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

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 beta-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 beta-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

Novel harmine analogues and the effect on dual specificity kinase activity of DYRK1A

Dual specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is encoded on human chromosome 21 and triplication of this specific region leads to a 1.5-fold increased expression of DYRK1A in Down syndrome. Today there is a growing interest in developing chemical probes for the functional characterization of kinases, but to date just a small fraction of the human kinome can be effectively targeted with selective small-molecule inhibitors to date. Harmine is one of the most potent and selective inhibitors of DYRK1A presently available. Nevertheless, its inhibitory effect on monoaminoxidase A (MAO A) limits the use of harmine as chemical probe in animal experiments and as a therapeutic agent.Therefore, the first part of this study aimed at the development of a harmine analogue to get an improved inhibitor of DYRK1A without MAO A inhibitory activity. Based on the known crystal structure of DYRK1A and on a model of the DYRK1A/harmine complex, diversely substituted harmine analogues have been synthesized and tested for their inhibitory potency for DYRK1A in in vitro assays. These experiments identified AnnH75 as one of the most potent newly synthesized inhibitors of DYRK1A with an IC50 value of 184 nM in radioactive assays, but without any inhibitory effect on MAO A. Kinome profiling against a panel of 300 kinases at a concentration of 1 μM revealed that AnnH75 inhibits only 4 off-target kinases by more than 90% (DYRK1B, CLK1, CLK4, Haspin) leading to a Gini value of 0.625. Cell based assays showed a high cell permeability and low cytotoxicity in HeLa and PC12 cells up to concentrations of 10 μM confirming AnnH75 as a usable, potent and specific inhibitor for investigations of DYRK1A function in biological systems, cell assays and animal testing.The dual specificity kinase activity is a defining feature of the DYRK family and is essentially linked to the characteristic autoactivation mechanism of DYRKs. The current model of the mechanism of tyrosine autophosphorylation in the DYRK family is based on a study of the Drosophila DYRKs dDYRK2 and MNB, but has not been verified for mammalian DYRK1A yet.Therefore, the second part of this study addressed to examine the mechanism of dual specificity kinase activity in mammalian DYRK1A. Additionally, this study investigated whether tyrosine phosphorylation is a strictly co-translational event or whether it can still take place in mature DYRKs and to study the potential of inhibitors to differentially target the tyrosine kinase form and the mature serine/ threonine-specific conformation of DYRK1A.In vitro translation experiments showed that wild type DYRK1A as well as the Y321F point mutant of DYRK1A retain the capacity of autophosphorylating tyrosine residues after termination of in vitro translation. Accordingly, tyrosine kinase activity of DYRK1A is not limited to a transient folding state that only exists during translation. Furthermore, it can be concluded that the ability of DYRK1A for posttranslational tyrosine autophosphorylation does not depend on the phosphorylation status of the conserved tyrosine 321 in the activation loop. Finally, the postulated differences of kinase inhibitor sensitivities for tyrosine autophosphorylation of mature DYRK1A and the translational intermediate form of DYRK1A was not confirmed in this study suggesting that the reported differences for DYRK2 are not conserved throughout the DYRK family

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

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

How to separate kinase inhibition from undesired monoamine oxidase a inhibition - the development of the DYRK1A inhibitor AnnH75 from the alkaloid harmine

The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure-activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors

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

<p>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.</p

Inhibition of SF3B1 phosphorylation by DYRK1A in HeLa cells.

<p>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. <b>A</b>, 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>B,</b> The column diagram summarizes the quantitative evaluation of 3–6 experiments for each compound (means + SD).</p

AnnH75 inhibits both threonine and tyrosine kinase activity of DYRK1A.

<p>A DYRK1A construct with an N-terminal StrepTag 2 (ST2-DYRK1Acat) was expressed in a cell-free <i>E</i>. <i>coli</i>-derived expression system. Coupled <i>in vitro</i> 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. <b>A</b>, Representative western blots. The asterisks mark unidentified bands. <b>B</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).</p

Inhibition of SEPT4 and tau phosphorylation by DYRK1A.

<p><b>A</b>, 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>B</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. <b>C</b>, 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.</p

Kinome selectivity of AnnH75 and published DYRK1A inhibitors.^a

<p>^a Structures are shown in Fig. D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132453#pone.0132453.s001" target="_blank">S1 File</a>.</p><p>^b IC₅₀ values are given because %-activity values are not given in the reference.</p><p>^c IC₅₀ value taken from Cuny et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132453#pone.0132453.ref038" target="_blank">38</a>]</p><p>Kinome selectivity of AnnH75 and published DYRK1A inhibitors.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132453#t002fn001" target="_blank">^a</a></p