A bivalent ß-carboline derivative inhibits macropinocytosis-dependent entry of pseudorabies virus by targeting the kinase DYRK1A.

Pseudorabies virus (PRV) has become a "new life-threatening zoonosis" since the human-originated PRV strain was first isolated in 2020. To identify novel anti-PRV agents, we screened a total of 107 ß-carboline derivatives and found 20 compounds displaying antiviral activity against PRV. Among them, 14 compounds showed better antiviral activity than acyclovir. We found that compound 45 exhibited the strongest anti-PRV activity with an IC50 value of less than 40 nM. Our in vivo studies showed that treatment with 45 significantly reduced the viral loads and protected mice challenged with PRV. To clarify the mode of action of 45, we conducted a time of addition assay, an adsorption assay, and an entry assay. Our results indicated that 45 neither had a virucidal effect nor affected viral adsorption while significantly inhibiting PRV entry. Using the FITC-dextran uptake assay, we determined that 45 inhibits macropinocytosis. The actin-dependent plasma membrane protrusion, which is important for macropinocytosis, was also suppressed by 45. Furthermore, the kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) was predicted to be a potential target for 45. The binding of 45 to DYRK1A was confirmed by drug affinity responsive target stability and cellular thermal shift assay. Further analysis revealed that knockdown of DYRK1A by siRNA suppressed PRV macropinocytosis and the tumor necrosis factor alpha-TNF-induced formation of protrusions. These results suggested that 45 could restrain PRV macropinocytosis by targeting DYRK1A. Together, these findings reveal a unique mechanism through which ß-carboline derivatives restrain PRV infection, pointing to their potential value in the development of anti-PRV agents.

Comparative analysis of the DYRK1A-SRSF6-TNNT2 pathway in myocardial tissue from individuals with and without Down syndrome.

Down syndrome (trisomy 21) is characterized by genome-wide imbalances that result in a range of phenotypic manifestations. Altered expression of DYRK1A in the trisomic context has been linked to some Down syndrome phenotypes. DYRK1A regulates the splicing of cardiac troponin (TNNT2) through a pathway mediated by the master splicing factor SRSF6. Here, we documented the expression of the DYRK1A-SRSF6-TNNT2 pathway in a collection of myocardial samples from persons with and without Down syndrome. Results suggest that "gene dosage effect" may drive the expression of DYRK1A mRNA but has no effect on DYRK1A protein levels in trisomic myocardium. The levels of phosphorylated DYRK1A-Tyr321 tended to be higher (~35%) in myocardial samples from donors with Down syndrome. The levels of phosphorylated SRSF6 were 2.6-fold higher in trisomic myocardium. In line, the expression of fetal TNNT2 variants was higher in myocardial tissue with trisomy 21. These data provide a representative picture on the extent of inter-individual variation in myocardial DYRK1A-SRSF6-TNNT2 expression in the context of Down syndrome.

Involvement of the dual-specificity tyrosine phosphorylation-regulated kinase 1A-alternative splicing factor-calcium/calmodulin-dependent protein kinase IIδ signaling pathway in myocardial infarction-induced heart failure of rats.

BACKGROUND: Alternative splicing factor (ASF)-regulated alternative splicing of calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) plays an important role in pathologic cardiac remodeling. ASF can be phosphorylated by dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A). This study aimed to investigate the possible involvement of the Dyrk1A-ASF-CaMKIIδ signaling pathway in the progression of myocardial infarction (MI)-induced heart failure (HF). METHODS AND RESULTS: MI in rats was induced by means of left anterior descending coronary artery ligation. Seven weeks after MI, the increase in left ventricular internal diameter at end-diastole (LVIDd), and the decrease in both ejection fraction (EF) and fractional shortening (FS) indicated that MI rats had developed HF. Quantitative real time reverse-transcription polymerase chain reaction indicated the dysregulation of CaMKIIδ alternative splicing, ie, up-regulation of CaMKIIδA and CaMKIIδC and down-regulation of CaMKIIδB in the hearts of HF rats. Electrophoresis and immunostaining revealed that HF activated the phosphorylation of ASF and affected its subcellular localization. Western blot analysis demonstrated a significant elevation in the activity and expression of Dyrk1A in HF rats. Inversely, treatment of MI-induced HF rats with Dyrk1A inhibitor, either harmine or EGCG, improved the symptoms of HF, reversed the molecular changes of Dyrk1A and ASF, and regulated alternative splicing of CaMKIIδ in HF rats. CONCLUSIONS: Enhanced activation of Dyrk1A-ASF-CaMKIIδ signaling pathway may underlie the mechanisms of HF after MI, and Dyrk1A inhibition may contribute to inactivation of this pathway and thereby retard the progression of MI-induced HF.

KAT7/HMGN1 signaling epigenetically induces tyrosine phosphorylation-regulated kinase 1A expression to ameliorate insulin resistance in Alzheimer's disease.

BACKGROUND: Epidemiological studies have revealed a correlation between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2D). Insulin resistance in the brain is a common feature in patients with T2D and AD. KAT7 is a histone acetyltransferase that participates in the modulation of various genes. AIM: To determine the effects of KAT7 on insulin patients with AD. METHODS: APPswe/PS1-dE9 double-transgenic and db/db mice were used to mimic AD and diabetes, respectively. An in vitro model of AD was established by Aß stimulation. Insulin resistance was induced by chronic stimulation with high insulin levels. The expression of microtubule-associated protein 2 (MAP2) was assessed using immunofluorescence. The protein levels of MAP2, Aß, dual-specificity tyrosine phosphorylation-regulated kinase-1A (DYRK1A), IRS-1, p-AKT, total AKT, p-GSK3ß, total GSK3ß, DYRK1A, and KAT7 were measured via western blotting. Accumulation of reactive oxygen species (ROS), malondialdehyde (MDA), and SOD activity was measured to determine cellular oxidative stress. Flow cytometry and CCK-8 assay were performed to evaluate neuronal cell death and proliferation, respectively. Relative RNA levels of KAT7 and DYRK1A were examined using quantitative PCR. A chromatin immunoprecipitation assay was conducted to detect H3K14ac in DYRK1A. RESULTS: KAT7 expression was suppressed in the AD mice. Overexpression of KAT7 decreased Aß accumulation and MAP2 expression in AD brains. KAT7 overexpression decreased ROS and MDA levels, elevated SOD activity in brain tissues and neurons, and simultaneously suppressed neuronal apoptosis. KAT7 upregulated levels of p-AKT and p-GSK3ß to alleviate insulin resistance, along with elevated expression of DYRK1A. KAT7 depletion suppressed DYRK1A expression and impaired H3K14ac of DYRK1A. HMGN1 overexpression recovered DYRK1A levels and reversed insulin resistance caused by KAT7 depletion. CONCLUSION: We determined that KAT7 overexpression recovered insulin sensitivity in AD by recruiting HMGN1 to enhance DYRK1A acetylation. Our findings suggest that KAT7 is a novel and promising therapeutic target for the resistance in AD.

DYRK1A-mediated PLK2 phosphorylation regulates the proliferation and invasion of glioblastoma cells.

Polo-like kinases (PLKs) are a family of serine-threonine kinases that exert regulatory effects on diverse cellular processes. Dysregulation of PLKs has been implicated in multiple cancers, including glioblastoma (GBM). Notably, PLK2 expression in GBM tumor tissue is lower than that in normal brains. Notably, high PLK2 expression is significantly correlated with poor prognosis. Thus, it can be inferred that PLK2 expression alone may not be sufficient for accurate prognosis evaluation, and there are unknown mechanisms underlying PLK2 regulation. In the present study, it was demonstrated that dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) interacts with and phosphorylates PLK2 at Ser358. DYRK1A-mediated phosphorylation of PLK2 increases its protein stability. Moreover, PLK2 kinase activity was markedly induced by DYRK1A, which was exemplified by the upregulation of alpha-synuclein S129 phosphorylation. Furthermore, it was found that phosphorylation of PLK2 by DYRK1A contributes to the proliferation, migration and invasion of GBM cells. DYRK1A further enhances the inhibition of the malignancy of GBM cells already induced by PLK2. The findings of the present study indicate that PLK2 may play a crucial role in GBM pathogenesis partially in a DYRK1A-dependent manner, suggesting that PLK2 Ser358 may serve as a therapeutic target for GBM.

Gene and protein interaction network analysis in the epithelial-mesenchymal transition of Hertwig's Epithelial Root Sheath reveals periodontal regenerative drug targets - An in silico study.

Background and aim: Hertwig's Εpithelial Root Sheath (HΕRS) has a major function in the developing tooth roots. Earlier research revealed that it undergoes epithelial-mesenchymal transition, a vital process for the morphogenesis and complete development of the tooth and its surrounding periodontium. Few studies have demonstrated the role of HERS in cementogenesis through ΕMΤ. The background of this in-silico system biology approach is to find a hub protein and gene involved in the EMT of HERS that may uncover novel insights in periodontal regenerative drug targets. Materials and methods: The protein and gene list involved in epithelial-mesenchymal transition were obtained from literature sources. The protein interaction was constructed using STRING software and the protein interaction network was analyzed. Molecular docking simulation checks the binding energy and stability of protein-ligand complex. Results: Results revealed the hub gene to be DYRK1A(Hepcidin), and the ligand was identified as isoetharine. SΤRIΝG results showed a confidence cutoff of 0.9 in sensitivity analysis with a condensed protein interaction network. Overall, 98 nodes from 163 nodes of expected edges were found with an average node degree of 11.9. Docking results show binding energy of -4.70, and simulation results show an RMSD value of 5.6 Å at 50 ns. Conclusion: Isoetharine could be a potential drug for periodontal regeneration.

Miltefosine as a PPM1A activator improves AD-like pathology in mice by alleviating tauopathy via microglia/neurons crosstalk.

Alzheimer's disease (AD) is a progressively neurodegenerative disease without effective treatment. Here, we reported that the levels of expression and enzymatic activity of phosphatase magnesium-dependent 1A (PPM1A) were both repressed in brains of AD patient postmortems and 3 × Tg-AD mice, and treatment of adeno-associated virus (AAV)-ePHP-overexpression (OE)-PPM1A for brain-specific PPM1A overexpression or the new discovered PPM1A activator Miltefosine (MF, FDA approved oral anti-leishmanial drug) for PPM1A enzymatic activation improved the AD-like pathology in 3 × Tg-AD mice. The mechanism was intensively investigated by assay against the 3 × Tg-AD mice with brain-specific PPM1A knockdown (KD) through AAV-ePHP-KD-PPM1A injection. MF alleviated neuronal tauopathy involving microglia/neurons crosstalk by both promoting microglial phagocytosis of tau oligomers via PPM1A/Nuclear factor-κb (NF-κB)/C-X3-C Motif Chemokine Receptor 1 (CX3CR1) signaling and inhibiting neuronal tau hyperphosphorylation via PPM1A/NLR Family Pyrin Domain Containing 3 (NLRP3)/tau axis. MF suppressed microglial NLRP3 inflammasome activation by both inhibiting NLRP3 transcription via PPM1A/NF-κB/NLRP3 pathway in priming step and promoting PPM1A binding to NLRP3 to interfere NLRP3 inflammasome assembly in assembly step. Our results have highly addressed that PPM1A activation shows promise as a therapeutic strategy for AD and highlighted the potential of MF in treating this disease.

Elucidating Pathogenic Mechanisms of Early-onset Alzheimer's Disease in Down Syndrome Patients.

Down syndrome (DS) patients demonstrate the neuropathology of Alzheimer's disease (AD) characterized by the formation of senile plaques and neurofibrillary tangles by age 40-50 years. It has been considered for a number of years that 1.5-fold expression of the gene for the amyloid precursor protein (APP) located on chromosome 21 leading to overproduction of amyloid-ß peptide (Aß) results in the early onset of AD in adults with DS. However, the mean age of onset of familial AD with the Swedish mutation on APP which has high affinity for ß-secretase associated with a dramatic increase in Aß production is about 55 years. This paradox indicates that there is a poor correlation between average ages of AD onset and the theoretical amount of Aß production and that there are factors exacerbating AD on chromosome 21. We therefore focused on dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), since overexpressing transgenic mice show AD-like brain pathology. The overexpression of DYRK1A caused suppression of the activity of neprilysin (NEP), which is a major Aß-degrading enzyme in the brain, and phosphorylation at the NEP cytoplasmic domain. NEP activity was markedly reduced in fibroblasts derived from DS patients compared with that in fibroblasts derived from healthy controls. This impaired activity of NEP was rescued by DYRK1A inhibition. These results show that DYRK1A overexpression causes suppression of NEP activity through its phosphorylation in DS patients. Our results suggest that DYRK1A inhibitors could be effective against AD not only in adults with DS but also in sporadic AD patients.