Phosphorylation-regulated Kinase Research Articles

Straightforward Access to a New Class of Dual DYRK1A/CLK1 Inhibitors Possessing a Simple Dihydroquinoline Core.

The DYRK (Dual-specificity tyrosine phosphorylation-regulated kinase) family of protein kinases is involved in the pathogenesis of several neurodegenerative diseases. Among them, the DYRK1A protein kinase is thought to be implicated in Alzheimer's disease (AD) and Down syndrome, and as such, has emerged as an appealing therapeutic target. DYRKs are a subset of the CMGC (CDK, MAPKK, GSK3 and CLK) group of kinases. Within this group of kinases, the CDC2-like kinases (CLKs), such as CLK1, are closely related to DYRKs and have also sparked great interest as potential therapeutic targets for AD. Based on inhibitors previously described in the literature (namely TG003 and INDY), we report in this work a new class of dihydroquinolines exhibiting inhibitory activities in the nanomolar range on hDYRK1A and hCLK1. Moreover, there is overwhelming evidence that oxidative stress plays an important role in AD. Pleasingly, the most potent dual kinase inhibitor 1p exhibited antioxidant and radical scavenging properties. Finally, drug-likeness and molecular docking studies of this new class of DYRK1A/CLK1 inhibitors are also discussed in this article.

In vivo drug discovery for increasing incretin-expressing cells identifies DYRK inhibitors that reinforce the enteroendocrine system

Analogs of the incretin hormones Gip and Glp-1 are used to treat type 2 diabetes and obesity. Findings in experimental models suggest that manipulating several hormones simultaneously may be more effective. To identify small molecules that increase the number of incretin-expressing cells, we established a high-throughput invivo chemical screen by using the gip promoter to drive the expression of luciferase in zebrafish. All hits increased the numbers of neurogenin 3-expressing enteroendocrine progenitors, Gip-expressing K-cells, and Glp-1-expressing L-cells. One of the hits, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor, additionally decreased glucose levels in both larval and juvenile fish. Knock-down experiments indicated that nfatc4, a downstream mediator of DYRKs, regulates incretin+ cell number in zebrafish, and that Dyrk1b regulates Glp-1 expression in an enteroendocrine cell line. DYRK inhibition also increased the number of incretin-expressing cells in diabetic mice, suggesting a conserved reinforcement of the enteroendocrine system, with possible implications for diabetes.

Inhibition of Dyrk1A Attenuates LPS-Induced Neuroinflammation via the TLR4/NF-κB P65 Signaling Pathway.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) is a highly conserved protein kinase, playing a key role in the regulation of physiological brain functions and pathological processes. In Alzheimer's disease (AD), Dyrk1A promotes hyperphosphorylation of tau protein and abnormal aggregation of amyloid-β protein (Aβ). This study investigated the role of Dyrk1A in regulating neuroinflammation, another critical factor that contributes to AD. In the present study, we used an immortalized murine BV2 microglia cell line induced by lipopolysaccharide (LPS) to study neuroinflammation. The expression and activity of Dyrk1A kinase were both increased by inflammation. Dyrk1A inhibition using harmine or siRNA silencing significantly reduced the production of proinflammatory factors in LPS-stimulated BV2 cells. Reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), and nitric oxide (NO), as well as the expression of the inflammatory proteins, cyclooxygenase 2 (COX2), and inducible nitric synthase (iNOS), were attenuated. In vivo, in ICR mice injected with LPS into the left lateral cerebral ventricle, harmine (20 mg/kg) administration decreased the expression of inflammatory proteins in the cortex and hippocampus of mice brain. In addition, immunohistochemical detection of ionized calcium-binding adapter molecule 1 (Iba1) and Nissl staining showed that harmine significantly attenuated microglia activation and neuronal damage in the CA1 region of hippocampus. Further mechanistic studies indicated that Dyrk1A suppression may be related to inhibition of the TLR4/NF-κB signaling pathway in LPS-induced neuroinflammation. Taken together, our studies suggest that Dyrk1A may be a novel target for the treatment of neurodegenerative diseases with an inflammatory component.

In-silico study for African plants with possible beta-cell regeneration effect through inhibition of DYRK1A

The continuous destruction of normal insulin-producing pancreatic beta-cells is a contributing factor in all common forms of diabetes, due to insufficient production of insulin, especially in type 1 diabetes. There are attempts at beta-cells transplantation, but the cost and availability of donors pose a great challenge to the process. Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase A (DYRK1A) plays a crucial role in beta-cells destruction. Our research targets to identify plants that can be utilized as a possible alternative approach to beta-cell replacement through a pharmacologically induced regeneration of new beta-cells in-silico. The 3D structure DYRK1A and 6511 phytochemicals were obtained from the Protein Data Bank and the African Natural Products Database respectively. They were duly prepared for molecular docking simulations (MDS). MDS was implemented, after validation of docking protocols, in AutoDock-Vina®, with virtual screening scripts. Phytocompounds with good binding affinities for DYRK1A were selected as frontrunners. The compounds were screened for toxicity, Lipinski’s rule confirmation with Data Warrior software followed by kinase inhibitory bioactivity prediction with the Molinspiration Chemoinformatics web tool. Twelve phytocompounds were found to be predictably highly active in-silico against DYRK1A with good drug-like property based on Lipinski’s rule, non-mutagenic, non-tumorigenic, no reproductive effect, and non-irritant, with high predicted bioactivity. In-silico active phytocompounds against DYRK1A with their plant sources and physicochemical parameters were identified. Further studies will be carried out in-vitro and in-vivo to validate the results of this study using plants containing the identified phytocompounds.

Protein Kinase MpYAK1 Is Involved in Meristematic Cell Proliferation, Reproductive Phase Change and Nutrient Signaling in the Liverwort Marchantia polymorpha.

Plant growth and development are regulated by environmental factors, including nutrient availability and light conditions, via endogenous genetic signaling pathways. Phosphorylation-dependent protein modification plays a major role in the regulation of cell proliferation in stress conditions, and several protein kinases have been shown to function in response to nutritional status, including dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). Although DYRKs are widely conserved in eukaryotes, the physiological functions of DYRKs in land plants are still to be elucidated. In the liverwort Marchantia polymorpha, a model bryophyte, four putative genes encoding DYRK homologous proteins, each of which belongs to the subfamily yet another kinase 1 (Yak1), plant-specific DYRK, DYRK2, or pre-mRNA processing protein 4 kinase, were identified. MpYAK1-defective male and female mutant lines generated by the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system showed smaller sizes of thalli than did the wild-type plants and repressed cell divisions in the apical notch regions. The Mpyak1 mutants developed rhizoids from gemmae in the gemma cup before release. The Mpyak1 lines developed sexual organs even in non-inductive short-day photoperiod conditions supplemented with far-red light. In nitrogen (N)-deficient conditions, rhizoid elongation was inhibited in the Mpyak1 mutants. In conditions of aeration with 0.08% CO2 (v/v) and N depletion, Mpyak1 mutants accumulated higher levels of sucrose and lower levels of starch compared to the wild type. Transcriptomic analyses revealed that the expression of peroxidase genes was differentially affected by MpYAK1. These results suggest that MpYAK1 is involved in the maintenance of plant growth and developmental responses to light conditions and nutrient signaling.

DYRK1A reinforces epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma via cooperatively activating STAT3 and SMAD

BackgroundHepatocellular carcinoma (HCC) accounts for the majority of liver cancer cases, while metastasis is considered the leading cause of HCC-related death. However, the currently available treatment strategies for efficient suppression of metastasis are limited. Therefore, novel therapeutic targets to inhibit metastasis and effectively treat HCC are urgently required.MethodsWound healing and Transwell assays were used to determine the migration and invasion abilities of HCC cells in vitro. Quantitative real-time PCR (qRT-PCR), protein array, immunofluorescence, and immunoprecipitation experiments were used to study the mechanism of DYRK1A-mediated metastasis. A tail vein metastasis model and H&E staining were utilized to assess metastatic potential in vivo.ResultsThe results of the current study demonstrated that dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) was upregulated in HCC tissues compared with normal liver tissues. Additionally, the level of DYRK1A was increased in primary HCC tissues of patients with metastasis compared with those of patients without metastasis, and DYRK1A overexpression correlated with worse outcomes in liver cancer patients. Gain- and loss-of-function studies suggested that DYRK1A enhanced the invasion and migration abilities of HCC cells by promoting epithelial-mesenchymal transition (EMT). Regarding the promoting effect of DYRK1A on cell invasion, the results showed that DYRK1A was coexpressed with TGF-β/SMAD and STAT3 signalling components in clinical tumour samples obtained from patients with HCC. DYRK1A also activated TGF-β/SMAD signalling by interacting with tuberous sclerosis 1 (TSC1) and enhanced metastasis of HCC cells by activating STAT3. Furthermore, DYRK1A promoted EMT by cooperatively activating STAT3/SMAD signalling.ConclusionOverall, the present study not only uncovered the promoting effect of DYRK1A on HCC metastasis and revealed the mechanism but also provided a new approach to predict and treat metastatic HCC.

Targeting dual-specificity tyrosine phosphorylation-regulated kinase 2 with a highly selective inhibitor for the treatment of prostate cancer

Prostate cancer (PCa) is one of the most prevalent cancers in men worldwide, and hormonal therapy plays a key role in the treatment of PCa. However, the drug resistance of hormonal therapy makes it urgent and necessary to identify novel targets for PCa treatment. Herein, dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is found and confirmed to be highly expressed in the PCa tissues and cells, and knock-down of DYRK2 remarkably reduces PCa burden in vitro and in vivo. On the base of DYRK2 acting as a promising target, we further discover a highly selective DYRK2 inhibitor YK-2-69, which specifically interacts with Lys-231 and Lys-234 in the co-crystal structure. Especially, YK-2-69 exhibits more potent anti-PCa efficacy than the first-line drug enzalutamide in vivo. Meanwhile, YK-2-69 displays favorable safety properties with a maximal tolerable dose of more than 10,000 mg/kg and pharmacokinetic profiles with 56% bioavailability. In summary, we identify DYRK2 as a potential drug target and verify its critical roles in PCa. Meanwhile, we discover a highly selective DYRK2 inhibitor with favorable druggability for the treatment of PCa.

Sexually dimorphic DYRK1A overexpression on postnatal day 15 in the Ts65Dn mouse model of Down syndrome: Effects of pharmacological targeting on behavioral phenotypes

The neurotypical spatiotemporal patterns of gene expression are disrupted in Down syndrome (DS) by trisomy of human chromosome 21 (Hsa21), resulting in altered behavioral development and brain circuitry. The Ts65Dn DS mouse model exhibits similar phenotypes to individuals with DS due to three copies of approximately one-half of the genes found on Hsa21. Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1a (Dyrk1a), one of these triplicated genes, is an attractive target to normalize brain development due to its influence in cellular brain deficits seen in DS. We hypothesized that postnatal development of DYRK1A expression is dysregulated in trisomic animals, and found significant overexpression of DYRK1A in the hippocampus, cerebral cortex, and cerebellum at postnatal day (P) 15 in male—but not female—Ts65Dn mice. We then hypothesized the existence of sex-dependent effects of trisomy on neurobehavioral attributes during P16–17, and that administration of a DYRK1A inhibitor (CX-4945, ~75 mg/kg) beginning on P14 would normalize aberrant behavior in trisomic animals. Both male and female trisomic mice given control injections of phosphate buffered saline (PBS) displayed sustained levels of locomotor activity over a 10-minute test in contrast to the PBS-treated euploid animals that showed significant within-session habituation. Trisomic animals were more persistent in choosing to remain in home shavings in a preference test. Treatment with CX-4945 failed to confirm therapeutic effects. CX-4945 prevented growth, and both CX-4945 and its 10% dimethyl sulfoxide vehicle affected locomotor activity in trisomic and euploid groups, indicating a non-specific disruption of behavior. Despite the negative outcomes for CX-4945, the novel demonstration of sexually dimorphic DYRK1A expression in trisomic animals at P15 supports the broader hypothesis that overexpression of trisomic genes in DS can vary with age, sex, and brain region. Identifying the developmental timing of periods of dysregulated DYRK1A may be important for understanding individual differences in neurodevelopmental trajectories in DS and for developing effective therapeutic interventions targeting DYRK1A.

CsPOM1, a DYRK Family Kinase, Plays Diverse Roles in Fungal Development, Virulence, and Stress Tolerance in the Anthracnose Pathogen Colletotrichum scovillei.

Colletotrichum scovillei is the major anthracnose fungus of sweet pepper and chili pepper (Capsicum annuum L.), causing significant losses in the yield and quality of the pepper fruits. Molecular mechanisms governing development and pathogenicity have been widely studied in many foliar fungal pathogens, but the information on fruit diseases is still limited. In this study, we determined the functional roles of the dual-specificity tyrosine phosphorylation-regulated kinase CsPOM1 in C. scovillei. Knockout mutant for CsPOM1 gene was obtained via homology-dependent gene replacement. The ΔCspom1 mutant exhibited a reduction in vegetative growth on osmotic stress, surface hydrophobicity, and conidiation compared with wild-type. Conidia of the ΔCspom1 mutant were already two-celled before inoculation on an induction surface, indicating that CsPOM1 negatively regulates conidial cell division. The ΔCspom1 mutant, similar to wild-type, formed appressoria on the plant surface, but was significantly reduced on hydrophobic coverslips, probably due to a defect in the recognition of surface hydrophobicity. Treatment of conidia with cutin monomers restored appressorium formation on hydrophobic coverslips in the ΔCspom1 mutant. On pepper fruits, the ΔCspom1 mutant exhibited delayed penetration and invasive growth, leading to significantly reduced virulence. Collectively, the results showed that CsPOM1 is important for stress tolerance, conidiation, surface hydrophobicity, appressorium formation, and virulence in C. scovillei.

Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry.

The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process. These studies collectively further expand our understanding of DYRK2 and provide a valuable tool to pinpoint its biological function.

Inhibition of DYRK1a Enhances Cardiomyocyte Cycling After Myocardial Infarction.

DYRK1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) contributes to the control of cycling cells, including cardiomyocytes. However, the effects of inhibition of DYRK1a on cardiac function and cycling cardiomyocytes after myocardial infarction (MI) remain unknown. We investigated the impacts of pharmacological inhibition and conditional genetic ablation of DYRK1a on endogenous cardiomyocyte cycling and left ventricular systolic function in ischemia-reperfusion (I/R) MI using αMHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (RLTG) (denoted αDKRC::RLTG) and αMHC-Cre::Fucci2aR::DYRK1aflox/flox mice. We observed that harmine, an inhibitor of DYRK1a, improved left ventricular ejection fraction (39.5±1.6% and 29.1±1.6%, harmine versus placebo, respectively), 2 weeks after I/R MI. Harmine also increased cardiomyocyte cycling after I/R MI in αDKRC::RLTG mice, 10.8±1.5 versus 24.3±2.6 enhanced Green Fluorescent Protein (eGFP)+ cardiomyocytes, placebo versus harmine, respectively, P=1.0×10-3. The effects of harmine on left ventricular ejection fraction were attenuated in αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes. The conditional cardiomyocyte-specific genetic ablation of DYRK1a in αMHC-Cre::Fucci2aR::DYRK1aflox/flox (denoted DYRK1a k/o) mice caused cardiomyocyte hyperplasia at baseline (210±28 versus 126±5 cardiomyocytes per 40× field, DYRK1a k/o versus controls, respectively, P=1.7×10-2) without changes in cardiac function compared with controls, or compensatory changes in the expression of other DYRK isoforms. After I/R MI, DYRK1a k/o mice had improved left ventricular function (left ventricular ejection fraction 41.8±2.2% and 26.4±0.8%, DYRK1a k/o versus control, respectively, P=3.7×10-2). RNAseq of cardiomyocytes isolated from αMHC-Cre::Fucci2aR::DYRK1aflox/flox and αMHC-Cre::Fucci2aR mice after I/R MI or Sham surgeries identified enrichment in mitotic cell cycle genes in αMHC-Cre::Fucci2aR::DYRK1aflox/flox compared with αMHC-Cre::Fucci2aR. The pharmacological inhibition or cardiomyocyte-specific ablation of DYRK1a caused baseline hyperplasia and improved cardiac function after I/R MI, with an increase in cell cycle gene expression, suggesting the inhibition of DYRK1a may serve as a therapeutic target to treat MI.

Combination of DYRK2 and TERT Expression Is a Powerful Predictive Marker for Early-stage Breast Cancer Recurrence.

DYRK2 is a dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase induces degradation of telomerase reverse transcriptase (TERT). The expression of both proteins in breast cancer were investigated as predictors of recurrence. Two hundred and twenty-one patients with early breast cancer treated at our institute between 2000 and 2009, were included. We used immunohistochemical analyses to measure the expression of DYRK2 and TERT and correlated it with clinicopathological factors and survival. DYRK2 and TERT were positive in 58 (26%) and 86 (39%) of 221 patients, respectively. There was no correlation between DYRK2 and TERT expression and clinicopathological factors. Better disease-free survival was observed in the DYRK2-positive group (p=0.032), and poorer disease-free survival was noted in the TERT-positive group (p=0.023). The DYRK2-positive TERT-negative group exhibited significantly better disease-free survival than the other groups (p=0.006). The combination of DYRK2 and TERT may be a powerful tool to stratify breast cancer patients.

The chromosome 21 kinase DYRK1A: emerging roles in cancer biology and potential as a therapeutic target

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a serine/threonine kinase that belongs to the DYRK family of proteins, a subgroup of the evolutionarily conserved CMGC protein kinase superfamily. Due to its localization on chromosome 21, the biological significance of DYRK1A was initially characterized in the pathogenesis of Down syndrome (DS) and related neurodegenerative diseases. However, increasing evidence has demonstrated a prominent role in cancer through its ability to regulate biologic processes including cell cycle progression, DNA damage repair, transcription, ubiquitination, tyrosine kinase activity, and cancer stem cell maintenance. DYRK1A has been identified as both an oncogene and tumor suppressor in different models, underscoring the importance of cellular context in its function. Here, we review mechanistic contributions of DYRK1A to cancer biology and its role as a potential therapeutic target.

QSAR modeling, molecular docking, ADMET prediction and molecular dynamics simulations of some 6-arylquinazolin-4-amine derivatives as DYRK1A inhibitors

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase with diverse functions in neuronal development and adult brain physiology. QSAR study was performed for a diverse set of thirty-three6-arylquinazolin-4-amine derivatives as DYRK1A inhibitors. The QSAR model with HOMO, LUMO, qCsub, SAG and MW descriptors showed satisfactory internal and external validation parameters (Rtrain2=0.867,Radj2=0.831, QLOO2= 0.755,F=24.682 andRpred2 =0.856). The excellent statistical results obtained for the developed model, strongly suggest that the model is reasonable for the prediction of the activity of new inhibitors. Therefore, we have designed several new potent DYRK1A inhibitors and validated their inhibitory activity by molecular docking methods using Autodock tools. Based in silico analysis, two novel compounds N2 and N11have been designed, which they have been identified hydrophobic and hydrogen bonds, with the binding pockets of active site4YLJ. Furthermore, the two newly designed compounds exhibited good ADMET properties, their molecular dynamics studies have shown a favorable energetic state and dynamic stability, characterized by root mean square deviation (RMSD), root mean square fluctuation (RMSF) and hydrogen bond. The outcomes of this study can be used to show the interest of DYRK1A inhibition for Alzheimer treatment.

Novel and Potential Small Molecule Scaffolds as DYRK1A Inhibitors by Integrated Molecular Docking-Based Virtual Screening and Dynamics Simulation Study.

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a novel, promising and emerging biological target for therapeutic intervention in neurodegenerative diseases, especially in Alzheimer’s disease (AD). The molMall database, comprising rare, diverse and unique compounds, was explored for molecular docking-based virtual screening against the DYRK1A protein, in order to find out potential inhibitors. Ligands exhibiting hydrogen bond interactions with key amino acid residues such as Ile165, Lys188 (catalytic), Glu239 (gk+1), Leu241 (gk+3), Ser242, Asn244, and Asp307, of the target protein, were considered potential ligands. Hydrogen bond interactions with Leu241 (gk+3) were considered key determinants for the selection. High scoring structures were also docked by Glide XP docking in the active sites of twelve DYRK1A related protein kinases, viz. DYRK1B, DYRK2, CDK5/p25, CK1, CLK1, CLK3, GSK3β, MAPK2, MAPK10, PIM1, PKA, and PKCα, in order to find selective DYRK1A inhibitors. MM/GBSA binding free energies of selected ligand–protein complexes were also calculated in order to remove false positive hits. Physicochemical and pharmacokinetic properties of the selected six hit ligands were also computed and related with the proposed limits for orally active CNS drugs. The computational toxicity webserver ProTox-II was used to predict the toxicity profile of selected six hits (molmall IDs 9539, 11352, 15938, 19037, 21830 and 21878). The selected six docked ligand–protein systems were exposed to 100 ns molecular dynamics (MD) simulations to validate their mechanism of interactions and stability in the ATP pocket of human DYRK1A kinase. All six ligands were found to be stable in the ATP binding pocket of DYRK1A kinase.

Rational Design and Identification of Harmine-Inspired, N-Heterocyclic DYRK1A Inhibitors Employing a Functional Genomic In Vivo Drosophila Model System.

Deregulation of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) plays a significant role in developmental brain defects, early-onset neurodegeneration, neuronal cell loss, dementia, and several types of cancer. Herein, we report the discovery of three new classes of N-heterocyclic DYRK1A inhibitors based on the potent, yet toxic kinase inhibitors, harmine and harmol. An initial in vitro evaluation of the small molecule library assembled revealed that the core heterocyclic motifs benzofuranones, oxindoles, and pyrrolones, showed statistically significant DYRK1A inhibition. Further, the utilization of a low cost, high-throughput functional genomic in vivo model system to identify small molecule inhibitors that normalize DYRK1A overexpression phenotypes is described. This in vivo assay substantiated the in vitro results, and the resulting correspondence validates generated classes as architectural motifs that serve as potential DYRK1A inhibitors. Further expansion and analysis of these core compound structures will allow discovery of safe, more effective chemical inhibitors of DYRK1A to ameliorate phenotypes caused by DYRK1A overexpression.

Dual-specificity Tyrosine Phosphorylation-regulated Kinase Inhibitor ID-8 Promotes Human Somatic Cell Reprogramming by Activating PDK4 Expression.

Human induced pluripotent stem cells (hiPSCs) hold great potentials in disease modeling, drug screening and cell therapy. However, efficiency and costs of hiPSCs preparation still need to be improved. We screened the compounds that target signaling pathways, epigenetic modifications or metabolic-process regulation to replace the growth factors. After small molecule treatment, TRA-1-60, which is a cell surface antigen expressed by human embryonic stem cells (hESCs), staining was performed to quantify the efficiency of somatic cell reprogramming. Next, small molecule cocktail-induced ESCs or iPSCs were examined with pluripotent markers expression. Finally, Genome-wide gene expression profile was analyzed by RNA-seq to illustrate the mechanism of human somatic cell reprogramming. Here, we found that a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor ID-8 robustly enhanced human somatic cell reprogramming by upregulation of pyruvate dehydrogenase kinase 4 (PDK4) and activation of glycolysis. Furthermore, we identified a novel growth-factor-free hiPSC generation system using small molecules ID-8 (I) and TGFβ signal pathway agonist Kartogenin (K). Importantly, we developed IK medium combined with low-dose bFGF to support the long-term expansion of human pluripotent stem cells. IK-iPSCs showed pluripotency and normal karyotype. Our studies may provide a novel growth-factor-free culture system to facilitate the generation of hiPSCs for multiple applications in regenerative medicine. In Brief Xu et at. found that a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor ID-8 robustly enhanced human somatic cell reprogramming by upregulation of PDK4 and activation of glycolysis. Furthermore, we established a novel growth-factor-free hiPSC generation system using small molecules ID-8/Kartogenin (IK). IK medium combined with Low-dose bFGF (IKB medium) supported the long-term expansion of human pluripotent stem cells. Highlights ID-8 Enhanced Reprogramming of Human Fibroblasts and Astrocytes Establishment of the Growth-factor-free Reprogramming System Using Small Molecule Compounds IK IKB Medium Maintained the Long-term Expansion of Human Pluripotent Stem Cells ID-8 Promoted Human Somatic Cell Reprogramming by Activating PDK4 Expression.

In-silico Study for African Plants with Possible Beta-Cell Regeneration Effect through Inhibition of DYRK1A

Background: The continuous destruction of normal insulin-producing pancreatic beta cells is a contributing factor in all common forms of diabetes, due to insufficient production of insulin, especially in type 1 diabetes. There are attempts to betacells transplantation, but the cost and availability of donors pose a great challenge to the process. Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase A plays a crucial role in beta-cells destruction. Aims: Our research targets to identify plants for that can be utilized possible alternative approach of beta-cell replacement through a pharmacologically induced regeneration of new beta cells in-silico. Methods and Material: The 3D structure Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase A and 6511 phytochemicals were obtained from Protein Databank and African Natural Products Database respectively. They were appropriately prepared for molecular docking simulations. Molecular docking simulations were implemented, after validation of docking protocols, in AutoDock-Vina®, using virtual screening scripts. Phytocompounds with good binding affinities for Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase A were selected as frontrunners. The compounds were screened for toxicity and Lipinski’s rule confirmation using Datawarrior and then kinase inhibitory bioactivity prediction using Molinspiration. Results: Twelve phytocompounds were found to be predictably highly active in-silico against Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase A, druglike based on Lipinski’s rule, non-mutagenic, non-tumorigenic, no reproductive effect and non-irritant, with high bioactivity prediction. Conclusions: In-silico active phytocompounds against Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase A with their plant sources and physicochemical parameters were identified. Further studies will be carried out in-vitro and in-vivo in to validate the results of this study using plants containing the identified phytocompounds.

Suppression of DYRK1A/B Drives Endoplasmic Reticulum Stress-mediated Autophagic Cell Death Through Metabolic Reprogramming in Colorectal Cancer Cells.

We previously identified KS40008 (4-(3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzene-1,2-diol), a novel inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase family (DYRK) 1A/B, which exhibited high enzymatic activity and cell proliferation-inhibitory effects in colorectal cancer (CRC) cell lines. In the present study, we aimed to elucidate the antitumor mechanisms of KS40008. To assess the cytotoxicity of KS40008, we utilized a human cell line and organoid model and performed a CCK-8 assay and real-time cell analysis. Mitochondrial function was determined through mitochondrial staining, mito-stress test, and glycolysis test. In addition, we investigated the mechanisms of cancer cell death induced by KS40008 through immunoblotting, real-time quantitative polymerase chain reaction, reactive oxygen species staining, and immunofluorescence staining. KS40008 exhibited significant cytotoxicity in CRC and non-CRC cell lines, and organoid models compared to 5-fluorouracil, a conventional chemotherapeutic drug. Moreover, KS40008-induced inhibition of DYRK1A/B led to mitochondrial dysfunction and endoplasmic reticulum stress, promoting autophagic cancer cell death. KS40008 exerts antitumor activity through the inhibition of DYRK1A/B. Here, we demonstrated a mechanism by which KS40008 affects endoplasmic reticulum stress-mediated autophagy through the induction of mitochondrial stress, leading to cytotoxicity in CRC.

Abstract P242: Preclinical efficacy landscape of the pan-CLK/DYRK inhibitor Cirtuvivint (SM08502)

Abstract A monumental challenge facing cancer therapy is the vast mechanistic diversity of tumors among and even within cancer patients. This severely limits the number of patients that respond to any given therapy and the durability of responses that do occur. Deep phenotypic profiling of almost 9000 patient samples indicates perturbation of alternative pre-mRNA splicing (AS) is often a root cause of cancer. This offers an extraordinary opportunity to restore health through normalization of AS in diseased tissue. The bottleneck to progress has been finding and drugging the right targets. The CLK/DYRK kinases modulate AS efficiency and specificity, and therefore, targeting them offers the potential to address this bottleneck. The isoquinoline cirtuvivint is a potent ATP-competitive inhibitor of the Cdc2-like kinases (CLK1-4) and the dual-specificity tyrosine phosphorylation-regulated kinases (DYRK1-4), with activity against only a minimal number of the remaining members of the CMGC-family kinases or the kinome as a whole. Cellular target engagement assays indicated biological IC50s below 0.06 μM across the CLK/DYRK target class. Thus, cirtuvivint has utility for robust chemical evaluation of the overarching contribution of CLK/DYRK family activity to tumor biology in general and context- dependent AS in particular. To help assess the breadth and depth of CLK/DYRK dependencies in human cancers, the consequences of pan-CLK/DYRK inhibition on growth and survival of 154 cancer cell line models, 46 PDX models, and 43 CDX models were tested. EC50s in cell viability assays ranged from 0.014 to 0.73 μM in culture with strong effects observed in subsets of cell lines across all lineages tested. Molecular profiles associated with high sensitivity to cirtuvivint included somatic mutations in the RBM10 splicing factor. Tumor growth inhibition assays with daily dosing at exposures about 2X below the MTD resulted in significant disease control (≥75% TGI) in 15/46 PDX and 18/43 CDX models. Together, these results indicate broad cancer relevance, at least in the preclinical setting, and are consistent with a common reliance on CLK/DYRK-dependent alternative splicing among otherwise highly mechanistically heterogeneous disease. Citation Format: Carine Bossard, Emily Creger, Elizabeth A. McMillan, Carsten Merkwirth, Maureen Ibanez, Deepti Bhat, Josh Stewart, Brian Eastman, Chi-Ching Mak, Vishal Deshmukh, Michael A. White. Preclinical efficacy landscape of the pan-CLK/DYRK inhibitor Cirtuvivint (SM08502) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P242.