T cell modulation plays a fundamental role to adaptive and innate immunity, which aids the recognition and defense against pathogens while also maintaining self-tolerance. Numerous molecular pathways participate in this process including thymic selection, T cell receptor and antigen-presenting cells cross linkage, along with co-stimulatory signaling cascades. The present review demonstrates a holistic analysis of various classic and novel mechanisms that govern T cell regulation and emerging therapeutic applications. Recent advancements have introduced novel roles in the journey of T cell modulation that can have a pivotal impact on the understanding of this process; for example, phase separation of the linker for activation of T cells, and the newer application of chimeric antigen receptor (CAR) T cell therapy in autoimmune diseases. While discoveries of proximal and distal signal transduction pathways have contributed to the comprehension of T cell anergy, cytokine-mediated differentiation and the delicate balance between immune activation and tolerance, there are still unresolved debates about further molecular mechanisms. There are also still questions about the long-term side effects of CAR-T cell therapy. Deeper research and analysis are required to further aid the understanding and use of this novel therapeutic approach.

A well-recognized molecular entity involved in pain-related neuroplasticity is the N-methyl-D-aspartate receptor (NMDAR), which is crucial for developing chronic pain. Likewise, the pannexin 1 (Panx1) channel has been described as necessary for initiating and maintaining neuropathic pain, driving nociceptive signals dependent on spinal NMDAR through several possible mechanisms. Through behavioral, pharmacological, and molecular approaches, our study in male rats has revealed several key findings: (1) neurons located in spinal cord laminae I and II express functional Panx1 channels in both neuropathic and sham rats. These channels can open (indicated by YOPRO-1 uptake) through the stimulation of NMDARs with intrathecal NMDA; (2) intrathecal NMDA leads to increased expression of pSrc and pPanx1 in dorsal horn neurons. This elevation exacerbates existing mechanical hyperalgesia in nerve-injured rats; (3) inhibition of Src with intrathecal PP2 or blockade of Panx1 with intrathecal 10 Panx effectively mitigates NMDA-induced effects and reduces the spontaneous mechanical hyperalgesia of nerve-injured rats. Notably, while 10 Panx successfully alleviates hyperalgesia, it does not alter pSrc expression; and (4) NMDA-stimulated YOPRO-1 uptake in neurons of laminae I-II of spinal cord slices were prevented by the NMDAR antagonist D-AP5, the Src inhibitor PP2 (but not PP3), as well as with the 10 Panx and carbenoxolone. Therefore, NMDAR activation in dorsal horn neurons triggers an NMDAR-Src-Panx1 signaling pathway, where Panx1 acts as an enhancing effector in neuropathic pain. This implies that disrupting the NMDAR-Panx1 communication (eg, through Src inhibitors and/or Panx1 blockers) may offer a valuable strategy for managing some forms of chronic pain.

Aging affects the reparative potency of mesenchymal stem/stromal cells (MSCs) by diminishing their proliferation and differentiation capability; making them unsuitable for regenerative purposes. Earlier we showed that MSCs acquire the expression of CD45 as a consequence of aging, and this increased expression is associated with downregulated expression of osteogenic markers and upregulated expression of adipogenic and osteoclastogenic markers. However, whether CD45 is actively involved in the aging-mediated deregulated differentiation in the MSCs was not elucidated.

In the present study, we showed that pharmacological inhibition of CD45-specific phosphatase activity in the aged MSCs restores their differentiation potential to young-like. Investigation of the molecular mechanism involved in the process showed that several regulatory kinases like p38, p44/42, Src, and GSK3β are in their dephosphorylated form in the aged MSCs, and importantly, this status gets reversed by the application of a CD45-specific PTP inhibitor. Conversely, pharmacological inhibition of these kinases in young MSCs imposes an aged-like gene expression profile on them. Additionally, we also showed that the secretome of aged MSCs affects the viability and differentiation of primary chondrocytes, and this detrimental effect is reversed by treating aged MSCs with the PTP inhibitor. Our data demonstrate that the aging-mediated expression of CD45 in MSCs alters their differentiation profile by dephosphorylating several kinases and treating the aged MSCs with a CD45 PTP activity inhibitor rejuvenates them.

CD45 can be used as an aging marker for mesenchymal stem cells. Alteration of CD45 phosphatase activity could have significant implications for the use of MSCs in regenerative medicine.

BAM15 is a novel mitochondrial uncoupling agent derived from a synthetic source, that has been wildly explored for its ability to enhance mitochondrial respiration and metabolic flexibility. In this study, we investigated the underlying mechanisms of BAM15 on atherosclerosis (AS) through experimental validation, RNA-seq and molecular docking. The results showed that oral administration of BAM15 suppressed atherosclerosis in western diet (WD)-fed ApoE(-/-) mice and significantly improved the hyperlipidemia. And the increased serum ALT, AST and liver TC, TG, ALT, AST in ApoE(-/-) mice were reduced by BAM15 treatment. In in vitro experiments BAM15 inhibited RAW264.7 macrophages invasive ability and reduced palmitic acid-induced lipid accumulation. RNA-seq results confirmed the differential genes after BAM15 treatment and 140 common targets were identified by intersecting with AS-related targets. A protein-protein interaction (PPI) network analysis high-lighted IL1A, SRC and CSF3 as key targets of BAM15 against AS, which is further verified by molecular docking and western blot. Molecular dynamics analysis results confirmed that BAM15 exhibits strong affinity with the IL-1α, SRC and CSF3 proteins. This study indicates that BAM15 inhibits atherosclerosis through a multi-molecular mechanism, and we propose it as a novel anti-atherosclerotic drug.

S-Palmitoylation is a reversible post-translational modification that tunes the localization, stability, and function of an impressive array of proteins including ion channels, G-proteins, and synaptic proteins. Indeed, altered protein palmitoylation is linked to various human diseases including cancers, neurodevelopmental and neurodegenerative diseases. As such, strategies to selectively manipulate protein palmitoylation with enhanced temporal and subcellular precision are sought after to both delineate physiological functions and as potential therapeutics. Here, we develop chemogenetically and optogenetically inducible engineered depalmitoylases to manipulate the palmitoylation status of target proteins. We demonstrate that this strategy is programmable allowing selective depalmitoylation in specific organelles, triggered by cell-signaling events, and of individual protein complexes. Application of this methodology revealed bidirectional tuning of neuronal excitability by distinct depalmitoylases. Overall, this strategy represents a versatile and powerful method for manipulating protein palmitoylation in live cells, providing insights into their regulation in distinct physiological contexts.

Disease-Associated Microglia (DAM) are a focus in Alzheimer's disease (AD) research due to their central involvement in the response to amyloid-beta plaques. Microglial Toll-like receptor 4 (TLR4) is instrumental in the binding of fibrillary amyloid proteins, while Lyn kinase (Lyn) is a member of the Src family of non-receptor tyrosine kinases involved in immune signaling. Lyn is a novel, non-canonical, intracellular adaptor with diverse roles in cell-specific signaling which directly binds to TLR4 to modify its function. Lyn can be activated in response to TLR4 stimulation, leading to phosphorylation of various substrates and modulation of inflammatory and phagocytosis signaling pathways. Here, we investigated the TLR4-Lyn interaction in neuroinflammation using WT, 5XFAD, and 5XFAD x Lyn-/- mouse models by western blotting (WB), co-immunoprecipitation (co-IP), immunohistochemistry (IHC) and flow cytometric (FC) analysis. A spatial transcriptomic analysis of microglia in WT, 5XFAD, and 5XFAD x Lyn-/- mice revealed essential genes involved in neuroinflammation, Aβ phagocytosis, and neuronal damage. Finally, we explored the effects of a synthetic, TLR4-Lyn modulator protein (TLIM) through an in vitro AD model using primary murine microglia. Our WB, co-IP, IHC, and FC data show an increased, novel, direct protein-protein interaction between TLR4 and Lyn kinase in the brains of 5XFAD mice compared to WT. Furthermore, in the absence of Lyn (5XFAD x Lyn-/- mice); increased expression of protective Syk kinase was observed, enhanced microglial Aβ phagocytosis, increased astrocyte activity, decreased neuronal dystrophy, and a further increase in the cell survival signaling and protective DAM population was noted. The DAM population in 5XFAD mice which produce more inflammatory cytokines and phagocytose more Aβ were observed to express greater levels of TLR4 and Lyn. Pathway analysis comparison between WT, 5XFAD, and 5XFAD x Lyn-/- mice supported these findings via our microglial spatial transcriptomic analysis. Finally, we created an in vitro co-culture system with primary murine microglial and primary murine hippocampal cells exposed to Aβ as a model of AD. When these co-cultures were treated with our TLR4-Lyn Interaction Modulators (TLIMs), an increase in Aβ phagocytosis and a decrease in neuronal dystrophy was seen. Lyn kinase has a central role in modulating TLR4-induced inflammation and Syk-induced protection in a 5XFAD mouse model. Our TLIMs ameliorate AD sequalae in an in vitro model of AD and could be a promising therapeutic strategy to treat AD.

Stemona tuberosa is widely recognized for its traditional applications as an anti-cancer agent. This study aimed to assess the anti-cancer properties of S. tuberosa in human lung adenocarcinoma A549 cells. Among the various solvent extracts of S. tuberosa, the methanolic extract showed the highest toxicity against A549 cells. The S. tuberosa extract elicited cytotoxic effects and suppressed colony formation in A549 cells in a dose-dependent manner. S. tuberosa activity was further supported by AO/EtBr staining, increased caspase 3/6 activity, upregulation of pro-apoptotic genes, DNA damage, and elevated lipid peroxidation, with decreasing antioxidant levels. LC-MS analysis identified 80 predominant secondary metabolites in the methanolic extracts of S. tuberosa. A network pharmacology study identified SRC as the primary target of compounds identified from S. tuberosa. SRC protein is crucial for advancing lung cancer because of its function in cell proliferation, survival, and metastasis. Among the various compounds identified from S. tuberosa extract, 4-Azatricyclo [4.3.1.13,8] undecan-5-one (ADE) (- 10.88 kcal/mol) and Dihydro-normorphine, 3-desoxy- (DNY) (- 10.83 kcal/mol) exhibited notable binding affinities for SRC. Further analysis using molecular dynamics simulations (100 ns) validated the stability of SRC-ligand complexes, with RMSD of 1.8 and 2.2 Å for ADE and DNY, respectively, alongside the establishment of essential hydrogen bonds with pivotal residues, including ASP408, ALA403, and THR438. Finally, gmx._MMPBSA showed favourable ΔGbind values for ADE (- 15.06 ± 0.11 kcal/mol) and DNY (- 15.66 ± 0.25 kcal/mol), which highlights the significant potential of ADE and DNY as effective SRC inhibitors, suggesting S. tuberosa as a novel candidate for cancer therapy.

Small molecule kinase inhibitors are crucial in the treatment of tumors, and the development of novel inhibitors is a primary approach to combat the continuous emergence of drug resistance. Macrocyclization has emerged as a cutting-edge strategy to enhance the potency, selectivity, and pharmacokinetic properties of these inhibitors by altering their biological and physicochemical characteristics compared to their acyclic counterparts.

The present article provides a comprehensive overview of the recent advancements in macrocyclic small molecule inhibitors and their inhibitory activities against various cancer cells, which have been patented since 2019.

To date, small-molecule kinase inhibitors have demonstrated remarkable therapeutic efficacy in clinical settings. Recent patents have primarily focused on addressing challenges associated with resistance mutations. Despite the significant success achieved in developing selective kinase agents, the identification of new targets and emergence of novel mutations necessitate the development of novel small-molecule inhibitors. Macrocyclic compounds possess distinctive conformational constraints, enhanced inhibitor potency and selectivity, as well as favorable pharmacokinetic properties, rendering them safe, efficient, selective, low-toxicity agents with unique structural characteristic.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of the T cell receptor signaling pathway and is therefore a target of interest for immunooncology. Nonselective HPK1 inhibitors may affect other kinase components of T cell activation, blunting the beneficial impact of enhanced T cell activity that results from HPK1 inhibition itself. Here, we report the discovery of pyrazine carboxamide HPK1 inhibitors and their optimization through structure-based drug design to afford a highly selective HPK1 inhibitor, compound 24 (AZ3246). This compound induces IL-2 secretion in T cells with an EC50 of 90 nM without inhibiting antagonistic kinases, exhibits pharmacokinetic properties consistent with oral dosing, and demonstrates antitumor activity in the EMT6 syngeneic mouse model.

Decidualization, the transformation of human endometrial stromal cells from a fibroblast-like to a rounded morphology, is crucial for creating a receptive intrauterine environment that supports successful embryo implantation. While decidual markers such as insulin-like growth factor-binding protein 1 and prolactin are well studied, the specific signaling mechanisms underlying morphological changes during decidualization remain unclear. In this study, we identified the phosphatidic acid (PA)-Src-focal adhesion kinase (FAK)-RhoA/Rho-associated protein kinase (ROCK) signaling pathway as a critical regulator of cytoskeletal rearrangement during PA-induced decidualization in human endometrial stromal cells. PA, a product of phospholipase D1, activates FAK, initiating a cascade of events involving Src-family kinases and RhoA signaling, ultimately leading to the cytoskeletal changes necessary for decidualization. Our in vitro experiments showed that PA-induced decidualization involved the formation of stress fibers mediated by ROCK activation. The traditional decidual markers, insulin-like growth factor-binding protein 1 and prolactin, did not significantly influence these morphological changes, suggesting that the PA-induced pathway operates independently of these markers. In vivo studies in ovariectomized mice demonstrated that PA injection into the uterine horn increased the uterine cavity weight and wall thickness, reinforcing the role of PA in promoting decidualization. These findings highlight the importance of the PA-Src-FAK-RhoA-ROCK pathway in regulating cytoskeletal dynamics during decidualization and suggest potential therapeutic targets for addressing implantation-associated infertility.

A potentially promising approach to targeted cancer prevention in genetically at-risk populations is the pharmacological upregulation of DNA repair pathways. SMUG1 is a base excision repair enzyme that ameliorates adverse genotoxic and mutagenic effects of hydrolytic and oxidative damage to pyrimidines. Here we describe the discovery and initial cellular activity of a small-molecule activator of SMUG1. Screening of a kinase inhibitor library and iterative rounds of structure-activity relationship studies produced compound 40 (SU0547), which activates SMUG1 by as much as 350 ± 60 % in vitro at 100 nM, with an AC50 of 4.3 ± 1.1 µM. To investigate the effect of compound 40 on endogenous SMUG1, we performed in vitro cell-based experiments with 5-hydroxymethyl-2'-deoxyuridine (5-hmdU), a pyrimidine oxidation product that is selectively removed by SMUG1. In several human cell lines, compound 40 at 3-5 µM significantly reduces the cytotoxicity of 5-hmdU and decreases levels of double-strand breaks induced by the damaged nucleoside. We conclude that the SMUG1 activator compound 40 is a useful tool to study the mechanisms of 5-hmdU toxicity and the potentially beneficial effects of suppressing damage to pyrimidines in cellular DNA.

Endometriosis affects over 190 million women globally, and effective therapies are urgently needed to address the burden of endometriosis on women's health. Using an artificial intelligence (AI)-driven target discovery platform, two unreported therapeutic targets, guanylate-binding protein 2 (GBP2) and hematopoietic cell kinase (HCK) are identified, along with a drug repurposing target, integrin beta 2 (ITGB2) for the treatment of endometriosis. GBP2, HCK, and ITGB2 are upregulated in human endometriotic specimens. siRNA-mediated knockdown of GBP2 and HCK significantly reduced cell viability and proliferation while stimulating apoptosis in endometrial stromal cells. In subcutaneous and intraperitoneal endometriosis mouse models, siRNAs targeting GBP2 and HCK notably reduced lesion volume and weight, with decreased proliferation and increased apoptosis within lesions. Both subcutaneous and intraperitoneal administration of Lifitegrast, an approved ITGB2 antagonist, effectively suppresses lesion growth. Collectively, these data present Lifitegrast as a previously unappreciated intervention for endometriosis treatment and identify GBP2 and HCK as novel druggable targets in endometriosis treatment. This study underscores AI's potential to accelerate the discovery of novel drug targets and facilitate the repurposing of treatment modalities for endometriosis.

Diabetic kidney disease is a significant complication of diabetes mellitus, and exposure to certain chemicals may play a role in its development. Triphenyl phosphate (TPHP) is commonly used in plastics and flame retardants. This study aims to investigate the potential impact of TPHP and its metabolite diphenyl phosphate (DPHP) on diabetic kidney disease using various methods, including network toxicology, molecular docking, and cell experiments like CCK8 assay and real-time-PCR. The research examined the relationship between urinary DPHP levels and kidney function in American adults using data from the National Health and Nutrition Examination Survey (NHANES) from 2017 to March 2020. Additionally, the study explored the targets of action for TPHP and DPHP using network toxicity analysis, conducted protein interaction analysis, and explored the functional aspects of action through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. Furthermore, the study identified key proteins involved in the action and conducted experimental verification by treating cells with TPHP and DPHP. Toxicity analysis showed that TPHP could cause dose-dependent toxicity in mouse podocyte clone 5 (MPC5) and mouse mesangial cells (MES13). The study also detected mRNA expression of core targets molecularly docked with TPHP and DPHP using real-time-PCR. The results indicated statistically significant regulation of most core targets by TPHP and DPHP in MPC5, MES13, and human kidney-2 cells.

Glaucoma, a leading cause of irreversible blindness, is characterised by progressive optic nerve damage, with elevated intraocular pressure (IOP) and extracellular matrix (ECM) remodelling in the lamina cribrosa (LC) contributing to its pathophysiology. While current treatments focus on IOP reduction, they fail to address the underlying fibrotic changes that perpetuate neurodegeneration. The Src proto-oncogene, a non-receptor tyrosine kinase, has emerged as a key regulator of cellular processes, including fibroblast activation, ECM deposition, and metabolism, making it a promising target for glaucoma therapy. Beyond its well-established roles in cancer and fibrosis, Src influences pathways critical to trabecular meshwork function, aqueous humour outflow, and neurodegeneration. However, the complexity of Src signalling networks remains a challenge, necessitating further investigation into the role of Src in glaucoma pathogenesis. This paper explores the therapeutic potential of Src inhibition to mitigate fibrotic remodelling and elevated IOP in glaucoma, offering a novel approach to halting disease progression.

Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are major forms of nephrotic syndrome that remain difficult to treat. MCD and FSGS have distinct but also overlapping clinical, histological, metabolic, and molecular features. Effective use of immunosuppressive drugs, activated immune cells, altered cytokine profiles, and upregulated signaling pathways suggest a link between immune dysfunction and nephrotic syndrome, but the exact mechanism of immunopathogenesis is unclear. Immune dysfunction is an area of ongoing research for identifying novel molecular targets for treating nephrotic syndrome. However, the available animal models do not directly address the role of immune dysfunction in nephrotic syndrome.

Genetic analysis indicates that heterogeneous genes related to the podocyte-specific proteins may indirectly cause damage to filtration barrier and influence the onset and progression of nephrotic syndrome. SH3BP2 protein regulates several pathways through its role as a scaffold for many signaling mediators and enzymes. SH3BP2 is expressed in immune as well as in nonimmune cells including podocytes. The role of SH3BP2 is discussed in the context of cells and molecules of adaptive and innate immune systems. Available information on the importance of SH3BP2 in diseases other than nephrotic syndrome and its role in the immunopathogenesis of human nephrotic syndrome are summarized. We outline the key features of a transgenic mouse strain with a gain-in-function mutation (Sh3bp2 KI/KI ) as a potential model to study immunopathogenesis of nephrotic syndrome.

Non-receptor, non-catalytic proteins such as SH3BP2 are a novel group of proteins that regulate the innate and adaptive immune responses in nephrotic syndrome. New evidence suggests a critical role of SH3BP2 in immunopathogenesis of nephrotic syndrome. Our recent results demonstrate that transgenic mice (Sh3bp2 KI/KI ) with a gain-in-function mutation will likely be a unique model to study immunopathogenesis of nephrotic syndrome.

Leishmania is responsible for a neglected tropical disease affecting millions of people around the world and could potentially spread more due to climate change. This disease not only leads to significant morbidity but also imposes substantial social and economic burdens on affected populations, often exacerbating poverty and health disparities. Despite the complexity and effectiveness of the immune response, the parasite has developed various strategies to evade detection and manipulates host cells in favor of its replication. These evasion strategies start at early stages of the infection by hijacking immune receptors to silence critical cellular response that would otherwise limit the pathogen's propagation. Among these receptors, Fc receptors have emerged as a significant player in the immune evasion strategies employed by microorganisms, as they could promote inhibitory pathways. This review explores the potential role of one of these immune receptors, the FcγRIIA, in leishmaniasis and how this parasite may use it and the signaling pathways downstream to evade the host immune response. By understanding the potential interactions between Leishmania and immune receptors such as FcγRIIA, we may identify novel targets for therapeutic intervention aimed to enhance the host immune response and reduce the burden of this disease.

IgA nephropathy (IgAN) is a leading cause of renal failure, but its pathogenesis remains unclear, complicating diagnosis and treatment. The invasive nature of renal biopsy highlights the need for non-invasive diagnostic biomarkers. Bulk RNA sequencing (RNA-seq) of urine offers a promising approach for identifying molecular changes relevant to IgAN.

We performed bulk RNA-seq on 53 urine samples from 11 untreated IgAN patients and 11 healthy controls, integrating these data with public renal RNA-seq, microarray, and scRNA-seq datasets. Machine learning was used to identify key differentially expressed genes, with protein expression validated by immunohistochemistry (IHC) and drug-target interactions explored via molecular docking.

Urine RNA-seq analysis revealed differential expression profiles, from which TYROBP and HCK were identified as key biomarkers using machine learning. These biomarkers were validated in both a test cohort and an external validation cohort, demonstrating strong predictive accuracy. scRNA-seq confirmed their cell-specific expression patterns, correlating with renal function metrics such as GFR and serum creatinine. IHC further validated protein expression, and molecular docking suggested potential therapeutic interactions with IgAN treatments.

TYROBP and HCK are promising non-invasive urinary biomarkers for IgAN. Their predictive accuracy, validated through machine learning, along with IHC confirmation and molecular docking insights, supports their potential for both diagnostic and therapeutic applications in IgAN.

Laminopathies represent a wide range of genetic disorders caused by mutations in gene-encoding proteins of the nuclear lamina. Altered nuclear mechanics have been associated with laminopathies, given the key role of nuclear lamins as mechanosensitive proteins involved in the mechanotransduction process. To shed light on the nuclear partners cooperating with altered lamins, we focused on Src tyrosine kinase, known to phosphorylate proteins of the nuclear lamina. Here, we demonstrated a tight relationship between lamin A/C and Src in skin fibroblasts from two laminopathic patients, assessed by advanced imaging-based microscopy techniques. With confocal laser scanning and Stimulated Emission Depletion (STED) microscopy, a statistically significant higher co-distribution between the two proteins was observed in patients' fibroblasts. Furthermore, the time-domain fluorescence lifetime imaging microscopy, combined with Förster resonance energy transfer detection, demonstrated a decreased lifetime value of Src (as donor fluorophore) in the presence of lamin A/C (as acceptor dye) in double-stained fibroblast nuclei in both healthy cells and patients' cells, thereby indicating a molecular interaction that resulted significantly higher in laminopathic cells. All these results demonstrate a molecular interaction between Src and lamin A/C in healthy fibroblasts and their aberrant interaction in laminopathic nuclei, thus creating the possibilities of new diagnostic and therapeutic approaches for patients.

The development of antiviral strategies is a key task of biomedical research, but broad-spectrum virus inhibitors are scarce. Here we show that fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors reduce infection of several cell types with DNA and RNA viruses by blocking early stages of infection, but not viral cell association. Unexpectedly, their antiviral activity was largely independent of FGFR kinase inhibition. RNA profiling showed upregulation of interferon response genes by FGFR inhibitors, but their expression did not correlate with the antiviral activity in infected cells. Using bioinformatics analysis of kinome data, targeted kinase assays, siRNA-mediated knock-down and pharmacological inhibition experiments, we show that blockade of Src family kinases, in particular Lyn, is mainly responsible for the antiviral activity of FGFR inhibitors. These results identify FGFR inhibitors as broad-spectrum antiviral agents and suggest the poorly studied Lyn kinase as a promising target for the treatment of viral infections.

Vascular retinopathy, characterized by abnormal blood vessel growth in the retina, frequently results in vision impairment or loss. Neovascular tufts, a distinctive pathologic feature of this condition, are highly leaky blood vessel structures, exacerbating secondary complications. Despite their clinical significance, the mechanisms underlying tuft development are not fully elucidated, posing challenges for effective management and treatment of vascular retinopathy. This study investigates the role of cellular (c)-Src in neovascular tuft formation. Although c-Src is a pivotal regulator in developmental angiogenesis within the retinal vasculature, its specific role in governing pathologic retinal angiogenesis remains to be fully understood. Herein, the oxygen-induced retinopathy model was used for neovascular tuft formation in both Cre-mediated vascular-specific c-Src knockout mice and wild-type littermates. High-resolution imaging and analysis of isolated retinas were conducted. c-Src depletion demonstrated a significant reduction in neovascular tufts within the oxygen-induced retinopathy model. This decrease in tuft formation was observed independently of any alterations in cell death, cell proliferation, or cell adhesion, and the absence of c-Src did not impact tuft pericyte coverage and junctional morphology. These findings underline the critical role of c-Src in the pathogenesis of neovascular tufts in vascular retinopathy. Understanding the molecular mechanisms involving c-Src may offer valuable insights for the development of targeted therapies aimed at mitigating vision-threatening complications associated with retinopathy.

Background: Lupus Nephritis (LN) is the primary causation of kidney injury in systemic lupus erythematosus (SLE). Ferroptosis is a programmed cell death. Therefore, understanding the crosstalk between LN and ferroptosis is still a significant challenge. Methods: We obtained the expression profile of LN kidney biopsy samples from the Gene Expression Omnibus database and utilised the R-project software to identify differentially expressed genes (DEGs). Then, we conducted a functional correlation analysis. Ferroptosis-related genes (FRGs) and differentially expressed genes (DEGs) crossover to select FRGs with LN. Afterwards, we used CIBERSORT to assess the infiltration of immune cells in both LN tissues and healthy control samples. Finally, we performed immunohistochemistry on LN human renal tissue. Results: 10619 DEGs screened from the LN biopsy tissue were identified. 22 hub-ferroptosis-related genes with LN (FRGs-LN) were screened out. The CIBERSORT findings revealed that there were significant statistical differences in immune cells between healthy control samples and LN tissues. Immunohistochemistry further demonstrated a significant difference in HRAS, TFRC, ATM, and SRC expression in renal tissue between normal and control groups. Conclusion: We developed a signature that allowed us to identify 22 new biomarkers associated with FRGs-LN. These findings suggest new insights into the pathology and therapeutic potential of LN ferroptosis inhibitors and iron chelators.

Anthraquinones have attracted considerable interest in the realm of cancer treatment owing to their potent anticancer properties. This study evaluates the potential of a series of new anthraquinone derivatives as anticancer agents for non-small-cell lung cancer (NSCLC). The compounds were subjected to a range of tests to assess their cytotoxic and apoptotic properties, ability to inhibit colony formation, pro-DNA damage functions, and capacity to inhibit the activity of tyrosine kinase proteins (PTKs). Based on the research findings, it has been discovered that most active derivatives (i84, i87, and i90) possess a substantial capability to impede the viability of NSCLC while having mostly a negligible effect on the human kidney cell line. Moreover, the anthraquinones displayed pro-apoptotic and genotoxic attributes while blocking the phosphorylation of multiple PTKs. Collectively, our findings indicate that these derivatives may demonstrate promising potential as effective anticancer agents for lung cancer treatment.

Collective cell migration is key during development, wound healing, and metastasis and relies on coordinated cell behaviors at the group level. Src kinase is a key signalling protein for the physiological functions of epithelia, as it regulates many cellular processes, including adhesion, motility, and mechanotransduction. Its overactivation is associated with cancer aggressiveness. Here, we take advantage of optogenetics to precisely control Src activation in time and show that its pathological-like activation slows the collective rotation of epithelial cells confined into circular adhesive patches. We interpret velocity, force, and stress data during period of non-activation and period of activation of Src thanks to a hydrodynamic description of the cell assembly as a polar active fluid. Src activation leads to a 2-fold decrease in the ratio of polar angle to friction, which could result from increased adhesiveness at the cell-substrate interface. Measuring internal stress allows us to show that active stresses are subdominant compared to traction forces. Our work reveals the importance of fine-tuning the level of Src activity for coordinated collective behaviors.

Thirteen new 4,7-disubstituted pyrimido[4,5-d]pyrimidines were synthesized via a straightforward methodology starting from thiourea. The anti-proliferative activity of these compounds was evaluated across a diverse panel of eight cancer cell lines, with derivatives 7d and 7h showing efficacy against several hematological cancer types. Furthermore, all compounds were assessed for their antiviral potency against a panel of viruses. Compounds featuring a cyclopropylamino group and an aminoindane moiety exhibited remarkable efficacy against human coronavirus 229E (HCoV-229E). These findings highlight the pyrimidino[4,5-d]pyrimidine scaffold as an interesting framework for the design of novel antiviral agents against HCoVs, with compounds 7a, 7b, and 7f emerging as strong candidates for further investigation.

Src kinase is one of the key regulators of cellular metabolism and is dysregulated in numerous diseases, including cancer, neurodegenerative diseases, and particularly Alzheimer's disease. Despite its therapeutic importance, its full-length structure has never been obtained before, as it contains an intrinsically disordered regulatory region, SH4UD. The SH4UD region is crucial for Src activation, functional dimerization, and regulation by other kinases. In this study, we used the replica exchange molecular dynamics approach with a hybrid temperature and Hamiltonian tempering to obtain the conformational ensemble of full-length Src kinase in its non-phosphorylated state and in the presence of its two key regulatory phosphorylations: pY419 and pY530. The representative structures and simulation trajectories of non-phosphorylated pY419 and pY530 Src are available in open access. We demonstrate that pY419 phosphorylation, which is associated with Src activation, enhances its motility, whereas inhibited pY530 Src preserves relatively compact conformation. This study also provides insights into how SH4UD contributes to Src substrate binding, dimerization, and autophosphorylation, highlighting the putative role of 14-RRR-16 in this process.

20-Hydroxyeicosatetraenoic acid (20-HETE) is associated with secondary damage in traumatic brain injury (TBI) of the immature brain. Microglial activation is pivotal in this process. However, the underlying mechanism of action remains unknown. While 20-HETE interacts with G protein-coupled receptor 75 (GPR75) in some pathological processes, their interaction in brain tissue remains uncertain. This study aimed to investigate whether 20-HETE can activate microglia by binding to GPR75 in TBI of the immature brain. Drug affinity responsive molecular target stability (DARTS) assays, cycloheximide (CHX) chase assays, and auto-dock assays were employed to analyze the interaction between 20-HETE and GPR75. The expression levels of cytochrome P450 4A (CYP4A) and GPR75 in activated microglia in an immature brain TBI model were observed by western blot and multiple immunofluorescence staining. The effects of different levels of 20-HETE expression and lentivirus-mediated GPR75 gene silencing on 20-HETE-induced inflammatory factor release from BV-2 cells were observed by enzyme-linked immunoassay (ELISA). The phosphorylation levels of the downstream Src kinase, epidermal growth factor receptor (EGFR), and nuclear factor (NF)-κB were assessed using western blot. Cell viability and apoptosis were detected by CCK-8 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. 20-HETE bound to the GPR75 protein and inhibited its degradation. GPR75 gene silencing reversed the 20-HETE-induced inflammatory activation of BV-2 cells, effectively inhibiting the activation of the Src/EGFR/NF-κB pathway and the effects of 20-HETE on cell viability and the apoptosis rate. In contrast, overexpression of GPR75 had the opposite effect. In addition, after immature brain TBI, the 20-HETE and GPR75 expression levels were upregulated in microglia, with significant activation of the Src/EGFR/NF-κB pathway. Inhibition of 20-HETE synthesis with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) produced the opposite effect. 20-HETE regulates the Src/EGFR/NF-κB signaling pathway via GPR75 to activate microglia, promoting immature brain TBI. These findings offer a novel target for promoting the brain injury effect of 20-HETE.

Protein-protein interactions (PPIs) play pivotal roles in directing T cell fate. One key player is the non-receptor tyrosine protein kinase Lck that helps to transduce T cell activation signals. Lck is mediated by other proteins via interactions that are inadequately understood. Here, we use the deep learning method AF2Complex to predict PPIs involving Lck, by screening it against ∼1,000 proteins implicated in immune responses, followed by extensive structural modeling for selected interactions. Remarkably, we describe how Lck may be specifically targeted by a palmitoyltransferase using a phosphotyrosine motif. We uncover "hotspot" interactions between Lck and the tyrosine phosphatase CD45, leading to a significant conformational shift of Lck for activation. Lastly, we present intriguing interactions between the phosphotyrosine-binding domain of Lck and the cytoplasmic tail of the immune checkpoint LAG3 and propose a molecular mechanism for its inhibitory role. Together, this multifaceted study provides valuable insights into T cell regulation and signaling.

Glioblastoma (GBM) is the most frequent and aggressive primary brain cancer. The Src inhibitor, TAT-Cx43266-283, exerts antitumor effects in in vitro and in vivo models of GBM. Because addressing the mechanism of action is essential to translate these results to a clinical setting, in this study we carried out an unbiased proteomic approach. Data-independent acquisition mass spectrometry proteomics allowed the identification of 190 proteins whose abundance was modified by TAT-Cx43266-283. Our results were consistent with the inhibition of Src as the mechanism of action of TAT-Cx43266-283 and unveiled antitumor effectors, such as p120 catenin. Changes in the abundance of several proteins suggested that TAT-Cx43266-283 may also impact the brain microenvironment. Importantly, the proteins whose abundance was reduced by TAT-Cx43266-283 correlated with an improved GBM patient survival in clinical datasets and none of the proteins whose abundance was increased by TAT-Cx43266-283 correlated with shorter survival, supporting its use in clinical trials.

The kinase domains of receptor tyrosine kinases (RTKs) are highly conserved, yet they are able to discriminate among potential substrates to selectively activate downstream signaling pathways. In this study, we tested the importance of catalytic domain specificity by creating two series of chimeric RTKs. In one set, the kinase domain of insulin-like growth factor I receptor (IGF1R) was replaced by the kinase domains from insulin receptor (IR), macrophage stimulating protein 1 receptor/Ron (Ron) or Src. In the other set of chimeras, the kinase domain of epidermal growth factor receptor (EGFR) was similarly replaced by the kinase domains of IR, Ron, or Src. We expressed the wild-type and chimeric forms of the receptors in mammalian cells. For some signaling events, such as recognition of IRS1, the identity of the tyrosine kinase catalytic domain did not appear to be crucial. In contrast, recognition of some sites, such as the C-terminal autophosphorylation sites on EGFR, did depend on the identity of the kinase domain. Our data also showed that ligand dependence was lost when the native kinase domains were replaced by Src, suggesting that the identity of the kinase domains could be important for proper receptor regulation. Overall, the results are consistent with the idea that the fidelity of RTK signaling depends on co-localization and targeting with substrates, as well as on the intrinsic specificity of the kinase domain.

Tyrosine kinases (TKs) are catalytic enzymes activated by auto-phosphorylation that function by phosphorylating tyrosine residues on downstream substrates. Tyrosine kinase inhibitors (TKIs) have been heavily exploited as cancer therapeutics, primarily due to their role in autophagy, blood vessel remodeling and inflammation. This suggests tyrosine kinase inhibition as an appealing therapeutic target for exploiting convergent mechanisms across several neurodegenerative disease (NDD) pathologies. The overlapping mechanisms of action between neurodegeneration and cancer suggest that TKIs may play a pivotal role in attenuating neurodegenerative processes, including degradation of misfolded or toxic proteins, reduction of inflammation and prevention of fibrotic events of blood vessels in the brain. In this review, we will discuss the distinct roles that select TKs have been shown to play in various disease-associated processes, as well as identify TKs that have been explored as targets for therapeutic intervention and associated pharmacological agents being investigated as treatments for NDDs.

As a founding member of the Src family of kinases, Src has been confirmed to participate in the regulation of immune responses, integrin signaling, and motility. Ducks are usually asymptomatic carriers of RNA viruses such as Newcastle disease virus and avian influenza virus, which can be deadly to chickens. The beneficial role of Src in modulating the immune response remains largely unknown in ducks. Here, we characterized the duck Src and found that it contains a 192-base-pair 5' untranslated region, a 1602-base-pair coding region, and a 2541-base-pair 3' untranslated region, encoding 533 amino acid residues. Additionally, duSrc transcripts were significantly activated in duck tissues infected by Newcastle disease virus compared to controls. The duSrc transcripts were notably widespread in all tissues examined, and the expression level was higher in liver, blood, lung, pancreas, and thymus. Moreover, we found the expression levels of IFN-β, NF-κB, IRF3, and Src were significantly increased in DEFs after infection with 5'ppp dsRNA, but there was no significant difference before and after treatment in DF1 cells. Furthermore, overexpression of duSrc followed by stimulation with 5'ppp dsRNA led to an elevation of IFN-β levels. The SH3 and PTKc domains of duSrc contributed to promoting the activity of IFN-β and NF-κB in DEFs stimulated by 5'ppp dsRNA.

Intricate signaling systems are required to maintain homeostasis and promote differentiation in the epidermis. Receptor tyrosine kinases are central in orchestrating these systems in epidermal keratinocytes. In particular, EPHA2 and EGFR transduce distinct signals to dictate keratinocyte fate, yet how these cell communication networks are integrated has not been investigated. Our work shows that loss of EPHA2 impairs keratinocyte stratification, differentiation, and barrier function. To determine the mechanism of this dysfunction, we drew from our proteomics data of potential EPHA2 interacting proteins. We identified EGFR as a high-ranking EPHA2 interactor and subsequently validated this interaction. We found that when EPHA2 is reduced, EGFR activation and downstream signaling are intensified and sustained. Evidence indicates that prolonged SRC association contributes to the increase in EGFR signaling. We show that hyperactive EGFR signaling underlies the differentiation defect caused by EPHA2 knockdown because EGFR inhibition restores differentiation in EPHA2-deficient 3-dimensional skin organoids. Our data implicate a mechanism whereby EPHA2 restrains EGFR signaling, allowing for fine tuning in the processes of terminal differentiation and barrier formation. Taken together, we purport that crosstalk between receptor tyrosine kinases EPHA2 and EGFR is critical for epidermal differentiation.

Lck, a member of the Src kinase family, is a non-receptor tyrosine kinase involved in immune cell activation, antigen recognition, tumor growth, and cytotoxic response. The enzyme has usually been linked to T lymphocyte activation upon antigen recognition. Lck activation is central to CD4, CD8, and NK activation. However, recently, it has become clearer that activating the enzyme in CD8 cells can be independent of antigen presentation and enhance the cytotoxic response. The role of Lck in NK cytotoxic function has been controversial in a similar fashion as the role of the enzyme in CAR T cells. Inhibiting tyrosine kinases has been a highly successful approach to treating hematologic malignancies. The inhibitors may be useful in treating other tumor types, and they may be useful to prevent cell exhaustion. New, more selective inhibitors have been documented, and they have shown interesting activities not only in tumor growth but in the treatment of autoimmune diseases, asthma, and graft vs. host disease. Drug repurposing and bioinformatics can aid in solving several unsolved issues about the role of Lck in cancer. In summary, the role of Lck in immune response and tumor growth is not a simple event and requires more research.

Hepatocellular carcinoma (HCC) is a disease of high unmet medical need that has become a global health problem. The development of targeted therapies for HCC has been hindered by the incomplete understanding of HCC pathogenesis and the limited number of relevant preclinical animal models. We recently unveiled a previously uncharacterized YES kinase (encoded by YES1)-dependent oncogenic signaling pathway in HCC. To model this subset of HCC, we established a series of syngeneic cell lines from liver tumors of transgenic mice expressing activated human YES. The resulting cell lines (referred to as HepYF) were enriched for expression of stem cell and progenitor markers, proliferated rapidly, and were characterized by high SRC family kinase (SFK) activity and activated mitogenic signaling pathways. Transcriptomic analysis indicated that HepYF cells are representative of the most aggressive proliferation class G3 subgroup of HCC. HepYF cells formed rapidly growing metastatic tumors upon orthotopic implantation into syngeneic hosts. Treatment with sorafenib or the SFK inhibitor dasatinib markedly inhibited the growth of HepYF tumors. The new HepYF HCC cell lines provide relevant preclinical models to study the pathogenesis of HCC and test novel small-molecule inhibitor and immunotherapy approaches.

Remarkable differences exist in the outcome of systemic cancer therapies. Lymphomas and leukemias generally respond well to systemic chemotherapies, while solid cancers often fail. We engineered different human cancer cells lines to uniformly express a modified herpes simplex virus thymidine kinase TK.007 as a suicide gene when ganciclovir (GCV) is applied, thus in theory achieving a similar response in all cell lines.

Fifteen different cell lines were engineered to express the TK.007 gene. XTT-cell proliferation assays were performed and the IC50-values were calculated. Functional kinome profiling, mRNA sequencing, and bottom-up proteomics analysis with Ingenuity pathway analysis were performed.

GCV potency varied among cell lines, with lymphoma and leukemia cells showing higher susceptibility than solid cancer cells. Functional kinome profiling implies a contribution of the SRC family kinases and decreased overall kinase activity. mRNA sequencing highlighted alterations in the MAPK pathways and bottom-up proteomics showed differences in apoptotic and epithelial junction signaling proteins.

The histogenetic origin of cells influenced the susceptibility of human malignant cells towards cytotoxic agents with leukemias and lymphomas being more sensitive than solid cancer cells.