Drug Affinity Responsive Target Stability Research Articles

Sodium danshensu modulates skeletal muscle fiber type formation and metabolism by inhibiting pyruvate kinase M1

Sodium Danshensu (SDSS) is extracted from Salvia miltiorrhiza and has many pharmacological effects. However, little is known about its effects on muscle fiber formation and metabolism. Here, we aimed to investigated the role and molecular mechanisms of SDSS in modulating the formation of skeletal muscle fiber. C2C12 cells were incubated in differentiation medium with or without SDSS for 4 days. C57BL/6 mice were orally administered SDSS by gavage once a day for 8 weeks. Grip strength, treadmill, muscle weight, western blotting, qPCR, immunofluorescence staining and H&E staining were performed. SDSS target proteins were searched through drug affinity responsive target stability (DARTS) and mass spectrometry analysis. Furthermore, molecular docking was carried out for Pyruvate kinase M1 (PKM1). The effect of PKM1 on myosin heavy chain (MyHCs) gene expression was verified by knockdown of PKM1 experiment. SDSS induced oxidative muscle fiber-related gene expression, and inhibited glycolytic fiber-related gene expression in C2C12 cells. Muscle mass, the percentage of slow oxidative fibers, succinic dehydrogenase activity, muscle endurance, glucose tolerance, and the expression of the MyHC1 and MyHC2a genes increased while MyHC2b expression, lactate dehydrogenase activity, and the percentage of glycolytic muscle fibers decreased in SDSS-treated mice. Mechanistically, SDSS bound to the pyruvate kinase PKM1 and significantly repressed its activity. PKM1 inhibited MyHC1 and MyHC2a expression but promoted MyHC2b expression. SDSS also significantly attenuated the effects of PKM1 on muscle fiber-related gene expression in C2C12 cells. Our findings indicate that SDSS promotes muscle fiber transformation from the glycolytic type to the oxidative type by inhibiting PKM1 activity, which provide a new idea for treating muscle atrophy, muscle metabolism diseases and improving animal meat production.

Binding with HSP90β, cimifugin ameliorates fibrotic cataracts in vitro and in vivo by inhibiting TGFβ signaling pathways

Fibrotic cataracts, the most frequent complications after phacoemulsification, cannot be cured by drugs in clinic. The primary mechanism underlying the disease is the epithelial-mesenchymal transition (EMT). Cimifugin is a natural monomer component of traditional Chinese medicines. Previous researches have demonstrated the effect of cimifugin inhibiting EMT in the lung. The purpose of this work is to evaluate the impact of cimifugin on EMT in the lens and elucidate its precise mechanism. The pathogenesis of fibrotic cataracts was simulated using TGFβ2-induced cell model of EMT and the injury-induced anterior subcapsular cataract animal model. Through H&E staining and immunofluorescence of mice eyeballs, we discovered that cimifugin can inhibit the expansion of fibrotic lesions in vivo. Furthermore, at mRNA and protein levels, we confirmed that cimifugin can allay EMT of lens epithelial cells (LECs) in vitro and in vivo. Additionally, the inhibition of cimifugin on the activation of TGFβ-related signaling pathways was certified by immunoblot. HSP90β, the target of cimifugin, was predicted by network pharmacology and verified by drug affinity responsive target stability, the cellular thermal shift assay, and microscale thermophoresis. Moreover, co-immunoprecipitation revealed the interaction between HSP90β and TGFβRII. Together, our findings showed that by weakening the binding of HSP90β and TGFβRII, cimifugin suppressed the TGFβ signaling pathways to alleviate fibrotic cataracts. Cimifugin is a promising medication for the treatment of fibrotic cataracts.

Shikonin induces ferroptosis in osteosarcomas through the mitochondrial ROS-regulated HIF-1α/HO-1 axis

BackgroundThe most common malignant bone tumour is osteosarcoma, which has an unsatisfactory prognosis and unsatisfactory treatment. Ferroptosis shows promise as an effective OS therapy. A substance extracted from the Lithospermum erythrorhizon, Shikonin (SHK), inhibits a number of tumours, including ovarian, gastric, and lung cancers. However, whether SHK induces OS ferroptosis and its mechanisms are not clear. PurposeOur study is aimed at investigating whether SHK causes ferroptosis and elucidating its molecular mechanism. MethodsCell counting Kit-8, cell cycle and cell apoptosis assay were utilised to assess therapeutic effect of SHK against OS. Normal cells, including H9C2, AML-12 and HK-2, haematoxylin and eosin staining and mice serum biomarker tests were used to assess SHK biosafety. Malondialdehyde (MDA) levels, the glutathione (GSH)/oxidized glutathione (GSSG) ratio, reactive oxygen species (ROS), lipid peroxide (LPO), and intracellular Fe2+ detection, quantitative reverse transcription PCR (qRT-PCR), Western blotting (WB) and rescue experiments were employed to confirm whether SHK induced ferroptosis in OS cells. Molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay were used to evaluate the direct binding between SHK and hypoxia-inducible factor-1α (HIF-1α). Protein stability and degradation analysis, small RNA interference, flow cytometry, qRT-PCR, and WB were used to investigate the molecular mechanism of ferroptosis. In vivo xenografts of nude mouse was used to study the anti-OS effect of SHK. ResultsSHK significantly reduced OS cell viability and induced apoptosis and G2/M arrest. SHK increased intracellular levels of MDA, ROS, LPO, and Fe2+ while simultaneously reducing the GSH/GSSG ratio and GPX4 and SLC7A11 expression. CETSA and DARTS results demonstrated that SHK did not bind targetly to HIF-1α. Instead, mitochondrial ROS (MitoROS) promoted HIF-1α expression, resulting in HO-1 overexpression, excess Fe2+ production, ROS accumulation and GPX depletion, and ferroptosis. Furthermore, inhibition of MitoROS using Mito-TEMPO downregulated HIF-1α/HO-1 axis and mitigated the SHK-induced ferroptosis. In vivo, SHK effectively suppressed OS growth with favourable biosafety, confirming the molecular mechanism underlying SHK-induced ferroptosis in OS. ConclusionWe observe that HIF-1α/HO-1 axis is the crucial factor in SHK-induced OS ferroptosis. Importantly, we demonstrate that HIF-1α is indirectly regulated by MitoROS rather than SHK bound directly to HIF-1α. Our research suggest that SHK is a potential drug candidate for OS treatment and may help in identifying novel therapeutic targets for OS.

Integrated network pharmacology, metabolomics and molecular docking analysis to reveal the mechanisms of quercetin in the treatment of hyperlipidemia

Hyperlipidemia (HLP) is a significant contributor to cardiovascular diseases. Quercetin (QUE), a naturally occurring flavonoid with diverse bioactivities, has garnered attention due to its potential therapeutic effects. However, the precise mechanisms underlying the effects of QUE on HLP remain unclear. In this study, an ultra-high-performance liquid chromatography-quadrupole/electrostatic field Orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive-MS) metabolomics strategy was employed to obtain metabolite profiles, and potential biomarkers were identified following data analysis. Network pharmacology and Drug Affinity Responsive Target Stability (DARTS) assays were utilized to explore the potential targets of QUE for HLP treatment. The results of metabolomics and network pharmacology were then integrated to identify the key targets and metabolic pathways involved in the therapeutic action of the QUE against HLP. Molecular docking and experimental validation were performed to confirm these key targets. A comprehensive database search identified 138 QUE-HLP-related targets. A protein-protein interaction (PPI) network was constructed using STRING, and the shared targets were filtered with Cytoscape. Among these, AKT1, TNF, VEGFA, mTOR, SREBP1, and SCD emerged as potential therapeutic targets. These findings were validated using in vitro cell experiments. Additionally, the mechanism of action of QUE against HLP was evaluated by integrating network pharmacology with metabolomics, identifying two metabolomic pathways crucial to HLP treatment. DARTS experiments confirmed the stable binding of QUE to FASN, p-mTOR, SREBP1, and p-AKT. In HepG2 cells treated with palmitic acid (PA), QUE significantly reduced the mRNA expression of ACLY, ACACA, FASN, and SCD (p < 0.05). Western blot analysis revealed that PA significantly increased protein expression of p-mTOR, SREBP1, FASN, and p-AKT (p < 0.05). In summary, our study provides novel insights into the protective mechanisms of QUE against HLP and offers valuable information regarding its potential benefits in clinical treatment.

Penfluridol inhibits melanoma growth and metastasis through enhancing von Hippel‒Lindau tumor suppressor-mediated cancerous inhibitor of protein phosphatase 2A (CIP2A) degradation.

Melanoma's high metastatic potential, especially to the brain, poses significant challenges to patient survival. The blood‒brain barrier (BBB) is a major obstacle to the effective treatment of melanoma brain metastases. We screened antipsychotic drugs capable of crossing the BBB and identified penfluridol (PF) as the most active candidate. PF reduced melanoma cell viability and induced apoptosis. In animal models, PF effectively inhibited melanoma growth and metastasis to the lung and brain. Using immunoprecipitation combined with high-resolution mass spectrometry, and other techniques such as drug affinity responsive target stability, we identified CIP2A as a direct binding protein of PF. CIP2A is highly expressed in melanoma and its metastases, and is linked to poor prognosis. PF can restore Protein Phosphatase 2A activity by promoting CIP2A degradation, thereby inhibiting several key oncogenic pathways, including AKT and c-Myc. Additionally, von Hippel‒Lindau (VHL) is the endogenous E3 ligase for CIP2A, and PF enhances the interaction between VHL and CIP2A, promoting the ubiquitin‒proteasome degradation of CIP2A, thereby inhibiting melanoma growth and metastasis. Overall, this study not only suggests PF's potential in treating melanoma and its brain metastases but also highlights CIP2A degradation as a therapeutic strategy for melanoma.

Panaxatriol exerts anti-senescence effects and alleviates osteoarthritis and cartilage repair fibrosis by targeting UFL1

IntroductionOsteoarthritis (OA), the most common degenerative joint disease, can eventually lead to disability. However, no safe or effective intervention is currently available. Therefore, there is an urgent need to develop effective drugs that reduce cartilage damage and treat OA. ObjectivesThis study aimed to ascertain the potential of panaxatriol, a natural small molecule, as a therapeutic drug for alleviating the progression of OA. MethodsAn in vitro culture of human cartilage explants and C28/I2 human chondrocytes and an in vivo surgically induced OA mouse model were used to evaluate the chondroprotective effect of panaxatriol. The Drug Affinity Responsive Target Stability assay, CRISPR-Cas9 assay, Whole-transcriptome RNA sequencing analysis and agonist or antagonist assays were used to identify the target and potential signaling pathways of panaxatriol. Poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) was used to construct the sustained-release system of panaxatriol. ResultsPanaxatriol protected against OA by regulating chondrocyte metabolism. Ubiquitin-fold modifier 1-specific E3 ligase 1 (UFL1) was identified as a novel target of panaxatriol. Whole transcriptome RNA sequencing showed that UFL1 was closely related to cell senescence. Panaxatriol inhibited chondrocyte senescence through UFL1/forkhead box O1 (FOXO1)/P21 and UFL1/NF-κB/SASPs signaling pathways. It also could inhibit fibrocartilage formation during cartilage repair via the UFL1/FOXO1/Collagen 1 signaling pathway. Finally, we constructed a sustained-release system for panaxatriol based on PLGA-PEG, which reduced the number of intra-articular injections, thereby alleviating joint swelling and injury. ConclusionsPanaxatriol exerts anti-senescence effects and has the potential to delay OA progression and reduce cartilage repair fibrosis by targeting UFL1.

Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway

BackgroundFibrosis cataract occurs in patients receiving cataract extraction. Still, no medication that can cure the disease exists in clinical. This study aims to investigate the effects and mechanisms of Entrectinib on fibrotic cataract in vitro and in vivo.MethodsThe human lens cells line SRA 01/04 and C57BL/6J mice were applied in the study. Entrectinib was used in animals and cells. Cataract severity was assessed by slit lamp and Hematoxylin and Eosin staining. Expression of alpha-smooth muscle actin, fibronectin, and collagen I were examined by real-time quantitative PCR, western blotting, and immunofluorescence. Cell proliferation was evaluated by Cell Counting Kit-8. Cell migration was measured by wound healing and transwell assays. Molecular docking, Drug Affinity Responsive Target Stability, and Cellular Thermal Shift Assay were applied to seek and certify the target of Entrectinib treating fibrosis cataract.ResultsEntrectinib can ameliorate fibrotic cataract in vitro and in vivo. At the RNA and the protein levels, the expression of alpha-smooth muscle actin, collagen I, and fibronectin can be downgraded by Entrectinib, while E-cadherin can be upregulated. The migration and proliferation of cells were inhibited by Entrectinib. Mechanistically, Entrectinib obstructs TGFβ2/Smad and TGFβ2/non-Smad signaling pathways to hinder the fibrosis cataract by targeting PYK2 protein.ConclusionsTargeting with PYK2, Entrectinib can block TGF-β2/Smad and TGF-β2/non-Smad signaling pathways, lessen the activation of EMT, and alleviate fibrosis cataract. Entrectinib may be a potential treatment for fibrosis cataract in clinic.

Anti-inflammation Mechanisms of Flavones Are Highly Sensitive to the Position Isomers of Flavonoids: Acacetin vs Biochanin A.

Acacetin (ACA) and biochanin A (BCA) are isomeric monomethoxyflavones with different structural positions of the 4'-methoxy-phenyl group. Both of them are present in many commonly consumed foods, such as citrus fruits and vegetables, and have been discovered with anti-inflammatory activities, but their mechanisms of action are not clearly elucidated at the molecular level. Herein, we reported the structure-activity relationship of ACA and BCA regarding their potency in inhibiting nitric oxide (NO) production, proinflammatory enzyme expression, and mRNA expression of proinflammatory cytokines in the lipopolysaccharide (LPS)-induced RAW 264.7 cells. Furthermore, transcriptome analysis was conducted to map out the overall pathways impacted by these two isomers. Our results showed that ACA possessed higher suppressive activity in nitric oxide (NO) production (IC50 = 23.93 ± 1.74 μM for ACA and IC50 = 71.41 ± 8.07 μM for BCA), inducible nitric oxide synthase (iNOS) expression, and proinflammatory interleukin (IL)-1β mRNA expression, but lower inhibitory activity in IL-6 mRNA expression as compared with BCA. Although two compounds were revealed to bind to c-Src protein according to drug affinity responsive target stability (DARTS) and western blotting results, molecular docking and molecular dynamics (MD) simulation showed that they had different binding sites, which subsequently resulted in different signaling transduction. ACA primarily exerted anti-inflammatory activity through the mitogen-activated protein kinase (MAPK) signaling pathway, the tumor necrosis factor (TNF) signaling pathway, and the hypoxia-inducible factor (HIF)-1 signaling pathway, while BCA was particularly involved in the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway, the nuclear factor kappa B (NF-κB) signaling pathway, the TNF signaling pathway, and the toll-like receptor (TLR) signaling pathway. Moreover, two isomers remained stable in the cell culture medium and did not encounter the biotransformation process in RAW 264.7 cells. However, BCA exhibited insufficient cellular uptake ability and transport efficiency in macrophages compared to ACA. Taken together, ACA is more promising for controlling inflammation and worthy of further study for human use.

Catalpol ameliorates liver fibrosis via inhibiting aerobic glycolysis by EphA2/FAK/Src signaling pathway

BackgroundHepatic fibrosis is a pathological process in a variety of acute or chronic liver injuries. Catalpol (CAT), an iridoid glycoside found in Rehmannia glutinosa, has several pharmacological properties, including anti-inflammatory, antidiabetic and anti-fibrotic effects. Nevertheless, there is currently no report on whether CAT regulates the aerobic glycolysis of hepatic stellate cells (HSCs) to inhibit liver fibrosis. ObjectiveThis study aimed to investigate the protective effects of CAT on hepatic fibrosis and elucidate its underlying mechanisms. MethodsTo explore whether CAT improved liver fibrosis in vivo and in vitro, hepatic fibrosis was induced to mice by intraperitoneally injecting carbon tetrachloride (CCl4). Additionally, LX-2 cells were stimulated with transforming growth factor-β (TGF-β) to simulate fibrosis in vitro. Serum markers of liver injury were examined by using an automatic biochemical analyzer. Histopathological staining, Immunofluorescence (IF) staining, Western blot (WB) analysis, co-immunoprecipitation (Co-IP), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), etc. were employed to identify the targeting between CAT and EphA2 and detect the expression of aerobic glycolysis related proteins, fiber markers and signaling pathways that are responsible for CAT's anti-fibrotic effects of CAT. ResultsResults showed that CAT significantly inhibited hepatic injury, fibrogenesis and inflammation in mice treated with CCl4. This was demonstrated by the enhancement of fibrosis markers, liver function indices, and histopathology. In addition, CAT significantly inhibited the activation of HSCs in TGF-β-induced LX-2 cells, as indicated by decreased proliferation, migration, and expression of collagen I and a-SMA. The study results also suggested that CAT may exert anti-fibrotic effects by inhibiting glycolysis in activated HSCs and in CCl4-treated mice. Mechanistically, CAT directly targets Ephrin type-A receptor 2 (EphA2) to reduce binding with focal adhesion kinases (FAK) and significantly inhibits the FAK/Src pathway. In addition, the pharmacological inhibition of EphA2 cannot further increase the therapeutic effects of CAT on liver fibrosis in vivo and in vitro. ConclusionThe study findings generally demonstrated that CAT presented a novel therapeutic method to treat hepatic fibrosis; this method which inhibits the aerobic glycolysis of activated HSCs through the EphA2/FAK/Src signaling pathway.

IGF-1 and Glucocorticoid Receptors Are Potential Target Proteins for the NGF-Mimic Effect of β-Cyclocitral from Lavandula angustifolia Mill. in PC12 Cells.

In the present study, the PC12 cells as a bioassay system were used to screen the small molecules with nerve growth factor (NGF)- mimic effect from Lavandula angustifolia Mill. The β-Cyclocitral (β-cyc) as an active compound was discovered, and its chemical structure was also determined. Furthermore, we focused on the bioactive and action mechanism of this compound to do an intensive study with specific protein inhibitors and Western blotting analysis. The β-cyc had novel NGF-mimic and NGF-enhancer effects on PC12 cells, while the insulin-like growth factor-1 receptor (IGF-1R)/phosphatidylinositol 3 kinase, (PI3K)/serine/threonine-protein kinase (AKT), and glucocorticoid receptor (GR)/phospholipase C (PLC)/protein kinase C (PKC) signaling pathways were involved in the bioactivity of β-cyc. In addition, the important role of the rat sarcoma (Ras)/protooncogene serine-threonine protein kinase (Raf) signaling pathway was observed, although it was independent of tyrosine kinase (Trk) receptors. Moreover, the non-label target protein discovery techniques, such as the cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS), were utilized to make predictions of its target protein. The stability of IGF-R and GR, proteins for temperature and protease, was dose-dependently increased after treatment of β-cyc compared with control groups, respectively. These findings indicated that β-cyc promoted the neuron differentiation of PC12 cells via targeting IGF-1R and GR and modification of downstream signaling pathways.

Corilagin alleviated intestinal ischemia-reperfusion injury by modulating endoplasmic reticulum stress via bonding with Bip

BackgroundIntestinal ischemia-reperfusion (II/R) injury is a common clinical emergency with high morbidity and mortality. Given the absence of efficacious prophylactic and therapeutic interventions and specific drugs, sustained efforts are essential to develop new targeted drugs. Corilagin, a naturally polyphenolic tannic acid widespread in longan, rambutan and many other edible economic crops with medicinal properties in China, is of interest due to its multiple bioactivities, including the potential to mitigate II/R injuries. Nevertheless, a clear understanding of its molecular targets and the intricate mechanisms against II/R injury remains obscure and requires further elucidation. ObjectiveThis study aimed to investigate corilagin's pharmacological impact and molecular mechanism for II/R injury. MethodsAn animal II/R model was established by clamping superior mesenteric artery (SMA), and the therapeutic efficacy of corilagin against II/R was evaluated by biochemical and pathological analysis. Next, integrated transcriptomic and proteomic analyses was performed to identify key targets. Moreover, endoplasmic reticulum stress (ERS) damage was respectively observed by transmission electron microscope (TEM), immunohistochemistry, TUNEL, flow cytometry and western blotting (WB). Finally, molecular docking, molecular dynamics (MD) simulation, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) assays were utilized to assess the interaction between corilagin and binding immunoglobulin protein (Bip, Grp78 or Hspa5), and co-IP assay was conducted to investigate the interaction between Bip and its substrate proteins. ResultsCorilagin exhibited robust protection against II/R injuries, effectively alleviating intestinal tissue damage and oxidative stress induced by II/R. The modulation of ERS as a potential regulatory mechanism was investigated through an integrated transcriptomic and proteomic analysis, identifying Bip as a key target contributing to corilagin's protective effects. Further experimental evidence using molecular docking, MD simulation, CETSA, and DARTS assays confirmed the potentially direct interaction of corilagin with Bip. This interaction promoted the ubiquitin-dependent degradation of the Bip-substrate complex, thereby suppressing ERS-related signalling pathways, including the IRE1 branch, PERK branch, and ATF6 branch, to alleviate tissue damage. ConclusionThis study confirmed that corilagin could selectively bind to Bip, facilitating its ubiquitin-dependent recognition and degradation, thereby inhibiting severe endoplasmic reticulum stress signalling and alleviating II/R injury. A detailed mechanistic insight into the action mode of corilagin had been proposed, supporting its potential usage as an ERS inhibitor.

Diterpene glycosides from Fructus Rubi ameliorates benign prostatic hyperplasia in rats through the androgen and TGF-β/Smad signaling pathway

Fructus Rubi (FR), a food material with medicinal value, is used in traditional Chinese medicine (TCM) for treatment of various kidney-related problems, such as impotence, spermatorrhea, and frequent urination. It is also frequently used to produce diverse functional foods in China. The purpose of this research was to assess the therapeutic effects of FR diterpene glycosides on RWPE-1 epithelial cell (RWPE-1), a human normal prostatic epithelial cell, and benign prostate hyperplasia (BPH) rats, both of which had been exposed to dihydrotestosterone (DHT) and testosterone propionate (TP), respectively, and to investigate the mechanism of action. Target proteins that could stably bind to certain diterpene glycosides were screened through drug affinity responsive target stability combined with mass spectrometry (DARTS/MS). DHT-induced RWPE-1cells were used to detect drug activity. TP was subcutaneously injected to induce BPH in rats. The extract of diterpene glycosides from FR (FDS) was orally administered for 28 days. The DHT levels in the serum and prostate tissue of the rats were measured through enzyme-linked immunosorbent assay (ELISA), and to analyze cell proliferation and epithelial-mesenchymal transition (EMT), the protein expression of prostate-specific antigen (PSA), androgen receptor (AR), steroid 5α-reductase type 2 (SRD5A2), proliferating cell nuclear antigen (PCNA), S100 calcium-binding protein A2 (S100A2), transforming growth factor-β1 (TGF-β1), E-cadherin, vimentin, and Smad4 was determined through western blotting (WB), immunohistochemistry (IHC), or immunofluorescence (IF). FDS reduced the proliferation of DHT-induced RWPE-1cells. It also significantly inhibited rat prostate enlargement; decreased DHT levels in the serum and prostate tissue; inhibited the protein expression of AR, PSA, PCNA, S100A2, TGF-β1, E-cadherin, and Smad4; and increased the protein expression of E-cadherin. The present study is the first to report that diterpene glycosides isolated from FR inhibited BPH at the cellular level, regulated the proliferation of prostate cells through the androgen signaling pathway, and prevented EMT in the prostate through the S100A2-mediated TGF-β/Smad signaling pathway. These results indicate that FDS is a promising multitarget therapy for BPH.

Chlorquinaldol Alleviates Lung Fibrosis in Mice by Inhibiting Fibroblast Activation through Targeting Methionine Synthase Reductase.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with limited treatment options. Thus, it is essential to investigate potential druggable targets to improve IPF treatment outcomes. By screening a curated library of 201 small molecules, we have identified chlorquinaldol, a known antimicrobial drug, as a potential antifibrotic agent. Functional analyses have demonstrated that chlorquinaldol effectively inhibits the transition of fibroblasts to myofibroblasts in vitro and mitigates bleomycin-induced pulmonary fibrosis in mice. Using a mass spectrometry-based drug affinity responsive target stability strategy, we revealed that chlorquinaldol inhibited fibroblast activation by directly targeting methionine synthase reductase (MTRR). Decreased MTRR expression was associated with IPF patients, and its reduced expression in vitro promoted extracellular matrix deposition. Mechanistically, chlorquinaldol bound to the valine residue (Val-467) in MTRR, activating the MTRR-mediated methionine cycle. This led to increased production of methionine and s-adenosylmethionine, counteracting the fibrotic effect. In conclusion, our findings suggest that chlorquinaldol may serve as a novel antifibrotic medication, with MTRR-mediated methionine metabolism playing a critical role in IPF development. Therefore, targeting MTRR holds promise as a therapeutic strategy for pulmonary fibrosis.

Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake

ObjectiveGlucose-1,6-bisphosphate (G-1,6-BP), a byproduct of glycolysis that is synthesized by phosphoglucomutase 2 like 1 (PGM2L1), is particularly abundant in neurons. G-1,6-BP is sensitive to the glycolytic flux, due to its dependence on 1,3-bisphosphoglycerate as phosphate donor, and the energy state, due to its degradation by inosine monophosphate-activated phosphomannomutase 1. Since the exact role of this metabolite remains unclear, our aim was to elucidate the specific function of G-1,6-BP in the brain. MethodsThe effect of PGM2L1 on neuronal post-ischemic viability was assessed by siRNA-mediated knockdown of PGM2L1 in primary mouse neurons. Acute mouse brain slices were used to correlate the reduction in G-1,6-BP upon ischemia to changes in carbon metabolism by 13C6-glucose tracing. A drug affinity responsive target stability assay was used to test if G-1,6-BP interacts with the mitochondrial pyruvate carrier (MPC) subunits in mouse brain protein extracts. Human embryonic kidney cells expressing a MPC bioluminescence resonance energy transfer sensor were used to analyze how PGM2L1 overexpression affects MPC activity. The effect of G-1,6-BP on mitochondrial pyruvate uptake and oxygen consumption rates was analyzed in isolated mouse brain mitochondria. PGM2L1 and a predicted upstream kinase were overexpressed in a human neuroblastoma cell line and G-1,6-BP levels were measured. ResultsWe found that G-1,6-BP in mouse brain slices was quickly degraded upon ischemia and reperfusion. Knockdown of PGM2L1 in mouse neurons reduced post-ischemic viability, indicating that PGM2L1 plays a neuroprotective role. The reduction in G-1,6-BP upon ischemia was not accompanied by alterations in glycolytic rates but we did see a reduced 13C6-glucose incorporation into citrate, suggesting a potential role in mitochondrial pyruvate uptake or metabolism. Indeed, G-1,6-BP interacted with both MPC subunits and overexpression of PGM2L1 increased MPC activity. G-1,6-BP, at concentrations found in the brain, enhanced mitochondrial pyruvate uptake and pyruvate-induced oxygen consumption rates. Overexpression of a predicted upstream kinase inhibited PGM2L1 activity, showing that besides metabolism, also signaling pathways can regulate G-1,6-BP levels. ConclusionsWe provide evidence that G-1,6-BP positively regulates mitochondrial pyruvate uptake and post-ischemic neuronal viability. These compelling data reveal a novel mechanism by which neurons can couple glycolysis-derived pyruvate to the tricarboxylic acid cycle. This process is sensitive to the glycolytic flux, the cell's energetic state, and upstream signaling cascades, offering many regulatory means to fine-tune this critical metabolic step.

Naoxintong capsule accelerates mitophagy in cerebral ischemia-reperfusion injury via TP53/PINK1/PRKN pathway based on network pharmacology analysis and experimental validation

Ethnopharmacological relevanceThe incidence and mortality of cerebrovascular diseases are increasing year by year. Cerebral ischemia-reperfusion injury (CIRI) is common in patients with ischemic stroke. Naoxintong (NXT) is composed of a variety of Chinese medicines and has the ability to treat CIRI. Aim of the studyThe aim of this study is to investigate whether NXT regulates mitophagy in CIRI based on network pharmacology analysis and experimental validation. Materials and methodsOxygen and glucose deprivation/re-oxygenation (OGD/R, 2/22 h) model of PC12 cells and transient middle cerebral artery occlusion (tMCAO, 2/22 h) model of rats were established. Pharmacodynamic indicators include neurological deficit score, 2,3,5-triphenyte-trazoliumchloride (TTC) staining, hematoxylin-eosin (HE) staining and cell viability. Network pharmacology was used to predict pharmacological mechanisms. Pharmacological mechanism indexes include transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), immunohistochemistry (IHC), western blot (WB) and immunofluorescence (IF). Kevetrin (an agonists of p53) and pifithrin-α (an inhibitor of p53) used to detect the key role of p53 in mitophagy of NXT. ResultsNXT (1% serum containing NXT and 110 mg/kg) improved the damage of OGD/R PC12 cells and tMCAO rats, and this protective effect was related to the anti-oxidation and ability to promote mitophagy of NXT. NXT and pifithrin-α increased the expression of promoting-mitophagy targets (PINK1, PRKN and LC3B) and inhibited the expression of inhibiting-mitophagy targets (p52) via restraining p53, and finally accelerated mitophagy caused by CIRI. ConclusionThis study demonstrates that NXT promotes mitophagy in CIRI through restraining p53 and promoting PINK1/PRKN in vivo and in vitro.

Loratadine Derivative Lo-7: A Weapon against Drug-Resistant Enterococcus and Streptococcal Infections.

The primary obstacles in the management of Enterococcus and Streptococcal infections are drug resistance and biofilm formation. Our study revealed that loratadine at a concentration of ≥25 μM exhibited significant inhibitory effects on biofilm formation in 167 clinical strains of Enterococcus faecalis and 15 clinical isolates of Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus pneumoniae. Additionally, the antibiofilm activity against E. faecalis and Streptococcal was demonstrated by several loratadine derivatives with altered side-chain carbamate moieties. This study investigated the antibacterial activity of the loratadine derivative Lo-7 against clinical strains of S. agalactiae and S. pyogenes, with minimum inhibitory concentrations ranging from 12.5 to 25 μM. The findings revealed that a low concentration of loratadine derivative Lo-7 (3.125 μM) significantly augmented the bactericidal efficacy of vancomycin against multidrug-resistant (MDR) S. agalactiae, both in vitro and in vivo. The loratadine derivative Lo-7, even at low concentrations, demonstrated significant efficacy in eliminating intracellular MDR S. agalactiae within macrophages, potentially indicating a unique advantage over vancomycin, linezolid, and loratadine. Mechanistically, exposure to the loratadine derivative Lo-7 resulted in membrane depolarization without affecting membrane permeability in S. agalactiae. The potential targeting of the SecG subunit of the SecYEG membrane-embedded channel by the loratadine derivative Lo-7 in S. agalactiae was identified through quantitative proteomics, a drug affinity responsive target stability assay, and molecular docking.

Targeting and degradation of OTUB1 by Erianin for antimetastasis in esophageal squamous cell carcinoma

BackgroundMetastasis is a major contributor to mortality in patients with esophageal squamous cell carcinoma (ESCC); effective treatment is currently lacking. Erianin, a bioactive ingredient of traditional Chinese medicine, Dendrobium chrysotoxum, has anti-tumor activity against multiple human tumors. However, the effect and associated underlying mechanism of Erianin on ESCC antimetastasis remain unclear. PurposeTo investigate the anti-metastatic properties of Erianin in ESCC both in vitro and in vivo and associated molecular mechanisms. MethodsWound healing assay, Transwell assay, CCK-8 assay, immunohistochemistry, and lung metastasis mouse model were carried out to examine ESCC cell migration and viability in vitro and in vivo. Drug affinity responsive target stability (DARTS), cellular thermal migration assay (CETSA), molecular docking, and Surface plasmon resonance (SPR) assay were used to confirm Erianin binding to ovarian tumor ubiquitin aldehyde-binding protein 1 (OTUB1) protein. Protein stability assay, cell transfection, and western blotting were used to confirm Erianin-mediated degradation of OTUB1 and Snail via the ubiquitin-proteasome pathway. qRT-PCR and western blotting were used to assess OTUB1expression in ESCC tissues. ResultsErianin suppressed the migration/invasion of ESCC cells without modulating cell viability in vitro and in vivo, bound to OTUB1 through DARTS, CETSA, and molecular docking, and SPR assay, and enhanced OTUB1 degradation via the ubiquitin-proteasome system. Moreover, Erianin inhibited the ESCC epithelial–mesenchymal transition by enhancing the ubiquitination and degradation of Snail via targeting OTUB1. ConclusionErianin inhibited ESCC metastasis through ubiquitination and degradation of Snail via targeting OTUB1. Our findings suggest Erianin as a novel OTUB1 inhibitor for preventing ESCC metastasis.

Fucoxanthin Mitigates High-Fat-Induced Lipid Deposition and Insulin Resistance in Skeletal Muscle through Inhibiting PKM1 Activity.

Glucose and lipid metabolism dysregulation in skeletal muscle contributes to the development of metabolic disorders. The efficacy of fucoxanthin in alleviating lipid metabolic disorders in skeletal muscle remains poorly understood. In this study, we systematically investigated the impact of fucoxanthin on mitigating lipid deposition and insulin resistance in skeletal muscle employing palmitic acid-induced lipid deposition in C2C12 cells and ob/ob mice. Fucoxanthin significantly alleviated PA-induced skeletal muscle lipid deposition and insulin resistance. In addition, fucoxanthin prominently upregulated the expression of lipid metabolism-related genes (Pparα and Cpt-1), promoting fatty acid β-oxidation metabolism. Additionally, fucoxanthin significantly increased the expression of Pgc-1α and Tfam, elevated the mtDNA/nDNA ratio, and reduced ROS levels. Further, we identified pyruvate kinase muscle isozyme 1 (PKM1) as a high-affinity protein for fucoxanthin by drug affinity-responsive target stability and LC-MS and confirmed their robust interaction by CETSA, microscale thermophoresis, and circular dichroism. Supplementation with pyruvate, the product of PKM1, significantly attenuated the beneficial effects of fucoxanthin on lipid deposition and insulin resistance. Mechanistically, fucoxanthin reduced glucose glycolysis rate and enhanced mitochondrial biosynthesis and fatty acid β-oxidation through inhibiting PKM1 activity, thereby alleviating lipid metabolic stress. These findings present a novel clinical strategy for treating metabolic diseases using fucoxanthin.

Berberine alleviates ulcerative colitis by inhibiting inflammation through targeting IRGM1

BackgroundBerberine (BBR), the main active component of Coptis chinensis Franch., has a variety of pharmacological effects, notably anti-inflammatory, which make it a potential treatment for ulcerative colitis (UC). Nevertheless, the specific target and the mode of action of BBR against UC are still unclear. PurposeHere, we aim to identify BBR's anti-inflammatory target and its mode of action in UC treatment. MethodsThe therapeutic effects of BBR and Coptis chinensis Franch. extract were first assessed in UC mice. Then, stable isotope labeling using amino acids in cell culture-activity-based protein profiling (SILAC-ABPP) was applied to identify the anti-inflammatory target proteins of BBR in an inflammation model of RAW264.7 cells stimulated by LPS. Molecular docking, drug affinity responsive target stability (DARTS), molecular dynamics simulation, cellular thermal shift assay (CETSA), and biological layer interference (BLI) measurement were employed to study the interaction between BBR and its targets. Lentiviral transfection was used to knock down the target protein and investigate BBR's anti-inflammatory mechanism. ResultsBBR and Coptis chinensis Franch. extracts both significantly alleviated UC in mice. SILAC-ABPP identified IRGM1 as BBR's anti-inflammatory target, with its overexpression reduced by BBR treatment in both RAW264.7 cell inflammation models stimulated by LPS and UC mice. BBR significantly reduced inflammatory cytokines in LPS-induced RAW264.7 cells by blocking the PI3K/AKT/mTOR pathway. Knockdown of IRGM1 weakened BBR's effects on cytokine expression and pathway regulation. ConclusionFor the first time, IRGM1 was identified as the direct anti-inflammatory target of BBR. BBR has the potential to inhibit IRGM1 expression in vitro as well as in vivo. The molecular mechanism of BBR's anti-inflammatory activity was inhibiting the PI3K/AKT/mTOR pathway by targeting IRGM1.

Nitidine chloride inhibits mTORC1 signaling through ATF4-mediated Sestrin2 induction and targets IGF2R for lysosomal degradation

AimsNitidine chloride (NC), a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb.) DC, exhibits multiple bioactivities, including antitumor, anti-inflammatory, and other therapeutic effects. However, the primary targets of NC and the mechanism of action (MOA) have not been explicitly defined. MethodsWe explored the effects of NC on mTORC1 signaling by immunoblotting and fluorescence microscopy in wild-type and gene knockout cell lines generated by the CRISPR/Cas9 gene editing technique. We identified IGF2R as a direct target of NC via the drug affinity-responsive target stability (DARTS) method. We investigated the antitumor effects of NC using a mouse melanoma B16 tumor xenograft model. Key findingsNC inhibits mTORC1 activity by targeting amino acid-sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction. NC directly binds to IGF2R and promotes its lysosomal degradation. Moreover, NC displayed potent cytotoxicity against various cancer cells and inhibited B16 tumor xenografts. SignificanceNC inhibits mTORC1 signaling through nutrient sensing and directly targets IGF2R for lysosomal degradation, providing mechanistic insights into the MOA of NC.