Cellular Thermal Shift Assay Research Articles

Lycopene from tomatoes and tomato products exerts renoprotective effects by ameliorating oxidative stress, apoptosis, pyroptosis, fibrosis, and inflammatory injury in calcium oxalate nephrolithiasis: the underlying mechanisms.

Several mechanisms underlying nephrolithiasis, one of the most common urological diseases, involve calcium oxalate formation, including oxidative stress, inflammatory reactions, fibrosis, pyroptosis, and apoptosis. Although lycopene has strong antioxidant activity, its protective effects against CaOx-induced injury have not yet been reported. This study aimed to systematically investigate the protective effects of lycopene and explore its mechanisms and molecular targets. Crystal deposition, renal function, oxidative stress, inflammatory response, fibrosis, pyroptosis, and apoptosis were assessed to evaluate the renoprotective effects of lycopene against crystal formation in a CaOx rat model and oxalate-stimulated NRK-52E and HK-2 cells. Lycopene markedly ameliorated crystal deposition, restored renal function, and suppressed kidney injury by reducing oxidative stress, apoptosis, inflammation, fibrosis, and pyroptosis in the rats. In cell models, lycopene pretreatment reversed reactive oxygen species increase, apoptotic damage, intracellular lactate dehydrogenase release, cytotoxicity, pyroptosis, and extracellular matrix deposition. Network pharmacology and proteomic analyses were performed to identify lycopene target proteins under CaOx-exposed conditions, and the results showed that Trappc4 might be a pivotal target gene for lycopene, as identified by cellular thermal shift assay and surface plasmon resonance analyses. Based on molecular docking, molecular dynamics simulations, alanine scanning mutagenesis, and saturation mutagenesis, we observed that lycopene directly interacts with Trappc4 via hydrophobic bonds, which may be attributed to the PHE4 and PHE142 residues, preventing ERK1/2 or elevating AMPK signaling pathway phosphorylation events. In conclusion, lycopene might ameliorate oxalate-induced renal tubular epithelial cell injury via the Trappc4/ERK1/2/AMPK pathway, indicating its potential for the treatment of nephrolithiasis.

Matrine induces ferroptosis in cervical cancer through activation of piezo1 channel.

Cervical cancer, which is a significant public health concern in women, currently lacks effective therapeutic drugs. Matrine, a constituent of the traditional Chinese herb Sophora flavescentis Radix, is known for its anti-cervical cancer properties and ability to induce programmed cell death. The induction of cancer cell ferroptosis, which is a novel cell death pattern, can become an effective clinical therapy for tumor in the future. However, the effect of matrine on ferroptosis in cervical cancer remains to be elucidated. In this study, we investigated whether matrine induces ferroptosis in cervical cancer and elucidated the underlying mechanisms. We established an SiHa-derived tumor-bearing mouse model using CB17 severe combined immunodeficient (SCID) mice and administered a group of matrine (25, 50, and 75mg/kg) and cisplatin (2mg/kg). We meticulously tracked alterations in body weight and tumor size and evaluated liver and kidney health using haematoxylin and eosin (H&E) staining. Using Gene Expression Omnibus (GEO) Dataset (GSE201309), we evaluated the relationship between the effects of matrine on malignant tumor cells and ferroptosis. In vitro, tetrazolium-based colorimetric (MTT), lactate dehydrogenase (LDH) and colony formation assays were used to study the effects of matrine on SiHa cell activity and cytotoxicity. We assessed ferroptosis-related protein abundance using western blotting and ferroptosis-related indices in cells using confocal immunofluorescence microscopy. The interaction of matrine with a protein linked to ferroptosis was studied using cellular thermal shift assay (CETSA). The effects of matrine on Piezo1 expression were investigated using calcium imaging. We also used Piezo1-specific siRNA to explore the role of Piezo1 in ferroptosis. Matrine administration effectively inhibited tumor growth in a SiHa-derived tumor-bearing mouse model without inducing noticeable harm. The analysis results of GEO data set show matrine-induced effects in tumor cells were indeed involved in the process of ferroptosis. Treatment with matrine resulted in a significant reduction in GPX4 protein levels and a concurrent increase in lipid peroxide and Fe2+ content, suggesting matrine-induced modulation of ferroptosis. Matrine promoted SiHa cell death in vitro, as evidenced by the results of MTT and LDH assays. Cell death coincides with increases in intracellular Fe2+, reactive oxygen species (ROS), and lipid peroxides. Our study also revealed significant upregulation of Piezo1 expression through the action of matrine, whereas transferrin receptor (Tfr) and System Xc- (xCT) expression and interaction remained unaffected. We provided further evidence that matrine induces calcium influx through the Piezo1 channel, thereby potentially influencing ferroptosis. Transfection with Piezo1 siRNA reversed the effects of matrine in SiHa cell. Our findings indicate that matrine exerts a protective effect against cervical cancer by inducing ferroptosis through the activation of Piezo1, but not xCT or Tfr.

Corynoline inhibits esophageal squamous cell carcinoma growth via targeting Pim-3

BackgroundEsophageal squamous cell carcinoma (ESCC) is an aggressive and deadly malignancy characterized by late-stage diagnosis, therapy resistance, and a poor 5-year survival rate. Finding novel therapeutic targets and their inhibitors for ESCC prevention and therapy is urgently needed. MethodsWe investigated the proviral integration site for maloney murine leukemia virus 3 (Pim-3) protein levels using immunohistochemistry. Using Methyl Thiazolyl Tetrazolium and clone formation assay, we verified the function of Pim-3 in cell proliferation. The binding and inhibition of Pim-3 by corynoline were verified by computer docking, pull-down assay, cellular thermal shift assay, and kinase assay. Cell proliferation, Western blot, and a patient-derived xenograft tumor model were performed to elucidate the mechanism of corynoline inhibiting ESCC growth. Results: Pim-3 was highly expressed in ESCC and played an oncogenic role. The augmentation of Pim-3 enhanced cell proliferation and tumor development by phosphorylating mitogen-activated protein kinase 1 at T185 and Y187. The deletion of Pim-3 induced apoptosis with upregulated cleaved caspase-9 and lower Bcl2 associated agonist of cell death phosphorylation at S112. Additionally, binding assays demonstrated corynoline directly bound with Pim-3, inhibiting its activity, and suppressing ESCC growth. ConclusionsOur findings suggest that Pim-3 promotes ESCC progression. Corynoline inhibits ESCC progression through targeting Pim-3.

Targeting the smooth muscle cell Keap1-Nrf2-GSDMD-pyroptosis axis by cryptotanshinone prevents abdominal aortic aneurysm formation.

Rationale: Abdominal aortic aneurysm (AAA) is an inflammatory, fatal aortic disease that currently lacks any effective drugs. Cryptotanshinone (CTS) is a prominent and inexpensive bioactive substance derived from Salvia miltiorrhiza Bunge, a well-known medicinal herb for treating cardiovascular diseases through its potent anti-inflammatory properties. Nevertheless, the therapeutic effect of CTS on AAA formation remains unknown. Methods: To investigate the therapeutic effect of CTS in AAA, variety of experimental approaches were employed, majorly including AAA mouse model establishment, real-time polymerase chain reaction (PCR), RNA sequencing, western blot, co-immunoprecipitation, scanning/transmission electron microscopy (SEM/TEM), enzyme-linked immunosorbent assay (ELISA), seahorse analysis, immunohistochemistry, and confocal imaging. Results: In this study, we demonstrated that CTS suppressed the formation of AAA in apolipoprotein E knock-out (ApoE-/-) mice infused with Ang II. A combination of network pharmacology and whole transcriptome sequencing analysis indicated that activation of the Keap1-Nrf2 pathway and regulation of programmed cell death in vascular smooth muscle cells (VSMCs) are closely linked to the anti-AAA effect of CTS. Mechanistically, CTS promoted the transcription of Nrf2 target genes, particularly Hmox-1, which prevented the activation of NLRP3 and GSDMD-initiated pyroptosis in VSMCs, thereby mitigating VSMC inflammation and maintaining the VSMC contractile phenotype. Subsequently, by utilizing molecular docking, together with the cellular thermal shift assay (CETSA) and isothermal titration calorimetry (ITC), a particular binding site was established between CTS and Keap1 at Arg415. To confirm the binding site, site-directed mutagenesis was performed, which intriguingly showed that the Arg415 mutation eliminated the binding between CTS and the Keap1-Nrf2 protein and abrogated the antioxidant and anti-pyroptosis effects of CTS. Furthermore, VSMC-specific Nrf2 knockdown in mice dramatically reversed the protective action of CTS in AAA and the inhibitory effect of CTS on VSMC pyroptosis. Conclusion: Naturally derived CTS exhibits promising efficacy as a treatment drug for AAA through its targeting of the Keap1-Nrf2-GSDMD-pyroptosis axis in VSMCs.

Dual inhibitory potential of ganoderic acid A on GLUT1/3: computational and in vitro insights into targeting glucose metabolism in human lung cancer.

Human glucose transporters (GLUTs) facilitate the uptake of hexoses into cells. In cancer, the increased proliferation necessitates higher expression of GLUTs, with particular emphasis on GLUT1 and GLUT3. Thus, inhibiting GLUTs holds promise as an anticancer therapy by starving these cells of fuel. Ganoderic acid A (GAA), a triterpene found in Ganoderma lucidum, has anticancer and antidiabetic properties. Recent studies show that GAA reduces glucose uptake in cancer cells, which indicates that GAA may affect GLUT1/GLUT3 by inhibiting glucose uptake. Therefore, this study aimed to inspect whether GAA could target GLUT1/GLUT3 and play an inhibitory role in changing their endofacial and exofacial conformations. To this end, AlphaFold2 was employed to model the endofacial and exofacial conformations of GLUT3 and GLUT1, respectively. Molecular docking, molecular dynamics simulation, cell viability, cellular thermal shift assays (CETSA), glucose uptake, qPCR, and western blotting were harnessed. In comparison to the endofacial (cytochalasin B) and exofacial (phloretin) GLUT1/3 inhibitors, the computational findings unveiled GAA's capacity to bind and stabilize GLUT1/3 in their two conformational states, with a preference for binding the endofacial conformation. A low, non-cytotoxic dose of GAA thermally stabilized both transporters and inhibited glucose uptake in human lung cancer cells, similar to cytochalasin B and phloretin. In conclusion, this study has unearthed novel functionalities of GAA, suggesting its potential utility in cancer therapy by targeting glucose metabolism.

Quercetin relieves coronary atherosclerosis via regulating fibroblast growth factor 2.

This study aims to investigate the role of quercetin in coronary atherosclerosis and explore its possible mechanisms. Hematoxylin-eosin (H&E), immunohistochemical (IHC), and aniline blue staining were used to analyze the pathological changes in the cross-section of the aorta. Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), Swiss Target Prediction, and PubChem were utilized to predict and screen the bioactive ingredients of traditional Chinese medicine (Huanglian, Yuxingcao, and Jinyinhua) for coronary atherosclerosis. Inflammatory factors and vascular protection parameters were quantitatively detected using ELISA and western blot. The proliferation and migration of vascular smooth muscle cells (VSMC) were evaluated using the Cell Counting Kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), and wound healing assays. The targets of quercetin were predicted using DisGeNET, Matascape, SWISSMODEL, cellular thermal shift assay (CETSA), and fluorescence titrimetric methods. Based on our findings, quercetin was identified as the active component of Huanglian, Yuxingcao, and Jinyinhua that exerted a positive effect on coronary atherosclerosis. In vivo and in vitro data demonstrated that quercetin improved the pathological changes in model mice and inhibited the proliferation, migration, and inflammatory response of VSMC cells. Specifically, we found that fibroblast growth factor 2 (FGF2) is a direct target of quercetin, and overexpression of FGF2 attenuated the anti-atherosclerosis function of quercetin. Overall, our study confirms the functional role of the quercetin-FGF2 axis in the progression of coronary atherosclerosis, providing a potential target for its treatment.

Matrine Targets BTF3 to Inhibit the Growth of Canine Mammary Tumor Cells.

The canine mammary tumor model is more suitable for studying human breast cancer, and the safety concentrations of matrine and the biotin-labeled matrine probe were determined in canine primary mammary epithelial cells, and then selected canine mammary tumor cell lines CHMm and CHMp were incubated with matrine, and cell viability was detected by CCK-8. The biotin-labeled matrine probe was used to pull-down the targets of matrine in canine mammary tumor cells, and the targets were screened in combination with activity-based protein profiling (ABPP) and Genecards database, and verified by qPCR and western blot. The results showed that the maximum non-cytotoxic concentrations of matrine and biotin-labeled matrine probe in canine primary mammary epithelial cells were 250 μg/mL and 500 μg/mL, respectively. Matrine and biotin-labeled matrine probe had a proliferation inhibitory effect time-dependently on CHMm and CHMp cells within a safe concentration range, and induced autophagy in cells. Then BTF3 targets were obtained by applying ABPP and Genecards screening. Cellular thermal shift assay (CETSA) findings indicated that matrine could increase the heat stability of BTF3 protein. Pull-down employing biotin-labeled matrine probe with CHMm and CHMp cell lysates revealed that BTF3 protein was detected in the biotin-labeled matrine probe group and that BTF3 protein was significantly decreased by the addition of matrine. The qPCR and western blot findings of CHMm and CHMp cells treated with matrine revealed that matrine decreased the expression of the BTF3 gene and protein with the extension of the action time, and the impact was more substantial at the protein level, respectively.

Anemoside B4, a new pyruvate carboxylase inhibitor, alleviates colitis by reprogramming macrophage function.

Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), whichwas found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to itsmolecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA),a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation andoxidative stress. These findings shed more light on thepossibilities ofPC as a potential new target for treating colitis.

Alisol B regulates AMPK/mTOR/SREBPs via directly targeting VDAC1 to alleviate hyperlipidemia

BackgroundThe occurrence of hyperlipidemia is significantly influenced by lipid synthesis, which is regulated by sterol regulatory element binding proteins (SREBPs), thus the development of drugs that inhibit lipid synthesis has become a popular treatment strategy for hyperlipidemia. Alisol B (ALB), a triterpenoid compound extracted from Alisma, has been reported to ameliorate no-nalcoholic steatohepatitis (NASH) and slow obesity. However, the effect of ALB on hyperlipidemia and mechanism are unclear. PurposeTo examine the therapeutic impact of ALB on hyperlipidemia whether it inhibits SREBPs to reduce lipid synthesis. Study DesignHepG2, HL7702 cells, and C57BL/6J mice were used to explore the effect of ALB on hyperlipidemia and the molecular mechanism in vivo and in vitro. MethodsHyperlipidemia models were established using western diet (WD)-fed mice in vivo and oleic acid (OA)-induced hepatocytes in vitro. Western blot, real-time PCR and other biological methods verified that ALB regulated AMPK/mTOR/SREBPs to inhibit lipid synthesis. Cellular thermal shift assay (CETSA), molecular dynamics (MD), and ultrafiltration-LC/MS analysis were used to evaluate the binding of ALB to voltage-dependent anion channel protein-1 (VDAC1). ResultsALB decreased TC, TG, LDL-c, and increased HDL-c in blood, thereby ameliorating liver damage. Gene set enrichment analysis (GSEA) indicated that ALB inhibited the biosynthesis of cholesterol and fatty acids. Consistently, ALB inhibited the protein expression of n-SREBPs and downstream genes. Mechanistically, the impact of ALB on SREBPs was dependent on the regulation of AMPK/mTOR, thereby impeding the transportation of SREBPs from endoplasmic reticulum (ER) to golgi apparatus (GA). Further investigations indicated that the activation of AMPK by ALB was independent on classical upstream CAMKK2 and LKB1. Instead, ALB resulted in a decrease in ATP levels and an increase in the ratios of ADP/ATP and AMP/ATP. CETSA, MD, and ultrafiltration-LC/MS analysis indicated that ALB interacted with VDAC1. Molecular docking revealed that ALB directly bound to VDAC1 by forming hydrogen bonds at the amino acid sites S196 and H184 in the ATP-binding region. Importantly, the thermal stabilization of ALB on VDAC1 was compromised when VDAC1 was mutated at S196 and H184, suggesting that these amino acids played a crucial role in the interaction. ConclusionOur findings reveal that VDAC1 serves as the target of ALB, leading to the inhibition of lipid synthesis, presents potential target and candidate drugs for hyperlipidemia.

Identification and structural validation of purine nucleoside phosphorylase from Plasmodium falciparum as a target of MMV000848

About 247 million cases of malaria occurred in 2021 with Plasmodium falciparum accounting for the majority of 619,000 deaths. In the absence of a widely available vaccine, chemotherapy remains crucial to prevent, treat, and contain the disease. The efficacy of several drugs currently used in the clinic is likely to suffer from the emergence of resistant parasites. A global effort to identify lead compounds led to several initiatives such as the Medicine for Malaria Ventures (MMV), a repository of compounds showing promising efficacy in killing the parasite in cell-based assays. Here, we used mass spectrometry coupled with cellular thermal shift assay to identify putative protein targets of MMV000848, a compound with an invitro EC50 of 0.5μM against the parasite. Thermal shift assays showed a strong increase of P.falciparum purine nucleoside phosphorylase (PfPNP) melting temperature by up to 15 °C upon incubation with MMV000848. Binding and enzymatic assays returned a KD of 1.52± 0.495μM and an IC50 value of 21.5± 2.36μM. The inhibition is competitive with respect to the substrate, as confirmed by a cocrystal structure of PfPNP bound with MMV000848 at the active site, determined at 1.85Å resolution. In contrast to transition states inhibitors, MMV000848 specifically inhibits the parasite enzyme but not the human ortholog. An isobologram analysis shows subadditivity with immucillin H and with quinine respectively, suggesting overlapping modes of action between these compounds. These results point to PfPNP as a promising antimalarial target and suggest avenues to improve inhibitor potency.

Strictosamide ameliorates LPS-induced acute lung injury by targeting ERK2 and mediating NF-κB signaling pathway

Ethnopharmacological relevanceAcute lung injury (ALI) ranks among the deadliest pulmonary diseases, significantly impacting mortality and morbidity. Presently, the primary treatment for ALI involves supportive therapy; however, its efficacy remains unsatisfactory. Strictosamide (STR), an indole alkaloid found in the Chinese herbal medicine Nauclea officinalis (Pierre ex Pit.) Merr. & Chun (Wutan), has been found to exhibit numerous pharmacological properties, particularly anti-inflammatory effects. Aim of the studyThis study aimes to systematically identify and validate the specific binding proteins targeted by STR and elucidate its anti-inflammatory mechanism in lipopolysaccharide (LPS)-induced ALI. Materials and methodsBiotin chemical modification, protein microarray analysis and network pharmacology were conducted to screen for potential STR-binding proteins. The binding affinity was assessed through surface plasmon resonance (SPR), cellular thermal shift assay (CETSA) and molecular docking, and the anti-inflammatory mechanism of STR in ALI treatment was assessed through in vivo and in vitro experiments. ResultsBiotin chemical modification, protein microarray and network pharmacology identified extracellular-signal-regulated kinase 2 (ERK2) as the most important binding proteins among 276 candidate STR-interacting proteins and nuclear factor-kappaB (NF-κB) pathway was one of the main inflammatory signal transduction pathways. Using SPR, CETSA, and molecular docking, we confirmed STR's affinity for ERK2. In vitro and in vivo experiments demonstrated that STR mitigated inflammation by targeting ERK2 to modulate the NF-κB signaling pathway in LPS-induced ALI. ConclusionsOur findings indicate that STR can inhibit the NF-κB signaling pathway to attenuate LPS-induced inflammation by targeting ERK2 and decreasing phosphorylation of ERK2, which could be a novel strategy for treating ALI.

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.

Development of Mitophagy-inducing Small Molecules as Therapeutics for Aging and Alzheimer's Disease

Alzheimer's disease (AD) is the leading cause of dementia in those over the age of65. The underlying molecular basis for the disease remains elusive. Rather than simply viewing individual diseases in isolation, researchers have turned to studying the contribution of common age-related processes. One such process is loss in mitochondrial function, normally maintained by defective mitochondrial clearance via mitophagy. Mitophagy slows with age and with various age-related conditions including AD. An agent which has received considerable attention is urolithin A (UA) which has recently been reported to elicit neuroprotection in a variety of AD models. As part of an independent small molecule screen, we identified a second structurally-related compound we named ‘mitophagy inducing compound’ (MIC). Using C. elegans as an initial validation tool, we showed that both compounds significantly improve mitochondrial function, protect against neuronal cell loss, and extend lifespan. These effects were translatable to both in vitro and in vivo mammalian models. To identify potential drug targets, we performed Cellular Thermal Shift Assay (CETSA). Preliminary data from knockdown of identified targets suggest the involvement of a mito-hormetic mechanism which acts to restore overall mitochondrial health. These studies may have important implications for how these compounds elicit their effects.

Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications

The cellular thermal shift assay (CETSA®) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA® has been used for semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based readouts using cell lines. However, there has been no quantitative evaluation of CETSA® in unprocessed human whole blood, which is preferred for clinical applications. Here we report two separate assay formats – Alpha CETSA® and MSD CETSA® – that require less than 100 μL of whole blood per sample without PBMC isolation. We chose RIPK1 as a proof-of-concept target and, by measuring engagement of seven different inhibitors, demonstrate high assay sensitivity and robustness. These quantitative CETSA® platforms enable possible applications in preclinical pharmacokinetic-pharmacodynamic studies, and direct target engagement with small molecules in clinical trials.

Mangiferin attenuates osteoporosis by inhibiting osteoblastic ferroptosis through Keap1/Nrf2/SLC7A11/GPX4 pathway

BackgroundFerroptosis is a crucial contributor to impaired osteoblast function in osteoporosis. Mangiferin, a xanthonoid glucoside isolated from mangoes, exhibits anti-osteoporosis effects. However, its potential mechanism is not fully understood. PurposeThis study explores the potencies of mangiferin on osteoblastic ferroptosis and deciphers its direct target in the context of solute carrier family 7-member 11 (SLC7A11)/glutathione peroxidases 4 (GPX4) pathway. MethodsIn vivo models include bilateral ovariectomy induced osteoporosis mice, iron-dextran induced iron-overloaded mice, and nuclear factor-erythroid 2-related factor 2 (Nrf2)-knockout mice. Mice are orally administrated mangiferin (10, 50 or 100 mg.kg−1.d−1) for 12 weeks. In vitro osteoblast models include iron-dextran induced iron-overloaded cells, erastin induced ferroptosis cells, and gene knockout cells. RNA sequencing is applied for investigating the underlying mechanisms. The direct target of mangiferin is studied using a cellular thermal shift assay, silico docking, and surface plasmon resonance. ResultsMangiferin promotes bone formation and inhibits ferroptosis in vivo models (osteoporosis mice, iron-overloaded mice) and in vitro models (ferroptosis osteoblast, iron-overloaded osteoblasts). Mechanismly, mangiferin directly binds to the kelch-like ECH-associated protein 1 (Keap1) and activates the downstream Nrf2/SLC7A11/GPX4 pathway in both the in vivo and in vitro models. Mangiferin failed to restore the osteoporosis and ferroptosis in Nrf2-knockout mice. Silencing Nrf2, SLC7A11 or GPX4 abolished the anti-ferroptosis effect of mangiferin in erastin-induced cells. Addition of the ferroptosis agonist RSL-3 also blocked the protective effects of mangiferin on iron-overloaded cells. Furthermore, mangiferin had better effects on osteogenesis than the ferroptosis inhibitor (ferrostatin-1) and the Nrf2 agonists (sulforaphane, dimethyl fumarate, and bardoxolone). ConclusionsWe identify for the first time mangiferin as a ferroptosis inhibitor and a direct Keap1 conjugator that promotes bone formation and alleviates osteoporosis. This work also provides a potentially practical pharmacological approach for treating ferroptosis-driven diseases.

Ginsenoside Rb2 inhibits p300-mediated SF3A2 acetylation at lysine 10 to promote Fscn1 alternative splicing against myocardial ischemic/reperfusion injury

IntroductionGinsenosides (GS) derived from Panax ginseng can regulate protein acetylation to promote mitochondrial function for protecting cardiomyocytes. However, the potential mechanisms of GS for regulating acetylation modification are not yet clear. ObjectivesThis study aimed to explore the potential mechanisms of GS in regulating protein acetylation and identify ginsenoside monomer for fighting myocardial ischemia-related diseases. MethodsThe 4D-lable free acetylomic analysis was employed to gain the acetylated proteins regulated by GS pretreatment. The co-immunoprecipitation assay, immunofluorescent staining, and mitochondrial respiration measurement were performed to detect the effect of GS or ginsenoside monomer on acetylated protein level and mitochondrial function. RNA sequencing, site-specific mutation, and shRNA interference were used to explore the downstream targets of acetylation modificationby GS. Cellular thermal shift assay and surface plasmon resonance were used for identifying the binding of ginsenoside with target protein. ResultsIn the cardiomyocytes of normal, oxygen glucose deprivation and/or reperfusion conditions, the acetylomic analysis identified that the acetylated levels of spliceosome proteins were inhibited by GS pretreatment and SF3A2 acetylation at lysine 10 (K10) was significantly decreased as a potential target of GS. Ginsenoside Rb2 was identified as one of the active ginsenoside monomers for reducing the acetylation of SF3A2 (K10), which enhanced mitochondrial respiration against myocardial ischemic injury in in vivo and in vitro experiments. RNA-seq analysis showed that ginsenoside Rb2 promoted alternative splicing of mitochondrial function-related genes and the level of fascin actin-bundling protein 1 (Fscn1) was obviously upregulated, which was dependent on SF3A2 acetylation. Critically, thermodynamic, kinetic and enzymatic experiments demonstrated that ginsenoside Rb2 directly interacted with p300 for inhibiting its activity. ConclusionThese findings provide a novel mechanism underlying cardiomyocyte protection of ginsenoside Rb2 by inhibiting p300-mediated SF3A2 acteylation for promoting Fscn1 expression, which might be a promising approach for the prevention and treatment of myocardial ischemic diseases.

Zuogui pill disrupt the malignant cycle in breast cancer bone metastasis through the Piezo1-Notch-1-GPX4 pathway and active molecules fishing

Background: Breast cancer bone metastasis is closely associated with the bone microenvironment. Zuogui Pill (ZGP), a clinically approved formulation in China, effectively regulates the bone microenvironment for the prevention and treatment of osteoporosis.Purpose: Few reports have utilized the ZGP for bone metastasis models. This study investigated the intervention and bone-protective properties of ZGP against breast cancer bone metastasis, explored the potential mechanism, and screened for its active compositions by molecules fishing.Methods: To investigate the intervention efficacy of ZGP and its protein-level mechanism of action, the mouse bone metastasis model and in vitro cell co-culture model were constructed. Affinity ultrafiltration, molecular docking, cellular thermal shift assay and physical scale detection were used to investigate the affinity components of the RANKL protein in ZGP.Results: The administration of ZGP combined with zoledronic acid inhibited the development of tumors and secondary lung metastasis in mice. This translated to a prolonged survival period and enhanced quality of life. ZGP could disrupt the malignant cycle by modulating the Piezo1-Notch-1-GPX4 signaling pathway in the "bone-cancer" communication in the cell co-culture model. Furthermore, 25 chemical components of ZGP were identified, with 10 active compounds exhibiting significant affinity for the RANKL protein.Conclusion: The findings of this work highlighted ZGP's potential for intervening in the progression of breast cancer bone metastasis. Thus, this investigation served as an experimental foundation for expanding the application scope of ZGP and for advancing drug development efforts in bone metastasis treatment.

EGFR/MAPK signaling pathway acts as a potential therapeutic target for sulforaphane-rescued heart tube malformation induced by various concentrations of PhIP exposure

Background2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyrimidine (PhIP) is a known carcinogen generated mainly from cooking meat and environmental pollutants. It is worth exploring the potential of natural small-molecule drugs to protect against adverse effects on embryonic development. PurposeIn this study, we investigated the potential toxicological effects of PhIP on embryonic heart tube formation and the effect of Sulforaphane (SFN) administration on the anti-toxicological effects of PhIP on embryonic cardiogenesis. Study design and methodsFirst, the chicken embryo model was used to investigate the different phenotypes of embryonic heart tubes induced by various concentrations of PhIP exposure. We also proved that SFN rescues PhIP-induced embryonic heart tube malformation. Second, immunofluorescence, western blot, Polymerase Chain Reaction (PCR) and flow cytometry experiments were employed to explore the mechanisms by which SFN protects cardiac cells from oxidative damage in the presence of PhIP. We used RNA-seq analysis, molecular docking, in situ hybridization, cellular thermal shift assay and solution nuclear magnetic resonance spectroscopy to explore whether SFN protects cardiogenesis through the EGFR/MAPK signaling pathway. ResultsThe study showed that PhIP might dose-dependently interfere with the C-looping heart tube (mild) or the fusion of a pair of bilateral endocardial tubes (severe) in chick embryos, while SFN administration prevented cardiac cells from oxidative damage in the presence of high-level PhIP. Furthermore, we found that excessive reactive oxygen species (ROS) production and subsequent apoptosis were not the principal mechanisms by which low-level PhIP induced malformation of heart tubes. This is due to PhIP-disturbed Mitogen-activated protein kinase (MAPK) signaling pathway could be corrected by SFN administration. ConclusionsThis study provided novel insight that PhIP exposure could increase the risk of abnormalities in early cardiogenesis and that SFN could partially rescue various concentrations of PhIP-induced abnormal heart tube formation by targeting EGFR and mediating EGFR/MAPK signaling pathways.

Kaempferol-3-O-sophoroside (PCS-1) contributes to modulation of depressive-like behaviour in C57BL/6J mice by activating AMPK.

Kaempferol-3-O-sophoroside (PCS-1) is the main component in Crocus sativus (Saffron), a herb with mood-enhancing properties. AMP-activated protein kinase (AMPK) is a potential therapeutic target for depression. This study explores the antidepressive-like properties of PCS-1 and its AMPK activation to confirm AMPK as a target for antidepression. Corticosterone (CORT)-induced PC12 cell injury served as an in vitro model to evaluate the neuroprotective effect of PCS-1. Neuro-2a cells and primary neurons were utilized to evaluate the synaptogenesis role of PCS-1. CORT-induced mouse depression model and chronic unpredictable mild stress (CUMS) model were used to assess the antidepressive-like properties of PCS-1 through behavioural tests, magnetic resonance imaging, and biochemical index measurements. Western blot and immunofluorescence assays were used to study the mechanisms of PCS-1. Cellular thermal shift assay was used to confirm the binding target. PCS-1 (12.5-50 μM) ameliorated CORT-induced PC12 cell damage, oxidative stress and inflammation. PCS-1 alone promoted an increase in synapses in Neuro-2a cells and primary neurons. Oral administration of PCS-1 (10 and 20 mg·kg-1 ) ameliorated weight loss, dyskinesia, and hippocampal volume reduction induced by CORT and CUMS. PCS-1 bound to AMPK to improve the expression of brain-derived neurotrophic factor (BDNF) and induce autophagy. PCS-1 binds to AMPK to promote BDNF production and autophagy enhancement, ultimately achieving antidepressant effects. This study provides support for the clinical application of saffron petals and provides further evidence for AMPK as a potential target for antidepression.

Cucurbitacin E reduces IL-1β-induced inflammation and cartilage degeneration by inhibiting the PI3K/Akt pathway in osteoarthritic chondrocytes

BackgroundOsteoarthritis is a degenerative joint disease. Cartilage degeneration is the earliest and most important pathological change in osteoarthritis, and persistent inflammation is one of the driving factors of cartilage degeneration. Cucurbitacin E, an isolated compound in the Cucurbitacin family, has been shown to have anti-inflammatory effects, but its role and mechanism in osteoarthritic chondrocytes are unclear.MethodsFor in vitro experiments, human chondrocytes were stimulated with IL-1β, and the expression of inflammatory genes was measured by Western blotting and qPCR. The expression of extracellular matrix proteins was evaluated by immunofluorescence staining, Western blotting and saffron staining. Differences in gene expression between cartilage from osteoarthritis patients and normal cartilage were analysed by bioinformatics methods, and the relationship between Cucurbitacin E and its target was analysed by a cellular thermal shift assay, molecular docking analysis and molecular dynamics simulation. For in vivo experiments, knee osteoarthritis was induced by DMM in C57BL/6 mouse knee joints, and the effect of Cucurbitacin E on knee joint degeneration was evaluated.ResultsThe in vitro experiments confirmed that Cucurbitacin E effectively inhibited the production of the inflammatory cytokine interleukin-1β(IL-1β) and cyclooxygenase-2 (COX-2) by IL-1β-stimulated chondrocytes and alleviates extracellular matrix degradation. The in vivo experiments demonstrated that Cucurbitacin E had a protective effect on the knee cartilage of C57BL/6 mice with medial meniscal instability in the osteoarthritis model. Mechanistically, bioinformatic analysis of the GSE114007 and GSE117999 datasets showed that the PI3K/AKT pathway was highly activated in osteoarthritis. Immunohistochemical analysis of PI3K/Akt signalling pathway proteins in pathological slices of human cartilage showed that the level of p-PI3K in patients with osteoarthritis was higher than that in the normal group. PI3K/Akt were upregulated in IL-1β-stimulated chondrocytes, and Cucurbitacin E intervention reversed this phenomenon. The cellular thermal shift assay, molecular docking analysis and molecular dynamics experiment showed that Cucurbitacin E had a strong binding affinity for the inhibitory target PI3K. SC79 activated Akt phosphorylation and reversed the effect of Cucurbitacin E on IL-1β-induced chondrocyte degeneration, demonstrating that Cucurbitacin E inhibits IL-1β-induced chondrocyte inflammation and degeneration by inhibiting the PI3K/AKT pathway.ConclusionCucurbitacin E inhibits the activation of the PI3K/AKT pathway, thereby alleviating the progression of OA. In summary, we believe that Cucurbitacin E is a potential drug for the treatment of OA.