Protein Kinase Family Research Articles

Identification of YBX2 and TSKS As STK33 Interacting Proteins in Testicular Germ Cells.

Spermiogenesis is a unique process, in which round spermatids undergo morphological changes to form spermatozoa. Serine/Threonine Kinase 33 (STK33), a member of the serine/threonine protein kinase family, plays a pivotal role in spermiogenesis, manifested by the infertile phenotype of Stk33 knockout mice and patients carrying STK33 mutations. To date, the mechanism by which STK33 promotes spermiogenesis is not fully understood. Here we aimed to identify germ cell-specific proteins that interact with STK33. Using immunoprecipitation and mass spectrometry, 13 proteins were identified that potentially interact with STK33 in testicular germ cells. By comparing the expression patterns of the candidate genes in testicular germ cells, we selected Y-Box Binding Protein 2 (YBX2) and Testis Specific Serine Kinase Substrate (TSKS) for validation. When co-expressed in cultured cells, TSKS was immunoprecipitated by STK33, and vice versa. Furthermore, STK33 was recruited to the TSKS foci, likely through interaction with TSKS. Although proximity ligation assay demonstrated that STK33 and YBX2 form the complex in germ cells, their interaction was not recapitulated in cultured cells. Phosphorylation assays showed that STK33 was unable to phosphorylate both YBX2 and TSKS in vitro. Overall, these results suggest that STK33 regulates spermiogenesis through TSKS and YBX2, which warrants further investigation in vivo.

Overexpression of Drosophila NUAK or Constitutively-Active Formin-Like Promotes the Formation of Aberrant Myofibrils.

Muscle development and maintenance is central to the normal functioning of animals. Muscle tissues exhibit high levels of activity and require the dynamic turnover of proteins. An actomyosin scaffold functions with additional proteins comprising the basic contractile subunit of striated muscle, known as the sarcomere. Drosophila muscles are similar to vertebrate muscles in composition and they share a similar mechanism of development. Drosophila NUAK (NUAK) is the homolog of NUAK1 and NUAK2 in vertebrates. NUAK belongs to the family of AMP-activated protein kinases (AMPKs), a group of proteins with broad and overlapping cellular targets. Here we confirm that NUAK dynamically modulates larval muscle sarcomere size as upregulation of NUAK produces longer sarcomeres, including increased thin filament lengths. Furthermore, NUAK overexpression results in aberrant myofibers above the nuclei plane, upregulation of Formin-like (Frl), and an increase in newly synthesized proteins at sites consistent with actin filament assembly. Expression of constitutively-active Frl also produces aberrant myofibers similar to NUAK overexpression. These results taken together strongly suggest a functional link between NUAK and Frl in myofibril formation in an invivo setting.

The eukaryotic elongation factor 2 kinase inhibitor, A484954, induces hypoglycaemic and hypotensive effects.

Eukaryotic elongation factor 2 kinase (eEF2K) belongs to the Ca2+/calmodulin-dependent protein kinase family. We previously revealed that A484954, a selective eEF2K inhibitor, caused hypotensive and diuretic effects via the production of nitric oxide (NO) in spontaneously hypertensive rats. Otsuka Long-Evans Tokushima Fatty (OLETF) rats are hypertensive because of obesity and type 2 diabetes. Because an NO synthase inhibitor was reported to increase the expression of sodium glucose co-transporter 2 (SGLT2), we hypothesised that A484954 causes not only hypotensive but also hypoglycaemic effects via NO production in OLETF rats. To test the hypothesis, we examined the effects of A484954 administration on hyperglycaemia and hypertension in OLETF rats. OLETF rats were given an intraperitoneal injection of A484954 (2.5 mg kg-1day-1) for 7 days. Then, we measured blood and urinary glucose level, urine output, systolic blood pressure and ventricular contractility. We also conducted Western blotting and isometric tension measurements. A484954 induced a decrease in blood glucose, an increase in urinary glucose excretion, and a decrease in protein expression of kidney SGLT2. In addition, A484954 induced a decrease in systolic blood pressure, an NO-dependent vasorelaxation, and a diuretic effect. Further, A484954 enhanced left ventricular contractility. We, for the first time, revealed that (1) A484954 caused hypoglycaemic effects through increasing urinary glucose excretion via decreasing SGLT2, (2) A484954 improved diabetic complication, including hypertension, through vasorelaxation and diuresis via NO production, and (3) A484954 had a positive inotropic effect.

Gynostemma pentaphyllum extract ameliorates motor dysfunc-tion in mouse Parkinson's disease model through inhibiting neuronal apoptosis.

To investigate the protective effects and underlying mechanisms of Gynostemma pentaphyllum extract on motor dysfunction in mouse model of Parkinson's disease (PD). Eighty C57BL/6 male mice were randomly divided into five groups: control group, PD model group, levodopa treatment group (positive control group), low-dose GP treatment group (LD-GP group), and high-dose GP treatment group (HD-GP group), with 16 mice per group. The PD model was induced by injection of 6-hydroxydopamine into the substantia nigra pars reticulata in mice of last 5 groups. Two weeks after 6-hydroxydopamine modeling, mice in positive control group received introperitoneal injection of levodopa 10 mg·kg－1·d－1, mice in LD-GP and HD-GP groups received oral 100 mg·kg－1·d－1 or 200 mg·kg－1·d－1 for 3 weeks, respectively. After 3-week-treatment, the effects of GP on motor dysfunction in 6-OHDA-induced PD mice were assessed using open field and CatWalk gait tests, while the effects on muscle strength were evaluated by forelimb grip strength. Immunofluorescence staining was used to detect the number of tyrosine hydroxylase (TH) positive neurons. The levels of dopamine and serotonin in midbrain were determined by enzyme-linked immunosorbent assay. In addition, Western blotting was performed to detect the expression of mitogen-activated protein kinase (MAPK) family proteins such as p- extracellular signal-regulated kinase (ERK)1/2, p-p38 and p-c-Jun N-terminal kinase (JNK)1/2 , and mitochondrial apoptosis pathway proteins such as B-cell lymphoma (Bcl)-2, Bcl-2 associated X protein (Bax), and cleaved- cysteine aspartic acid specific protease (caspase)-3. Behavioral experiments showed that GP significantly improved the spontaneous activity and motor coordination of PD mice (P<0.05). And the forelimb grip strength was also increased by GP treatment (P<0.05), compared with PD model group. In addition, compared with model group, the number of TH-positive neurons in substantia nigra pars reticulata region, the levels of dopamine and serotonin in midbrain and the expression of p-ERK1/2 were significantly increased by GP treatment (all P<0.05), whereas the expression of p-p38 and p-JNK1/2, the ratio of Bax/Bcl-2 and cleaved-caspase 3/caspase 3 were significantly decreased (all P<0.05). The results indicate that GP might increase dopamine and serotonin levels in midbrain and promoted the survival of dopaminergic neurons in substantia nigra pars reticulata by regulating the expression of phosphorylation of MAPK family proteins and the expression of mitochondrial apoptosis-related proteins, and then ameliorate motor deficits in PD mice.

Unraveling the role of RIPKs in diabetic kidney disease and its therapeutic perspectives.

Nephropathy is the microvascular complication of diabetes mellitus and is the leading cause of chronic kidney disease. This review discusses the implications of receptor-interacting protein kinase (RIPK) family members and their regulation of inflammation and cell death pathways in the initiation and progression of diabetic kidney disease. Hyperglycemia leads to reactive oxygen species (ROS) generation and RIPK1 overexpression, the first regulator of necroptosis. Further, RIPK1 can form complex I to promote nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathway activation or complex II to cause programmed cell death in the kidneys. The rise in RIPK1 level upon ROS generation declines the apoptosis regulators' level while the necroptosis regulators' level is boosted. Necroptosis is a programmed or controlled necrosis-type cell death pathway executed by RIPK1, RIPK3, and mixed lineage kinase domain-like (MLKL) proteins, and recent research suggests its importance in diabetic nephropathy. In necroptosis, RIPK1 and RIPK3 interrelate with their RIP homotypic interaction motif (RHIM) domains and cause the recruitment of MLKL. Next, MLKL gets oligomerized, migrate towards the plasma membrane, and causes its rupture. We emphasized different research studies on drugs highlighting the nephroprotective effects via regulating the RIPKs. We hope that the conclusions of this review may provide new strategies for diabetic kidney disease treatment and promising targets for drug development based on necroptosis.

Genome-wide identification of protein kinase family in Populus tomentosa: Functional evidence for causative protein kinase in secondary cell wall biosynthesis

The protein kinase (PK) superfamily holds paramount importance in the plant kingdom, orchestrating myriad cellular processes integral to plant growth, development, as well as resilience against environmental stresses. Nonetheless, the extant biological data about the PK superfamily in perennial woody species remains sparse. In this study, our comprehensive genomic survey of Populus tomentosa unveiled an expansive repertoire of 1543 PK genes, meticulously classified into nine discrete groups and 125 specialized subfamilies. A collinearity analysis revealed that whole genome duplication events served as the preeminent driving forces for PK superfamily expansion. Utilizing cluster analysis of expression profiling in different tissues, we identified 248 PK genes distributed across two clusters implicated in secondary cell wall (SCW) biosynthesis and lignification. Additionally, an association mapping study conducted within a natural population of P. tomentosa pinpointed 176 significant associations, corresponding to 67 PK genes, which were linked to SCW compositional attributes. These findings underscore the genetic determinants shaping PK-mediated modulation of SCW biosynthesis in Populus species. Of particular note, the causative PK gene PtoCMGC48 emerged as a linchpin, exhibiting a pronounced signal associated with lignin content and was found to interact with and phosphorylate the SCW-related transcription factor PtoVND6-C2. This discovery posits a novel molecular paradigm wherein PKs orchestrate SCW biosynthesis via targeted phosphorylation events in P. tomentosa. Overall, our findings lay the groundwork for a deeper appreciation of PK gene functionalities embedded within the complex network governing SCW formation.

Salt-inducible kinases (SIKs) in cancer: mechanisms of action and therapeutic prospects.

Salt-inducible kinases (SIKs), a group of serine/threonine kinases in the adenosine monophosphate-activated protein kinase (AMPK) family, exist in three isoforms: SIK1, SIK2 and SIK3. These kinases are crucial in various physiological processes. Emerging evidence indicates that dysregulation of SIK expression and activation significantly contributes to carcinogenesis by promoting cellular proliferation, metabolic dysregulation, metastasis and chemoresistance through the modulation of crucial signaling pathways. The role of SIKs in cancer progression and metastasis involves complex mechanisms that vary among cancer types. Additionally, research on SIK inhibitors suggests that targeting these kinases might offer promising avenues for improving cancer treatment outcomes.

Novel kinase regulators of extracellular matrix internalisation identified by high-content screening modulate invasive carcinoma cell migration.

The interaction between cancer cells and the extracellular matrix (ECM) plays a pivotal role in tumour progression. While the extracellular degradation of ECM proteins has been well characterised, ECM endocytosis and its impact on cancer cell progression, migration, and metastasis is poorly understood. ECM internalisation is increased in invasive breast cancer cells, suggesting it may support invasiveness. However, current high-throughput approaches mainly focus on cells grown on plastic in 2D, making it difficult to apply these to the study of ECM dynamics. Here, we developed a high-content screening assay to study ECM uptake, based on the of use automated ECM coating for the generation of highly homogeneous ECM a pH-sensitive dye to image ECM trafficking in live cells. We identified that mitogen-activated protein kinase (MAPK) family members, MAP3K1 and MAPK11 (p38β), and the protein phosphatase 2 (PP2) subunit PPP2R1A were required for the internalisation of ECM-bound α2β1 integrin. Mechanistically, we show that down-regulation of the sodium/proton exchanger 1 (NHE1), an established macropinocytosis regulator and a target of p38, mediated ECM macropinocytosis. Moreover, disruption of α2 integrin, MAP3K1, MAPK11, PPP2R1A, and NHE1-mediated ECM internalisation significantly impaired cancer cell migration and invasion in 2D and 3D culture systems. Of note, integrin-bound ECM was targeted for lysosomal degradation, which was required for cell migration on cell-derived matrices. Finally, α2β1 integrin and MAP3K1 expression were significantly up-regulated in pancreatic tumours and correlated with poor prognosis in pancreatic cancer patients. Strikingly, MAP3K1, MAPK11, PPP2R1A, and α2 integrin expression were higher in chemotherapy-resistant tumours in breast cancer patients. Our results identified the α2β1 integrin/p38 signalling axis as a novel regulator of ECM endocytosis, which drives invasive migration and tumour progression, demonstrating that our high-content screening approach has the capability of identifying novel regulators of cancer cell invasion.

Genome-wide identification and analysis of expression patterns of the ABC1K gene family members in Medicago sativa.

The ABC1K (activity of bc1 complex kinase) atypical protein kinase family regulates diverse physiological functions in plants, including the development, growth, and response of plants to various stress stimuli. However, to date, only a few members of the alfalfa (Medicago sativa) ABC1K gene family have been identified, which severely limits the exploration of the functional mechanism of alfalfa ABC1K. Here, we identified 22 ABC1K genes from the alfalfa genome and categorized them into four types on the basis of phylogenetic analysis results and gene structure. We then characterized the physical and biochemical properties, chromosomal location, subcellular localization, cis-regulatory elements, and conserved motifs of these genes. Transcript profiling analysis confirmed that MsABC1Ks were widely expressed in various alfalfa tissues, with tissue-specific expression. We also found that salt and drought conditions significantly regulated MsABC1K gene expression, thus indicating that MsABC1K genes perform critical functions in alfalfa's response to abiotic stress. In summary, the findings of our study serve as an important basis to enhance the stress resistance of alfalfa and provide valuable insights to better comprehend the functions of the MsABC1K gene family.

LDC000067, a CDK9 inhibitor, unveils promising results in suppressing influenza virus infections in vitro and in vivo.

Influenza virus infections continue to pose a significant threat to public health. Current anti-influenza drugs target viral proteins; however, the emergence of resistant strains has hampered their effectiveness. Fortunately, as with most viruses, influenza virus depends on various host factors during its replication cycle and in pathogenicity. Therefore, the manipulation of key host factors for viral replication to combat influenza has garnered increased attention due to its lesser tendency to induce viral mutation. Cyclin-dependent kinases (CDKs) are a family of protein kinases that regulate various cellular processes, including the cell cycle and transcription. While the specific involvement of CDKs in the transcription of influenza virus genes is less extensively studied than their roles in the cell cycle, some evidence suggests their potential contributions as anti-influenza drugs. Here, we report that LDC000067 (LDC), a highly specific CDK9 inhibitor, not only strongly suppressed influenza virus replication in vitro and in vivo but also emerged as a potential candidate for anti-influenza virus agents. Further investigation revealed that inhibition of CDK9 by LDC treatment and CDK9 silencing disrupts viral RNA transcription and the nuclear import of vRNPs, significantly suppressing influenza virus replication. Mechanistically, we showed that LDC treatment and CDK9 silencing reduce Pol II expressions, a requisite host protein for viral RNA transcription. Altogether, our findings indicate that CDK9 could be a promising target for developing antivirals against influenza virus infections, and LDC, with its strong anti-influenza properties, instills confidence in its potential as an effective anti-influenza agent.

Pharmacoinformatics-based prediction of Checkpoint Kinase-1 inhibitors from Momordica charantia Linn. for cancer

Checkpoint kinase 1 (Chk-1), a serine/threonine kinase family protein, is an emerging target in cancer research owing to its crucial role in cell cycle arrest. Therefore, we aimed to predict potential Chk-1 inhibitors from Momordica charantia Linn., using high-throughput molecular docking. We used a graph theoretical network approach to determine the target protein, Chk-1. Among 86 compounds identified from M. charantia L., five molecules such as α-spinasterol (-9.7kcal × mol−1), stigmasterol (-9.6kcal × mol−1), stigmasta-7,22,25-trienol (-9.5kcal × mol−1), campesterol (-9.5kcal × mol−1), and stigmasta-7,25-dien-3beta-ol (-9.5kcal × mol−1) and standard drug CCT245737 (-8.3kcal×mol-1) displayed highest binding affinity with Chk-1. Besides, pharmacokinetic studies have demonstrated the non-toxic and drug-like properties of these compounds. Furthermore, molecular dynamics (MD) simulation studies confirmed the strong intermolecular interactions and stability of the compounds with Chk-1. The estimation of binding free-energy derived from molecular docking was fully recognized by the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) produced from the MD simulation paths. Altogether, these five compounds may serve as effective inhibitors of Chk-1, thereby could be used to develop new medications for cancer treatment.

Role of Rho-associated kinases and their inhibitor fasudil in neurodegenerative diseases.

Neurodegenerative diseases (NDDs) are prevalent in the elderly. The pathogenesis of NDDs is complex, and currently, there is no cure available. With the increase in aging population, over 20 million people are affected by common NDDs alone (Alzheimer's disease and Parkinson's disease). Therefore, NDDs have profound negative impacts on patients, their families, and society, making them a major global health concern. Rho-associated kinases (ROCKs) belong to the serine/threonine protein kinases family, which modulate diverse cellular processes (e.g., apoptosis). ROCKs may elevate the risk of various NDDs (including Huntington's disease, Parkinson's disease, and Alzheimer's disease) by disrupting synaptic plasticity and promoting inflammatory responses. Therefore, ROCK inhibitors have been regarded as ideal therapies for NDDs in recent years. Fasudil, one of the classic ROCK inhibitor, is a potential drug for treating NDDs, as it repairs nerve damage and promotes axonal regeneration. Thus, the current review summarizes the relationship between ROCKs and NDDs and the mechanism by which fasudil inhibits ROCKs to provide new ideas for the treatment of NDDs.

Benchmarking Cross-Docking Strategies in Kinase Drug Discovery.

In recent years, machine learning has transformed many aspects of the drug discovery process, including small molecule design, for which the prediction of bioactivity is an integral part. Leveraging structural information about the interactions between a small molecule and its protein target has great potential for downstream machine learning scoring approaches but is fundamentally limited by the accuracy with which protein-ligand complex structures can be predicted in a reliable and automated fashion. With the goal of finding practical approaches to generating useful kinase-inhibitor complex geometries for downstream machine learning scoring approaches, we present a kinase-centric docking benchmark assessing the performance of different classes of docking and pose selection strategies to assess how well experimentally observed binding modes are recapitulated in a realistic cross-docking scenario. The assembled benchmark data set focuses on the well-studied protein kinase family and comprises a subset of 589 protein structures cocrystallized with 423 ATP-competitive ligands. We find that the docking methods biased by the cocrystallized ligand, utilizing shape overlap with or without maximum common substructure matching, are more successful in recovering binding poses than standard physics-based docking alone. Also, docking into multiple structures significantly increases the chance of generating a low root-mean-square deviation (RMSD) docking pose. Docking utilizing an approach that combines all three methods (Posit) into structures with the most similar cocrystallized ligands according to the maximum common substructure (MCS) proved to be the most efficient way to reproduce binding poses, achieving a success rate of 70.4% across all included systems. The studied docking and pose selection strategies, which utilize the OpenEye Toolkits, were implemented into pipelines of the KinoML framework, allowing automated and reliable protein-ligand complex generation for future downstream machine learning tasks. Although focused on protein kinases, we believe that the general findings can also be transferred to other protein families.

An updated review of small-molecule HPK1 kinase inhibitors (2016-present).

Hematopoietic progenitor kinase 1 (HPK1) is a serine-threonine kinase specific to hematopoiesis and a member of the MAP4K family of Ste20-related protein kinases. Targeting HPK1 to ameliorate T cell exhaustion and enhance T cell functions is a promising strategy for clinical immunotherapies. Numerous studies have reported the progress in developing effective HPK1 inhibitors and elucidating their mechanisms of action. However, most inhibitors affect multiple signaling pathways, resulting in unintended side effects that limit their clinical development and application. Herein, we reviewed HPK1-related signaling pathways, clinical candidates and recent advances in small-molecule inhibitors targeting HPK1. Additionally, we present our perspectives on current challenges and potential future research field, hoping to provide inspiration for the development of novel HPK1 inhibitors.

COMPUTATIONAL SCREENING OF POTENT ANTI-INFLAMMATORY COMPOUNDS FOR HUMAN MITOGEN-ACTIVATED PROTEIN KINASE: A COMPREHENSIVE AND COMBINED IN SILICO APPROACH

Objective: Inflammatory diseases have a serious impact on one’s life and represent a diverse group of ailments stemming from various causes and presenting in various forms. p38α of the mitogen-activated protein kinase family plays a crucial role in regulating inflammation, where the activation of this kinase initiates a cascade of events resulting in the production of proinflammatory mediators and cellular stress responses. In this context, attempts were made to identify potent small-molecule inhibitors of p38α and assess their binding affinity through molecular docking studies. Methods: From comprehensive reviews of several published reports, a few compounds, such as P38, P39, VPC00628, and N17, have shown substantial inhibitory activity toward p38α at various concentrations. Hence, these four compounds were chosen as lead compounds, and small-molecule libraries were constructed on the basis of their structural similarity. Next, virtual screening docking was performed to investigate the inhibitory potency of the four libraries toward the p38α isoforms (DFG-out and DFG-in), providing insights into their potential mechanisms of action. Results: In addition, a comprehensive analysis of physicochemical and pharmacokinetic properties was also performed for the identified hits from each library. Our findings have shown that, compared with those of the p38α DFG-in motif, the binding energies of the p38α DFG-out motif are greater. Conclusion: Furthermore, a few compounds from each library presented binding energies higher than those of their respective lead compounds, confirming their potential as novel therapeutic agents against inflammation.

Expression Analysis of the Extensive Regulation of Mitogen-Activated Protein Kinase (MAPK) Family Genes in Buckwheat (Fagopyrum tataricum) During Organ Differentiation and Stress Response

The mitogen-activated protein kinase (MAPK) signaling cascade is a unique and relatively conserved signaling pathway in eukaryotes, transmitting extracellular signals into cells through successive phosphorylation and eliciting appropriate responses from the organism. While its mechanism in plant immune response has been partially elucidated in Arabidopsis, it has been rarely examined in Tartary buckwheat (Fagopyrum tataricum). Based on the conserved MAPK domain, we identified 16 MAPK family genes in Tartary buckwheat. The FtMAPKs have similar structures and motif compositions, indicating that this gene family is conserved yet functionally diverse. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, we observed significant expression variation in 15 genes across different organs, except for FtMAPK12. FtMAPK9 showed specific expression in vegetative organs, FtMAPK4 in reproductive organs, and FtMAPK1 and FtMAPK10 in leaves and flowers, respectively, indicating their regulatory roles in Tartary buckwheat development. Following drought and salt stress treatments, 12 and 14 FtMAPKs, respectively, showed significantly altered expression in leaves exhibiting notable biological oxidation. Among these, FtMAPK3, FtMAPK4, and FtMAPK8 demonstrated highly significant changes across both treatments. Transcriptome analysis confirmed these findings, suggesting that these three genes play pivotal roles in Tartary buckwheat’s response to abiotic stress and hold potential for molecular breeding improvements.

Discovery of the first selective and potent PROTAC degrader for the pseudokinase TRIB2

Pseudokinase TRIB2, a member of the CAMK Ser/Thr protein kinase family, regulates various cellular processes through phosphorylation-independent mechanisms. Dysregulation of TRIB2 has been implicated in promoting tumor growth, metastasis, and therapy resistance, making it a promising target for cancer treatment. In this study, we designed and synthesized a series of TRIB2 PROTAC degraders by conjugating a TRIB2 binder 1 with VHL or CRBN ligands via linkers of varying lengths and compositions. Among these compounds, 5k demonstrated potent TRIB2 degradation with a DC50 value of 16.84 nM (95 % CI: 13.66–20.64 nM) in prostate cancer PC3 cells. Mechanistic studies revealed that 5k directly interacted with TRIB2, selectively inducing its degradation through a CRBN-dependent ubiquitin-proteasomal pathway. Moreover, 5k outperformed the TRIB2 binder alone in inhibiting cell proliferation and inducing apoptosis, confirming that TRIB2 protein degradation could be a promising therapeutic strategy for TRIB2-associated cancers. Additionally, compound 5k also serves as an effective tool for probing TRIB2 biology.

ERK5 is essential for early porcine embryonic development by maintaining Endoplasmic Reticulum homeostasis

Extracellular signal-regulated kinase 5 (ERK5), a mitogen-activated protein kinase (MAPK) family member, plays an important role in various biological processes, such as proliferation, apoptosis, differentiation, survival, and cell regulation. However, studies on the effects of ERK5 on porcine preimplantation embryos are limited. In this study, to determine the function of ERK5 during porcine embryo development, ERK5 function was inhibited by adding the ERK5 inhibitor JWG-071. The ERK5 mRNA and protein expression levels tended to decrease from the 4-cell stage compared to the 1-cell and 2-cell stages, suggesting that ERK5 is the maternal gene. During embryonic development in pigs, adding 5 μM of JWG-071 significantly reduced the phosphorylation of ERK5 and the blastocyst development rate (control: 53.44 ± 8.38 %; treatment: 26.65 ± 3.40 %). Additionally, ERK5 inhibition increased the expression of UPR-related proteins, glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP) by inducing ER stress. Compared to the control group, the expression of the autophagy-related proteins LC3 and ATG7 was significantly increased in the ERK5 inhibition group, indicating that the inhibition of ERK5 induced autophagy. In addition, ERK5 inhibition increased the expression of BAX, a pro-apoptotic gene, resulting in apoptosis. In conclusion, the results show that ERK5 inhibition during porcine embryonic development induces autophagy and apoptosis by increasing ER stress, resulting in a negative effect on embryonic development in pigs.

Selective Covalent Inhibiting JNK3 by Small Molecules for Parkinson's Diseases

Abstractc‐Jun N‐terminal kinases (JNKs) including JNK1/2/3 are key members of mitogen‐activated protein kinase family. Wherein JNK3 is specifically expressed in brain and emerges as therapeutic target, especially for neurodegenerative diseases. However, developing JNK3 selective inhibitors as chemical probes to investigate its therapeutic potential in diseases remains challenging. Here, we adopted the covalent strategy for identifying JNK3‐selective covalent inhibitor JC16I, with high inhibitory activity against JNK3. Despite targeting a conserved cysteine in the vicinity of ATP pocket in JNK family, JC16I exerted a greater than 160‐fold selectivity for JNK3 over JNK1/2. Importantly, even at low concentration, JC16I showed enhanced and long‐lasting inhibition against cellular JNK3. In addition, its alkyne‐containing probe JC‐P1 could label JNK3 in SH‐SY5Y cell lysate and living cells, with good proteome‐wide selectivity. JC16I selectively suppressed the abnormal activation of JNK3 signaling and sufficiently exhibited neuroprotective effect in Parkinson's diseases (PD) models. Overall, our findings highlight the potential of developing isoform‐selective and cell‐active JNK3 inhibitors by covalent drug design strategy targeting a conserved cysteine. This work not only provides a valuable chemical probe for JNK3‐targeted investigations in vitro and in vivo but also opens new avenues for the treatment of PD.

Selective Covalent Inhibiting JNK3 by Small Molecules for Parkinson's Diseases.

c-Jun N-terminal kinases (JNKs) including JNK1/2/3 are key members of mitogen-activated protein kinase family. Wherein JNK3 is specifically expressed in brain and emerges as therapeutic target, especially for neurodegenerative diseases. However, developing JNK3 selective inhibitors as chemical probes to investigate its therapeutic potential in diseases remains challenging. Here, we adopted the covalent strategy for identifying JNK3-selective covalent inhibitor JC16I, with high inhibitory activity against JNK3. Despite targeting a conserved cysteine in the vicinity of ATP pocket in JNK family, JC16I exerted a greater than 160-fold selectivity for JNK3 over JNK1/2. Importantly, even at low concentration, JC16I showed enhanced and long-lasting inhibition against cellular JNK3. In addition, its alkyne-containing probe JC-P1 could label JNK3 in SH-SY5Y cell lysate and living cells, with good proteome-wide selectivity. JC16I selectively suppressed the abnormal activation of JNK3 signaling and sufficiently exhibited neuroprotective effect in Parkinson's diseases (PD) models. Overall, our findings highlight the potential of developing isoform-selective and cell-active JNK3 inhibitors by covalent drug design strategy targeting a conserved cysteine. This work not only provides a valuable chemical probe for JNK3-targeted investigations in vitro and in vivo but also opens new avenues for the treatment of PD.