Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A Inhibitors Research Articles

Global phosphoproteomics reveals DYRK1A regulates CDK1 activity in glioblastoma cells

Both tumour suppressive and oncogenic functions have been reported for dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Herein, we performed a detailed investigation to delineate the role of DYRK1A in glioblastoma. Our phosphoproteomic and mechanistic studies show that DYRK1A induces degradation of cyclin B by phosphorylating CDC23, which is necessary for the function of the anaphase-promoting complex, a ubiquitin ligase that degrades mitotic proteins. DYRK1A inhibition leads to the accumulation of cyclin B and activation of CDK1. Importantly, we established that the phenotypic response of glioblastoma cells to DYRK1A inhibition depends on both retinoblastoma (RB) expression and the degree of residual DYRK1A activity. Moderate DYRK1A inhibition leads to moderate cyclin B accumulation, CDK1 activation and increased proliferation in RB-deficient cells. In RB-proficient cells, cyclin B/CDK1 activation in response to DYRK1A inhibition is neutralized by the RB pathway, resulting in an unchanged proliferation rate. In contrast, complete DYRK1A inhibition with high doses of inhibitors results in massive cyclin B accumulation, saturation of CDK1 activity and cell cycle arrest, regardless of RB status. These findings provide new insights into the complexity of context-dependent DYRK1A signalling in cancer cells.

DYRK1A Negatively Regulates CDK5-SOX2 Pathway and Self-Renewal of Glioblastoma Stem Cells.

Glioblastoma display vast cellular heterogeneity, with glioblastoma stem cells (GSCs) at the apex. The critical role of GSCs in tumour growth and resistance to therapy highlights the need to delineate mechanisms that control stemness and differentiation potential of GSC. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) regulates neural progenitor cell differentiation, but its role in cancer stem cell differentiation is largely unknown. Herein, we demonstrate that DYRK1A kinase is crucial for the differentiation commitment of glioblastoma stem cells. DYRK1A inhibition insulates the self-renewing population of GSCs from potent differentiation-inducing signals. Mechanistically, we show that DYRK1A promotes differentiation and limits stemness acquisition via deactivation of CDK5, an unconventional kinase recently described as an oncogene. DYRK1A-dependent inactivation of CDK5 results in decreased expression of the stemness gene SOX2 and promotes the commitment of GSC to differentiate. Our investigations of the novel DYRK1A-CDK5-SOX2 pathway provide further insights into the mechanisms underlying glioblastoma stem cell maintenance.

Identification of harmine and β-carboline analogs from a high-throughput screen of an approved drug collection; profiling as differential inhibitors of DYRK1A and monoamine oxidase A and for in vitro and in vivo anti-cancer studies

DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is highly expressed in glioma, an aggressive brain tumor, and has been proposed as a therapeutic target for cancer. In the current study, we have used an optimized and validated time-resolved fluorescence energy transfer (TR-FRET)-based DYRK1A assay for high-throughput screening (HTS) in 384-well format. A small-scale screen of the FDA-approved Prestwick drug collection identified the β-carboline, harmine, and four related analogs as DYRK1A inhibitors. Hits were confirmed by dose response and in an orthogonal DYRK1A assay. Harmine's potential therapeutic use has been hampered by its off-target activity for monoamine oxidase A (MAO-A) which impacts multiple nervous system targets. Selectivity profiling of harmine and a broader collection of analogs allowed us to map some divergent SAR (structure-activity relationships) for the DYRK1A and MAO-A activities. The panel of harmine analogs had varying activities in vitro in glioblastoma (GBM)cell lines when tested for anti-proliferative effects using a high content imaging assay. In particular, of the identified analogs, harmol was found to have the best selectivity for DYRK1A over MAO-A and, when tested in a glioma tumor xenograft model, harmol demonstrated a better therapeutic window compared to harmine.

SM04755, a small-molecule inhibitor of the Wnt pathway, as a potential topical treatment for tendinopathy.

The Wnt pathway is upregulated in tendinopathy, affecting inflammation and tenocyte differentiation. Given its potential role in tendinopathy, this signaling pathway may be a relevant target for treatment. The current study examined the therapeutic potential of SM04755, a topical, small‐molecule Wnt pathway inhibitor, for the treatment of tendinopathy using in vitro assays and animal models. In vitro, SM04755 decreased Wnt pathway activity, induced tenocyte differentiation, and inhibited catabolic enzymes and pro‐inflammatory cytokines in human mesenchymal stem cells, rat tendon‐derived stem cells, and human peripheral blood mononuclear cells. Evaluation of the mechanism of action of SM04755 by biochemical profiling and computational modeling identified CDC‐like kinase 2 (CLK2) and dual‐specificity tyrosine phosphorylation‐regulated kinase 1A (DYRK1A) as molecular targets. CLK and DYRK1A inhibition by siRNA knockdown or pharmacological inhibition induced tenocyte differentiation and reduced tenocyte catabolism. In vivo, topically applied SM04755 showed therapeutically relevant exposure in tendons with low systemic exposure and no detectable toxicity in rats. Moreover, SM04755 showed reduced tendon inflammation and evidence of tendon regeneration, decreased pain, and improved weight‐bearing function in rat collagenase‐induced tendinopathy models compared with vehicle control. Together, these data demonstrate that CLK2 and DYRK1A inhibition by SM04755 resulted in Wnt pathway inhibition, enhanced tenocyte differentiation and protection, and reduced inflammation. SM04755 has the potential to benefit symptoms and modify disease processes in tendinopathy.

Small Molecule Inhibitors of DYRK1A Identified by Computational and Experimental Approaches.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase with diverse functions in cell regulation. Abnormal expression and activity of DYRK1A contribute to numerous human malignancies, Down syndrome, and Alzheimer’s disease. Notably, DYRK1A has been proposed as a potential therapeutic target for the treatment of diabetes because of its key role in pancreatic β-cell proliferation. Consequently, DYRK1A is an attractive drug target for a variety of diseases. Here, we report the identification of several DYRK1A inhibitors using our in-house topological water network-based approach. All inhibitors were further verified by in vitro assay.

The novel DYRK1A inhibitor KVN93 regulates cognitive function, amyloid-beta pathology, and neuroinflammation

Regulating amyloid beta (Aβ) pathology and neuroinflammatory responses holds promise for the treatment of Alzheimer's disease (AD) and other neurodegenerative and/or neuroinflammation-related diseases. In this study, the effects of KVN93, an inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase-1A (DYRK1A), on cognitive function and Aβ plaque levels and the underlying mechanism of action were evaluated in 5x FAD mice (a mouse model of AD). KVN93 treatment significantly improved long-term memory by enhancing dendritic synaptic function. In addition, KVN93 significantly reduced Aβ plaque levels in 5x FAD mice by regulating levels of the Aβ degradation enzymes neprilysin (NEP) and insulin-degrading enzyme (IDE). Moreover, Aβ-induced microglial and astrocyte activation were significantly suppressed in the KVN-treated 5xFAD mice. KVN93 altered neuroinflammation induced by LPS in microglial cells but not primary astrocytes by regulating TLR4/AKT/STAT3 signaling, and in wild-type mice injected with LPS, KVN93 treatment reduced microglial and astrocyte activation. Overall, these results suggest that the novel DYRK1A inhibitor KVN93 is a potential therapeutic drug for regulating cognitive/synaptic function, Aβ plaque load, and neuroinflammatory responses in the brain.

The green tea polyphenol epigallocatechin-3-gallate (EGCG) restores CDKL5-dependent synaptic defects in vitro and in vivo

CDKL5 deficiency disorder (CDD) is a rare X-linked neurodevelopmental disorder that is characterised by early-onset seizures, intellectual disability, gross motor impairment, and autistic-like features. CDD is caused by mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene that encodes a serine/threonine kinase with a predominant expression in the brain. Loss of CDKL5 causes neurodevelopmental alterations in vitro and in vivo, including defective dendritic arborisation and spine maturation, which most likely underlie the cognitive defects and autistic features present in humans and mice.Here, we show that treatment with epigallatocathechin-3-gallate (EGCG), the major polyphenol of green tea, can restore defects in dendritic and synaptic development of primary Cdkl5 knockout (KO) neurons. Furthermore, defective synaptic maturation in the hippocampi and cortices of adult Cdkl5-KO mice can be rescued through the intraperitoneal administration of EGCG, which is however not sufficient to normalise behavioural CDKL5-dependent deficits. EGCG is a pleiotropic compound with numerous cellular targets, including the dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) that is selectively inhibited by EGCG. DYRK1A controls dendritic development and spine formation and its deregulation has been implicated in neurodevelopmental and degenerative diseases. Treatment with another DYRK1A inhibitor, harmine, was capable of correcting neuronal CDKL5-dependent defects; moreover, DYRK1A levels were upregulated in primary Cdkl5-KO neurons in concomitance with increased phosphorylation of Tau, a well-accepted DYRK1A substrate. Altogether, our results indicate that DYRK1A deregulation may contribute, at least in part, to the neurodevelopmental alterations caused by CDKL5 deficiency.

Pharmacologic and genetic approaches define human pancreatic β cell mitogenic targets of DYRK1A inhibitors.

Small molecule inhibitors of dual specificity, tyrosine phosphorylation-regulated kinase 1A (DYRK1A), including harmine and others, are able to drive human β cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only or the most important target is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human β cell RNA sequencing, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B. Simultaneous silencing of both DYRK1A and DYRK1B yields greater β cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to 7 targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human β cell proliferation, and we provide clarity for future efforts in structure-based drug design for human β cell regenerative drugs.

Licocoumarone induces BxPC-3 pancreatic adenocarcinoma cell death by inhibiting DYRK1A

Protein kinases play an indispensable role in signaling pathways that regulate tumor cell functions, which represent potent therapeutic targets in cancers. Dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) as a serine/threonine kinase has recently been reported to be upregulated in pancreatic ductal adenocarcinoma (PDAC) and show protumorigenic effect. By activity-guided phytochemical investigation of the extracts from Glycyrrhiza uralensis Fisch, we expect to find the effective constituents that can suppress pancreatic cancer cell proliferation and/or induce cells apoptotic by inhibiting DYRK1A. Eight isopentenyl-substituted compounds (1–8), including four coumarins (1–4), one benzofuran (5), and three flavonoids (6–8), were isolated and identified from G. uralensis Fisch. Among them, licocoumarone (LC, 5) showed effective inhibitory activity against DYRK1A with an IC50 value of 12.56 μM. Molecular docking analysis suggested that LC completely occupied the whole pocket of DYRK1A and formed obvious hydrophobic interactions and hydrogen bonds with DYRK1A residues. Further in vitro validation, including Microscale Thermophoresis (MST) and drug affinity responsive target stability (DARTS) techniques, demonstrated the specific combining capacity of LC to DYRK1A. Meanwhile, LC induced significant cytotoxicity against DYRK1A-overexpressing BxPC-3 cells with an IC50 value of 50.77 μM. Mechanism studies revealed that LC reduced c-MET protein level by inhibiting DYRK1A. These findings provide preliminary evidences that LC as a natural DYRK1A inhibitor suppresses human pancreatic adenocarcinoma BxPC-3 cell proliferation and induces cell apoptotic, which might present new options and possibilities for targeted therapies in pancreatic cancer therapy.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A ameliorates insulin resistance in neurons by up-regulating IRS-1 expression

Insulin resistance in the brain is a pathological mechanism that is shared between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Although aberrant expression and phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to insulin resistance, the underlying mechanism remains elusive. In this study, we used several approaches, including adeno-associated virus-based protein overexpression, immunoblotting, immunoprecipitation, immunohistochemistry, and in situ proximal ligation assays, to investigate the function of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) in IRS-1 regulation and the downstream insulin signaling in neurons. We found that DYRK1A overexpression up-regulated IRS-1 expression by slowing turnover of the IRS-1 protein. We further observed that DYRK1A directly interacted with IRS-1 and phosphorylated IRS-1's multiple serine residues. Of note, DYRK1A and IRS-1 were coordinately up-regulated in the prefrontal cortex of db/db mice brain. Furthermore, DYRK1A overexpression ameliorated chronic high insulin-induced insulin resistance in SH-SY5Y cells as well as in primary rat neurons. These findings suggest that DYRK1A protects against insulin resistance in the brain by elevating IRS-1 expression.

Generation of highly potent DYRK1A-dependent inducers of human β-Cell replication via Multi-Dimensional compound optimization

Small molecule stimulation of β-cell regeneration has emerged as a promising therapeutic strategy for diabetes. Although chemical inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is sufficient to enhance β-cell replication, current lead compounds have inadequate cellular potency for in vivo application. Herein, we report the clinical stage anti-cancer kinase inhibitor OTS167 as a structurally novel, remarkably potent DYRK1A inhibitor and inducer of human β-cell replication. Unfortunately, OTS167’s target promiscuity and cytotoxicity curtails utility. To tailor kinase selectivity towards DYRK1A and reduce cytotoxicity we designed a library of fifty-one OTS167 derivatives based upon a modeled structure of the DYRK1A-OTS167 complex. Indeed, derivative characterization yielded several leads with exceptional DYRK1A inhibition and human β-cell replication promoting potencies but substantially reduced cytotoxicity. These compounds are the most potent human β-cell replication-promoting compounds yet described and exemplify the potential to purposefully leverage off-target activities of advanced stage compounds for a desired application.

Upregulated Expression of MicroRNA-204-5p Leads to the Death of Dopaminergic Cells by Targeting DYRK1A-Mediated Apoptotic Signaling Cascade.

MicroRNAs (miRs) downregulate or upregulate the mRNA level by binding to the 3′-untranslated region (3′UTR) of target gene. Dysregulated miR levels can be used as biomarkers of Parkinson’s disease (PD) and could participate in the etiology of PD. In the present study, 45 brain-enriched miRs were evaluated in serum samples from 50 normal subjects and 50 sporadic PD patients. The level of miR-204-5p was upregulated in serum samples from PD patients. An upregulated level of miR-204-5p was also observed in the serum and substantia nigra (SN) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Expression of miR-204-5p increased the level of α-synuclein (α-Syn), phosphorylated (phospho)-α-Syn, tau, or phospho-tau protein and resulted in the activation of endoplasmic reticulum (ER) stress in SH-SY5Y dopaminergic cells. Expression of miR-204-5p caused autophagy impairment and activation of c-Jun N-terminal kinase (JNK)-mediated apoptotic cascade in SH-SY5Y dopaminergic cells. Our study using the bioinformatic method and dual-luciferase reporter analysis suggests that miR-204-5p positively regulates mRNA expression of dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) by directly interacting with 3′UTR of DYRK1A. The mRNA and protein levels of DYRK1A were increased in SH-SY5Y dopaminergic cells expressing miR-204-5p and SN of MPTP-induced PD mouse model. Knockdown of DYRK1A expression or treatment of the DYRK1A inhibitor harmine attenuated miR-204-5p-induced increase in protein expression of phospho-α-Syn or phospho-tau, ER stress, autophagy impairment, and activation of JNK-mediated apoptotic pathway in SH-SY5Y dopaminergic cells or primary cultured dopaminergic neurons. Our results suggest that upregulated expression of miR-204-5p leads to the death of dopaminergic cells by targeting DYRK1A-mediated ER stress and apoptotic signaling cascade.

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.

Modulation of the Wnt pathway through inhibition of CLK2 and DYRK1A by lorecivivint as a novel, potentially disease-modifying approach for knee osteoarthritis treatment

Wnt pathway upregulation contributes to knee osteoarthritis (OA) through osteoblast differentiation, increased catabolic enzymes, and inflammation. The small-molecule Wnt pathway inhibitor, lorecivivint (SM04690), which previously demonstrated chondrogenesis and cartilage protection in an animal OA model, was evaluated to elucidate its mechanism of action. Biochemical assays measured kinase activity. Western blots measured protein phosphorylation in human mesenchymal stem cells (hMSCs), chondrocytes, and synovial fibroblasts. siRNA knockdown effects in hMSCs and BEAS-2B cells on Wnt pathway, chondrogenic genes, and LPS-induced inflammatory cytokines was measured by qPCR. Invivo anti-inflammation, pain, and function were evaluated following single intra-articular (IA) lorecivivint or vehicle injection in the monosodium iodoacetate (MIA)-induced rat OA model. Lorecivivint inhibited intranuclear kinases CDC-like kinase 2 (CLK2) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Lorecivivint inhibited CLK2-mediated phosphorylation of serine/arginine-rich (SR) splicing factors and DYRK1A-mediated phosphorylation of SIRT1 and FOXO1. siRNA knockdowns identified a role for CLK2 and DYRK1A in Wnt pathway modulation without affecting β-catenin with CLK2 inhibition inducing early chondrogenesis and DYRK1A inhibition enhancing mature chondrocyte function. NF-κB and STAT3 inhibition by lorecivivint reduced inflammation. DYRK1A knockdown was sufficient for anti-inflammatory effects, while combined DYRK1A/CLK2 knockdown enhanced this effect. In the MIA model, lorecivivint inhibited production of inflammatory cytokines and cartilage degradative enzymes, resulting in increased joint cartilage, decreased pain, and improved weight-bearing function. Lorecivivint inhibition of CLK2 and DYRK1A suggested a novel mechanism for Wnt pathway inhibition, enhancing chondrogenesis, chondrocyte function, and anti-inflammation. Lorecivivint shows potential to modify structure and improve symptoms of knee OA.

Design, Synthesis, and In Vitro Biological Activities of a Bio-Oxidizable Prodrug to Deliver Both ChEs and DYRK1A Inhibitors for AD Therapy.

Despite their side effects, cholinesterase (ChE) inhibitors remain the only approved drugs to treat Alzheimer’s disease patients, along with the N-methyl-d-aspartate (NMDA) receptor antagonist memantine. In the last few years, the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) has also been studied as a promising target for the development of new drugs for this pathology. In this context, and based on our previous characterization of bio-oxidizable prodrugs of potent acetylcholinesterase (AChE) inhibitors, we envisioned a strategy involving the synthesis of a bio-oxidizable prodrug of both ChE and DYRK1A inhibitors. To this end, we fixed our interest on a known potent inhibitor of DYRK1A, namely INDY. The designed prodrug of both ChE and DYRK1A inhibitors was successfully synthesized, connecting both inhibitors by a carbonate link. This prodrug and its corresponding drug were then evaluated as ChEs and DYRK1A inhibitors. Remarkably, in vitro results were in accordance with the starting hypothesis, showing a relative inactivity of the prodrug against DYRK1A and ChEs and a potent inhibition of ChEs by the oxidized form. Molecular docking and kinetic studies of ChE inhibition by the active compound are also discussed in this report.

New pyrido[3,4-g]quinazoline derivatives as CLK1 and DYRK1A inhibitors: synthesis, biological evaluation and binding mode analysis

Cdc2-like kinase 1 (CLK1) and dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) are involved in the regulation of alternative pre-mRNA splicing. Dysregulation of this process has been linked to cancer progression and neurodegenerative diseases, making CLK1 and DYRK1A important therapeutic targets. Here we describe the synthesis of new pyrido[3,4-g]quinazoline derivatives and the evaluation of the inhibitory potencies of these compounds toward CDK5, CK1, GSK3, CLK1 and DYRK1A. Introduction of aminoalkylamino groups at the 2-position resulted in several compounds with low nanomolar affinity and selective inhibition of CLK1 and/or DYRK1A. Their evaluation on several immortalized or cancerous cell lines showed varying degree of cell viability reduction. Co-crystal structures of CLK1 with two of the most potent compounds revealed two alternative binding modes of the pyrido[3,4-g]quinazoline scaffold that can be exploited for future inhibitor design.

The Chromosome 21 Kinase DYRK1A and Its Substrate FOXO1 Constitute a Novel Therapeutic Pathway in B-ALL

Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase 1A (DYRK1A) is a serine/threonine kinase that regulates diverse pathways such as splicing, cell cycle, differentiation, apoptosis, and transcription. DYRK1A is encoded within the Down syndrome (DS) critical region of chromosome 21, underlying its importance in DS-related pathologies, such as Alzheimer's disease. Children with DS have an increased risk of developing hematologic malignancies, namely acute megakaryoblastic leukemia (DS-AMKL) and B-cell acute lymphoblastic leukemia (DS-ALL). We previously reported that DYRK1A promotes DS-AMKL by regulating subcellular localization of its substrate NFAT. In a subsequent study, we examined its role in normal hematopoiesis and found that DYRK1A is necessary for B and T cell development through phosphorylation and destabilization of Cyclin D3. Dyrk1a-deficient large pre-B cells and double negative thymocytes are unable to enter quiescence for maturation. Despite elevated levels of Cyclin D3, however, these cells lose proliferative capacity due to a block at the G2-M transition. This observation suggests that DYRK1A inhibition may exhibit anti-tumor activity in lymphocytes by first stimulating exit from quiescence but then blocking repeated rounds of cell division.Notably, DYRK1A is overexpressed in acute leukemias, including both T-ALL and B-ALL, relative to normal hematopoietic counterparts. Moreover, overexpression of dominant-negative DYRK1A-K188R impairs proliferation in human B-ALL cell lines, suggesting that DYRK1A kinase activity is required for B-ALL growth. In order to assess the physiologic relevance of targeting DYRK1A in vivo, we generated a murine model of B-ALL with a floxed Dyrk1a allele and observed significant survival advantages with homozygous (p=0.0045) and heterozygous deletion (p=0.0015). Additionally, both B-ALL cell lines and patient samples were sensitive to EHT1610, a potent and selective DYRK1 inhibitor. Relevant to the localization of DYRK1A on chromosome 21, DS-ALL samples were especially sensitive to kinase inhibition. EHT1610 also conferred synergistic growth inhibition of B-ALL cells when combined with cytotoxic chemotherapy drugs used in traditional ALL treatment regimens, such as dexamethasone, methotrexate and cytarabine.We next aimed to elucidate the mechanism by which DYRK1A inhibition cause a failure of G2-M progression. Using global and directed phosphoproteomic studies, we identified several DYRK1A substrates in pre-B cells that are involved in cell cycle, splicing, transcriptional regulation, and RNA metabolism. In addition to Cyclin D3, a notable substrate is FOXO1, an indispensable transcription factor in B lymphopoiesis. We observed that inhibition of DYRK1A led to an accumulation of FOXO1 in the nucleus of large pre-B cells despite intact PI3K/Akt signaling, which is the predominant negative regulator of FOXO1. Treatment of pre-B cells with AS1842856, an inhibitor of FOXO1 nuclear translocation, rescued the G2-M blockade and proliferative impairment induced by EHT1610 treatment. Despite FOXO1 acting as a tumor suppressor in normal lymphocytes, B-ALL cell lines and patient samples were paradoxically sensitive to FOXO1 inhibition, suggesting a unique requirement in the survival of B-ALL cells. This may be due to regulation of DNA damage, as DYRK1A inhibition alone led to negligible changes in gamma-H2AX foci, whereas FOXO1 inhibition increased DNA damage. When DYRK1A and FOXO1 were inhibited in combination, we observed a synergistic accumulation of DNA damage along with cell death in B-ALL cell lines.Finally, as both EHT1610 and AS1842856 are potent inhibitors of B-ALL cell growth in vitro, we assessed their in vivo efficacy. Both EHT1610 and AS1842856 significantly increased survival in xenograft models of B-ALL (p=0.0002 and p=0.001, respectively). We therefore conclude that both DYRK1A and its substrate FOXO1 are therapeutic targets in B-ALL. Importantly, EHT1610 represents the first selective DYRK1A inhibitor with suitable in vivo activity. Ultimately, we have determined that the DYRK1A pathway is integral to the maintenance of normal and malignant B-lymphopoiesis, the latter which can be effectively targeted through 1) a primary proliferative impairment, 2) sensitization to cell cycle-dependent chemotherapy, and 3) downstream inhibition of DYRK1A substrates such as FOXO1. DisclosuresLee:AbbVie: Employment. Bourquin:Amgen: Other: Travel Support. Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Novel selective thiadiazine DYRK1A inhibitor lead scaffold with human pancreatic β-cell proliferation activity

The Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A) is an enzyme that has been implicated as an important drug target in various therapeutic areas, including neurological disorders (Down syndrome, Alzheimer's disease), oncology, and diabetes (pancreatic β-cell expansion). Current small molecule DYRK1A inhibitors are ATP-competitive inhibitors that bind to the kinase in an active conformation. As a result, these inhibitors are promiscuous, resulting in pharmacological side effects that limit their therapeutic applications. None are in clinical trials at this time. In order to identify a new DYRK1A inhibitor scaffold, we constructed a homology model of DYRK1A in an inactive, DFG-out conformation. Virtual screening of 2.2 million lead-like compounds from the ZINC database, followed by in vitro testing of selected 68 compounds revealed 8 hits representing 5 different chemical classes. We chose to focus on one of the hits from the computational screen, thiadiazine 1 which was found to inhibit DYRK1A with IC50 of 9.41 μM (Kd = 7.3 μM). Optimization of the hit compound 1, using structure-activity relationship (SAR) analysis and in vitro testing led to the identification of potent thiadiazine analogs with significantly improved binding as compared to the initial hit (Kd = 71–185 nM). Compound 3–5 induced human β-cell proliferation at 5 μM while showing selectivity for DYRK1A over DYRK1B and DYRK2 at 10 μM. This newly developed DYRK1A inhibitor scaffold with unique kinase selectivity profiles has potential to be further optimized as novel therapeutics for diabetes.

Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A) Inhibitors as Potential Therapeutics.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a member of an evolutionarily conserved family of protein kinases that belongs to the CMGC group of kinases. DYRK1A, encoded by a gene located in the human chromosome 21q22.2 region, has attracted attention due to its association with both neuropathological phenotypes and cancer susceptibility in patients with Down syndrome (DS). Inhibition of DYRK1A attenuates cognitive dysfunctions in animal models for both DS and Alzheimer's disease (AD). Furthermore, DYRK1A has been studied as a potential cancer therapeutic target because of its role in the regulation of cell cycle progression by affecting both tumor suppressors and oncogenes. Consequently, selective synthetic inhibitors have been developed to determine the role of DYRK1A in various human diseases. Our perspective includes a comprehensive review of potent and selective DYRK1A inhibitors and their forthcoming therapeutic applications.

Indole-3-Carbonitriles as DYRK1A Inhibitors by Fragment-Based Drug Design.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a potential drug target because of its role in the development of Down syndrome and Alzheimer’s disease. The selective DYRK1A inhibitor 10-iodo-11H-indolo[3,2-c]quinoline-6-carboxylic acid (KuFal194), a large, flat and lipophilic molecule, suffers from poor water solubility, limiting its use as chemical probe in cellular assays and animal models. Based on the structure of KuFal194, 7-chloro-1H-indole-3-carbonitrile was selected as fragment template for the development of smaller and less lipophilic DYRK1A inhibitors. By modification of this fragment, a series of indole-3-carbonitriles was designed and evaluated as potential DYRK1A ligands by molecular docking studies. Synthesis and in vitro assays on DYRK1A and related protein kinases identified novel double-digit nanomolar inhibitors with submicromolar activity in cell culture assays.