Affinity Responsive Target Stability Assay Research Articles

Erianin alleviates LPS-induced acute lung injury via antagonizing P-selectin-mediated neutrophil adhesion function

Ethnopharmacological relevanceDendrobium officinale Kimura et Migo, known as "Tiepi Shihu" in traditional Chinese medicine, boasts an extensive history of medicinal use documented in the Chinese Pharmacopoeia. "Shen Nong Ben Cao Jing" records D. officinale as a superior herbal medicine for fortifying "Yin" and invigorating the five viscera. Erianin, a benzidine compound, emerges as a prominent active constituent derived from D. officinale, with the pharmacological efficacy of D. officinale closely linked to the anti-inflammatory properties of erianin. Aim of the studyAcute lung injury (ALI) is a substantial threat to global public health, while P-selectin stands out as a promising novel target for treating acute inflammatory conditions. This investigation aims to explore the therapeutic potential of erianin in ALI treatment and elucidate the underlying mechanisms. Experimental designThe effectiveness of erianin in conferring protection against ALI was investigated through comprehensive histopathological and biochemical analyses of lung tissues and bronchoalveolar lavage fluid (BALF) in an in vivo model of LPS-induced ALI in mice. The impact of erianin on fMLP-induced neutrophil chemotaxis was quantitatively assessed using the Transwell and Zigmond chamber, respectively. To determine the therapeutic target of erianin and elucidate their binding capability, a series of sophisticated assays were employed, including drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and molecular docking analyses. ResultsErianin demonstrated a significant alleviation of LPS-induced acute lung injury, characterized by reduced total cell and neutrophil counts and diminished total protein contents in BALF. Moreover, erianin exhibited a capacity to decrease proinflammatory cytokine production in both lung tissues and BALF. Notably, erianin effectively suppressed the activation of NF-κB signaling in the lung tissues of LPS- challenged mice; however, it did not exhibit in vitro inhibitory effects on inflammation in LPS-induced human pulmonary microvascular endothelial cells (HPMECs). Additionally, erianin blocked the adhesion and rolling of neutrophils on HPMECs. While erianin did not influence endothelial P-selectin expression or cytomembrane translocation, it significantly reduced the ligand affinity between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). ConclusionsErianin inhibits P-selectin-mediated neutrophil adhesion to activated endothelium, thereby alleviating ALI. The present study highlights the potential of erianin as a promising lead for ALI treatment.

Unraveling the enigma: Molecular mechanisms of berberrubine-induced nephrotoxicity reversed by its parent form berberine

BackgroundBerberine is an isoquinoline alkaloid that is extensively applied in the clinic due to its potential therapeutic effects on dysentery and infectious diarrhoea. Its main metabolite, berberrubine, a promising candidate for ameliorating hyperlipidaemia, has garnered more attention than berberine. However, our study revealed that berberrubine induces severe kidney damage, while berberine was proven to be safe. PurposeHerein, we explored the opposite biological effects of these two compounds on the kidney and elucidated their underlying mechanisms. MethodsFirst, integrated metabolomic and proteomic analyses were conducted to identify relevant signalling pathways. Second, a click chemistry method combined with a cellular thermal shiftassay, a drug affinity responsive target stability assay, and microscale thermophoresis were used to identify the direct target proteins. Moreover, a mutation experiment was performed to study the specific binding sites. ResultsAnimal studies showed that berberrubine, but not berberine, induced severe chronic, subchronic, and acute nephrotoxicity. More importantly, berberine reversed the berberrubine-reduced nephrotoxicity. The results indicated that the cPLA2 signalling pathway was highly involved in the nephrotoxicity induced by berberrubine. We further confirmed that the direct target of berberrubine is the BASP1 protein (an upstream factor of cPLA2 signalling). Moreover, berberine alleviated nephrotoxicity by binding cPLA2 and inhibiting cPLA2 activation. ConclusionThis study is the first to revel the opposite biological effects of berberine and its metabolite berberrubine in inducing kidney injury. Berberrubine, but not berberine, shows strong nephrotoxicity. The cPLA2 signalling pathway can be activated by berberrubine through targeting of BASP1, while berberine inhibits this pathway by directly binding with cPLA2. Our study paves the way for studies on the exact molecular targets of herbal ingredients. We also demonstrated that natural small molecules and their active metabolites can have opposite regulatory roles in vivo through the same signalling pathway.

Structurally Diverse Alkaloids with Anti-Renal-Fibrosis Activity from the Centipede Scolopendra subspinipes mutilans.

Twelve new alkaloids, scolopenolines A-L (1-7, 9-11, 13, 14), along with six known analogues, were isolated from Scolopendra subspinipes mutilans, identified by analysis of spectroscopic data and quantum chemical and computational methods. Scolopenoline A (1), a unique guanidyl-containing C14 quinoline alkaloid, features a 6/6/5 ring backbone. Scolopenoline B (2) is a novel sulfonyl-containing heterodimer comprising quinoline and tyramine moieties. Scolopenoline G (7) presents a rare C12 quinoline skeleton with a 6/6/5 ring system. Alkaloids 1, 8, 10, and 15-18 display anti-inflammatory activity, while 10 and 16-18 also exhibit anti-renal-fibrosis activity. Drug affinity responsive target stability and RNA-interference assays show that Lamp2 might be a potentially important target protein of 16 for anti-renal-fibrosis activity.

Flavokawain B inhibits NF-κB inflammatory signaling pathway activation in inflammatory bowel disease by targeting TLR2

Inflammatory bowel disease (IBD) is characterized by recurrent inflammatory reactions in the intestinal mucosa, including ulcerative colitis (UC) and Crohn's disease (CD). The expression of Toll-like receptor 2 (TLR2) has been observed to increase during the progression of IBD. Flavokawain B (FKB), a natural chalcone with potent anti-inflammatory activity, exerts its effects through inhibition of the NF-κB pathway. In this study, we aimed to investigate the effects and mechanisms of FKB targeting TLR2 in IBD. C57BL/6 J mice were treated with 2.5% dextran sulfate sodium (DSS) for 7 days, with administration of FKB or TLR2 inhibitor C29 starting on day 2 to establish the model of IBD. In vitro, bone marrow-derived macrophages (BMDMs) were stimulated with the TLR2 agonist Pam3CSK4 to explore the therapeutic effect of FKB and its pharmacological mechanism. Compared with the model group, the FKB-treated group showed significant reductions in colitis-related injuries in the IBD mouse model, including weight gain, increased colon length and reduced inflammation. FKB decreased the formation of TLR2-MyD88 complex by targeting TLR2, leading to suppression of downstream NF-κB signaling pathway. Similar therapeutic effects were observed in the C29-treated group. Additionally, in vitro data suggested that FKB exerted its anti-inflammatory effect by targeting TLR2 and inhibiting Pam3CSK4-induced activation of the NF-κB pathway. The anti-inflammatory effects of FKB were demonstrated through drug affinity responsive target stability assay and cellular thermal shift assay, revealing its binding affinity to TLR2. By inhibiting the activation of the TLR2/NF-κB signaling pathway, FKB effectively prevented DSS-induced IBD and exhibited promising potential as a therapeutic candidate for IBD treatment.

Hydrangea paniculata coumarins alleviate adriamycin-induced renal lipotoxicity through activating AMPK and inhibiting C/EBPβ

Ethnopharmacological relevanceThroughout Chinese history, Hydrangea paniculata Siebold has been utilized as a traditional medicinal herb to treat a variety of ailments associated to inflammation. In a number of immune-mediated kidney disorders, total coumarins extracted from Hydrangea paniculata (HP) have demonstrated a renal protective effect. Aim of the studyTo investigate renal beneficial effect of HP on experimental Adriamycin nephropathy (AN), and further clarify whether reversing lipid metabolism abnormalities by HP contributes to its renoprotective effect and find out the underlying critical pathways. Materials and methodsAfter establishment of rat AN model, HP was orally administrated for 6 weeks. Biochemical indicators related to kidney injury were determined. mRNAs sequencing using kidney tissues were performed to clarify the underlying mechanism. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, western blot, molecular docking, and drug affinity responsive target stability (DARTS) assay was carried out to further explore and confirm pivotal molecular pathways and possible target by which HP and 7-hydroxylcoumarin (7-HC) played their renal protection effect via modulating lipid metabolism. ResultsHP could significantly improve renal function, and restore renal tubular abnormal lipid metabolism and interstitial fibrosis in AN. In vitro study demonstrated that HP and its main metabolite 7-HC could reduce ADR-induced intracellular lipid deposition and fibrosis characteristics in renal tubular cells. Mechanically, HP and 7-HC can activate AMP-activated protein kinase (AMPK) via direct interaction, which contributes to its lipid metabolism modulation effect. Moreover, HP and 7-HC can inhibit fibrosis by inhibiting CCAAT/enhancer binding protein beta (C/EBPβ) expression in renal tubular cells. Normalization of lipid metabolism by HP and 7-HC further provided protection of mitochondrial structure integrity and inhibited the nuclear factor kappa-B (NF-κB) pathway. Long-term toxicity using beagle dogs proved the safety of HP after one-month administration. ConclusionCoumarin derivates from HP alleviate adriamycin-induced lipotoxicity and fibrosis in kidney through activating AMPK and inhibiting C/EBPβ.

Gigantol ameliorates DSS-induced colitis via suppressing β2 integrin mediated adhesion and chemotaxis of macrophage

Ethnopharmacological relevanceDendrobium, recognized as “Shihu” in traditional Chinese medicine, holds a rich history of medicinal utilization documented in the Chinese Pharmacopoeia. Ancient texts like “Shen Nong Ben Cao Jing” extol Dendrobium's virtues as a superior herbal medicine fortifying “Yin” and invigorating the five viscera. Dendrobium is extensively employed for the treatment of gastrointestinal inflammatory disorders, showcasing significant therapeutic efficacy, particularly against ulcerative colitis (UC), within the realm of Chinese ethnopharmacology. Dendrobium plays crucial pharmacological roles due to its rich content of polysaccharides, alkaloids, phenanthrenes, and bibenzyls. Gigantol, a prominent bibenzyl compound, stands out as one of the most vital active constituents within Dendrobium, the gigantol content of Dendrobium leaves can reach approximately 4.79 μg/g. Its significance lies in being recognized as a noteworthy anti-inflammatory compound derived from Dendrobium. Aim of the studyGiven the pivotal role of gigantol as a primary active substance in Dendrobium, the therapeutic potential of gigantol for gastrointestinal diseases remains enigmatic. Our present investigation aimed to evaluate the therapeutic effects of gigantol on dextran sulfate sodium (DSS)-induced colitis and reveal its potential mechanism in countering UC activity. Materials and methodsThe protective efficacy of gigantol against colitis was assessed by examining the histopathological changes and conducting biochemical analyses of colon from DSS-challenged mice. Assessments focused on gigantol's impact on improving the intestinal epithelial barrier and its anti-inflammatory effects in colonic tissues of colitis mice. Investigative techniques included the exploration of the macrophage inflammatory signaling pathway via qPCR and Western blot analyses. In vitro studies scrutinized macrophage adhesion, migration, and chemotaxis utilizing transwell and Zigmond chambers. Furthermore, F-actin and Rac1 activation assays detailed cellular cytoskeletal remodeling. The potential therapeutic target of gigantol was identified and validated through protein binding analysis, competitive enzyme-linked immunosorbent assay (ELISA), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay. The binding sites between gigantol and its target were predicted via molecular docking. ResultsGigantol ameliorated symptoms of DSS-induced colitis, rectified damage to the intestinal barrier, and suppressed the production of pro-inflammatory cytokines in colonic tissues. Intriguingly, gigantol significantly curtailed NF-κB signaling activation in the colons of DSS-induced colitis mice. Notably, gigantol impaired the β2 integrin-dependent adhesion and migratory capacity of RAW264.7 cells. Moreover, gigantol notably influenced the cytoskeleton remodeling of RAW264.7 cells by suppressing Vav1 phosphorylation and Rac1 activation. Mechanistically, gigantol interacted with β2 integrin, subsequently diminishing binding affinity with intercellular adhesion molecule-1 (ICAM-1). ConclusionsIn conclusion, these findings elucidate that gigantol ameliorates DSS-induced colitis by antagonizing β2 integrin-mediated macrophage adhesion, migration, and chemotaxis, thus it may impede macrophage recruitment and infiltration into colonic tissues. This study suggests that gigantol shows promise as a viable candidate for clinical colitis therapy.

Abstract 2096: A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy

Abstract Background: Ferroptosis is a novel form of cell death driven by iron-dependent lipid peroxidation, which disrupts cellular membranes resulting in immune-activating death. A variety of tumor types, particularly those associated with enhanced lipid and/or iron content, or which experience increased oxidative stress (including drug tolerant persister cells), are sensitive to ferroptosis, rendering ferroptosis a promising therapeutic strategy for cancer. However, currently available ferroptosis inducers show limited activity in vivo, suggesting a need for novel molecular targets to validate the clinical utility of ferroptosis induction. Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of VHL resulting in constitutive activation of HIF-1α and HIF-2α, which contribute to disease progression, including by promoting lipid accumulation that sensitizes ccRCC cells to ferroptosis. Methods: We performed a high-throughput screen to identify small molecule inhibitors of HIF-2α followed by structure-activity-relationship studies for validation. The molecular target of these inhibitors was identified using the Drug Affinity Responsive Target Stability assay, and target validation was confirmed using siRNA and NMR. In vitro efficacy was determined in a panel of cancer cell lines and in vivo anti-tumor efficacy was verified using mouse models of cancer. Results: We identified a novel molecule, KD061, which decreases HIF-1/2α and induces ferroptosis in vivo through oral administration by targeting Iron Sulfur Cluster Assembly 2 (ISCA2). ISCA2 is a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex and ISCA2 inhibition either pharmacologically or using siRNA triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 inhibition also decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation. These studies reveal a novel function of ISCA2 in extra-mitochondrial Fe-S assembly that may be limiting in cancer due to the increased need for these proteins in DNA replication and maintenance. Outside of ccRCC, a 92-cell line screen reveals a variety of tumor types sensitive to ISCA2 inhibition including melanoma and clear cell ovarian cancer. Treatment of tumor-bearing mice with KD061 resulted in > 65% tumor growth inhibition, associated with significantly increased lipid peroxidation, thus confirming ferroptosis in vivo, and with decreased HIF-1/2α. The anti-tumor efficacy of KD061 exceeded that of α-PD1 and was well tolerated at the therapeutic dose. Conclusions: We describe the first orally available inducer of ferroptosis with significant anti-tumor efficacy in ccRCC and other tumor types. Citation Format: Yangsook Song Green, Maria Ferreira dos Santos, Simone Ciofi-Baffoni, Xiaohui Liu, Mei Yee Koh. A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2096.

Oxidative tryptamine dimers from Corynebacterium durum directly target survivin to induce AIF-mediated apoptosis in cancer cells

Accumulating evidence indicates that microbial communities in the human body crucially affect health through the production of chemical messengers. However, the relationship between human microbiota and cancer has been underexplored. As a result of a biochemical investigation of the commensal oral microbe, Corynebacterium durum, we identified the non-enzymatic transformation of tryptamine into an anticancer compound, durumamide A (1). The structure of 1 was determined using LC-MS and NMR data analysis as bis(indolyl)glyoxylamide, which was confirmed using one-pot synthesis and X-ray crystallographic analysis, suggesting that 1 is an oxidative dimer of tryptamine. Compound 1 displayed cytotoxic activity against various cancer cell lines with IC50 values ranging from 25 to 35 μM. A drug affinity responsive target stability assay revealed that survivin is the direct target protein responsible for the anticancer effect of 1, which subsequently induces apoptosis-inducing factor (AIF)-mediated apoptosis. Inspired by the chemical structure and bioactivity of 1, a new derivative, durumamide B (2), was synthesized using another indole-based neurotransmitter, serotonin. The anticancer properties of 2 were similar to those of 1; however, it was less active. These findings reinforce the notion of human microbiota-host interplay by showing that 1 is naturally produced from the human microbial metabolite, tryptamine, which protects the host against cancer.

Effect of resveratrol on herpesvirus encephalitis: Evidences for its mechanisms of action

BackgroundHerpes simplex virus type 1 (HSV-1)-induced herpes simplex encephalitis (HSE) has a high mortality rate in clinically immunocompromised patients, while recovered patients often experience neurological sequelae due to neuroinflammation. Nucleoside drugs and nucleoside analogues such as acyclovir and ganciclovir are mainly used in clinical treatment, and the emergence of resistant viral strains makes the development of new anti-herpesvirus encephalitis drugs urgent. Resveratrol is a multifunctional, plant-derived bioactive compound and its antiviral potential is attracting much attention. PurposeThis study aimed to investigate the anti-HSV-1 mechanism of resveratrol in microglial cells and in the HSE mouse model. MethodsThe antiviral effect of resveratrol on HSV-1 infection was investigated by plaque assay, virus titer, immunofluorescence, Western blot and time-of-addition assay. The influence of resveratrol on stimulator of interferon gene (STING)/Nuclear Factor kappa B (NF-κB) signaling pathway-mediated neuroinflammation was examined by Western blot, RT-qPCR and ELISA. The interaction between resveratrol and STING/heat shock protein 90 beta (HSP90β) was evaluated by molecular modeling, co-immunoprecipitation, and drug affinity responsive target stability assay. The therapeutic effect of resveratrol on HSE was evaluated in the HSE mouse model by analyzing weight loss, neurodegenerative symptoms and histopathological scores. ResultsResveratrol inhibited the early process of HSV-1 infection, and interfered with the STING/NF-κB signaling pathway to attenuate HSV-1-induced neuroinflammation and microglial M1 polarization, independent of its classical target Sirtuin1. Mechanistically, resveratrol completely bound to Glu515 and Lys491 of HSP90β, thus disrupting the HSP90β-STING interaction and promoting STING degradation. Resveratrol also significantly alleviated viral encephalitis and neuroinflammation caused by HSV-1 in the HSE mouse model. ConclusionResveratrol acted as a non-classical HSP90β inhibitor, binding to the STING-HSP90β interaction site to promote STING degradation and attenuate HSV-1-induced encephalitis and neuroinflammation. These findings suggest the alternative strategy of targeting HSP90β and resveratrol-mediated inhibition of HSP90β as a potential antiviral approach.

A novel DDIT3 activator dehydroevodiamine effectively inhibits tumor growth and tumor cell stemness in pancreatic cancer

BackgroundThe existence of pancreatic cancer stem cells (PCSCs) results in limited survival benefits from current treatment options. There is a scarcity of effective agents for treating pancreatic cancer patients. Dehydroevodiamine (DeHE), a quinazoline alkaloid isolated from the traditional Chinese herb Evodiae fructus, exhibited potent inhibition of pancreatic ductal adenocarcinoma (PDAC) cell proliferation and tumor growth both in vitro and in vivo. MethodsThe cytotoxic effect of DeHE on PDAC cells was assessed using CCK-8 and colony formation assays. The antitumor efficacy of DeHE were appraised in human PANC-1 xenograft mouse model. Sphere formation assay and flow cytometry were employed to quantify the tumor stemness. RNA‐Seq analysis, drug affinity responsive target stability assay (DARTS), and RNA interference transfection were conducted to elucidate potential signaling pathways. Western blotting and immunohistochemistry were utilized to assess protein expression levels. ResultsDeHE effectively inhibited PDAC cell proliferation and tumor growth in vitro and in vivo, and exhibited a better safety profile compared to the clinical drug gemcitabine (GEM). DeHE inhibited PCSCs, as evidenced by its suppression of self-renewal capabilities of PCSCs, reduced the proportion of ALDH+ cells and downregulated stemness-associated proteins (Nanog, Sox-2, and Oct-4) both in vitro and in vivo. Furthermore, there is potential involvement of DDIT3 and its downstream DDIT3/TRIB3/AKT/mTOR pathway in the suppression of stemness characteristics within DeHE-treated PDAC cells. Additionally, results from the DARTS assay indicated that DeHE interacts with DDIT3, safeguarding it against degradation mediated by pronase. Notably, the inhibitory capabilities of DeHE on PDAC cell proliferation and tumor stemness were partially restored by siDDIT3 or the AKT activator SC-79. ConclusionIn summary, our study has identified DeHE, a novel antitumor natural product, as an activator of DDIT3 with the ability to suppress the AKT/mTOR pathway. This pathway is intricately linked to tumor cell proliferation and stemness characteristics in PDAC. These findings suggest that DeHE holds potential as a promising candidate for the development of innovative anticancer therapeutics.

Jin-Gui-Shen-Qi Wan alleviates fibrosis in mouse diabetic nephropathy via MHC class II

Ethnopharmacological relevanceJin-Gui-Shen-Qi Wan (JGSQW) is a traditional Chinese medicine formula that has been traditionally used to alleviate urinary system ailments such as frequent urination and polyuria. Clinical studies have indicated that when combined with hypoglycaemic drugs, JGSQW exhibits a synergistic effect and can improve diabetic nephropathy (DN), yet its underlying mechanism and targets remain unclear. Aim of the studyThis study aims to investigate the therapeutic efficacy of JGSQW and its underlying mechanisms using a DN db/db mouse model. Materials and methodsUltrahigh-performance liquid chromatography coupled with mass spectrometry was utilized to analyse the primary active compounds, blood levels, and pharmacokinetics of JGSQW. Additionally, the therapeutic effects of JGSQW and metformin on blood glucose levels, lipid levels, renal function, and renal pathology in diabetic nephropathy mice were investigated using a db/db mouse model. Proteomic analysis was carried out to identify the primary target of JGSQW in treating DN. The mechanism of action was verified by western blotting, immunohistochemistry, and immunofluorescence. Then, molecular docking and molecular dynamics, transfection, drug affinity responsive target stability (DARTS) assay and cell thermal migration assay (CETSA) further validated the targeted binding effect. ResultsJGSQW combined with metformin significantly improved the blood glucose levels, blood lipids, renal function, and renal pathology of DN mice. JGSQW mainly exerted its therapeutic effect on DN by targeting major histocompatibility complex class II (MHC class II) molecules. Immunohistochemistry results showed that JGSQW inhibited the expression of collagen I, fibronectin, and alpha smooth muscle actin (α-SMA) expression. Immunofluorescence and Western blot results showed that JGSQW inhibited the expression of H2-Ab1 and H2-Aa, which are MHC class II molecules, thereby suppressing CD4+ T-cell infiltration and improving diabetic kidney fibrosis. The binding ability of paeoniflorin to H2-Aa was predicted and verified by molecular, DARTS, and CETSA assays. Treatment with 80 μM paeoniflorin effectively alleviated high glucose-induced injury in the MPC-5 injury model. H2-Aa was overexpressed at this model concentration, and Western blotting further confirmed that paeoniflorin reduced glomerular podocyte fibrosis by regulating H2-Aa. ConclusionsJGSQW combined with metformin may have a synergistic effect to alleviates renal fibrosis in diabetic nephropathy by downregulating immune complex MHC class II molecules and attenuating the antigen presentation effect of MHC class II on CD4.

Unveiling Vacuolar H+-ATPase Subunit a as the Primary Target of the Pyridinylmethyl-Benzamide Fungicide, Fluopicolide.

An estimated 240 fungicides are presently in use, but the direct targets for the majority remain elusive, constraining fungicide development and efficient resistance monitoring. In this study, we found that Pcα-actinin knockout did not influence the sensitivity of Phytophthora capsici to fluopicolide, which is a notable oomycete inhibitor. Using a combination of Bulk Segregant Analysis Sequencing and Drug Affinity Responsive Target Stability (DARTS) assays, the vacuolar H+-ATPase subunit a (PcVHA-a) was pinpointed as the target protein of fluopicolide. We also confirmed four distinct point mutations in PcVHA-a responsible for fluopicolide resistance in P. capsici through site-directed mutagenesis. Molecular docking, ATPase activity assays, and a DARTS assay suggested a fluopicolide-PcVHA-a interaction. Sequence analysis and further molecular docking validated the specificity of fluopicolide for oomycetes or fish. These findings support the claim that PcVHA-a is the target of fluopicolide, proposing vacuolar H+-ATPase as a promising target for novel fungicide development.

Formononetin, a bioactive isoflavonoid constituent from Astragalus membranaceus (Fisch.) Bunge, ameliorates type 1 diabetes mellitus via activation of Keap1/Nrf2 signaling pathway: An integrated study supported by network pharmacology and experimental validation

Ethnopharmacological relevanceType 1 diabetes mellitus (T1DM) results from insulin deficiency due to the destruction of pancreatic β-cells. Previously, our studies showed that inhibition of Keap1/Nrf2 signaling pathway promoted the onset of T1DM, which suggests that finding drugs that can activate the Keap1/Nrf2 signaling may be a promising therapeutic strategy for the T1DM treatment. Astragalus membranaceus (Fisch.) Bunge is a common traditional Chinese medicine that has been frequently applied in Chinese clinics for the treatment of diabetes and other diseases. Formononetin (FMNT), one of the major isoflavonoid constituents isolated from this herbal medicine, possesses diverse pharmacological benefits and T1DM therapeutic potential. However, the exact molecular mechanisms underlying the action of FMNT in ameliorating T1DM have yet to be fully elucidated. Aims of the studyThis study is to investigate the regulation of FMNT on the Keap1/Nrf2 signaling pathway to ameliorate T1DM based on network pharmacology approach combined with experimental validation. Materials and methodsA mouse-derived pancreatic islet β-cell line (MIN6) was used for the in vitro studies. An alloxan (ALX)-induced T1DM model in wild-type and Nrf2 knockout (Nrf2−/−) C57BL/6J mice were established for the in vivo experiments. The protective effects of FMNT against ALX-stimulated MIN6 cell injury were evaluated using MTT, EdU, apoptosis and comet assays. The levels of blood glucose in mice were measured by using a blood monitor and test strips. The protein expression was detected by Western blot analysis. Furthermore, the binding affinity of FMNT to Keap1 was evaluated using cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and solvent-induced protein precipitation (SIP) assay. The interaction pattern between FMNT and Keap1 was assessed by molecular docking and molecular dynamics simulation techniques. ResultsNetwork pharmacology analysis revealed that FMNT exerted its therapeutic effect against T1DM by mainly regulating oxidative stress response-associated signaling molecules and pathways, such as Nrf2 regulating anti-oxidant/detoxification enzymes and Keap1-Nrf2 signaling pathway. The in vivo results showed that FMNT significantly deceased the ALX-induced high blood glucose levels and conversely increased the ALX-induced low insulin contents. In vitro, FMNT markedly protected MIN6 cells from ALX-induced cytotoxicity, proliferation inhibition and DNA damage and reduced the ALX-stimulated cell apoptosis. FMNT also inhibited ALX-induced overproduction of intracellular ROS to alleviate oxidative stress. In addition, FMNT could bind to Keap1 to notably activate the Keap1/Nrf2 signaling to upregulate Nrf2 expression and promote the Nrf2 translocation from the cytoplasm to the nucleus, resulting in enhancing the expression of antioxidant proteins HO-1 and NQO1. Inhibition of Keap1/Nrf2 signaling by ALX was also markedly abolished in the cells and mice exposed to FMNT. Moreover, these effects of FMNT in ameliorating T1DM were not observed in Nrf2−/− mice. ConclusionsThis study demonstrates that FMNT could bind to Keap1 to activate the Keap1/Nrf2 signaling to prevent intracellular ROS overproduction, thereby attenuating ALX-induced MIN6 cell injury and ameliorating ALX-stimulated T1DM. Results from this study might provide evidence and new insight into the therapeutic effect of FMNT and indicate that FMNT is a promising candidate agent for the treatment of T1DM in clinics.

Phenylacetyl glutamine (PAGln) enhances cardiomyocyte death after myocardial infarction through β1 adrenergic receptor.

This study aims to explore the roles of phenylacetyl glutamine (PAGln) on myocardial infarction (MI) pathogenesis. Here, using targeted metabolomics analysis, it was found that the plasma metabolite PAGln was upregulated in coronary artery disease (CAD) patients and MI mice and could be an independent risk factor for CAD. In vivo and in vitro functional experiments revealed that PAGln pretreatment enhanced MI-induced myocardial injury and cardiac fibrosis, as evident by the increased infarct size, cardiomyocyte death, and the upregulated expression of cardiac fibrosis markers (Col1a1 and α-SMA). Combined with RNA-sequencing analysis and G protein-coupled receptor (GPCR) inhibitor, we found that the GPCR signaling activation is essential for PAGln-mediated effects on cardiomyocyte death. Furthermore, drug affinity responsive target stability and cellular thermal shift assay demonstrated that PAGln could interact with β1-adrenergic receptor (AR). Moreover, β1-AR blocker treatment indeed extended the cardiac remodeling after PAGln-enhanced MI. These results suggest that PAGln might be a potential therapeutic target for extending the cardiac remodeling window in MI patients that signals via β1-AR.

Luteolin ameliorates DSS-induced colitis in mice via suppressing macrophage activation and chemotaxis

ObjectivesLuteolin, known for its multifaceted therapeutic properties against inflammatory diseases, holds potential for addressing the unmet need for effective treatments in ulcerative colitis (UC), a prevalent subtype of inflammatory bowel disease (IBD). This study aimed to comprehensively assess luteolin's therapeutic efficacy in a dextran sulfate sodium (DSS)-induced colitis mouse model, shedding light on its anti-UC mechanisms. MethodsOur investigation encompassed in vivo assessments of luteolin's therapeutic potential against DSS-induced colitis through rigorous histopathological examination and biochemical analyses. Furthermore, we scrutinized luteolin's anti-inflammatory prowess in vitro using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary peritoneal macrophages. Additionally, we quantitatively evaluated the impact of luteolin on C–C motif chemokine ligand 2 (CCL2)-induced macrophage migration employing Transwell and Zigmond chambers. Furthermore, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and molecular docking were employed to identify potential therapeutic targets of luteolin and investigate their binding sites and interaction patterns. ResultsLuteolin demonstrated therapeutic potential against DSS-induced colitis by ameliorating colitis symptoms, restoring intestinal barrier integrity, and inhibiting proinflammatory cytokine production in the colonic tissues. Moreover, luteolin demonstrated robust anti-inflammatory activity in vitro, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary peritoneal macrophages. Notably, luteolin suppressed the phosphorylation of IKKα/β, IκBα, and p65, along with preventing IκBα degradation in LPS-treated RAW264.7 cells and peritoneal macrophages. Furthermore, luteolin impaired the migratory behavior of RAW264.7 cells and peritoneal macrophages, as evidenced by reduced migration distance and velocity of luteolin-treated macrophages. Mechanistically, luteolin was found to antagonize IKKα/β, subsequently inhibiting IKKα/β phosphorylation and the activation of NF-κB signaling. ConclusionLuteolin emerges as a promising lead compound for the clinical therapy of colitis by virtue of its ability to ameliorate DSS-induced colitis, antagonize IKKα/β, suppress NF-κB signaling, and impede macrophage activation and migration.

A natural sesquiterpene lactone isolinderalactone attenuates lipopolysaccharide-induced inflammatory response and acute lung injury through inhibition of NF-κB pathway and activation Nrf2 pathway in macrophages

Isolinderalactone is the main sesquiterpene lactone isolated from Lindera aggregata, a traditional Chinese medicine widely used to treat pain and inflammation. Although isolinderalactone has been demonstrated to possess anti-cancer effect, its anti-inflammatory activity and underlying mechanism has not been well characterized. Herein, isolinderalactone was able to significantly inhibit the production of NO and PGE2 by reducing the expressions of iNOS and COX2 in LPS-stimulated RAW264.7 macrophages and BMDMs, and decreased the mRNA levels of IL-1β, IL-6, and TNF-α in LPS-induced RAW264.7 cells. In vivo, isolinderalactone effectively alleviated LPS-induced acute lung injury (ALI), which manifested as reduction in pulmonary inflammatory infiltration, myeloperoxidase activity, and production of PGE2, IL-1β, IL-6, TNF-α, and malondialdehyde. Furthermore, isolinderalactone inhibited phosphorylation of IKKα/β, phosphorylation and degradation of IκBα, and nuclear translocation of NF-κB p65, thereby blocking NF-κB pro-inflammatory pathway. Meanwhile, isolinderalactone reduced the intracellular ROS through promoting the activation of Nrf2-HMOX1 antioxidant axis. By using drug affinity responsive target stability assay and molecular docking, isolinderalactone was found to covalently interact with IKKα/β and Keap1, which may contribute to its anti-inflammatory action. Additionally, a thiol donor β-mercaptoethanol significantly abolished isolinderalactone-mediated anti-inflammatory action in vitro, indicating the crucial role of the unsaturated lactone of isolinderalactone on its anti-inflammatory effects. Taken together, isolinderalactone protected against LPS-induced ALI in mice, which may be associated with its inhibition of NF-κB pathway and activation of Nrf2 signaling in macrophages.

BNTA attenuates temporomandibular joint osteoarthritis progression by directly targeting ALDH3A1: An in vivo and in vitro study

BNTA is known to have a therapeutic effect on knee osteoarthritis and inflammatory osteoclastogenesis. However, the protective effect of BNTA regarding temporomandibular mandibular joint osteoarthritis (TMJOA) and its underlying mechanism and physiological target remains unclear. In the present study, BNTA ameliorated cartilage degradation and inflammation responses in monosodium iodoacetate (MIA)-induced TMJOA in vivo. In IL-1β-induced condylar chondrocytes, BNTA prevents oxidative stress, inflammatory responses and increasing synthesis of cartilage extracellular matrix through activating nuclear factor-E2-related factor 2 (NRF2) signaling. Suppression of NRF2 signaling abolishes the protective effect of BNTA in TMJOA. Notably, BNTA may bind directly to ALDH3A1 and act as a stabilizer, as evidenced by drug affinity responsive target stability assay (DARTS), cellular thermal shift assay (CETSA) and molecular docking results. Further investigation of the underlying molecular and cellular mechanism infers a positive correlation of ALDH3A1 regulating NRF2 signaling. In conclusion, BNTA may attenuate TMJOA progression via the ALDH3A1/NRF2 axis, inferring that BNTA is a therapeutic target for treating temporomandibular mandibular joint osteoarthritis.

Fighting drug-resistant lung cancer by induction of NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated ferroptosis.

Drug resistance is a major challenge in cancer treatment. The substrates of NAD(P)H:quinone oxidoreductase 1 (NQO1) show a promising anticancer effect in clinical trials. We previously identified a natural NQO1 substrate 2-methoxy-6-acetyl-7-methyljuglone (MAM) with a potent anticancer effect. The present study was designed to explore the efficacy of MAM in fighting against drug-resistant non-small cell lung cancer (NSCLC). The anticancer effect of MAM was evaluated in cisplatin-resistant A549 and AZD9291-resistant H1975 cells. The interaction of MAM with NQO1 was measured by cellular thermal shift assay and drug affinity responsive target stability assay. The NQO1 activity and expression were measured using NQO1 recombinant protein, Western blotting, and immunofluorescence staining assay. The roles of NQO1 were examined by NQO1 inhibitor, small interfering RNA (siRNA), and short hairpin RNA (shRNA). The roles of reactive oxygen species (ROS), labile iron pool (LIP), and lipid peroxidation were determined. MAM induced significant cell death in drug-resistant cells with similar potency to that of parental cells, which were completely abolished by NQO1 inhibitor, NQO1 siRNA, and iron chelators. MAM activates and binds to NQO1, which triggers ROS generation, LIP increase, and lipid peroxidation. MAM significantly suppressed tumor growth in the tumor xenograft zebrafish model. These results showed that MAM induced ferroptosis by targeting NQO1 in drug-resistant NSCLC cells. Our findings provided a novel therapeutic strategy for fighting against drug resistance by induction of NQO1-mediated ferroptosis.

Polyphyllin VII induces autophagy-dependent ferroptosis in human gastric cancer through targeting T-lymphokine-activated killer cell-originated protein kinase.

T-lymphokine-activated killer cell-originated protein kinase (TOPK) is a serine-threonine kinase that is overexpressed in gastric cancer (GC) and promotes tumor progression. Polyphyllin VII (PPVII), a pennogenin isolated from the rhizomes of Paris polyphylla, shows anticancer effects. Here, we explored the antitumor activity and mechanism of PPVII in GC. Ferroptosis was detected by transmission electron microscope, malondialdehyde, and iron determination assays. Autophagy and its upstream signaling pathway were detected by Western blot, and gene alterations. The binding of PPVII and TOPK was examined through microscale thermophoresis and drug affinity responsive target stability assays. An in vivo mouse model was performed to evaluate the therapeutic of PPVII. PPVII inhibits GC by inducing autophagy-mediated ferroptosis. PPVII promotes the degradation of ferritin heavy chain 1, which is responsible for autophagy-mediated ferroptosis. PPVII activates the Unc-51-like autophagy-activating kinase 1 (ULK1) upstream of autophagy. PPVII inhibits the activity of TOPK, thereby weakening the inhibition of downstream ULK1. PPVII stabilizes the dimer of the inactive form of TOPK by direct binding. PPVII inhibits tumor growth without causing obvious toxicity in vivo. Collectively, this study suggests that PPVII is a potential agent for the treatment of GC by targeting TOPK to activate autophagy-mediated ferroptosis.

Narirutin ameliorates alcohol-induced liver injury by targeting MAPK14 in zebrafish larvae

BackgroundAlcohol-associated liver disease (ALD) encompasses a range of hepatic abnormalities, including isolated alcoholic steatosis, steatohepatitis, and cirrhosis. The flavanone-7-O-glycoside narirutin (NRT), the primary flavonoid in citrus peel, has antioxidant, anti-inflammatory, and lipid-lowering activity. We investigated the effects of NRT on liver injury induced by alcohol and explored the underlying mechanisms. MethodsZebrafish larvae were used to investigate the effects of NRT on acute exposure to ethanol (EtOH). Liver phenotypic, morphological, and biochemical assessments were performed to evaluate the hepatoprotective effects of NRT. Network pharmacology and molecular docking analyses were conducted to identify candidate targets of NRT in EtOH-induced liver injury. A drug affinity responsive target stability (DARTS) assay was conducted to evaluate the binding of NRT to mitogen-activated protein kinase 14 (MAPK14). The mechanism of action of NRT was validated by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analysis. ResultsThe liver phenotypic, morphological, and biochemical assessments revealed that NRT has potential therapeutic effects against acute EtOH-induced liver injury. RT-qPCR confirmed that NRT reversed the change in the expression of genes related to oxidative stress, lipogenesis, and the endoplasmic reticulum (ER)/unfolded protein response pathway. Network pharmacology and molecular docking analyses identified potential targets of NRT’s protective effects and confirmed that NRT regulates the p38 MAPK signaling pathway by targeting mitogen-activated protein kinase 14 (MAPK14). ConclusionsNRT mitigates alcohol-induced liver injury by preventing lipid formation, protecting the antioxidant system, and suppressing ER stress–induced apoptosis through MAPK14 modulation.