Association between novel genetic variants of Notch signaling pathway genes and survival of hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: Although the Notch pathway plays an important role in formation and progression of hepatocellular carcinoma (HCC), few studies have reported the associations between functional genetic variants and the survival of hepatitis B virus (HBV)-related HCC. METHODS: In the present study, we performed multivariable Cox proportional hazard regression analysis to evaluate associations between 36,101 SNPs in 264 Notch pathway-related genes and overall survival (OS) of 866 patients with HBV-related HCC. RESULTS: It was found that three independent SNPs (NEURL1B rs4868192, CNTN1 rs444927 and FCER2 rs1990975) were significantly associated with the HBV-related HCC OS. The number of protective genotypes (NPGs) were significantly associated with better survival in a dose-response manner (ptrend <0.001). Compared with the model with sole clinical factors, the addition of protective genotypes to the predict models significantly increased the AUC, i.e., from 72.72% to 75.13% (p = 0.002) and from 72.04% to 74.76 (p = 0.004) for 3-year and 5-year OS, respectively. The expression quantitative trait loci (eQTL) analysis further revealed that the rs4868192 C allele was associated with lower mRNA expression levels of NEURL1B in the whole blood (p = 1.71 × 10-3), while the rs1990975 T allele was correlated with higher mRNA expression levels of FCER2 in the whole blood and normal liver tissues (p = 3.51 × 10-5 and 0.033, respectively). CONCLUSIONS: Three potentially functional SNPs of NEURL1B, CNTN1 and FCER2 may serve as potential prognostic biomarkers for HBV-related HCC.

Comparative characteristics of early-onset vs. late-onset advanced colorectal cancer: a nationwide study in China.

BACKGROUND: The incidence of early-onset colorectal cancer (EOCRC, diagnosed in patients under the age of 50 years) has been increasing around the world. Here, we aimed to systematically identify distinctive features of EOCRC. METHODS: From 2020 to 2021, we conducted a nationwide survey in 19 hospitals, collecting data on advanced CRC patients' demographics, clinical features, disease knowledge, medical experiences, expenditures, and health-related quality of life (HRQOL). We compared these features between EOCRC and late-onset colorectal cancer (LOCRC, ≥ 50 years old) groups and analyzed the association between EOCRC and HRQOL using multivariate linear regression. FINDINGS: In total, 991 patients with EOCRC and 3581 patients with LOCRC were included. Compared to the LOCRC group, the EOCRC group had higher levels of education, were more informed about the risk factors for CRC, were more likely to have widespread metastases throughout the body, were more inclined to undergo gene testing, and were more likely to opt for targeted therapy, radiotherapy, and chemotherapy. However, HRQOL in the EOCRC group was similar to that of the LOCRC group, and no significant association was observed between EOCRC and HRQOL (beta: -0.753, P value: 0.307). INTERPRETATION: In Chinese patients, EOCRC patients had more aggressive features. Despite undergoing more intensified treatments and gene testing, they had similar HRQOL compared with LOCRC. These findings advocate for a more tailored approach to treatment, especially for young CRC patients with advanced TNM stages and metastasis.

Chirality-Induced Crystallization and Defect Passivation of Perovskites: Toward High-Performance Solar Cells.

As an additive for perovskites, in addition to functional groups, the steric configuration of molecules is worthy of consideration because it influences perovskite crystallization, thus determining whether defect passivation is effective without any side effects. In this work, the chiral molecules l- and d-pyroglutamic acid (l-PA and d-PA) were chosen as additives for perovskite passivators to reveal the reasons for the differences in passivation between amino acids with different steric configurations. Functional groups, such as the C═O groups and N-H groups of l-PA and d-PA, can passivate the perovskite defects. However, l-PA exhibited a more distorted steric configuration, while d-PA was more planar, leading to differences in the distances between the two C═O groups. Taking the Pb-Pb bond length as a reference, the shorter distance between the two C═O groups of l-PA distorts the perovskite lattice structure, which results in poor device stability. Conversely, the similar distance between the two C═O groups of d-PA promoted the preferred orientational growth of the perovskite. Finally, the d-PA-doped device accomplished an excellent efficiency of 24.11% with an improved open-circuit voltage of 1.17 V. Furthermore, the efficiency of the unencapsulated d-PA-doped device was maintained at 93% in N2 for more than 3000 h and 74% after 500 h of operation at maximum power point tracking under continuous illumination.

zDHHC20-driven S-palmitoylation of CD80 is required for its costimulatory function.

CD80 is a transmembrane glycoprotein belonging to the B7 family, which has emerged as a crucial molecule in T cell modulation via the CD28 or CTLA4 axes. CD80-involved regulation of immune balance is a finely tuned process and it is important to elucidate the underlying mechanism for regulating CD80 function. In this study we investigated the post-translational modification of CD80 and its biological relevance. By using a metabolic labeling strategy, we found that CD80 was S-palmitoylated on multiple cysteine residues (Cys261/262/266/271) in both the transmembrane and the cytoplasmic regions. We further identified zDHHC20 as a bona fide palmitoyl-transferase determining the S-palmitoylation level of CD80. We demonstrated that S-palmitoylation protected CD80 protein from ubiquitination degradation, regulating the protein stability, and ensured its accurate plasma membrane localization. The palmitoylation-deficient mutant (4CS) CD80 disrupted these functions, ultimately resulting in the loss of its costimulatory function upon T cell activation. Taken together, our results describe a new post-translational modification of CD80 by S-palmitoylation as a novel mechanism for the regulation of CD80 upon T cell activation.

SynergyX: a multi-modality mutual attention network for interpretable drug synergy prediction.

Discovering effective anti-tumor drug combinations is crucial for advancing cancer therapy. Taking full account of intricate biological interactions is highly important in accurately predicting drug synergy. However, the extremely limited prior knowledge poses great challenges in developing current computational methods. To address this, we introduce SynergyX, a multi-modality mutual attention network to improve anti-tumor drug synergy prediction. It dynamically captures cross-modal interactions, allowing for the modeling of complex biological networks and drug interactions. A convolution-augmented attention structure is adopted to integrate multi-omic data in this framework effectively. Compared with other state-of-the-art models, SynergyX demonstrates superior predictive accuracy in both the General Test and Blind Test and cross-dataset validation. By exhaustively screening combinations of approved drugs, SynergyX reveals its ability to identify promising drug combination candidates for potential lung cancer treatment. Another notable advantage lies in its multidimensional interpretability. Taking Sorafenib and Vorinostat as an example, SynergyX serves as a powerful tool for uncovering drug-gene interactions and deciphering cell selectivity mechanisms. In summary, SynergyX provides an illuminating and interpretable framework, poised to catalyze the expedition of drug synergy discovery and deepen our comprehension of rational combination therapy.

[Zuogui Pills improve testicular development of offspring rats exposed to prenatal stress via Cx43].

This study aims to reveal the mechanism of prenatal stress in affecting the testicular development of offspring rats and the intervention effects of Zuogui Pills via connexin 43(Cx43). Forty pregnant SD rats were randomized into a blank control group, a mo-del group, a high-dose(18.9 g·kg~(-1)) Zuogui Pills group, a low-dose(9.45 g·kg~(-1)) Zuogui Pills group, and a vitamin E(1.44 mg·kg~(-1)) group. The other groups except the blank control group was subjected to chronic unpredictable mild stress for the modeling of prenatal stress. The model was evaluated by sucrose preference test, open field test, and enzyme-linked immunosorbent assay(ELISA) of the glucocorticoid level. ELISA was employed to measure the thyroxine 4(T4), testosterone(T), and follicle-stimulating hormone(FSH) levels to assess kidney deficiency. Hematoxylin-eosin(HE) staining was employed to evaluate the status of testicular germ cells. An automatic sperm analyzer was used to measure the sperm quality. Immunofluorescence double staining was employed to detect the expression of Cx43 and follicle-stimulating hormone receptor(FSHR) in the testes of offspring rats. The mRNA and protein levels of Cx43, FSHR, phosphatidylinositol 3-kinase(PI3K), and protein kinase B(Akt) were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot, respectively. Prenatal stress induced testicular development disorders in offspring rats. The HE staining results showed that on the day of birth, the model group had reduced seminiferous tubules in the testes, elevated FSH level in the serum, and lowered Cx43 level in the testicular tissue. Male offspring rats of 60 days old had reduced testicular spermatogenic function, decreased sperm quality, elevated FSH level and lowered T level in the serum, and down-regulated protein and mRNA levels of Cx43, FSHR, PI3K, and Akt in the testicular tissue. Zuogui Pills alleviated the abnormal development and dysfunction of testicles in the offspring rats caused by prenatal stress. In summary, Zuogui Pills may weaken the effects of prenatal stress on testicular development and spermatogenic function of offspring rats by activating the PI3K/Akt pathway to regulate Cx43 expression in the testicular tissue.

Enhancing the safety of CAR-T cell therapy: Synthetic genetic switch for spatiotemporal control.

Chimeric antigen receptor T (CAR-T) cell therapy is a promising and precise targeted therapy for cancer that has demonstrated notable potential in clinical applications. However, severe adverse effects limit the clinical application of this therapy and are mainly caused by uncontrollable activation of CAR-T cells, including excessive immune response activation due to unregulated CAR-T cell action time, as well as toxicity resulting from improper spatial localization. Therefore, to enhance controllability and safety, a control module for CAR-T cells is proposed. Synthetic biology based on genetic engineering techniques is being used to construct artificial cells or organisms for specific purposes. This approach has been explored in recent years as a means of achieving controllability in CAR-T cell therapy. In this review, we summarize the recent advances in synthetic biology methods used to address the major adverse effects of CAR-T cell therapy in both the temporal and spatial dimensions.

Nerve growth factor (Ngf) gene-driven semaphorin 3a (Sema3a) expression exacerbates thoracic aortic aneurysm dissection in mice.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease and currently there is no pharmacological therapy. Sympathetic nerve overactivity plays an important role in the development of TAAD. Sympathetic innervation is mainly controlled by nerve growth factor (NGF, a key neural chemoattractant) and semaphoring 3A (Sema3A, a key neural chemorepellent), while the roles of these two factors in aortic sympathetic innervation and especially TAAD are unknown. We hypothesized that genetically manipulating the NGF/Sema3A ratio by the Ngf -driven Sema3a expression approach may reduce aortic sympathetic nerve innervation and mitigate TAAD progression. A mouse strain of Ngf gene-driven Sema3a expression (namely NgfSema3a/Sema3a mouse) was established by inserting the 2A-Sema3A expression frame to the Ngf terminating codon using CRISPR/Cas9 technology. TAAD was induced by ß-aminopropionitrile monofumarate (BAPN) both in NgfSema3a/Sema3a mice and wild type (WT) littermates. Contrary to our expectation, the BAPN-induced TAAD was severer in NgfSema3a/Sema3a mice than in wild-type (WT) mice. In addition, NgfSema3a/Sema3a mice showed higher aortic sympathetic innervation, inflammation and extracellular matrix degradation than the WT mice after BAPN treatment. The aortic vascular smooth muscle cells isolated from NgfSema3a/Sema3a mice and pretreated with BAPN in vivo for two weeks showed stronger capabilities of proliferation and migration than that from the WT mice. We conclude that the strategy of Ngf -driven Sema3a expression cannot suppress but worsens the BAPN-induced TAAD. By investigating the aortic phenotype of NgfSema3a/Sema3a mouse strain, we unexpectedly find a path to exacerbate BAPN-induced TAAD which might be useful in future TAAD studies.

Functional genetic variants of the disulfidptosis-related INF2 gene predict survival of hepatitis B virus-related hepatocellular carcinoma.

Disulfidptosis is a novel form of programmed cell death involved in migration and invasion of cancer cells, but few studies investigated the roles of genetic variants in disulfidptosis-related genes in survival of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We used Cox proportional hazards regression analyses, Kaplan-Meier curves and receiver operating characteristic curves to assess effects of genetic variants in 14 disulfidptosis-related genes on overall survival of 866 HBV-HCC patients. The Bayesian false discovery probability was used for multiple testing corrections. We also investigated biological mechanisms of the significant variants through expression quantitative trait loci analyses using the data from publicly available databases, luciferase reporter assays and differential expression analyses. As a result, we identified two independently functional single nucleotide polymorphisms (SNPs) (INF2 rs4072285 G > A and INF2 rs4444271 A > T) that predicted overall survival of HBV-HCC patients, with adjusted hazard ratios of 1.60 (95% CI = 1.22-2.11, P = 0.001) and 1.50 (95% CI = 1.80-1.90, P < 0.001), respectively, after multiple testing correction. Luciferase reporter assays indicated that both INF2 rs4072285 A and INF2 rs4444271 T alleles increased INF2 mRNA expression levels (P < 0.001) that were also higher in HCC tumor tissues than in adjacent normal tissues (P < 0.001); such elevated INF2 expression levels were associated with a poorer survival of HBV-HCC patients (P < 0.001) in the TCGA database. In summary, this study supported that INF2 rs4072285 and INF2 rs4444271 may be novel biomarkers for survival of HBV-HCC patients.

Corrigendum to "Crystal structure of RNA helicase from Saint Louis encephalitis virus and discovery of its inhibitors" [Genes Dis 10 (2023) 389-392].

[This corrects the article DOI: 10.1016/j.gendis.2022.07.001.].

G protein-coupled estrogen receptor 1 ameliorates nonalcoholic steatohepatitis through targeting AMPK-dependent signaling.

Nonalcoholic fatty liver disease (NAFLD), especially nonalcoholic steatohepatitis (NASH), has emerged as a prevalent cause of liver cirrhosis and hepatocellular carcinoma, posing severe public health challenges worldwide. The incidence of NASH is highly correlated with an increased prevalence of obesity, insulin resistance, diabetes, and other metabolic diseases. Currently, no approved drugs specifically targeted for the therapies of NASH partially due to the unclear pathophysiological mechanisms. G protein-coupled estrogen receptor 1 (GPER1) is a membrane estrogen receptor involved in the development of metabolic diseases such as obesity and diabetes. However, the function of GPER1 in NAFLD/NASH progression remains unknown. Here, we show that GPER1 exerts a beneficial role in insulin resistance, hepatic lipid accumulation, oxidative stress, or inflammation in vivo and in vitro. In particular, we observed that the lipid accumulation, inflammatory response, fibrosis, or insulin resistance in mouse NAFLD/NASH models were exacerbated by hepatocyte-specific GPER1 knockout but obviously mitigated by hepatic GPER1 activation in female and male mice. Mechanistically, hepatic GPER1 activates AMP-activated protein kinase signaling by inducing cyclic AMP release, thereby exerting its protective effect. These data suggest that GPER1 may be a promising therapeutic target for NASH.

Regioselective Thiol-yne Reaction of Thiol with ((Methyl-d3)sulfonyl)ethyne: Synthesis of (2-((Methyl-d3)sulfonyl)vinyl)sulfides.

Herein, we have developed a new method for the synthesis of ((methyl-d3)sulfonyl)ethyne, which is cost-effective and environmentally friendly and can be synthesized at the gram level. As an ideal thiol-yne reagent, it can be reacted with various types of thiols to afford (Z)- and (E)-type vinyl sulfides under different conditions with high selectivity. In addition, it can complete the conformational transition from Z- to E-type products under suitable conditions, and can also carry out further derivatization smoothly. The deuterium content of all products was greater than 99%. The preliminary mechanistic studies support the visible light-mediated radical course, and herein provide a novel and efficient synthetic strategy for the direct introduction of deuterated methyl groups, enriching the methods for the construction of C-S bond-containing compounds.

Synaptotagmin-7 mediates cardiac hypertrophy by targeting autophagy.

Sustained cardiac hypertrophy damages the heart and weakens cardiac function, often leading to heart failure and even death. Pathological cardiac hypertrophy has become a central therapeutic target for many heart diseases including heart failure. However, the underlying mechanisms of cardiac hypertrophy, especially the involvement of autophagy program, are still ill-understood. Synaptotagmin-7 (Syt7), a multifunctional and high-affinity calcium sensor, plays a pivotal role in asynchronous neurotransmitter release, synaptic facilitation, and vesicle pool regulation during synaptic transmission. However, little is known about whether Syt7 is expressed in the myocardium and involved in the pathogenesis of heart diseases. Here we showed that Syt7 was significantly upregulated in Ang II-treated hearts and cardiomyocytes. Homozygous syt7 knockout (syt7-/-) mice exhibited significantly attenuated cardiac hypertrophy and fibrosis and improved cardiac function. We further found that Syt7 exerted a pro-hypertrophic effect by suppressing the autophagy process. In exploring the upstream mechanisms, microRNA (miR)-93 was identified to participate in the regulation of Syt7 expression. miR-93 protected hearts against Ang II-induced hypertrophy through targeting Syt7-autophagy pathway. In summary, our data reveal a new cardiac hypertrophy regulator and a novel hypertrophy regulating model composed of miR-93, Syt7 and autophagy program. These molecules may serve as potential therapeutic targets in the treatment of cardiac hypertrophy and heart failure.

Dapagliflozin Suppresses Isoprenaline-Induced Cardiac Hypertrophy Through Inhibition of Mitochondrial Fission.

ABSTRACT: Dapagliflozin (DAPA) is a novel oral hypoglycemic agent, and there is increasing evidence that DAPA has a protective effect against cardiovascular disease. The study aimed to investigate how DAPA inhibits cardiac hypertrophy and explore its potential mechanisms. By continuously infusing isoprenaline (ISO) for 2 weeks using a subcutaneous osmotic pump, a cardiac hypertrophic model was established in male C57BL/6 mice. On day 14 after surgery, echocardiography showed that left ventricle mass (LV mass), interventricular septum, left ventricle posterior wall diastole, and left ventricular posterior wall systole were significantly increased, and ejection fraction was decreased compared with control mice. Masson and Wheat Germ Agglutinin staining indicated enhanced myocardial fibrosis and cell morphology compared with control mice. Importantly, these effects were inhibited by DAPA treatment in ISO-induced mice. In H9c2 cells and neonatal rat cardiomyocytes, we found that mitochondrial fragmentation and mitochondrial oxidative stress were significantly augmented in the ISO-induced group. However, DAPA rescued the cardiac hypertrophy in ISO-induced H9c2 cells and neonatal rat cardiomyocytes. Mechanistically, we found that DAPA restored the PIM1 activity in ISO-induced H9c2 cells and subsequent increase in dynamin-associated protein 1 (Drp1) phosphorylation at S616 and decrease in Drp1 phosphorylation at S637 in ISO-induced cells. We found that DAPA mitigated ISO-induced cardiac hypertrophy by suppressing Drp1-mediated mitochondrial fission in a PIM1-dependent fashion.

Simultaneous Preparation of Sulfides/Selenides and Sulfones via Synergistic Nickel-Catalyzed Reductive Coupling and SN2 Reaction.

Sulfone compounds and thioether compounds are two highly valuable classes of compounds, but it is challenging to prepare sulfone and thioether compounds simultaneously and efficiently. Here we report that sulfides/selenides and sulfones can be obtained simultaneously using allyl bromide/benzyl bromide-activated alkyl bromides and thiosulfonates/selenosulfonates using a nickel-catalyzed reductive coupling and SN2 synergistic strategy, which is characterized by excellent atom and step economy, mild reaction conditions, broad functional group compatibility, and excellent yields.

Potentially Functional Genetic Variants in the NRF2 Signaling Pathway Genes are Associated With HBV-related Hepatocellular Carcinoma Survival.

The nuclear factor E2-related factor 2 (NRF2) signaling pathway is one of the most important cell defense pathways. However, it is unclear whether genetic variants in NRF2 signaling pathway genes are associated with the survival of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). In the present study, we utilized a new hypothesis-driven approach based on biological pathways to investigate the associations between 17919 single nucleotide polymorphisms (SNPs) in 137 NRF2 signaling pathway genes and the overall survival (OS) of 866 patients with HBV-related HCC. As a result, two independent SNPs with potential biological function were identified to be significantly associated with HBV-related HCC OS: [SLC2A9 rs28643326 T>C: hazard ratio (HR) = 0.74, 95% confidence interval (95% CI) = 0.62-0.89, P < 0.001 and SLC5A10 rs2472711 G>T: HR = 0.81, 95% CI = 0.71-0.93, P = 0.003, respectively]. The expression quantitative trait loci (eQTL) analysis further revealed that the rs28643326 C allele was significantly associated with increased levels of SLC2A9 mRNA expression (P < 0.001), and higher mRNA expression levels of SLC2A9 in adjacent normal liver tissues were associated with better survival. Although the association between the rs2472711 T allele and the mRNA expression of SLC5A10 was not statistically significant (P = 0.200), the fact that rs2472711 is located at the DNase I hypersensitivity site and is a marker for promoter and enhancer histones also suggests that it may have the function of regulating its corresponding gene expression. In conclusion, genetic variants of NRF2 signaling pathway genes may serve as potential prognostic biomarkers for HBV-related HCC and also provide a solid basis for further mechanistic exploration.

The Regulatory Effect of Receptor-Interacting Protein Kinase 3 on CaMKIIδ in TAC-Induced Myocardial Hypertrophy.

Necroptosis is a newly discovered mechanism of cell death, and its key regulatory role is attributed to the interaction of receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3. Ca2+/calmodulin-dependent protein kinase (CaMKII) is a newly discovered RIPK3 substrate, and its alternative splicing plays a fundamental role in cardiovascular diseases. In the present study, we aimed to explore the role and mechanism of necroptosis and alternative splicing of CaMKIIδ in myocardial hypertrophy. Transverse aortic constriction (TAC) was performed on wild-type and knockout mice to establish the model of myocardial hypertrophy. After 3 weeks, echocardiography, cardiac index, cross-sectional area of myocardial cells, hypertrophic gene expression, myocardial damage, and fibers were assessed. Moreover, we detected the levels of inflammatory factors (IL-6 and TNF-α) and examined the expressions of necroptosis-related proteins RIPK3, RIPK1, and phosphorylated MLKL. Meanwhile, we tested the expression levels of splicing factors ASF/SF2 and SC-35 in an attempt to explore CaMKII δ. The relationship between variable splicing disorder and the expression levels of splicing factors ASF/SF2 and SC-35. Further, we also investigated CaMKII activation, oxidative stress, and mitochondrial ultrastructure. In addition, wild-type mice were administered with a recombinant adeno-associated virus (AAV) carrying RIPK3, followed by TAC surgery to construct a model of myocardial hypertrophy, and the above-mentioned indicators were tested after 3 weeks. The results showed that RIPK3 deficiency could alleviate cardiac dysfunction, myocardial injury, aggravation of necrosis, and CaMKII activation induced by TAC surgery in mice with myocardial hypertrophy. Tail vein injection of AAV could reverse cardiac dysfunction, myocardial damage, aggravation of necrosis, and CaMKII activation in mice with myocardial hypertrophy. These results proved that RIPK3 could be used as a molecular intervention target for the prevention and treatment of myocardial hypertrophy.

Crystal structure of mRNA cap (guanine-N7) methyltransferase E12 subunit from monkeypox virus and discovery of its inhibitors.

In July 2022, the World Health Organization announced monkeypox as a public health emergency of international concern (PHEIC), and over 85,000 global cases have been reported currently. However, preventive and therapeutic treatments for the monkeypox virus (MPXV) remain limited. MPXV mRNA cap N7 methyltransferase (MTase) is composed of two subunits (E1 C-terminal domain (E1CTD) and E12) which are essential for the replication of MPXV. Here, we solved a 2.16 Å crystal structure of E12. We also docked the D1CTD of the vaccinia virus (VACV) corresponding to the E1CTD in MPXV with E12 and found critical residues at their interface. These residues were further used for drug screening. After virtual screening, the top 347 compounds were screened out and a list of top 20 potential MPXV E12 inhibitors were discovered, including Rutin, Quercitrin, Epigallocatechin, Rosuvastatin, 5-hydroxy-L-Tryptophan, and Deferasirox, etc., which were potential E12 inhibitors. Taking the advantage of the previously unrecognized special structure of MPXV MTase composing of E1CTD and E12 heterodimer, we screened for inhibitors targeting MTase for the first time based on the interface between the heterodimer of MPXV MTase. Our study may provide insights into the development of anti-MPXV drugs.

Identification of Potential JNK3 Inhibitors: A Combined Approach Using Molecular Docking and Deep Learning-Based Virtual Screening.

JNK3, a member of the MAPK family, plays a pivotal role in mediating cellular responses to stress signals, with its activation implicated in a myriad of inflammatory conditions. While JNK3 holds promise as a therapeutic target for neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases, there remains a gap in the market for effective JNK3 inhibitors. Despite some pan-JNK inhibitors reaching clinical trials, no JNK-targeted therapies have achieved market approval. To bridge this gap, our study introduces a sophisticated virtual screening approach. We begin with an energy-based screening, subsequently integrating a variety of rescoring techniques. These encompass glide docking scores, MM/GBSA, and artificial scoring mechanisms such as DeepDock and advanced Graph Neural Networks. This virtual screening workflow is designed to evaluate and identify potential small-molecule inhibitors with high binding affinity. We have implemented a virtual screening workflow to identify potential candidate molecules. This process has resulted in the selection of ten molecules. Subsequently, these ten molecules have undergone biological activity evaluation to assess their potential efficacy. Impressively, molecule compound 6 surfaced as the most promising, exhibiting a potent kinase inhibitory activity marked by an IC50 of 130.1 nM and a notable reduction in TNF-α release within macrophages. This suggests that compound 6 could potentially serve as an effective inhibitor for the treatment of neuroinflammation and neurodegenerative diseases. The prospect of further medicinal modifications to optimize compound 6 presents a promising avenue for future research and development in this field. Utilizing binding pose metadynamics coupled with molecular dynamics simulations, we delved into the explicit binding mode of compound 6 to JNK3. Such insights pave the way for refined drug development strategies. Collectively, our results underscore the efficacy of the hybrid virtual screening workflow in the identification of robust JNK3 inhibitors, holding promise for innovative treatments against neuroinflammation and neurodegenerative disorders.