Resveratrol targeting NRF2 disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage in mice pulmonary infection.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Polygonum cuspidatum Sieb. et Zucc (PC), known as 'Huzhang' in the Chinese Pharmacopoeia, has been traditionally employed for its anti-inflammatory, antiviral, antimicrobial, and other biological activities. Polydatin (PD) and its aglycone, resveratrol (RES), are key pharmacologically active components responsible for exerting anti-inflammatory and antioxidant effects. However, its specific targets and action mechanisms remain unclear. AIM OF THE STUDY: The equilibrium of the KEAP1-NRF2 system serves as the primary protective response to oxidative and electrophilic stresses within the body, particularly in cases of acute lung injury caused by pathogenic microbial infection. In this study, the precise mechanisms by which RES alleviates oxidative stress damage in conjunction with NRF2 activators are discussed. MATERIALS AND METHODS: The active components from PC were screened to evaluate their potential to inhibit reactive oxygen species (ROS) and activate antioxidant activity dependent on antioxidant response elements (ARE). RES was evaluated for its potential to alleviate the oxidative stress caused by pathogenic microbial infection. Functional probes were designed to study the RES distribution and identify its targets. A lipopolysaccharide (LPS)-induced oxidative injury model was used to evaluate the effects of RES on the KEAP1-NRF2/ARE pathway in RAW 264.7 cells. The interaction between RES and NRF2 was elucidated using drug-affinity responsive target stability (DARTS), cellular thermal shift assays (CETSA), co-immunoprecipitation (Co-IP), and microscale thermophoresis (MST) techniques. The key binding sites were predicted using molecular docking and validated in NRF2-knockdownand reconstructed cells. Finally, protective effects against pulmonary stress were verified in a mouse model of pathogenic infection. RESULTS: The accumulation of RES in lung macrophages disrupted the binding between KEAP1 and NRF2, thereby preventing the ubiquitination degradation of NRF2 through its interaction with Ile28 on the NRF2-DLG motif. The activation of NRF2 resulted in the upregulation of nuclear transcription, enhances the expression of antioxidant genes dependent on ARE, suppresses ROS generation, and ameliorates oxidative damage both in vivo and in vitro. CONCLUSION: These findings shed light on the potential of RES to mitigate oxidative stress damage caused by pathogenic microorganism-induced lung infections and facilitate the discovery of novel small molecule modulators targeting the KEAP1-NRF2 DLG motif interaction.

Ginsenoside CK targeting KEAP1-DGR/Kelch domain disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage.

BACKGROUND: Panax ginseng and Panax notoginseng as traditional Chinese medicines, are widely used in the treatment of qi deficiency, viral or bacterial infection, inflammation and cancer. Ginsenoside CK, an active metabolite of protopanoxadiol among the ginseng saponins, has been shown in previous studies to improve the organism's oxidative balance by regulating the KEAP1-NRF2/ARE pathway, thus slowing the progression of diseases. However, the specific targets and mechanisms of CK in improving oxidative stress remain unclear. PURPOSE: The aim of this study was to determine the potential therapeutic targets and molecular mechanisms of CK in improving oxidative stress injury both in vitro and in vivo. METHODS: LPS was used to induce oxidative damage in RAW 264.7 cells to evaluate the regulatory effects of CK on the KEAP1-NRF2/ARE pathway. Drug affinity responsive target stability technology (DARTS) combined with proteomics was employed to identify CK's potential target proteins. CK functional probe were designed to analyze the target protein using click chemistry. Furthermore, small molecule and protein interaction technologies were used to verify the mechanism, and computer dynamic simulation technology was used to analyze the interaction sites between CK and the target protein. The pharmacological effects and mechanism of CK in improving oxidative damage were verified in vivo by LPS-induced acute injury in mice and physical mechanical injury in rat soft tissues. RESULTS: KEAP1 was identified as the target protein that CK regulates to improve oxidative damage through the KEAP1-NRF2/ARE pathway. CK competitively binds to the DGR/Kelch domain of KEAP1, disrupting the binding between DLG peptide in NRF2 and KEAP1, thereby inhibiting the occurrence of oxidative damage induced by LPS or physical mechanical stress. CONCLUSIONS: CK functions as a natural KEAP1-NRF2 inhibitor, disrupting the binding between KEAP1 and NRF2-DLG motifs by targeting the DGR/Kelch domain of KEAP1, activating the antioxidant transcriptional program of NRF2, and reducing oxidative stress damage.

Fuziline Ameliorates Glucose and Lipid Metabolism by Activating Beta Adrenergic Receptors to Stimulate Thermogenesis.

Radix aconiti carmichaeli is a widely used traditional Chinese medicine that has been found to be effective in treating cardiovascular diseases and metabolic disorders. Patients with these diseases often experience a heat generation disorder, which is characterized by chilliness and can worsen the progression of the disease. This study established an in vitro screening model combining the examination of cellular mitochondrial membrane potential and mitochondrial temperature to screen drugs with thermogenic activity. After differentiation and determination of the content of characteristic metabolites of the drug-containing serum blood components, it was found that Fuziline (FZL) is the key thermogenic property in Radix aconiti carmichaeli, responsible for its thermogenic effects with a high relative importance of 33%. Experiments were conducted to evaluate the thermogenic activity of Radix aconiti carmichaeli and FZL in vivo by assessing temperature changes in various organs, including the rectum, liver, and brown adipose tissue. Moreover, the effects of intracellular ß3-adrenergic receptor (ß3-AR) agonistic effects were evaluated using transient ß3-AR transfection and dual-luciferase assay systems. The molecular mechanism by which FZL promotes thermogenesis and improves mitochondrial function was investigated by verifying the ß-adrenergic receptors (ß-AR) downstream signaling pathway. The results suggest that FZL activates ß-AR nonselectively, which in turn activates the downstream cAMP-PKA signaling pathway and leads to an increase in liver glycogenolysis and triglyceride hydrolysis, accompanied by enhancing mitochondrial energy metabolism. Consequently, the liver and brown adipose tissue receive energy to generate heat. In summary, these findings provide insight into the therapeutic application of Radix aconiti carmichaeli for metabolic disorders associated with heat generation disorders.

Astragaloside IV targets PRDX6, inhibits the activation of RAC subunit in NADPH oxidase 2 for oxidative damage.

BACKGROUND: Radix Astragali Mongolici, as a traditional Chinese medicine, is widely used in the treatment of qi deficiency, viral or bacterial infection, inflammation and cancer. Astragaloside IV (AST), a key active compound in Radix Astragali Mongolici, has been shown to reduce disease progression by inhibiting oxidative stress and inflammation. However, the specific target and mechanism of action of AST in improving oxidative stress are still unclear. PURPOSE: This study aims to explore the target and mechanism of AST to improve oxidative stress, and to explain the biological process of oxidative stress. METHODS: AST functional probes were designed to capture target proteins and combined with protein spectrum to analyze target proteins. Small molecule and protein interaction technologies were used to verify the mode of action, while computer dynamics simulation technology was used to analyze the site of interaction with the target protein. The pharmacological activity of AST in improving oxidative stress was evaluated in a mouse model of acute lung injury induced by LPS. Additionally, pharmacological and serial molecular biological approaches were used to explore the underlying mechanism of action. RESULTS: AST inhibits PLA2 activity in PRDX6 by targeting the PLA2 catalytic triad pocket. This binding alters the conformation and structural stability of PRDX6 and interferes with the interaction between PRDX6 and RAC, hindering the activation of the RAC-GDI heterodimer. Inactivation of RAC prevents NOX2 maturation, attenuates superoxide anion production, and improves oxidative stress damage. CONCLUSION: The findings of this research indicate that AST impedes PLA2 activity by acting on the catalytic triad of PRDX6. This, in turn, disrupts the interaction between PRDX6 and RAC, thereby hindering the maturation of NOX2 and diminishing the oxidative stress damage.

Okicamelliaside targets the N-terminal chaperone pocket of HSP90 disrupts the chaperone protein interaction of HSP90-CDC37 and exerts antitumor activity.

Heat shock protein 90 (HSP90) has been recognized as a crucial target in cancer cells. However, various toxic reactions targeting the ATP binding site of HSP90 may not be the best choice for HSP90 inhibitors. In this paper, an ellagic acid derivative, namely, okicamelliaside (OCS), with antitumor effects was found. To identify potential anti-cancer mechanisms, an OCS photosensitive probe was applied to target fishing and tracing. Chemical proteomics and protein-drug interaction experiments have shown that HSP90 is a key target for OCS, with a strong binding affinity (KD = 6.45 µM). Mutation analysis of the target protein and molecular dynamics simulation revealed that OCS could competitively act on the key Glu-47 site at the N-terminal chaperone pocket of HSP90, where the co-chaperone CDC37 binds to HSP90, affect its stability and reduce the ∆Gbind of HSP90-CDC37. It was demonstrated that OCS destroys the protein-protein interactions of HSP90-CDC37; selectively affects downstream kinase client proteins of HSP90, including CDK4, P-AKT473, and P-ERK1/2; and exerts antitumor effects on A549 cells. Furthermore, tumor xenograft experiments demonstrated high antitumor activity and low toxicity of OCS in the same way. Our findings identified a novel N-terminal chaperone pocket natural inhibitor of HSP90, that is, OCS, which selectively inhibits the formation of the HSP90-CDC37 protein complex, and provided further insight into HSP90 inhibitors for anti-cancer candidate drugs.

Dietary Flavone Baicalein Combinate with Genipin Attenuates Inflammation Stimulated by Lipopolysaccharide in RAW264.7 Cells or Pseudomonas aeruginosa in Mice via Regulating the Expression and Phosphorylation of AKT.

Mounting evidence has shown that single-targeted therapy might be inadequate to achieve satisfactory effects. Thus, drug combinations are gaining attention as they can regulate multiple targets to obtain more beneficial effects. Heat shock protein 90 (HSP90) is a molecular chaperone that assists the protein assembly and folding of client proteins and maintains their stability. Interfering with the interaction between HSP90 and its client proteins by inhibiting the latter's activity may offer a new approach toward combination therapy. The HSP90 client protein AKT plays an important role in the inflammatory response syndrome caused by infections. In this study, the dietary flavone baicalein was identified as a novel inhibitor of HSP90 that targeted the N-terminal ATP binding pocket of HSP90 and hindered the chaperone cycle, resulting in AKT degradation. Combining baicalein with genipin, which was extracted from Gardenia jasminoides, could inhibit the pleckstrin homology domain of AKT, significantly increasing the anti-inflammatory effects both in vitro and in vivo. This synergistic effect was attributed to the reduction in AKT expression and phosphorylation. Thus, elucidating the mechanism underlying this effect will provide a new avenue for the clinical application and development of synergistic anti-inflammatory drugs.

Integrated molecular network and HPLC-UV-FLD analysis to explore antioxidant ingredients in camellia nitidissima Chi.

At present, screening of active ingredients from natural products for pharmacological and clinical research is mostly time-consuming and costly. In this study, a molecular network (MN) guided high performance liquid chromatography-ultraviolet-fluorescence detector (HPLC-UV-FLD) method was carried out to profile the global antioxidant activity compounds, including the trace amount ingredients in Camellia nitidissima Chi (CNC). Firstly, HPLC-UV-FLD postcolumn derivatization system was utilized to screen the antioxidants. Then the MN of CNC was established via mass spectrometry (MS) data for getting the connection between ingredient structures. As a result, HPLC-UV-FLD indicated three antioxidant ingredients: gallic acid (126.3 mg/g), catechin (564.8 mg/g), and salicylic acid (24.3 mg/g). Combined with the MN, the actives' precise location and connection relationship were clarified based on the structural similarities. A new antioxidant ingredient, okicamelliaside, was suggested and evaluated at free radical scavenging and enzymatic protection. The novel method of activity and structural correlation analysis based on MN could provide a useful guide for screening trace active ingredients in natural products. PRACTICAL APPLICATION: Three main ingredients were screened out from Camellia nitidissima Chi by HPLC-UV-FLD postcolumn derivatization system. Integrated molecular network and HPLC-UV-FLD analysis, a new type of antioxidant okicamelliaside was selected. The novel method of activity and structural correlation analysis based on molecular network could provide a useful guide for screening trace active ingredients in natural products.

Ursolic acid reduces hepatocellular apoptosis and alleviates alcohol-induced liver injury via irreversible inhibition of CASP3 in vivo.

Alcoholic liver disease (ALD) is one of the pathogenic factors of chronic liver disease with the highest clinical morbidity worldwide. Ursolic acid (UA), a pentacyclic terpenoid carboxylic acid, has shown many health benefits including antioxidative, anti-inflammatory, anticancer, and hepatoprotective activities. We previously found that UA was metabolized in vivo into epoxy-modified UA containing an epoxy electrophilic group and had the potential to react with nucleophilic groups. In this study we prepared an alkynyl-modified UA (AM-UA) probe for tracing and capturing the target protein of UA from liver in mice, then investigated the mode by which UA bound to its target in vivo. By conducting proteome identification and bioinformatics analysis, we identified caspase-3 (CASP3) as the primary target protein of UA associated with liver protection. Molecule docking analysis showed that the epoxy group of the UA metabolite reacted with Cys-163 of CASP3, forming a covalent bond with CASP3. The binding mode of the UA metabolites (UA, CM-UA, and EM-UA) was verified by biochemical evaluation, demonstrating that the epoxy group produced by metabolism played an important role in the inhibition of CASP3. In alcohol-treated HepG2 cells, pretreatment with the UA metabolite (10 µM) irreversibly inhibited CASP3 activities, and subsequently decreased the cleavage of PARP and cell apoptosis. Finally, pre-administration of UA (20-80 mg· kg-1 per day, ig, for 1 week) dose-dependently alleviated alcohol-induced liver injury in mice mainly via the inhibition of CASP3. In conclusion, this study demonstrates that UA is a valuable lead compound for the treatment of ALD.

Lepidiline A Improves the Balance of Endogenous Sex Hormones and Increases Fecundity by Targeting HSD17B1.

SCOPE: Maca (Lepidium meyenii), a well-known plant from the Andean highlands of Peru, has been used widely as a nutritional supplement to increase sexual function and fecundity. However, the identity of its active ingredients and how they function remain unknown. METHODS AND RESULTS: Chemical substances in maca are identified by UPLC-Q-TOF, and the active ingredients are screened through HotMap coupled with an artificial neural network. Lepidiline A (LA), an imidazole alkaloid, is identified as the key active compound. LA affects the balance of endogenous sex hormones in mice and improves fecundity in Drosophila. Using a molecular LA probe, 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1) is revealed to be the potential target of LA using a fishing-rod strategy. It is demonstrated with experimental data that LA targets HSD17B1 to enhance the enzyme's activity and increases its bioconversion efficiency of actively formed sex hormones including estrogen to 17ß-estradiol and 4-androsten-3,7-dione to testosterone, which ultimately improves reproductive activity. CONCLUSION: LA improves the balance of endogenous sex hormones and increases fecundity by targeting HSD17B1. This underlying mechanism of action provides a useful insight into the application of maca in the regulation of dietary nutrition and healthy fertility.

Cinnamaldehyde Enhances Antimelanoma Activity through Covalently Binding ENO1 and Exhibits a Promoting Effect with Dacarbazine.

At present, melanoma is a common malignant tumor with the highest mortality rate of all types of skin cancer. Although the first option for treating melanoma is with chemicals, the effects are unsatisfactory and include poor medication response and high resistance. Therefore, developing new medicines or a novel combination approach would be a significant breakthrough. Here, we present cinnamaldehyde (CA) as a potential candidate, which exerted an antitumor effect in melanoma cell lines. Chemical biology methods of target fishing, molecular imaging, and live cell tracing by an alkynyl-CA probe revealed that the α-enolase (ENO1) protein was the target of CA. The covalent binding of CA with ENO1 changed the stability of the ENO1 protein and affected the glycolytic activity. Furthermore, our results demonstrated that dacarbazine (DTIC) showed a high promoting effect with CA for antimelanoma both in vivo and in vitro. The combination improved the DTIC cell cycle arrest in the S phase and markedly impacted melanoma growth. As a covalent inhibitor of ENO1, CA combined with DTIC may be beneficial in patients with drug resistance in antimelanoma therapy.

Apoptosis effect of Aegiceras corniculatum on human colorectal cancer via activation of FoxO signaling pathway.

Aegiceras corniculatum (L.) Blanco is known to exhibit anticancer effects against different types of cancer; however, to the best of our knowledge, the anticancer activity and underlying mechanisms of action of A. corniculatum leaf extract on colorectal cancer have not been elucidated. In the present study, colony-forming assay, western blot analysis, flow cytometry, and a xenograft model were used to investigate the effects of an n-butanol extract of A. corniculatum leaves (NACL) on colorectal cancer in vitro and in vivo. The results showed that NACL inhibits the viability and proliferation of colorectal cancer cells in a dose-dependent manner. Besides, NACL also induces cell apoptosis and cell cycle arrest by activating Forkhead box proteins and controlling the cell cycle checkpoint pathways, which are associated with the caspase-dependent mitochondrial apoptotic cascades and Bcl-2 family proteins. More importantly, the tumour sizes in HT-29 xenograft nude mice decreased after treatment with NACL in vivo. These findings indicate that A. corniculatum leaf extracts have potent anticancer activities across different colorectal and other solid tumour cell lines, via regulation of the cell cycle and apoptosis; thus, it has the potential to be developed as an anticancer agent to enhance clinical standards of care for patients with colorectal cancer.

Comprehensive TCM molecular networking based on MS/MS in silico spectra with integration of virtual screening and affinity MS screening for discovering functional ligands from natural herbs.

Accessing the rich source of compounds from natural herbs for use in the pharmaceutical industry using conventional bioassay-based screening platforms has low efficiency and is cost-prohibitive. In this study, we developed a new method involving traditional Chinese medicine (TCM) molecular networking and virtual screening coupled with affinity mass spectrometry (MN/VS-AM) for the efficient discovery of herb-derived ligands. The in silico MS/MS fragmentation database (ISDB) generated by molecular networking of TCM can rapidly identify compounds in complex herb extracts and perform compound activity mapping. Additionally, the pre-virtual screening conveniently includes candidate herbs with potential bioactivity, while affinity MS screening completely eliminates the requirement for a tedious pure compound preparation at the initial screening phase. After applying this approach, two types of compounds, isoamylene flavanonols and 20(s)-protopanoxadio saponins, which were confirmed to interact with the small GTPase of Ras, were successfully identified from a dozen anti-cancer TCM herbs. The results demonstrate that the modified screening strategy dramatically improved the accuracy and throughput sensitivity of ligand screening from herbal extracts. Graphical abstract.