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

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents

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

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

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

Selectivity profile of AnnH75.

<p>AnnH75 was profiled at a concentration of 1 μM against a panel of 300 protein kinases (see Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132453#pone.0132453.s001" target="_blank">S1 File</a> for the complete results). <b>A</b>, Target kinases inhibited by more than 50% are indicated. The kinome dendrogram was adapted and is reproduced courtesy of Cell Signaling Technology. <b>B</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).</p