Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo

Collapsin response mediator proteins (CRMPs) are a family of neuron-enriched proteins that regulate neurite outgrowth and growth cone dynamics. Here, we show that Cdk5 phosphorylates CRMP1, CRMP2, and CRMP4, priming for subsequent phosphorylation by GSK3 in vitro. In contrast, DYRK2 phosphorylates and primes CRMP4 only. The Cdk5 and DYRK2 inhibitor purvalanol decreases the phosphorylation of CRMP proteins in neurons, whereas CRMP1 and CRMP2, but not CRMP4, phosphorylation is decreased in Cdk5(-/-) cortices. Stimulation of neuroblastoma cells with IGF1 or TPA decreases GSK3 activity concomitantly with CRMP2 and CRMP4 phosphorylation. Conversely, increased GSK3 activity is not sufficient to increase CRMP phosphorylation. However, the growth cone collapse-inducing protein Sema3A increases Cdk5 activity and promotes phosphorylation of CRMP2 (but not CRMP4). Therefore, inhibition of GSK3 alters phosphorylation of all CRMP isoforms; however, individual isoforms can be differentially regulated by their respective priming kinase. This is the first GSK3 substrate found to be regulated in this manner and may explain the hyperphosphorylation of CRMP2 observed in Alzheimer's disease.     

Tau phosphorylation and neuronal apoptosis induced by the blockade of PP2A preferentially involve GSK3β

Overactivation of GSK3β (glycogen synthase kinase-3β) and downregulation of PP2A (protein phosphatase-2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation in Alzheimer’s disease (AD). GSK3β and PP2A signaling pathways were reported to be interconnected. Targeting tau kinases was suggested to represent a therapeutic strategy for AD. Here, tau phosphorylation and neuronal apoptosis were induced in cortical cultured neurons by the inhibition of PP2A by okadaic acid (OKA). In this in vitro model of ‘tau pathology’ and neurodegeneration, we tested whether GSK3β and other tau kinases including DYRK1A and CDK5 were implicated. Our results show that the inhibitors of GSK3β, lithium and 6-BIO (6-bromoindirubin-3′-oxime), prevented OKA-induced tau phosphorylation and neuronal apoptosis. The implication of GSK3β in these OKA-induced effects was confirmed by its silencing by hairpin siRNA. By contrast, inhibition of DYRK1A (dual-specificity tyrosine-phosphorylation regulated kinase-1A) and CDK5 (cyclin-dependent kinase-5) reversed OKA-induced tau phosphorylation at certain sites but failed to prevent neuronal apoptosis. These results indicate that OKA-induced effects, especially neuronal apoptosis, are preferentially mediated by GSK3β. Furthermore, since chronic exposure to lithium and 6-BIO might be deleterious for neurons, we tested the effect of a new 6-BIO derivative, 6-BIBEO (6-bromoindirubin-3′-(2-bromoethyl)-oxime), which is much less cytotoxic and more selectively inhibits GSK3β compared to lithium and 6-BIO. We show that 6-BIBEO efficiently reversed OKA-induced tau phosphorylation and neuronal apoptosis. It will be interesting to test neuroprotection by 6-BIBEO in an in vivo model of tau pathology and neurodegeneration.     

Insights into mechanism of pyrido[2,3‐d]pyrimidines as DYRK1A inhibitors based on molecular dynamic simulations

DYRK1A is characterized by the early development and regulation of neuronal proliferation, and its over expression gives rise to neurological abnormalities. As the promising DYRK1A inhibitors, the binding mechanism between DYRK1A and pyrido[2,3‐d]pyrimidines derivatives at molecular level are still veiled. In this article, it was achieved to get the structural insights into pyrido[2,3‐d]pyrimidines derivatives as DYRK1A inhibitors by means of comprehensive computational approaches involving molecular docking, molecular dynamics simulation, free energy calculation, and energy decomposition analysis. The calculated energy values were highly consistent with the experimental activities. Based on the individual energy terms analysis, the van der Waals interaction was the major leading force in the DYRK1A–ligand interaction. Lys188 was the important residue that formed the hydrogen bond, which improved the inhibitory activity. Furthermore, four novel inhibitors with higher predicted activity were designed based on the obtained findings and confirmed by molecular simulations. Our study is expected to provide significant drug design strategy for the development of more promising DYRK1A inhibitors. Proteins 2016; 84:1108–1123. © 2016 Wiley Periodicals, Inc.     

Activation, regulation, and inhibition of DYRK1A

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 associated with the pathology of neurodegenerative diseases and have been implicated 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 current knowledge about the initial self-activation of DYRK1A by tyrosine autophosphorylation and propose that this mechanism presents an ancestral feature of the CMGC group of kinases. However, tyrosine phosphorylation does not appear to regulate the enzymatic activity of DYRK1A. Control of DYRK1A may take place on the level of gene expression, interaction 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 β-carboline alkaloid harmine is currently the most selective and potent inhibitor of DYRK1A and has proven very useful in cellular assays.     

Unravelling the Selectivity of 6,7-Dimethyl Quinoxaline Analogs for Kinase Inhibition: An Insight towards the Development of Alzheimer's Therapeutics

Untangling the most selective kinase inhibitors via pharmacological intervention remains one of the challenging affairs to date. In accordance to this drift, herein we describe the design and synthesis of a set of new heterocyclic analogs consisting of 6,7-dimethyl Quinoxaline, appended to a connector, employing Schiff base strategy (Compounds I – IX ). The compounds were characterized by various spectroscopic techniques and the kinase inhibition assay were performed on few prime members of the CMGC family namely the GSK3β, DYRK1A and CLK1 receptors, respectively, that have been known to be directly involved in hyperphosphorylation of Tau. Interestingly the biological evaluation results revealed that Compounds IV and V , with bromo/chloro functionalities in the aromatic core were advantaged of being highly selective towards the target GSK3β over others. To strengthen our analysis, we adopted molecular modelling studies, where compounds IV/V were redocked in the same grid 4AFJ, as that of the reference ligand, 5-aryl-4-carboxamide-1,3-oxazole. Surprisingly, our investigation underpinned that for both the compounds IV/V , a primary H-bonding existed between the designed molecules ( IV/V ) and Val 135 residue in the receptor GSK3β, in line with the reference ligand. We attribute this interaction to instigate potency in the compounds. Indeed the other non-covalent interaction, between the derivative's aromatic nucleus and Arg 141/Thr 138 in the receptor GSK3β, might have been responsible for enhancing the selectivity in the targets. Overall, we feel that the present work depicts a logical demonstration towards fine tuning the efficacy of the inhibitors through systematic adjustment of electron density at appropriate positions in the aromatic ring be it the main quinoxaline or the other aromatic nucleus. Thus this pathway offers a convenient strategy for the development of efficient therapeutics for diversified neurodegenerative diseases like that of Alzheimer's.     

Tau pathology reduction with SM07883, a novel,potent, and selective oral DYRK1A inhibitor: A potential therapeutic for Alzheimer's disease

Dual‐specificity tyrosine phosphorylation‐regulated kinase‐1A (DYRK1A) is known to phosphorylate the microtubule‐associated tau protein. Overexpression is correlated with tau hyperphosphorylation and neurofibrillary tangle (NFT) formation in Alzheimer's disease (AD). This study assessed the potential of SM07883, an oral DYRK1A inhibitor, to inhibit tau hyperphosphorylation, aggregation, NFT formation, and associated phenotypes in mouse models. Exploratory neuroinflammatory effects were also studied. SM07883 specificity was tested in a kinase panel screen and showed potent inhibition of DYRK1A (IC₅₀ = 1.6 nM) and GSK‐3β (IC₅₀ = 10.8 nM) kinase activity. Tau phosphorylation measured in cell‐based assays showed a reduction in phosphorylation of multiple tau epitopes, especially the threonine 212 site (EC₅₀ = 16 nM). SM07883 showed good oral bioavailability in multiple species and demonstrated a dose‐dependent reduction of transient hypothermia‐induced phosphorylated tau in the brains of wild‐type mice compared to vehicle (47%, p < 0.001). Long‐term efficacy assessed in aged JNPL3 mice overexpressing the P301L human tau mutation (3 mg/kg, QD, for 3 months) exhibited significant reductions in tau hyperphosphorylation, oligomeric and aggregated tau, and tau‐positive inclusions compared to vehicle in brainstem and spinal cord samples. Reduced gliosis compared to vehicle was further confirmed by ELISA. SM07883 was well tolerated with improved general health, weight gain, and functional improvement in a wire‐hang test compared to vehicle‐treated mice (p = 0.048). SM07883, a potent, orally bioavailable, brain‐penetrant DYRK1A inhibitor, significantly reduced effects of pathological tau overexpression and neuroinflammation, while functional endpoints were improved compared to vehicle in animal models. This small molecule has potential as a treatment for AD.     

DYRK3 activation,engagement of protein kinase A/cAMP response element-binding protein,and modulation of progenitor cell survival

DYRKs are a new family of dual-specificity tyrosine-regulated kinases with emerging roles in cell growth and development. Recently, we discovered that DYRK3 is expressed primarily in erythroid progenitor cells and modulates late erythropoiesis. We now describe 1) roles for the DYRK3 YTY signature motif in kinase activation, 2) the coupling of DYRK3 to cAMP response element (CRE)-binding protein (CREB), and 3) effects of DYRK3 on hematopoietic progenitor cell survival. Regarding the DYRK3 kinase domain, intactness of Tyr(333) (but not Tyr(331)) within subdomain loop VII-VIII was critical for activation. Tyr(331) plus Tyr(333) acidification (Tyr mutated to Glu) was constitutively activating, but kinase activity was not affected substantially by unique N- or C-terminal domains. In transfected 293 and HeLa cells, DYRK3 was discovered to efficiently stimulate CRE-luciferase expression, to activate a CREB-Gal4 fusion protein, and to promote CREB phosphorylation at Ser(133). Interestingly, this CREB/CRE response was also supported (50% of wild-type activity) by a kinase-inactive DYRK3 mutant as well as a DYRK3 C-terminal region and was blocked by protein kinase A inhibitors, suggesting functional interactions between protein kinase A and DYRK3. Finally, DYRK3 expression in cytokine-dependent hematopoietic FDCW2 cells was observed to inhibit programmed cell death. Thus, primary new insight into DYRK3 kinase signaling routes, subdomain activities, and possible biofunctions is provided.     

Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells

The identification of phosphorylation state-dependent interacting proteins provides clues as to the function of the phosphorylation. Techniques such as yeast two hybrid and co-immunoprecipitation do not employ a single species of fully phosphorylated proteins. This is a particular problem for substrates of glycogen synthase kinase-3 (GSK3), where multiple Ser/Thr residues can be targeted, almost always subsequent to a priming phosphorylation by an alternative kinase. We previously identified the brain enriched collapsin response mediator proteins (CRMP2 and CRMP4) as physiological substrates of GSK3. Cdk5 phosphorylates CRMP2 at Ser522, priming for subsequent phosphorylation at three residues by GSK3 in vitro and in vivo. It is clear that phosphorylation of CRMP2 influences axonal growth; however, the molecular processes underlying this action are not fully established. In addition, the role of phosphorylation in other actions of CRMPs has not been elucidated. We developed a novel procedure to isolate CRMP2 and CRMP4 fully phosphorylated at four sites, namely, Ser522 (by CDK5), Ser518, Thr514, and Thr509 (by GSK3). These phosphoproteins were then used to identify binding partners in rat brain lysates in direct comparison with the non-phosphorylated isoforms. We validated the approach by confirming that a previously reported interaction with tubulin-beta is regulated by phosphorylation. We also show that CRMPs (CRMP1, CRMP2, and CRMP4) form heteromers and found that these complexes may also be regulated by phosphorylation. We identified DYRK and Pin1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin1 with phospho-CRMP4. Finally, we used this approach to identify the mitochondrial protein ANT as a novel CRMP2 and CRMP4 binding protein. We believe that this approach could be applied generally to the study of phosphorylation-dependent interactions.     

Pyridazinone derivatives: design, synthesis, and in vitro vasorelaxant activity

In an attempt to identify potential vasodilator-cardiotonic lead compounds, three series of pyridazinones were designed using three-dimensional pharmacophore developed with CATALYST software from a set of potent cyclic nucleotide phosphodiesterase III, cAMP PDEIII inhibitors. The features of the target compounds were based on the structures of many biologically active lead compounds with cAMP phosphodiesterase III inhibiting activity such as Milrinone and others. Compounds with higher fit scores to the developed pharmacophore were synthesized namely; 6-(3-ethoxycarbonyl-4-oxo-1,4-dihydroquinolin-6-yl)-4,5-dihydro-3(2H)-pyridazinones (3a and 3b), 6-[4-(2,6-disubstituted-quinolin-4-ylamino)phenyl]-4,5-dihydropyridazin-3(2H)-ones (5a-f), and 6-[3-(5-cyano-6-oxo-4-aryl-1,6-dihydro-2-pyridyl)phenylamino]-3(2H)pyridazinone (8a and 8b). The vasodilator activity of the newly synthesized compounds was examined on the isolated main pulmonary artery of the rabbit. Some of the tested compounds showed moderate vasorelaxant activity compared with standard drug, Milrinone.     

Hepatocyte-specific Dyrk1a gene transfer rescues plasma apolipoprotein A-I levels and aortic Akt/GSK3 pathways in hyperhomocysteinemic mice

Hyperhomocysteinemia, characterized by high plasma homocysteine levels, is recognized as an independent risk factor for cardiovascular diseases. The increased synthesis of homocysteine, a product of methionine metabolism involving B vitamins, and its slower intracellular utilization cause increased flux into the blood. Plasma homocysteine level is an important reflection of hepatic methionine metabolism and the rate of processes modified by B vitamins as well as different enzyme activity. Lowering homocysteine might offer therapeutic benefits. However, approximately 50% of hyperhomocysteinemic patients due to cystathionine-beta-synthase deficiency are biochemically responsive to pharmacological doses of B vitamins. Therefore, effective treatments to reduce homocysteine levels are needed, and gene therapy could provide a novel approach. We recently showed that hepatic expression of DYRK1A, a serine/threonine kinase, is negatively correlated with plasma homocysteine levels in cystathionine-beta-synthase deficient mice, a mouse model of hyperhomocysteinemia. Therefore, Dyrk1a is a good candidate for gene therapy to normalize homocysteine levels. We then used an adenoviral construct designed to restrict expression of DYRK1A to hepatocytes, and found decreased plasma homocysteine levels after hepatocyte-specific Dyrk1a gene transfer in hyperhomocysteinemic mice. The elevation of pyridoxal phosphate was consistent with the increase in cystathionine-beta-synthase activity. Commensurate with the decreased plasma homocysteine levels, targeted hepatic expression of DYRK1A resulted in elevated plasma paraoxonase-1 activity and apolipoprotein A-I levels, and rescued the Akt/GSK3 signaling pathways in aorta of mice, which can prevent homocysteine-induced endothelial dysfunction. These results demonstrate that hepatocyte-restricted Dyrk1a gene transfer can offer a useful therapeutic targets for the development of new selective homocysteine lowering therapy.     

Kinase inhibitions in pyrido[4,3-h] and [3,4-g]quinazolines: Synthesis,SAR and molecular modeling studies

New pyrido[3,4-g]quinazoline derivatives were prepared and evaluated for their inhibitory potency toward 5 protein kinases (CLK1, DYRK1A, GSK3, CDK5, CK1). A related pyrido[4,3-h]quinazoline scaffold with an angular structure was also synthesized and its potency against the same protein kinase panel was compared to the analogous pyrido[3,4-g]quinazoline. Best results were obtained for 10-nitropyrido[3,4-g]quinazoline 4 toward CLK1 with nanomolar activities.     

DYRK1A inhibition suppresses STAT3/EGFR/Met signalling and sensitizes EGFR wild‐type NSCLC cells to AZD9291

DYRK1A is considered a potential cancer therapeutic target, but the role of DYRK1A in NSCLC oncogenesis and treatment requires further investigation. In our study, high DYRK1A expression was observed in tumour samples from patients with lung cancer compared with normal lung tissues, and the high levels of DYRK1A were related to a reduced survival time in patients with lung cancer. Meanwhile, the DYRK1A inhibitor harmine could suppress the proliferation of NSCLC cells compared to that of the control. As DYRK1A suppression might be effective in treating NSCLC, we next explored the possible specific molecular mechanisms that were involved. We showed that DYRK1A suppression by siRNA could suppress the levels of EGFR and Met in NSCLC cells. Furthermore, DYRK1A siRNA could inhibit the expression and nuclear translocation of STAT3. Meanwhile, harmine could also regulate the STAT3/EGFR/Met signalling pathway in human NSCLC cells. AZD9291 is effective to treat NSCLC patients with EGFR‐sensitivity mutation and T790 M resistance mutation, but the clinical efficacy in patients with wild‐type EGFR remains modest. We showed that DYRK1A repression could enhance the anti‐cancer effect of AZD9291 by inducing apoptosis and suppressing cell proliferation in EGFR wild‐type NSCLC cells. In addition, harmine could enhance the anti‐NSCLC activity of AZD9291 by modulating STAT3 pathway. Finally, harmine could enhance the anti‐cancer activity of AZD9291 in primary NSCLC cells. Collectively, targeting DYRK1A might be an attractive target for AZD9291 sensitization in EGFR wild‐type NSCLC patients.     

Aristolactam BIII,a naturally derived DYRK1A inhibitor,rescues Down syndrome-related phenotypes

BackgroundDual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a significant pathogenic factor in Down syndrome (DS), wherein DYRK1A is overexpressed by 1.5-fold because of trisomy of human chromosome 21. Thus, DYRK1A inhibition is considered a therapeutic strategy to modify the disease.PurposeThis study aims to identify a novel DYRK1A inhibitor and validate its therapeutic potential in DS-related pathological conditions.Study designIn order to identify a novel DYRK1A inhibitor, we carried out two-step screening: a structure-based virtual screening of > 300,000 chemical library (first step) and cell-based nuclear factor of activated T-cells (NFAT)-response element (RE) promoter assay (second step). Primary hits were evaluated for their DYRK1A inhibitory activity using in vitro kinase assay and Tau phosphorylation in mammalian cells. Confirmed hit was further evaluated in pathological conditions including DYRK1A-overexpressing fibroblasts, flies, and mice.ResultsWe identified aristolactam BIII, a natural product derived from herbal plants, as a novel DYRK1A inhibitor. It potently inhibited the kinase activity of DYRK1A in vitro (IC₅₀ = 9.67 nM) and effectively suppressed DYRK1A-mediated hyperphosphorylation of Tau in mammalian cells. Aristolactam BIII rescued the proliferative defects of DYRK1A transgenic (TG) mouse-derived fibroblasts and neurological and phenotypic defects of DS-like Drosophila models. Oral administration of aristolactam BIII acutely suppressed Tau hyperphosphorylation in the brain of DYRK1A TG mice. In the open field test, aristolactam BIII significantly ameliorated the exploratory behavioral deficit of DYRK1A TG mice.ConclusionOur work revealed that aristolactam BIII as a novel DYRK1A inhibitor rescues DS phenotypes in cells and in vivo and suggested its therapeutic potential for the treatment of DYRK1A-related diseases.     

Synthesis and kinase inhibitory activity of novel substituted indigoids

The bis-indole indigoids are a promising protein kinase inhibitor scaffold to be further evaluated against the numerous human diseases that imply abnormal regulation of kinases including neurodegenerative disorders. In an effort to identify new pharmacological inhibitors of disease-relevant protein kinases with increased potency and selectivity, we designed, synthesized new 5,7-disubstituted or 6-substituted bis-indole derivatives. On the basis of our previous synthetic work, 22 selected compounds were tested on CDK1/cyclin B, CDK5/p25, DYRK1A, CK1, and GSK-3α/β kinases, five kinases involved in Alzheimer’s disease. Some of them were also evaluated for their cytotoxic and antiproliferative activities. 6-Nitro-3′-N-oxime-indirubin and 5-amino-3′-N-oxime-indirubin derivatives exhibited inhibitory activity in a submicromolar range against CDK1/cyclin B (0.18 and 0.1 μM, respectively), CK1 (0.6 μM and 0.13 μM) and GSK3 (0.04 μM and 0.36 μM).     

Dual-specificity tyrosine phosphorylation-regulated kinase 1A does not require tyrosine phosphorylation for activity in vitro

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene is localized in human chromosome 21, and its overexpression has been associated with the learning and memory deficits of Down syndrome. DYRK1A contains a Y319XY321 motif shared by all members of the DYRK protein kinase family. Residue Y321 in the motif is phosphorylated in DYRK1A prepared from Escherichia coli and from eukaryotic cells. It has been proposed that the YXY motif is an equivalent of the TXY motif, the activation loop, of mitogen-activated protein kinase and that phosphorylation at the motif is required for DYRK activity. In this study, the role of tyrosine phosphorylation in the activity of DYRK1A was investigated in detail. Wild-type DYRK1A with a reduced level of phosphotyrosine (pY) was prepared by treating E. coli-produced DYRK1A with two different protein tyrosine phosphatases. The resulting pY-depleted DYRK1A could not regain pY during autophosphorylation but was as active as the untreated control. These findings were further supported by the observation that DYRK1A retained significant enzymatic activity when both tyrosine residues in the YXY motif were replaced with either histidine or glutamine. Together, we conclude that tyrosine phosphorylation and tyrosine residues in the YXY motif are not directly involved in DYRK1A enzymatic activity in vitro.