenous substrate protein with similar potency. In conclusion, we have characterized an optimized β-carboline inhibitor as a highly selective chemical probe that complies with desirable properties of drug-like molecules and is suitable to interrogate the function of DYRK1A in biological studies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Inhibition of DYRK1A and related kinases by selected β-carbolines.

Kinase activities are given as the means of at least 3 measurements in the presence of 1 μM of the compounds (10 μM in HIPK2 assays) and are expressed as the percentage of the uninhibited control (Kinase-GLO assay). 5-iodotubercidin (IoT) served as a structurally unrelated control compound that inhibits all tested kinases.

Fig 2. Analysis of kinase inhibitor interactions.

A-D, Predicted binding modes of AnnH75 at DYRK1A and related kinases. The inhibitor is colored cyan and kinases are depicted as ribbon structures. Only relevant amino acid residues in the ATP binding pocket are shown for clarity. In B-D, residues different from DYRK1A are colored green and labeled. The distances of the two hydrogen bonds (red line) and the distance between the cyano group and the glycine of the P-loop (G166 in DYRK1A) are given in Angstrom. (DYRK1A, PDB ID 3ANR; DYRK2 PDB ID 4AZF; HIPK2, homology model, CLK1, PDB ID 2VAG). E, Comparison of amino acid residues relevant for AnnH75 binding. In the left, the relationship of the kinases is illustrated by the sequence identity of their catalytic domains. Residues generally conserved in protein kinases (G166, K188, D307) and the gatekeeper residue (F238) are highlighted by bold print. Residues different from DYRK1A are shown with their position in the sequence. Differences correlating with kinase resistance to AnnH75 are highlighted in red.

Fig 3. Selectivity profile of AnnH75.

AnnH75 was profiled at a concentration of 1 μM against a panel of 300 protein kinases (see Table A in S1 File for the complete results). A, Target kinases inhibited by more than 50% are indicated. The kinome dendrogram was adapted and is reproduced courtesy of Cell Signaling Technology. B, Calculation of the Gini coefficient as a measure of kinase selectivity. The Lorenz curve illustrates the degree to which the total inhibitory activity of a compound (i.e. the sum of inhibition of all tested kinases) is equally distributed among all tested kinases (bisector line, Gini coefficient of 0) or directed towards a single kinase (maximal selectivity, a Gini coefficient of 1).

Fig 4. Inhibition of SF3B1 phosphorylation by DYRK1A in HeLa cells.

HeLa cells expressing GFP-SF3B1-NT were treated with the indicated compounds for 18 h. The phosphorylation state of SF3B1 was determined by immunoblotting with pT434 antibody, and the results were normalized to the total amount of SF3B1 immunoreactivity. A, Representative western blots. AnnH79 is a harmine analogue that does not inhibit DYRK1A and was used as negative control. The vertical line indicates where irrelevant lanes were deleted from the final image. B, The column diagram summarizes the quantitative evaluation of 3–6 experiments for each compound (means + SD).

Fig 5. Inhibition of SEPT4 and tau phosphorylation by DYRK1A.

A, HeLa cells transiently expressing FLAG-SEPT4 were treated with AnnH31 or AnnH75 for 5 h before cells were lysed and analysed by immunoblotting with a FLAG-tag antibody. 5-iodotubercidin (IoT) served as positive control. Relative SEPT4 phosphorylation was calculated as the ratio of the intensities of the phosphorylated upper band and the lower band. B, HEK293 cells with constitutive expression of GFP-tau and regulatable expression of GFP-DYRK1A were treated with doxycyclin and the indicated inhibitors for 18 h. Phosphorylation of tau on Thr212 was detected with a phosphospecific antibody. Expression levels of GFP-tau and GFP-DYRK1A were assessed with a GFP antibody. For quantitative evaluation of DYRK1A inhibition, the basal pT212 signal in control cells not treated with doxycyclin (Ctrl) was subtracted from all values. C, Quantitative evaluation of three experiments each for SEPT4 and tau. All data were standardized to the level of phosphorylation in cells untreated with inhibitors. Error bars indicate SEM.

Fig 6. AnnH75 inhibits both threonine and tyrosine kinase activity of DYRK1A.

A DYRK1A construct with an N-terminal StrepTag 2 (ST2-DYRK1Acat) was expressed in a cell-free E. coli-derived expression system. Coupled in vitro transcription and translation reactions were incubated for 1 h in the presence of recombinant SF3B1-NT-His6 and AnnH75. Phosphorylation of tyrosines in DYRK1A and of Thr434 in SF3B1 was determined by immunoblotting with a phosphotyrosine-specific antibody (pTyr) and a pThr434-specific antibody. A, Representative western blots. The asterisks mark unidentified bands. B, Quantitative evaluation. Results were normalized to the total amount of DYRK1A or SF3B1, respectively, and are plotted relative to the phosphorylation in the untreated control samples (means +/- SEM, n = 3).