Dual-specificity Tyrosine Phosphorylation-regulated Kinase Research Articles

Unraveling the mechanistic interplay of mediators orchestrating the neuroprotective potential of harmine.

Neurodegenerative diseases (NDDs) encompass a range of conditions characterized by the specific dysfunction and continual decline of neurons, glial cells, and neural networks within the brain and spinal cord. The majority of NDDs exhibit similar underlying causes, including oxidative stress, neuroinflammation, and malfunctioning of mitochondria. Elevated levels of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), alongside decreased expression of brain-derived neurotrophic factor (BDNF) and glutamate transporter subtype 1 (GLT-1), constitute significant factors contributing to the pathogenesis of NDDs. Additionally, the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene has emerged as a significant target for the treatment of NDDs at the preclinical level. It significantly contributes to developmental brain defects, early onset neurodegeneration, neuronal loss, and dementia in Down syndrome. Moreover, an impaired ubiquitin-proteosome system (UPS) also plays a pathological role in NDDs. Malfunctioning of UPS leads to abnormal protein buildup or aggregation of α-synuclein. α-Synuclein is a highly soluble unfolded protein that accumulates in Lewy bodies and Lewy neurites in Parkinson's disease and other synucleinopathies. Recent research highlights the promising potential of natural products in combating NDDs relative to conventional therapies. Alkaloids have emerged as promising candidates in the fight against NDDs. Harmine is a tricyclic β-carboline alkaloid (harmala alkaloid) with one indole nucleus and a six-membered pyrrole ring. It is extracted from Banisteria caapi and Peganum harmala L. and exhibits diverse pharmacological properties, encompassing neuroprotective, antioxidant, anti-inflammatory, antidepressant, etc. Harmine has been reported to mediate its neuroprotective via reducing the level of inflammatory mediators, NADPH oxidase, AChE, BChE and reactive oxygen species (ROS). Whereas, it has been observed to increase the levels of BDNF, GLT-1 and anti-oxidant enzymes, along with protein kinase-A (PKA)-mediated UPS activation. This review aims to discuss the mechanistic interplay of various mediators involved in the neuroprotective effect of harmine.

Discovery of a novel chemotype as DYRK1A inhibitors against Alzheimer's disease: Computational modeling and biological evaluation

Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) plays an essential role in Tau and Aβ pathology closely related to Alzheimer's disease (AD). Accumulative evidence has demonstrated DYRK1A inhibition is able to reduce the pathological features of AD. Nevertheless, there is no approved DYRK1A inhibitor for clinical use as anti-AD therapy. This is somewhat due to the lack of effective and safe chemotypes of DYRK1A inhibitors. To address this issue, we carried out in silico screening, in vitro assays and in vivo efficacy evaluation with the aim to discover a new class of DYRK1A inhibitors for potential treatment of AD. By in silico screening, we selected and purchased 16 potential DYRK1A inhibitors from the Specs chemical library. Among them, compound Q17 (Specs ID: AO-476/40829177) potently inhibited DYRK1A. The hydrogen bonds between compound Q17 and two amino acid residues named GLU239 and LYS188, were uncovered by molecular docking and molecular dynamics simulation. The cell-based assays showed that compound Q17 could protect the SH-SY5Y human neuroblastoma cell line from okadaic acid (OA)-induced injury by targeting DYRK1A. More importantly, compound Q17 significantly improved cognitive dysfunction of 3 × Tg-AD mice, ameliorated pathological changes, and attenuated Tau hyperphosphorylation as well as Aβ deposition. In summary, our computational modeling strategy is effective to identify novel chemotypes of DYRK1A inhibitors with great potential to treat AD, and the identified compound Q17 in this study is worthy of further study.

Discovery of the First Potent DYRK2 Proteolysis Targeting Chimera Degraders.

Dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) has been identified as a promising oncogenic driver of several types of cancer and is considered to be a critical cancer therapeutic target. Several inhibitors of DYRK2 have been reported, but no degraders have been found yet. In this work, we designed and synthesized the first series of proteolysis-targeting chimeras (PROTACs) using curcumin and its analogs as warheads to target and degrade DYRK2. The results of degradation assays showed that the compound CP134 could effectively downregulate the intracellular DYRK2 level (DC50 = 1.607 μM). Further mechanism of action experiments revealed that CP134 induced DYRK2 degradation through the ubiquitin-proteasome system. Altogether, CP134 disclosed in this study is the first potent DYRK2 degrader, which could serve as a valuable chemical tool for further evaluation of its therapeutic potential, and our results broaden the substrate spectrum of PROTAC-based degraders for further therapeutic applications.

Dual targeting of inflammation and β-cell dysfunction for therapy of diabetes mellitus

Diabetes Mellitus (DM) is a chronic metabolic disorder characterized by β-cell loss and inflammation. From a therapeutic standpoint, it would be necessary to halt the uncontrolled inflammatory response and to enable the regeneration of the damaged β-cells. In this work, to achieve the latter two drug-loaded nanocarrier systems have been developed and evaluated in-vivo in DM mouse model. For β-cell proliferation, small chitosan (CS) nanoparticles (NPs) (201 ± 30 nm) were loaded with 4b, a selective inhibitor of DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A). To reduce inflammation, large poly lactic-co-glycolic acid (PLGA) microparticles (MPs) (1172 ± 77 nm) were encapsulated with BI-605906, an inhibitor of IKKβ (inhibitor of kappa B kinase beta), acting as an inhibitor of an inhibitor. The EE% values were 90 ± 4.2 % and 87 ± 4.46 % for 4b–CS–NPs and BI-605906-PLGA-MPs, respectively. In-vivo biodistribution studies indicated the ability of both particles to accumulate in the pancreas. For CS-NPs, this was achieved as a function of the increased vascular permeability in DM, whereas for PLGA-MPs, this was achieved as function of macrophage uptake. Treatment with 4b–CS–NPs (but not the unloaded drug) reduced blood glucose levels (BGLs) and elevated cyclin D1 (CD1) gene expression indicating the successful ability of the NP system in promoting β-cells proliferation. Although treatment with free BI-605906 showed a modest reduction in BGLs, BI-605906-PLGA-MPs showed no improvement in BGLs. The latter could possibly be attributed to sub-optimal dosing. These results indicate that 4b–CS–NPs have the potential to lower BGLs by promoting β-cell proliferation and restoring pancreatic function. Further optimization is needed for BI-605906-PLGA-MPs, which may enhance therapeutic outcome by dual therapy.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A promotes the inclusion of amyloid precursor protein exon 7

Extracellular amyloid plaques made of Amyloid-β (Aβ) derived from amyloid precursor protein (APP) is one of the major neuropathological hallmarks of Alzheimer’s disease (AD). There are three major isoforms of APP, APP770, APP751, and APP695 generated by alternative splicing of exons 7 and 8. Exon 7 encodes the Kunitz protease inhibitor (KPI) domain. Its inclusion generates APP isoforms containing KPI, APPKPI+, which is elevated in AD and Down syndrome (DS) brains and associated with increased Aβ deposition. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) phosphorylates many splicing factors and regulates the alternative splicing of pre-mRNA. It is upregulated in DS and AD brain. However, it is not yet clear whether Dyrk1A could regulate APP alternative splicing. In the present study, we overexpressed or knocked down Dyrk1A in cultured cells and observed that Dyrk1A promoted the inclusion of both APP exons 7 and 8. Moreover, a significant increase in APP exon7 inclusion was also detected in the forebrain and hippocampus of human Dyrk1A transgenic mice – Tg/Dyrk1A. Screening for splicing factors regulated by Dyrk1A revealed that serine/arginine-rich protein 9G8 inhibited APP exon7 inclusion and interacted with APP pre-mRNA. In vitro, expression of exon 7 facilitated APP cleavage. In human Dyrk1A transgenic mice, we also found an increase in Aβ production. These findings suggest that Dyrk1A inhibits the splicing factor 9G8 and promotes APP exon 7 inclusion, leading to more APPKPI+ expression and APP cleavage and potentially contributing to Aβ production in vivo.

Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

Melanoma is a type of skin cancer that originates in melanin-producing melanocytes. It is considered a multifactorial disease caused by both genetic and environmental factors, such as UV radiation. Dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) phosphorylates many substrates involved in signaling pathways, cell survival, cell cycle control, differentiation, and neuronal development. However, little is known about the cellular function of DYRK3, one of the five members of the DYRK family. Interestingly, it was observed that the expression of DYRK3, as well as p62 (a multifunctional signaling protein), is highly enhanced in most melanoma cell lines. This study aimed to investigate whether DYRK3 interacts with p62, and how this affects melanoma progression, particularly in melanoma cell lines. We found that DYRK3 directly phosphorylates p62 at the Ser-207 and Thr-269 residue. Phosphorylation at Thr-269 of p62 by DYRK3 increased the interaction of p62 with TRAF6, an already known activator of mTORC1 in the mTOR-involved signaling pathways. Moreover, the phosphorylation of p62 at Thr-269 promoted the activation of mTORC1. We also found that DYRK3-mediated phosphorylation of p62 at Thr-269 enhanced the growth of melanoma cell lines and melanoma progression. Conversely, DYRK3 knockdown or blockade of p62-T269 phosphorylation inhibited melanoma growth, colony formation, and cell migration. In conclusion, we demonstrated that DYRK3 phosphorylates p62, positively modulating the p62-TRAF6-mTORC1 pathway in melanoma cells. This finding suggests that DYRK3 suppression may be a novel therapy for preventing melanoma progression by regulating the mTORC1 pathway.

Human cytomegalovirus (CMV) dysregulates neurodevelopmental pathways in cerebral organoids

Human cytomegalovirus (CMV) infection is the leading non-genetic aetiology of congenital malformation in developed countries, causing significant fetal neurological injury. This study investigated potential CMV pathogenetic mechanisms of fetal neural malformation using in vitro human cerebral organoids. Cerebral organoids were permissive to CMV replication, and infection dysregulated cellular pluripotency and differentiation pathways. Aberrant expression of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK), sonic hedgehog (SHH), pluripotency, neurodegeneration, axon guidance, hippo signalling and dopaminergic synapse pathways were observed in CMV-infected organoids using immunofluorescence and RNA-sequencing. Infection with CMV resulted in dysregulation of 236 Autism Spectrum Disorder (ASD)-related genes (p = 1.57E-05) and pathways. This notable observation suggests potential links between congenital CMV infection and ASD. Using DisGeNET databases, 103 diseases related to neural malformation or mental disorders were enriched in CMV-infected organoids. Cytomegalovirus infection-related dysregulation of key cerebral cellular pathways potentially provides important, modifiable pathogenetic mechanisms for congenital CMV-induced neural malformation and ASD.

Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 3 Expression and Its Correlation with Prognosis and Growth of Serous Ovarian Cancer: Correlation of DYRK3 with Ovarian Cancer Survival.

Epithelial ovarian cancer, primarily serous ovarian cancer (SOC), stands as a predominant cause of cancer-related mortality among women globally, emphasizing the urgent need for comprehensive research into its molecular underpinnings. Within this context, the dual-specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3) has emerged as a potential key player with implications for prognosis and tumor progression. This study conducted a meticulous retrospective analysis of 254 SOC cases from our medical center to unravel the prognostic significance of DYRK3. Survival analyses underscored DYRK3 as an independent adverse prognostic factor in SOC, with a hazard ratio of 2.60 (95% CI 1.67-4.07, P < 0.001). Experimental investigations involved DYRK3 knockdown in serous ovarian cancer cell lines (CAOV3 and OVCAR-3) through a shRNA strategy, revealing substantial decreases in cell growth and invasion capabilities. Bioinformatics analyses further hinted at DYRK3's involvement in modulating the tumor immune microenvironment. In vivo experiments with DYRK3-knockdown cell lines validated these findings, demonstrating a notable restriction in the growth of ovarian cancer xenografts. Our findings collectively illuminate DYRK3 as a pivotal tumor-promoting oncogene in SOC. Beyond its adverse prognostic implications, DYRK3 knockdown exhibited promising therapeutic potential by impeding cancer progression and potentially influencing the tumor immune microenvironment. This study establishes a compelling foundation for further research into DYRK3's intricate role and therapeutic potential in ovarian cancer treatment. As we unravel the complexities surrounding DYRK3, our work not only contributes to the understanding of SOC pathogenesis but also unveils new prospects for targeted therapeutic interventions, holding promise for improved outcomes in ovarian cancer management.

Increased dosage of DYRK1A leads to congenital heart defects in a mouse model of Down syndrome.

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21). DS is a gene dosage disorder that results in multiple phenotypes including congenital heart defects. This clinically important cardiac pathology is the result of a third copy of one or more of the approximately 230 genes on Hsa21, but the identity of the causative dosage-sensitive genes and hence mechanisms underlying this cardiac pathology remain unclear. Here, we show that hearts from human fetuses with DS and embryonic hearts from the Dp1Tyb mouse model of DS show reduced expression of mitochondrial respiration genes and cell proliferation genes. Using systematic genetic mapping, we determined that three copies of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1a) gene, encoding a serine/threonine protein kinase, are associated with congenital heart disease pathology. In embryos from Dp1Tyb mice, reducing Dyrk1a gene copy number from three to two reversed defects in cellular proliferation and mitochondrial respiration in cardiomyocytes and rescued heart septation defects. Increased dosage of DYRK1A protein resulted in impairment of mitochondrial function and congenital heart disease pathology in mice with DS, suggesting that DYRK1A may be a useful therapeutic target for treating this common human condition.

Identification of selective dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors and their effects on tau and microtubule

The overexpression of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), commonly observed in neurodegenerative diseases like Alzheimer's disease (AD) and Down syndrome (DS), can induce the formation of neurofibrillary tangles (NFTs) and amyloid plaques. Hence, designing a selective DYRK1A inhibitor would result in a promising small molecule for treating neurodegenerative diseases. Developing selective inhibitors for DYRK1A has been a difficult challenge due to the highly preserved ATP-binding site of protein kinases. In this study, we employed a structure-based virtual screening (SBVS) campaign targeting DYRK1A from a database containing 1.6 million compounds. Enzymatic assays were utilized to verify inhibitory properties, confirming that Y020-3945 and Y020-3957 showed inhibitory activity towards DYRK1A. In particular, the compounds exhibited high selectivity for DYRK1A over a panel of 120 kinases, reduced the phosphorylation of tau, and reversed the tubulin polymerization for microtubule stability. Additionally, treatment with the compounds significantly reduced the secretion of inflammatory cytokines IL-6 and TNF-α activated by DYRK1A-assisted NFTs and Aβ oligomers. These identified inhibitors possess promising therapeutic potential for conditions associated with DYRK1A in neurodegenerative diseases. The results showed that Y020-3945 and Y020-3957 demonstrated structural novelty compared to known DYRK1A inhibitors, making them a valuable addition to developing potential treatments for neurodegenerative diseases.

DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner.

Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.

Discovery of Potent, Selective, and Orally Bioavailable DYRK2 Inhibitors for the Treatment of Prostate Cancer.

Prostate cancer (PCa) seriously threatens male health, and targeting dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) has been verified to reduce PCa burden, while the research progress on the DYRK2 inhibitors was relatively slow. In this work, we discovered DYRK2 inhibitor 12 (IC50 = 9681 nM) through virtual screening. Subsequently, we performed systematic structural optimization to obtain 54 (IC50 = 14 nM). Compound 54 exhibited high selectivity among 215 kinases and significantly suppressed the proliferation and metastasis of PCa cells in vitro. Moreover, compound 54 displayed high safety, favorable bioavailability, and potent tumor growth inhibitory activity in vivo, which could be used as a potential candidate in the discovery of novel anti-PCa drugs.

Discovery of novel harmine derivatives as GSK-3β/DYRK1A dual inhibitors for Alzheimer's disease treatment.

Multitarget-directed ligands (MTDLs) have recently attracted significant interest due to their superior effectiveness in multifactorial Alzheimer's disease (AD). Combined inhibition of two important AD targets, glycogen synthase kinase-3β (GSK-3β) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), may be a breakthrough in the treatment of AD. Based on our previous work, we have designed and synthesized a series of novel harmine derivatives, investigated their inhibition of GSK-3β and DYRK1A, and evaluated a variety of biological activities. The results of the experiments showed that most of these compounds exhibited good activity against GSK-3β and DYRK1A in vitro. ZLQH-5 was selected as the best compound due to the most potent inhibitory effect against GSK-3β and DYRK1A. Molecular docking studies demonstrated that ZLQH-5 could form stable interactions with the ATP binding pocket of GSK-3β and DYRK1A. In addition, ZLQH-5 showed low cytotoxicity against SH-SY5Y and HL-7702, good blood-brainbarrierpermeability, and favorable pharmacokinetic properties. More importantly, ZLQH-5 also attenuated the tau hyperphosphorylation in the okadaic acidSH-SY5Y cell model. These results indicated that ZLQH-5 could be a promising dual-target drug candidate for the treatment of AD.

Anti-Proliferative Potential of Cynaroside and Orientin—In Silico (DYRK2) and In Vitro (U87 and Caco-2) Studies

Luteolin derivates are plant compounds with multiple benefits for human health. Stability to heat and acid hydrolysis and high resistance to (auto)oxidation are other arguments for the laden interest in luteolin derivates today. The present study was designed to compare the in silico and in vitro anti-proliferative potential of two luteolin derivates, luteolin-7-O-glucoside/cynaroside (7-Lut) and luteolin-8-C-glucoside/orientin (8-Lut). In silico investigations were carried out on the molecular target, namely, the human dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) in association with its natural ligand, curcumin (PDB ID: 5ZTN), by CLC Drug Discovery Workbench v. 1.5.1. software and Molegro Virtual Docker (MVD) v. MVD 2019.7.0. software. In vitro studies were performed on two human tumor cell lines, glioblastoma (U87) and colon carcinoma (Caco-2), respectively. Altogether, docking studies have revealed 7-Lut and 8-Lut as effective inhibitors of DYRK2, even stronger than the native ligand curcumin; in vitro studies indicated the ability of both luteolin glucosides to inhibit the viability of both human tumor cell lines, up to 85% at 50 and 100 µg/mL, respectively; the most augmented cytotoxic and anti-proliferative effects were obtained for U87 exposed to 7-Lut (IC50 = 26.34 µg/mL). The results support further studies on cynaroside and orientin to create drug formulas targeting glioblastoma and colon carcinoma in humans.

DnaK promotes autophosphorylation of DYRK1A and its family kinases in Escherichia coli-based cell-free protein expression

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is one of the drug target kinases involved in neurological disorders. DYRK1A phosphorylates substrate proteins related to disease progression in an intermolecular manner. Meanwhile, DYRK1A intramolecularly phosphorylates its own residues on key segments during folding process, which is required for its activation and stabilization. To reproduce the autophosphorylation in vitro, DYRK1A was expressed in Escherichia coli-based cell-free protein synthesis system. Although this system was useful for investigating autophosphorylation of serine residue at position 97 (Ser97) in DYRK1A, only a small fraction of the synthesized protein was successfully autophosphorylated. In this study, we found that the addition of DnaK, a bacterial HSP70 chaperone, to cell-free expression of DYRK1A promoted its Ser97 autophosphorylation. Structure prediction with AlphaFold2 indicates that Ser97 forms a hydrogen bond within an α-helix structure, indicating a possibility that DnaK unfolds the α-helix and maintains the structure around Ser97 in a conformation susceptible to phosphorylation. In addition, DnaK promoted phosphorylation of DYRK1B and HIPK2, but not DYRK2 and DYRK4, suggesting a sequence selectivity in the action of DnaK. This study provides a facile method for promoting autophosphorylation of DYRK family kinases in cell-free protein expression.

Functional Roles of DYRK2 as a Tumor Regulator.

The dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) regulates the induction of apoptosis and DNA repair, metastasis inhibition, cell cycle G1/S transition, protein scaffold stability for E3 ligase complexes, and embryogenesis. Owing to these functions, DYRK2 is thought to regulate tumorigenesis, and its function in cancer has been investigated. Notably, DYRK2 has been reported to function as a tumor suppressor; however, it has also been reported to act as an oncogene in some cancers. This discrepancy makes it difficult to elucidate the conserved functions of DYRK2 in cancer. Here, we reviewed the functions of DYRK2 in various cancers. Patient tissue samples were evaluated for each cancer type. Although some studies have used cell lines and/or xenografts to elucidate the mechanism of DYRK2 function, these studies are not sufficient to understand the role of DYRK2 in cancers. In particular, studies using genetically modified mice would help us to understand the reported functional duality of DYRK2 in cancer.

An inhibitory circuit-based enhancer of DYRK1A function reverses Dyrk1a-associated impairment in social recognition

Heterozygous mutations in the dual-specificity tyrosine phosphorylation-regulated kinase 1a (Dyrk1a) gene define a syndromic form of autism spectrum disorder. The synaptic and circuit mechanisms mediating DYRK1A functions in social cognition are unclear. Here, we identify a social experience-sensitive mechanism in hippocampal mossy fiber-parvalbumin interneuron (PV IN) synapses by which DYRK1A recruits feedforward inhibition of CA3 and CA2 to promote social recognition. We employ genetic epistasis logic to identify a cytoskeletal protein, ABLIM3, as a synaptic substrate of DYRK1A. We demonstrate that Ablim3 downregulation in dentate granule cells of adult heterozygous Dyrk1a mice is sufficient to restore PV IN-mediated inhibition of CA3 and CA2 and social recognition. Acute chemogenetic activation of PV INs in CA3/CA2 of adult heterozygous Dyrk1a mice also rescued social recognition. Together, these findings illustrate how targeting DYRK1A synaptic and circuit substrates as "enhancers of DYRK1A function" harbors the potential to reverse Dyrk1a haploinsufficiency-associated circuit and cognition impairments.

Leucettinibs, a Class of DYRK/CLK Kinase Inhibitors Inspired by the Marine Sponge Natural Product Leucettamine B.

Dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) recently attracted attention due to their central involvement in various pathologies. We here describe a family of DYRK/CLK inhibitors derived from Leucettines and the marine natural product Leucettamine B. Forty-five N2-functionalized 2-aminoimidazolin-4-ones bearing a fused [6 + 5]-heteroarylmethylene were synthesized. Benzothiazol-6-ylmethylene was selected as the most potent residue among 15 different heteroarylmethylenes. 186 N2-substituted 2-aminoimidazolin-4-ones bearing a benzothiazol-6-ylmethylene, collectively named Leucettinibs, were synthesized and extensively characterized. Subnanomolar IC50 (0.5-20 nM on DYRK1A) inhibitors were identified and one Leucettinib was modeled in DYRK1A and co-crystallized with CLK1 and the weaker inhibited off-target CSNK2A1. Kinase-inactive isomers of Leucettinibs (>3-10 μM on DYRK1A), named iso-Leucettinibs, were synthesized and characterized as suitable negative control compounds for functional experiments. Leucettinibs, but not iso-Leucettinibs, inhibit the phosphorylation of DYRK1A substrates in cells. Leucettinibs provide new research tools and potential leads for further optimization toward therapeutic drug candidates.

Abstract 437: Computational discovery and preclinical validation of therapeutic leads with novel MOAs for Glioblastoma

Abstract Glioblastoma (GBM) is an aggressive, highly invasive tumor that has a 5-year survival rate of less than 5%, with median survival of approximately 15 months. Treatment options for patients with GBM are relatively unchanged over the past two decades. We present results from our early-stage GBM drug discovery program enabled by Symphony™, Aria Pharmaceutical’s artificial intelligence (AI) platform. Using patient-derived, unconnected multimodal data and a select chemical library of over 2 million characterized molecules as input, our AI platform built an interpretable in silico model of GBM disease biology and identified novel, high-value drug discovery hits with known pharmacological properties. Nine molecules with unique and novel MOAs (not clinically evaluated for GBM) were identified as drug discovery hits and evaluated in preclinical efficacy studies. Six out of the nine molecules showed anti-proliferative activity in 4 different GBM cell lines (U87MG, LN18, A172, and U118MG) (Table 1). The MOAs showing efficacy in this initial study were validated in a colony formation assay. Three target classes, stearoyl-CoA desaturase (SCD) inhibitor (MK8245 and MF-438), dual tyrosine-regulated kinase 3 (DYRK3) inhibitor (GSK626616), and heat shock protein 90 (HSP90) inhibitor (XL-888) potently inhibited colony formation in a 3D cell culture system. Pharmacokinetic studies showed that MF-438 had good metabolic stability and lower predicted clearance in human and mouse liver microsome assays. Further, MF-438 showed good exposure in plasma and brain extract (brain-to-plasma ratio of 2.2). SCD is a lipogenic enzyme that plays a key role in tumor lipid metabolism and membrane architecture and is often up-regulated in cancer. These studies demonstrate that the SCD1 inhibitor MF-438 can serve as a valuable tool compound that warrants further investigation as a potential treatment for GBM. Table 1. Molecules assessed in GBM cell lines Molecule Mechanism of Action (MOA) Proliferation Assay 3D Colony Formation Assay AS-605240 Phosphoinositide-3 kinase (PI3K), Dual specificity tyrosine-phosphorylation-regulated kinase (DYRK), and CDC like kinase (CLK1) inhibitor Positive Not tested (GSK626616 selected to investigate DYRK inhibition) GSK626616 Dual specificity tyrosine-phosphorylation-regulated kinase (DYRK) 3 inhibitor Not tested Positive Anilinopyrimidine1 Aurora A kinase (AURKA) and Lymphocyte-specific protein tyrosine kinase (LCK) inhibitor Positive Negative Entospletinib Spleen tyrosine kinase (SYK) inhibitor Negative Not tested T0901317 Liver X receptor (LXR) inhibitor Positive Negative MK-8245 Stearoyl-CoA desaturase (SCD) inhibitor Positive Positive MK-438 Stearoyl-CoA desaturase (SCD) inhibitor Not tested Positive Nedocromil Cysteinyl leukotriene receptor 1 (CYSLTR1) and 2 (CYSLTR2) inhibitor Negative Not tested DB07268 c-Jun N-terminal kinase (JNK1) inhibitor Positive Negative PF-670462 Casein kinase 1-e (CSNK1E) and 1-d (CSNK1D) inhibitor Negative Not tested XL888 Heat shock protein 90 (HSP90) inhibitor Positive Positive Citation Format: Hyekyoung Kim, Aaron C. Daugherty, Sana Mujahid, Isaac I. Hakim, Anjali Pandey, Inyong Bae, Jinhwa Lee. Computational discovery and preclinical validation of therapeutic leads with novel MOAs for Glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 437.

Quantitative Phosphoproteomics Reveals the Requirement of DYRK1-Mediated Phosphorylation of Ion Transport- and Cell Junction-Related Proteins for Notochord Lumenogenesis in Ascidian.

The dual-specificity tyrosine phosphorylation-regulated kinase (DYRK1) phosphorylates diverse substrates involved in various cellular processes. Here, we found that blocking the kinase activity of DYRK1 inhibited notochord development and lumenogenesis in ascidian Ciona savignyi. By performing phosphoproteomics in conjunction with notochord-specific proteomics, we identified 1065 notochord-specific phosphoproteins that were present during lumen inflation, of which 428 differentially phosphorylated proteins (DPPs) were identified after inhibition of DYRK1 kinase activity. These DPPs were significantly enriched in metal ion transmembrane transporter activity, protein transport and localization, and tight junction. We next analyzed the downregulated phosphoproteins and focused on those belonging to the solute carrier (SLC), Ras-related protein (RAB), and tight junction protein (TJP) families. In vivo phospho-deficient study showed that alanine mutations on the phosphosites of these proteins resulted in defects of lumenogenesis during Ciona notochord development, demonstrating the crucial roles of phosphorylation of transmembrane transport-, vesicle trafficking-, and tight junction-related proteins in lumen formation. Overall, our study provides a valuable data resource for investigating notochord lumenogenesis and uncovers the molecular mechanisms of DYRK1-mediated notochord development and lumen inflation.