Dual degradation mechanism of GPX4 degrader in induction of ferroptosis exerting anti-resistant tumor effect.

Targeting Glutathione peroxidase 4 (GPX4) has become a promising strategy for drug-resistant cancer therapy via ferroptosis induction. It was found that the GPX4 inhibitors such as RSL3 have GPX4 degradation ability via not only autophagy-lysosome pathway but also ubiquitin-proteasome system (UPS). Proteolysis targeting chimeras (PROTACs) using small molecule with both inhibition and degradation ability as the ligand of protein of interest (POI) have not been reported. To obtain better compounds with effective disturbance of GPX4 activity, and compare the difference between GPX4 inhibitors with degradation ability and their related PROTACs, we designed and synthesized a series of GPX4 degraders using PROTAC technology in terms of its excellent characteristics such as high efficiency and selectivity and the capacity of overcoming resistance. Hence, 8e was discovered as a potent and highly efficacious GPX4 degrader based upon the inhibitor RSL3. It was 2-3 times more potent than RSL3 in all the in vitro anti-tumor assays, indicating the importance of the PROTAC ternary complex of GPX4, 8e and E3 ligase ligand. 8e revealed better potency in resistant tumor cells than in wide type cells. Furthermore, we discovered for the first time that degrader 8e exhibit GPX4 degradation activity via ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway with UPS plays the major role in the process. Our data also suggested that 8e and RSL3 could potently induce ferroptosis of HT1080 cells via GPX4 inhibition and degradation. In summary, our data revealed that the GPX4 degrader 8e achieves better degradation and anti-tumor effects compared to its related GPX4 inhibitor RSL3. Thus, an efficient strategy to induce GPX4 degradation and subsequent ferroptosis was established in this study for malignant cancer treatment in the future.

PDTAC: Targeted Photodegradation of GPX4 Triggers Ferroptosis and Potent Antitumor Immunity.

Targeted degradation of proteins, especially those regarded as undruggable or difficult to drug, attracts wide attention to develop novel therapeutic strategies. Glutathione peroxidase 4 (GPX4), the key enzyme regulating ferroptosis, is currently a target with just covalent inhibitors. Here, we developed a targeted photolysis approach and achieved efficient degradation of GPX4. The photodegradation-targeting chimeras (PDTACs) were synthesized by conjugating a clinically approved photosensitizer (verteporfin) to noninhibitory GPX4-targeting peptides. These chimeras selectively degraded the target protein in both cell lysates and living cells upon red-light irradiation. The targeted photolysis of GPX4 resulted in dominant ferroptotic cell death in malignant cancer cells. Moreover, the dying cells resulting from the PDTACs exhibited potent immunogenicity in vitro and efficiently elicited antitumor immunity in vivo. Our approach therefore provides a novel method to induce GPX4 dysfunction based on noncovalent binding and specifically trigger immunogenic ferroptosis, which may boost the application of ferroptosis in cancer immunotherapy.

Non-enzymatic lipid peroxidation initiated by photodynamic therapy drives a distinct ferroptosis-like cell death pathway.

Ferroptosis is primarily triggered by a failure of the glutathione (GSH)-glutathione peroxidase 4 (GPX4) reductive system and associated overwhelming lipid peroxidation, in which enzymes regulating polyunsaturated fatty acid (PUFA) metabolism, and in particular acyl-CoA synthetase long chain family member 4 (ACSL4), are central. Here, we found that exogenous oxygen radicals generated by photodynamic therapy (PDT) can directly peroxidize PUFAs and initiate lipid autoxidation, coinciding with cellular GSH depletion. Different from canonical ferroptosis induced by RSL3 or erastin, PDT-initiated lipid peroxidation and ferroptotis-like cell death is independent of lipoxygenase (ALOXs) and ACSL4. Especially, this form of cell death modality can be triggered in malignant cells insensitive to or acquired resistance to canonical ferroptosis inducers. We also observed a distinct iron metabolism pathway in this PDT-triggered cell death modality, in which cytosolic labile iron is decreased probably due to its relocation to mitochondria. Inhibition of the mitochondrial Ca2+ and Fe2+ uniporter (MCU) effectively prevented PDT-triggered lipid peroxidation and subsequent cell death. Therefore, we tentatively term this distinct ferroptosis-like cell death as liperoptosis. Moreover, using the clinically approved photosensitizer Verteporfin, PDT inhibited tumor growth through inducing prevailing ferroptosis-like cell death in a mouse xenograft model. With its site-specific advantages, these findings highlight the value of using PDT to trigger lipid peroxidation and ferroptosis-like cell death in vivo, and will benefit exploring the exact molecular mechanism of immunological effects of PDT in cancer treatment.

pH-dependent rearrangement determines the iron-activation and antitumor activity of artemisinins.

The action mechanisms of artemisinins remains elusive for decades, and one long-standing question is whether the indispensable peroxide group is activated by iron or heme. Although heme usually reacts faster with artemisinins than iron does, we have found that rearrangement of dihydroartemisinin (DHA) into monoketo-aldehyde-peroxyhemiacetal (MKA) under physiological conditions can significantly enhance its reaction towards iron. The rearrangement is pH-dependent and the derived MKA is identified by LC-MS in the cellular metabolites of DHA in cancer cells. MKA reacts quickly with ferrous irons to afford reactive carbon-centered radicals and can inhibit enzyme activities in vitro. Moreover, MKA oxidizes ferrous irons to ferric irons, which may explain the effect of DHA on decreasing cellular labile iron pool (LIP). Both addition of exogenous iron and increase in LIP via triggering ferroptosis can enhance the cytotoxicity of DHA against cancer cells. While artesunate (ATS) can also decompose to MKA after hydrolyzing into DHA, the other artemisinins of lower antitumor activity, e.g. artemisinin (ART), artemether (ATM) and arteether (ATE), exhibit negligible hydrolysis and rearrangement under the same conditions. Our study reveals the vital role of molecular rearrangement to the activation and activity of artemisinins and provides a new strategy for designing antitumor molecules containing endoperoxide group.

Metabolomics analysis of serum reveals the effect of Danggui Buxue Tang on fatigued mice induced by exhausting physical exercise.

Danggui Buxue Tang (DBT), believed to invigorate 'Qi' (vital energy) and nourish 'Blood' (body circulation), is a traditional Chinese medicine formula. In this study, a metabolomics approach with gas chromatography coupled to mass spectrometry combined with pattern recognition was adopted to investigate the underlying mechanism of the antifatigue effect of DBT on fatigue of mice induced by weight-loaded forced swimming. Fourteen endogenous metabolites, up-regulated or down-regulated, were identified in the model mice by analysis tools of partial least-squares discriminant analysis (PLS-DA) and XCMS online software. Furthermore, the metabolites were reversed by DBT treatment, offering evidence for the antifatigue effect. In addition, intervention of DBT changed the levels of biochemical parameters. DBT showed obvious efficacy on the fatigued mice possibly by regulating the pathways of phenylalanine, tyrosine and tryptophan metabolism, glycine, serine, and threonine metabolism, glyoxylate and dicarboxylate metabolism, pyruvate metabolism, and TCA cycle. This study demonstrated that DBT has a good antifatigue effect and that metabolomics is a powerful means to gain insights into the therapeutic effect of traditional Chinese medicine formulas.

Metabonomic analysis of serum reveals antifatigue effects of Yi Guan Jian on fatigue mice using gas chromatography coupled to mass spectrometry.

Yi Guan Jian (YGJ), one of the most commonly used traditional Chinese medicines, has been reported to possess significant antifatigue effects. However, the mechanisms underlying its antifatigue effects remain largely unresolved. In this study, a metabonomics approach, involving gas chromatography coupled to mass spectrometry and a multivariate statistical technique, was developed to estimate the extent to which YGJ alleviated the exhausting swimming-induced fatigue of mice. High-dose treatment with YGJ significantly extended the swimming time of fatigued mice. Significant alterations of metabolites involving amino acids, organic acids and carbohydrates were observed in the serum of fatigued mice, which were reversed by YGJ treatment while biochemical indexes returned to normal. These metabolic changes suggest that the antifatigue effect of YGJ is associated with the impairement of amino acid, organic acids and carbohydrates. It also appears that YGJ can induce significant metabolic alterations independent of the exhausting swimming-induced metabolic changes. The significantly altered metabolites induced by YGJ intervention include l-2-amino-acetoacetate, taurine, fumaric acid, malic acid, oxoadipic acid and l-aspartate, all of which are associated with antifatigue properties. This suggests that YGJ exerts chemopreventive effects via antifatigue mechanisms.

[Anti-inflammation effect of Jinlingzi San in rat metabonomics based on 1H-NMR and LC-MS technology].

To further explore the regulatory effect of Jinlingzi San on in vivo inflammatory mechanism during inflammatory treatment, this study adopted 1H-NMR and LC-MS technology to analyze differences in in vivo metabolites of carrageen-induce rat foot swelling model. Besides, biomarkers related to inflammation models of Jinlingzi San in SD rats were discovered to speculate the regulatory mechanism of Jinlingzi San in resisting carrageen-induce inflammation. Through the analysis of detection spectrum, we found 18 biomarkers of metabolites(citrate, pyruvate, malic acid, succinate, glutamate, lysine, tartrate, 2-oxobutyric acid, glycine, guanosine, 9-cis-retinoic acid, triphosphate, inosine 5'-diphosphate, inosine diphosphate, tripolyphosphate, inorganic triphosphate, glycerophosphocholine, 21-deoxycortisol). Relevant pathway analysis results were TCA cycle, pyruvate metabolism, glycine, serine and threonine metabolism, and dicarboxylic acid metabolism. From the metabolic network, we can see that the anti-inflammatory effect of Jinlingzi San can regulate citric acid, succinic acid and glycine content to resist oxygen free radical and reduce body damage by ROS, so as to down-regulate inflammatory factors generated from body tissues and resist inflammation.

[Subacute toxicity metabonomics of Jinlingzi powder based on LC-MS].

To further understand the metabolic characteristics of Jinlingzi powder toxicity effect in rats and explore the effect of Jinlingzi powder on unknown biological pathways in the treatment process. In this experiment, the effect of three doses of Jinlingzi powder decoction on rat liver and kidney was investigated to explore the characteristics and rules of Jinlingzi powder on in vivo metabonomic changes in rats. First, urine and serum samples of the rats were used for LC-MS analysis. Under the XCMS online analysis, 44 differential substances were found in the identification of metabolites. Finally, Metpa was used for metabolic pathways enrichment and analysis, and five related metabolic pathways were obtained: steroid hormone biosynthesis, tryptophan metabolism, pentose and glucuronate interconversions, ascorbate and aldarate metabolism, as well as glutathione metabolism. Metabolic network diagram showed that the toxicity-related pathways were mainly associated with lysine metabolism in living organisms, glucuronic acid conversion, and hormone metabolism, especially the metabolism imbalance of lysine and glutathione would result in the disorder of energy metabolism or oxidative stress regulation, and thus inducing the damage in rats. Subacute toxicity test results for three doses groups (low, middle and high doses) showed that, Jinlingzi powder with doses of 19.7 g•kg⁻¹ and 39.4 g•kg⁻¹ caused obvious toxic effect, indicating Jinlingzi powder could produce toxic effect in vivo in a dose-dependent manner, and cause irreversible damage to the body.

The investigation of anti-inflammatory activity of Yi Guanjian decoction by serum metabonomics approach.

Yi Guanjian (YGJ), one of the Chinese herbal medicines most commonly used in western countries, reported to possess significant anti-inflammatary effects that inhibit the process of inflammation. However, the mechanisms underlying its anti-inflammation effects remain largely unresolved. This study was aimed to investigate the anti-inflammatory activity of YGJ and to explore its potential anti-inflammatory mechanisms by serum metabonomics approach. An xylene-induced mouse right-ear-edema model was used as an inflammatory response in vivo model. Ear edema, prostaglandin E2 (PGE2) and Tumor-Necrosis-Factor-alpha (TNF-α) were detected. Then, serum metabolic profiling was analyzed and pathway analysis performed on the biomarkers reversed after YGJ administration and further integration of metabolic networks. The results showed that YGJ alleviated ear edema and decreased serum PGE2 and TNF-α levels. Fourteen biomarkers were screened, and the levels were all reversed to different degrees after YGJ administration. These biomarkers were mainly related to linoleic acid metabolism, taurine and hypotaurine metabolism, glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism and citrate cycle (TCA cycle). In metabolic networks, glycine and pyruvate were node molecules. This indicated that YGJ could significantly inhibit inflammatory response triggered by acute local stimulation and exerted anti-inflammatory activity mainly by regulating node molecules.

Metabonomic analysis of biochemical changes in the plasma and urine of carrageenan-induced rats after treatment with Yi-Guan-Jian decoction.

The urinary and plasma metabonomics method based on a Agilent-1200 LC system coupled to an Agilent-6410 mass spectrometry (HPLC-MS/MS) had been established to investigate the anti-inflammatory activity of Yi-Guan-Jian (YGJ) decoction and explore its potential anti-inflammatory mechanism. Rat acute inflammation was induced by subcutaneous injection of carrageenan in hind paws. Multivariate statistical approaches, such as principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA) and XCMS online software were used to distinguish normal control group (NG), model group (MG), aspirin-treated group (AG) and Yi-Guan-Jian decoction group (YGJ), aimed at finding out the potential biomarkers. There was a clear separation among the four groups in PCA model. Twenty-five potential biomarkers had been identified using PCA, PLS-DA and XCMS online software. Lastly, we had an enrichment for the related metabolic pathways and screened out the pathways that influence the organism a lot in MetPA, then five mainly metabolism: tryptophan metabolism, lipid metabolism, oxidative stress, glyoxylate and dicarboxylate metabolism, taurine and hypotaurine metabolism were found. In this study, YGJ showed good anti-inflammatory effects and it could suppress the changes of pathologic inflammatory cytokines of carrageenan-induced rat paw edema (CIE). There might be a correlation between these results and the regulation of the disturbed metabolites in urine and plasma. This study demonstrates that metabonomics is a powerful methodology to gain insight into the mechanisms of traditional Chinese medicine (TCM) formula in therapy.