Báo cáo khoa học: Activation, regulation, and inhibition of DYRK1A Walter Becker1 and Wolfgang Sippl2 pptx

MINIREVIEW Activation, regulation, and inhibition of DYRK1A Walter Becker 1 and Wolfgang Sippl 2 1 Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Germany 2 Institute of Pharmacy, Martin-Luther-Universita ¨ t Halle-Wittenberg, Germany Introduction Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are a conserved family of eukaryotic kinases that are related to the cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinases (GSKs) and CDK-like kin- ases (CLKs), which are collectively termed the CMGC group. Two distinctive features of DYRK1A have originally stimulated the cloning and characterization of this founding member of the mammalian DYRK family. First, DYRK1A is a dual-specificity protein kinase that catalyses the phosphorylation of serine and threonine residues in its substrates as well as the auto- phosphorylation on a tyrosine residue in the activation loop [1,2]. Second, the human DYRK1A gene was identified as a Down syndrome candidate gene, because of its localization in the Down syndrome criti- cal region on human chromosome 21 [3] and due to the previous observation that the orthologous Drosophila kinase, minibrain (MNB), has an essential role in postembryonic neurogenesis [4]. Since then, multiple evidence has been obtained for a function of DYRK1A in neurodevelopment, supporting the hypothesis that DYRK1A contributes to the aberrant brain development underlying mental retardation Keywords CMGC kinases; dual-specificity; DYRK1A; harmine; INDY; kinase inhibitor; structural model; tyrosine autophosphorylation Correspondence W. Becker, Institute of Pharmacology and Toxicology, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany Fax: +49 241 80 82433 Tel: +49 241 80 89124 E-mail: wbecker@ukaachen.de Database Structural data is available at the Protein Data Bank under the accession numbers 2VX3 and 3KVW (Received 15 July 2010, revised 26 August 2010, accepted 2 September 2010) doi:10.1111/j.1742-4658.2010.07956.x Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase with diverse functions in neuronal development and adult brain physiology. Higher than normal levels of DYRK1A are associ- ated with the pathology of neurodegenerative diseases and have been impli- cated in some neurobiological alterations of Down syndrome, such as mental retardation. It is therefore important to understand the molecular mechanisms that control the activity of DYRK1A. Here we review the cur- rent knowledge about the initial self-activation of DYRK1A by tyrosine autophosphorylation and propose that this mechanism presents an ances- tral feature of the CMGC group of kinases. However, tyrosine phosphory- lation does not appear to regulate the enzymatic activity of DYRK1A. Control of DYRK1A may take place on the level of gene expression, inter- action with regulatory proteins and regulated nuclear translocation. Finally, we compare the properties of small molecule inhibitors that target DYRK1A and evaluate their potential application and limitations. The b-carboline alkaloid harmine is currently the most selective and potent inhibitor of DYRK1A and has proven very useful in cellular assays. Abbreviations CDK, cyclin-dependent kinase; CLK, CDK-like kinase; DMAT, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DYRK1A, dual-specificity tyrosine phosphorylation-regulated kinase 1A; EGCG, epigallocatechin-gallate; GSK, glycogen synthase kinase; MAPK, mitogen-activated protein kinase; MNB, protein kinase encoded by the minibrain gene from Drosophila; NFAT, nuclear factor of activated T-cells; SPRED, sprouty-related protein with an EVH1 domain; TBB, 4,5,6,7-tetrabromo-1H-benzotriazole. 246 FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS in Down syndrome [5,6]. More recently, overexpres- sion of DYRK1A has also been associated with neurodegenerative diseases [7]. The function of DYRK1A in neuronal development and in neurode- generation is covered by the two accompanying reviews within this minireview series [8,9]. Because the level of activity of DYRK1A is of key importance for its physiological and pathological effects, this review focuses on the molecular mechanisms of activation and regulation of DYRK1A. In addition, we provide an overview of the small molecule drugs that inhibit the catalytic activity of DYRK1A. Activation Most protein kinases function as molecular switches that can adopt distinct active and inactive conforma- tions. Conversion between these states is in many cases regulated by reversible phosphorylation of discrete ser- ine, threonine or tyrosine residues in the centrally located so-called ‘activation loop’. Phosphorylation of the activation loop stabilizes a conformation with an appropriately positioned substrate binding site [10]. Typically, this conformational change is driven by the electrostatic interactions between the phosphoresidue and a positively charged binding site named the RD pocket. Main contributors to the basic nature of this pocket are a conserved arginine (R) immediately pre- ceding the invariant aspartate (D) in the catalytic loop and a basic residue in the b9 strand [11] (Fig. 1). Kinases of the DYRK family depend on the phos- phorylation of an absolutely conserved tyrosine residue in the activation loop to achieve full activity [1,2]. This tyrosine (Y321 in DYRK1A) corresponds to Y185 in the doubly phosphorylated TEY motif of ERK2, a res- idue classified as a ‘secondary activation loop phos- phorylation site’ because it does not interact with the RD pocket [10]. Instead, the phosphorylated tyrosine forms salt bridges with two arginines in the P + 1 loop (R189 and R192 in ERK2). Many kinases of the CMGC group contain a tyrosine at the same position, either in combination with a threonine as the ‘primary phosphorylation site’ in a TxY motif, as in the MAP- Ks, or in the absence of a primary site, as in the DYRKs and GSK3a ⁄ b (Fig. 1). Although PRP4 and HIPK1-3 harbour phosphorylatable serine or threonine AB Fig. 1. Tyrosine phosphorylation in the activation loop of CMGC kinases. (A) CMGC kinases with a tyrosine phosphorylation site in the acti- vation loop belong to different branches of the CMGC group. Human Kinome provided courtesy of Cell Signaling Technology (http:// www.cellsignal.com). (B) Sequence motifs directly involved in the activation mechanism. Basic residues that interact with the primary phos- phorylation site (RD pocket, formed by the residue preceding the catalytic aspartic acid and the b9 strand) and the basic residues in the P + 1 loop that interact with the phosphotyrosine are shown in blue. In GSK3, the RD pocket binds a phosphorylated residue in the sub- strate. Two conserved cysteines in the DYRK family that may form a reversible disulfide bond are highlighted in yellow. The mechanism of tyrosine phosphorylation is indicated by symbols as indicated. For HIPK2, tyrosine phosphorylation was shown in a kinase-negative point mutant, but so far there is no evidence for an upstream kinase [80]. The TEY motif was shown to be essential for full activity of MOK, but as yet there is no direct evidence of tyrosine phosphorylation [81]. CLK1-4, ERK3-4, SRPK ⁄ MSSK1 and the CDK subfamily do not contain a tyrosine in the respective position of the activation loop. Evidence for tyrosine phosphorylation in the activation loop was extracted from the indicated references [82–86] or the PhosphoSitePlus database (P) (http://www.phosphosite.org). W. Becker and W. Sippl Activation, regulation, and inhibition of DYRK1A FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS 247 residues at the primary phosphorylation site, they lack the RD pocket, and phosphoproteomic studies (http://www.phosphosite.org) only provide strong evi- dence for the phosphorylation of the tyrosine in these kinases (Table 1). In DYRK1A, pY321 is engaged in the same interactions with the two arginines as pY185 in ERK2 (R325 and R328 in DYRK1A) (Fig. 2). Whereas the dual-phosphorylation of the MAP kinases is a classical paradigm for the on ⁄ off regulation by upstream protein kinases, tyrosine phosphorylation of DYRKs and GSK3 occurs by autophosphorylation and appears to be constitutive [2,13]. This one-off autoactivation takes place during or immediately after translation by an intramolecular reaction [14,15]. In Drosophila DYRK2 (dDYRK2), tyrosine autophos- phorylation depends on the presence of an N-terminal autophosphorylation accessory region. The N-terminal autophosphorylation accessory region is conserved in a subgroup of the DYRK family (class 2 DYRKs), but is not required for tyrosine autophosphorylation of MNB [16]. We have shown for mammalian DYRK1A that tyrosine autophosphorylation is an intrinsic capacity of the catalytic domain and does not depend on other domains or any cofactor [17]. Notably, a few kinases with the doubly phosphorylated TxY motif also activate by autophosphorylation (ERK7, CDKL5, Table 1), and some degree of activation loop auto- phosphorylation has historically even been observed in ERK1 and ERK2 [18,19]. Thus, a sometimes latent capacity of tyrosine autophosphorylation appears to be a characteristic of many CMGC group protein kin- ases and may be an evolutionarily ancestral feature. A very interesting intermediate mechanism exists in the closely related kinases ICK and MAK, which auto- phosphorylate the tyrosine in the TDY motif, but are fully active only after threonine phosphorylation by an upstream kinase [20,21]. Another variation of the theme is realized in the stress-activated kinase p38a, which can be activated either by upstream kinases or by regulated tyrosine autophosphorylation [22]. Fur- ther work is required to dissect the full spectrum of activation modes of the different CMGC group kinas- es. However, so far there is no evidence that the cata- lytic activity of any member of the DYRK family is reversibly regulated by phosphorylation ⁄ dephosphory- lation of the activation loop tyrosine. Mature DYRKs phosphorylate their substrates only on serine or threonine residues and cannot rephospho- rylate on tyrosine after phosphatase treatment [14,23]. How can a serine ⁄ threonine-specific protein kinase autophosphorylate on tyrosine? Lochhead et al. [14] proposed that only a translational folding intermediate of DYRKs with biochemical properties different from the mature kinase is capable of tyrosine phosphoryla- tion. This idea is supported by their finding that the tyrosine phosphorylation of newly synthesized Dro- sophila dDYRK2 and substrate phosphorylation by the mature kinase exhibit differential sensitivity towards kinase inhibitors. This result suggests that the ‘dual-specificity’ of DYRKs comes together with a ‘dual-sensitivity’ to kinase inhibitors [14]. There are several open questions concerning the acti- vation mechanism of DYRK1A. The substitution of the activation loop tyrosine by phenylalanine markedly reduces (> 80%) the catalytic activity of DYRK1A [1,2,24]. Surprisingly, dephosphorylation does not inac- tivate mature DYRK1A [23] and reduces the activity of Drosophila dDYRK2 and MNB by only  50% Table 1. DYRK1A inhibitors. List of small molecule drugs that have been used as DYRK1A inhibitors. Inhibitor IC 50 for DYRK1A a IC 50 for other kinases a Comments References Purvalanol 300 n M 100 nM CDK2 Inhibits tyrosine autophosphorylation of dDYRK2 [11,58] DMAT 120 n M 150 nM CK2 [75] TBB 4360 n M 150 nM CK2 990 n M DYRK2 Does not inhibit tyrosine autophosphorylation of dDYRK2 [11,75,76] Pyrazolidin-diones 18 and 21 600 n M ND IC 50 was determined in an autophosphorylation assay [55] TG003 12 n M b 930 lM 19 nM CLK1 130 n M DYRK1B Less potent inhibitor of DYRK1A than harmine in HeLa cells c [77,78] [12] INDY 240 n M 230 nM DYRK1B Structurally related with TG003 [12] EGCG 330 n M 1000 nM PRAK Non-ATP-competitive inhibitor [58,79] Harmine 80 n M b 33 nM 150 nM DYRK1B 900 n M DYRK2 800 n M DYRK3 IC 50 = 1900 nM for tyrosine auto-phosphorylation of DYRK1A [17,59] a IC 50 values were determined in in vitro kinase assays at variable reaction conditions. b Deviating results obtained by different assays. c M. Rahbari & W. Becker, unpublished results. Activation, regulation, and inhibition of DYRK1A W. Becker and W. Sippl 248 FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS [14]. It is conceivable that tyrosine phosphorylation is only required for the switch into the active conformation but not for maintaining this state. Alternatively, the stabilizing effect of the salt bridges between the phosp- hotyrosine with the two arginines in the P + 1 may be critical in living cells, but less so in in vitro assays. For comparison, it is interesting to consider the role of the phosphotyrosine (pY216) in GSK3b, which resembles the DYRKs in its autoactivation mechanism [13,15]. Analysis of crystal structures revealed that unphos- phorylated GSK3b, in stark contrast to unphosphory- lated ERK2, can acquire a catalytically active conformation [25,26]. However, dephosphorylation de- stabilizes the active conformation and leads to a loss of activity over time [13]. Regulation The available evidence suggests that DYRK1A is always catalytically active when isolated from tissues or cells. However, the role of protein kinases as cellu- lar regulators implies that their own activity is some- how regulated. Given that the activation loop phosphorylation is apparently constitutive, how then is the activity of DYRK1A modulated? Genetic evidence indicates that small changes in expression levels of DYRK1A have severe phenotypic consequences, as exemplified by patients with homozygous deficiency of DYRK1A [27] and transgenic animal models [28]. Thus, the function of DYRK1A may be controlled by more subtle changes in activity than the paradigmatic on ⁄ off switches as known from MAPKs, cAMP- dependent protein kinase, receptor tyrosine kinases and many other kinases [11]. Although our present understanding of the regulation of DYRK1A function is only rudimentary, we will discuss here potential mechanisms by which the function of DYRK1A may be controlled, including changes in expression levels, association with regulatory proteins, changes in subcel- lular localization. There is evidence for a regulation of DYRK1A on the level of gene expression and protein abundance. A recent study demonstrated circadian changes in DYRK1A levels in the mouse liver and identified DYRK1A as a novel component of the molecular clock [29]. Numerous microarray studies have revealed striking changes in DYRK1A mRNA levels in various systems of cellular differentiation and proliferation (for references see [30]). However, very little is known about the transcriptional regulation of DYRK1A gene expression. Activator protein 4 was described as a neg- ative regulator of DYRK1A expression in non-neural cells [31]. In the neuroblastoma cell line SH-SY5Y, an increase in DYRK1A mRNA was induced by the b-amyloid peptide, but nothing is known about the potential mechanism of regulation [32]. The transcrip- tion factor E2F1 increases DYRK1A mRNA levels by enhancing promoter activity [30]. In a recent report, nuclear factor of activated T-cells (NFATc1) was shown to upregulate DYRK1A levels in bone marrow macrophages [33]. This control mechanism forms part of a negative feedback loop, because NFATs are nega- tively regulated by DYRK1A [34]. The presence of potentially destabilizing AUUUA elements in the 3¢-UTR of the DYRK1A mRNA and the PEST region in the DYRK1A protein [1] supports the hypothesis that DYRK1A levels are subject to rapid changes. PEST sequences are rich in the amino acids proline (P), glutamate (E), serine (S) and threonine (T) and correlate with rapid protein turnover in eukaryotic cells, often by acting as degradation tags that direct proteins for proteasome-mediated destruction [35]. However, data on the half-life of DYRK1A or on the regulation of its degradation have not been reported. Several studies suggest a regulation of DYRK1A by interacting proteins. Binding of 14-3-3b to an autop- hosphorylated serine residue in the C-terminal domain of DYRK1A (Ser529, numbered Ser520 in the short splicing variant of DYRK1A) stimulates the catalytic activity of DYRK1A up to a factor of 2 [36]. Binding Fig. 2. Potential intramolecular disulfide bridge in DYRK1A observed in the X-ray structure of DYRK1A (PDB acc. 2VX3). The distance between the thiol groups of the two conserved cysteines (coloured green, C286 and C312 in DYRK1A) is  4.3 A ˚ , indicating the probability of forming a disulfide bridge in the oxidized form. In addition, arginines R325 and R328 interacting with the phos- phorylated Y321 are shown (hydrogen bonds are shown as dashed lines). W. Becker and W. Sippl Activation, regulation, and inhibition of DYRK1A FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS 249 of RanBPM (=RanBP9) to a neighbouring region in the C-terminal domain (550–563) negatively modulates DYRK1A activity [37]. The WD40 repeat protein, WDR68 (also called HAN11, official human gene sym- bol DCAF7), has repeatedly been found to be associ- ated with DYRK1A and ⁄ or DYRK1B and may function as a regulatory subunit [38–41]. Overexpres- sion of WDR68 inhibited the DYRK1A-mediated stim- ulation of GLI1-dependent reporter gene activity [39]. Recently, SPRED1 and SPRED2 (sprouty-related pro- tein with an EVH1 domain) were found to directly interact with the catalytic domain of DYRK1A and to inhibit the phosphorylation of the substrate proteins, Tau and STAT3 [42]. Interestingly, the inhibitory effect of the SPRED proteins appears to be due to competi- tion with the substrate proteins and not due to a gen- eral inhibition of catalytic activity. Further research is clearly required to elucidate the role of these interacting proteins in the regulation of DYRK1A. A novel regulatory mechanism was recently revealed for the DYRK2 orthologous kinase in Caenorhabditis elegans. In this case, the catalytically inactive ‘pseudo- phosphatases’ EGG-4 and EGG-5 inhibit substrate phosphorylation by MBK-2 by binding to the phosp- hotyrosine motif in the activation loop of the kinase [43,44]. Catalytically inactive tyrosine phosphatases also exist in mammals, and it will be exciting to see whether this mechanism applies to other members of the DYRK family, including DYRK1A. Regulated nuclear translocation has been reported for mammalian DYRK2 and the DYRK in budding yeast, Yak1p [45,46]. DYRK1A harbours a functional nuclear localization sequence in its N-terminal domain and has been found in both the nucleus and in the cytoplasm (see also the accompanying minireview by Wegiel et al. [9]), possibly depending on the cell type. Furthermore, DYRK1A phosphorylates both nuclear and cytoplasmic proteins [8,47]. DYRK1A controls nuclear import (GLI1) or export (FOXO1, NFAT) of several transcription factors [34,48,49], but the ques- tion whether DYRK1A itself undergoes cytoplasmic– nuclear shuttling has not yet been directly addressed. Interestingly, dynamic changes in the intracellular localization of the chicken DYRK1A orthologue, MNB, have been deduced from immunofluorescence analyses of developing neurons during brain develop- ment [5]. Finally, we want to propose a new potential mode of regulation of DYRKs that has not yet been experi- mentally tested. As we and others have previously noted [50,51], two cysteine residues are located in those positions that correspond to the RD pocket in kinases regulated by the primary phosphorylation site in the activation loop (RD kinases, Fig. 1). This unique structural peculiarity is conserved in DYRK family kinases from fungi, plants and animals, but cannot easily be explained by the catalytic mechanism or structural constraints and thus must have another essential function. In the three-dimensional structures of DYRK1A and DYRK2 (PDB acc. 2VX3; 2WO6; 3KVW; 3K2L), the thiol moieties of these cysteines are sufficiently close to each other ( 4.3 A ˚ in DYRK1A and DYRK2) to allow the formation of a disulfide bridge (Fig. 2). It is tempting to speculate that redox regulation of DYRK1A can occur through changes in the redox state of these cysteines. Redox modification at C286 and C312 in DYRK1A (-SH reduced to -S–S- oxidized state) could result in a conformational change, which could then somehow influence the cata- lytic activity of DYRK kinases. DYRK1A inhibitors Small cell-permeant inhibitors of protein kinases are important tools for studying intracellular signal trans- duction pathways. Although genetic techniques such as RNA interference offer an alternative to study kinase function, small-molecule inhibitors provide more rapid temporal control. Moreover, comparisons of genetic kinase knockouts with effects of inhibitors have often revealed major differences in the resulting phenotype [52]. The hypothesis that the elevated activity of DYRK1A contributes to the cognitive deficits in Down syndrome and the development of Alzheimer’s disease has stimulated interest in DYRK1A as a potential target for therapeutic inhibitors [53,54]. Synthetic inhibitors The first targeted approach to develop an inhibitor of DYRK1A resulted in the identification and optimiza- tion of pyrazolidinedione compounds that inhibit DYRK1A autophosphorylation with IC 50 values from 0.6–2.5 lm. [53,55]. These inhibitors remain to be fur- ther characterized for their specificity against a broad panel of kinases and their effects in cells or in experi- mental animals. Ogawa et al. recently reported the characterisation of a new benzothiazol inhibitor of DYRK1A, designated INDY, as well as the three- dimensional structure of the DYRK1A/INDY complex [PDB accession 3ANQ]. Furthermore, the authors demonstrate that harmine resembles TG003 and INDY in its capacity to inhibit CLKs at a level comparable with the inhibition of DYRK1A [12]. Several compounds originally designed to target other protein kinases were uncovered as fairly efficient inhibi- Activation, regulation, and inhibition of DYRK1A W. Becker and W. Sippl 250 FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS tors of DYRK1A (Table 1, Fig. 3). Purvalanol A had been developed as an inhibitor of CDKs, 2-dimethyla- mino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and 4,5,6,7-tetrabromo-1H-benzotriazole (TBB) as CK2 inhibitors, and TG003 as an inhibitor of CLKs. Obvi- ously, the promiscuous behaviour of these inhibitors limits their value as tools for the analysis of signalling pathways. Nevertheless, use of purvalanol A has guided the identification of DYRK1A as the kinase responsible for the phosphorylation of specific sites in MAP1B and caspase 9 [56,57]. A prodrug of INDY was successfully used to reverse malformations of Xenopus embryos induced by DYRK1A overexpression [12]. Furthermore, cross-reactivity with other kinases is not an issue in mechanistic studies with purified enzymes, such as the analysis of the autoactivation mechanism of dDYRK2 and MNB [14]. Natural compounds Two plant compounds, epigallocatechin-gallate (EGCG) and harmine, have been identified as DYRK1A inhibitors in selectivity profiling studies [58,59] (Table 1). EGCG, the major polyphenolic com- pound of green tea, inhibited DYRK1A rather specifi- cally among 29 kinases tested [58]. However, multiple and heterogeneous effects of EGCG on signalling path- ways have been described (e.g. [60–62]). Furthermore, EGCG exhibits complex pharmacokinetic properties and poor bioavailability [63], which limit its usefulness in cell and animal experiments. EGCG was used in cell culture studies to confirm the presumed role of DYRK1A in signalling events [56,64–66] and to rescue brain defects of DYRK1A-overexpressing mice [67]. Harmine is a b-carboline alkaloid that has long been known as a potent inhibitor of monoamine oxidase A (IC 50 =5nm) [68]. Harmine is produced by divergent plant species, including the South American vine Ban- isteriopsis caapi and the mideastern shrub Peganum harmala (Syrian rue). Banisteriopsis is a component of hoasca (also called ayahuasca or yage ´ ), an hallucino- genic brew of plant extracts used in shamanic rituals and South American sects for its visionary effects. The monoamine oxidase-inhibiting activity of harmine blocks the first pass metabolism of dimethyltryptamine and thereby allows the oral ingestion of this natural hallucinogenic. It is interesting to note that plasma lev- els of harmine in hoasca users are within a range ( 0.5 lm) [69] expected to cause a substantial inhibi- tion of DYRK1A. As a kinase inhibitor, harmine displays excellent specificity for DYRK1A among 69 protein kinases [59]. Obviously, side-effects on kinases not included in the screen cannot be excluded. Development of an optimized DYRK1A inhibitor on the basis of harmine as a lead structure appears feasible, because harmine is a rather small molecule (212 Da). The crystal struc- tures of DYRK1A complexed with an indazol inhibi- tor and DYRK2 complexed with an indirubin inhibitor have been solved recently [70]. Based on the analysis of the two highly similar 3D structures, only three amino acid residues in the binding pocket have been found to be different. These include V222, M240 and V306, which are substituted by the more spacious residues I212, L230 and I294 in DYRK2. Docking studies showed that harmine interacts with the residues of the ATP binding pocket and is involved in two hydrogen bonds – one to the hinge region (backbone NH of M240) and one to the conserved K188. Based on the model of the DYRK1A ⁄ harmine complex, it is suggested that the accessible volume of the ATP bind- ing pocket can accommodate substituents at the b-carboline structure (Fig. 4A). Interestingly, due to the substitution of L230 for M240 and I212 for V222, the binding pocket in DYRK2 is more restricted (Fig. 4B), resulting in a slightly different orientation of the docked harmine. For DYRK2 the observed bind- ing mode is less favourable, showing longer hydrogen bond distances between inhibitor and kinase (not shown). The closest relative of DYRK1A, DYRK1B, is inhibited at somewhat higher concentrations by har- mine (Table 1), but this difference is not sufficient to discriminate these kinases pharmacologically [17]. Harmine inhibits DYRK1A in cultured cells with simi- Fig. 3. Chemical structures of known DYRK1A inhibitors. W. Becker and W. Sippl Activation, regulation, and inhibition of DYRK1A FEBS Journal 278 (2011) 246–256 ª 2010 The Authors Journal compilation ª 2010 FEBS 251 lar potency as in vitro at concentrations where little toxicity is observed [17]. Effects of harmine on many other targets have been described, but generally require much higher concentrations than the inhibition of DYRK1A. The antidiabetic effect of harmine as an inducer of Id2 and PPARc expression in preadipocytes also required concentrations greater than 1 lm [70,71]. The molecular target responsible for these effects is not yet known. Harmine is arguably the most useful inhibitor of DYRK1A presently available and has been used in several studies to support a presumed cel- lular function of DYRK1A [57,72–74]. However, the inhibitory effect on monoamine oxidase clearly limits its use in animals and in experimental systems suscepti- ble to the influence of monoamine oxidase (e.g. brain slices). Specific inhibition of tyrosine autophosphorylation? As noted above, Lochhead et al. [14] provided evidence that the ‘dual-specificity’ of the Drosophila DYRKs is associated with a differential inhibitor sensitivity of the transitory folding intermediate and the mature conformation of the kinase. We have recently shown that harmine inhibits tyrosine auto- phosphorylation of mammalian DYRK1A much less potently than the phosphorylation of exogenous sub- strates [17]. However, it must be noted that these reactions are kinetically very different. It is possible that a much lower degree of catalytic activity is required for the intramolecular autophosphorylation (a first-order reaction) than the trans-phosphorylation of other substrates in a second-order reaction. The identification of an inhibitor that specifically or preferentially inhibits tyrosine autophosphorylation of DYRK1A versus serine ⁄ threonine phosphorylation of substrates could prove the concept of dual inhibitor sensitivity. This is important not only from a biochemical point of view, because the one-off auto- activation mechanism of the DYRKs suggests that inhibitors of tyrosine autophosphorylation should act irreversibly. However, this hypothesis remains to be experimentally substantiated. Acknowledgements We thank German Erlenkamp for excellent technical assistance. Financial support of our research on DYRK1A by the Deutsche Forschungsgemeinschaft and the Jerome Lejeune Foundation is gratefully acknowledged (WB). 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MINIREVIEW Activation, regulation, and inhibition of DYRK1A Walter Becker 1 and Wolfgang Sippl 2 1 Institute of Pharmacology and Toxicology, Medical Faculty of. between DYRK1A and DYRK2 (PDB acc. 2VX3 and 3KVW), illustrating differences in the ATP binding pocket. Activation, regulation, and inhibition of DYRK1A