Identifying eleven new ferroptosis inhibitors as neuroprotective agents from FDA-approved drugs.

With increasing evidence that ferroptosis is associated with diverse neurological disorders, targeting ferroptosis offers a promising avenue for developing effective pharmaceutical agents for neuroprotection. In this study, we identified ferroptosis inhibitors as neuroprotective agents from US Food and Drug Administration (FDA)-approved drugs. 1176 drugs have been screened against erastin-induced ferroptosis in HT22 cells, resulting in 89 ferroptosis inhibitors. Among them, 26 drugs showed significant activity with EC50 below10 µM. The most active ferroptosis inhibitor is lumateperone tosylate at nanomolar level. 11 drugs as ferroptosis inhibitors were not reported previously. Further mechanistic studies revealed that their mechanisms of actions involve free radical scavenging, Fe2+ chelation, and 15-lipoxygenase inhibition. Notably, the active properties of some drugs were firstly revealed here. These ferroptosis inhibitors increase the chemical diversity of ferroptosis inhibitors, and offer new therapeutic possibilities for the treatments of related neurological diseases.

Dexmedetomidine alleviates Hypoxia/reoxygenation-induced mitochondrial dysfunction in cardiomyocytes via activation of Sirt3/Prdx3 pathway.

BACKGROUND: Myocardial ischemia/reperfusion injury (MIRI) seriously threatens the health of people. The mitochondrial dysfunction in cardiomyocytes can promote the progression of MIRI. Dexmedetomidine (Dex) could alleviate the myocardial injury, which was known to reverse mitochondrial dysfunction in lung injury. However, the function of Dex in mitochondrial dysfunction during MIRI remains unclear. OBJECTIVE: To assess the function of Dex in mitochondrial dysfunction during MIRI. METHODS: To investigate the function of Dex in MIRI, H9C2 cells were placed in condition of hypoxia/reoxygenation (H/R). CCK8 assay was performed to test the cell viability, and the mitochondrial membrane potential was evaluated by JC-1 staining. In addition, the binding relationship between Sirt3 and Prdx3 was explored by Co-IP assay. Furthermore, the protein expressions were examined using western blot. RESULTS: Dex could abolish H/R-induced mitochondrial dysfunction in H9C2 cells. In addition, H/R treatment significantly inhibited the expression of Sirt3, while Dex partially restored this phenomenon. Knockdown of Sirt3 or Prdx3 obviously reduced the protective effect of Dex on H/R-induced mitochondrial injury. Meanwhile, Sirt3 could enhance the function of Prdx3 via deacetylation of Prdx3. CONCLUSION: Dex was found to attenuate H/R-induced mitochondrial dysfunction in cardiomyocytes via activation of Sirt3/Prdx3 pathway. Thus, this study might shed new lights on exploring new strategies for the treatment of MIRI.

Dexmedetomidine Promoted HSPB8 Expression via Inhibiting the lncRNA SNHG14/UPF1 Axis to Inhibit Apoptosis of Nerve Cells in AD : The Role of Dexmedetomidine in AD.

Dexmedetomidine (Dex) is reported to play a neuroprotective role in Alzheimer's disease (AD). However, the specific mechanism remains unclear. Figure out the underlying molecular mechanism of Dex regulating nerve cell apoptosis in the AD model. The AD model in vitro was established after SH-SY5Y cells were treated with Aß1 - 42 at (10 µM) for 24 h. The interaction among UPF1, lncRNA SNHG14, and HSPB8 was verified by RIP assay. Cell viability, apoptosis, the level of genes, and proteins were detected by CCK-8 assay, flow cytometry, Western blot, and qRT-PCR, respectively. Dex downregulated lncRNA SNHG14 level and inhibited apoptosis of nerve cells. LncRNA SNHG14 overexpression reversed the inhibitory effect of Dex on nerve cell apoptosis in the AD model. LncRNA SNHG14 attenuated HSPB8 mRNA stability by recruiting UPF1. HSPB8 overexpression inhibited apoptosis of nerve cells in the AD model. Moreover, HSPB8 knockdown reversed the inhibitory effect of Dex on nerve cell apoptosis in the AD model. Our study demonstrated that Dex promoted HSPB8 expression via inhibiting the lncRNA SNHG14/UPF1 axis to inhibit nerve cell apoptosis in AD.

Discovery of neo-Clerodane Diterpenoids from Ajuga campylantha as Neuroprotective Agents against Ferroptosis and Neuroinflammation.

Twelve new neo-clerodane diterpenoids, eight undescribed methoxy/ethoxy acetal analogues, and one new nor-iridane monoterpenoid were isolated from Ajuga campylantha. Their structures were elucidated using a combination of spectroscopic data, quantum chemical calculations, and X-ray crystallography. This research reveals the distinctive structural features of A. campylantha diterpenes, including distinct C rings and 4,18-double bonds, distinguishing them from diterpenes of other plants in the Ajuga genus. Compound 2 represents the first example of a 19(5→6)-abeo-clerodane formed through a Wagner-Meerwein rearrangement. The isolated compounds were assessed for their neuroprotective effects against RSL3-induced ferroptosis in HT22 cells and LPS-induced neuroinflammation in BV-2 cells. Notably, compound 7 inhibits ferroptosis (EC50 = 10 µM) with a potentially new mechanism of action. The preliminary structure-activity relationship studies revealed that the furan-clerodane diterpenoids possess potential ferroptosis inhibitory activity, while the lactone-clerodanes do not. This study represents the first report of furan-containing clerodanes within the Ajuga genus, providing fresh insights into the phytochemistry and pharmacological potential of A. campylantha.

Chemical constituents of Ajuga forrestii and their anti-ferroptosis activity.

Six new neoclerodane diterpenoids (1-6), along with ten known compounds (7-16), were isolated from Ajuga forrestii. Their structures were elucidated by HRESIMS, 1D and 2D NMR, ECD calculation, and single-crystal X-ray diffraction analysis. The structure of a known neoclerodane diterpene ajudecunoid C (6) was revised based on the reported NMR empirical rules. All the isolates were evaluated for their inhibitory effect on RSL3-induced ferroptosis in HT22 mouse hippocampal neuronal cells. Among them, compounds 8, 9, and 12 significantly inhibited RSL3-induced ferroptosis with EC50 values of 0.45 µM, 0.076 µM, and 0.14 µM.

Discovery and optimization of 2-(trifluoromethyl)benzimidazole derivatives as novel ferroptosis inducers in vitro and in vivo.

Ferroptosis is implicated in diverse human diseases. Ferroptosis inducers hold great potential for cancer therapy. The existing ferroptosis inducers, however, lack structural diversity, and only a few of them are suitable for in vivo applications. Herein, by phenotypic screenings, we discovered a new ferroptosis inducer FA-S, a 2-(trifluoromethyl)benzimidazole derivative, from which a series of its analogs were designed and synthesized to improve the activity. This produced the most potent compound FA16 with single-digit micromolar activity of ferroptosis induction and satisfactory metabolic stability. Further studies demonstrated that FA16 induced ferroptosis by inhibiting cystine/glutamate antiporter (system Xc-). It is noteworthy that analogue FA16 has more favorable metabolic stability than the classic system Xc- inhibitor erastin, which is not suitable for in vivo studies. FA16 significantly inhibited tumor growth in the HepG2 xenograft model by inducing ferroptosis. This work provides new ferroptosis inducers with a novel scaffold, but also a promising lead for hepatocellular carcinoma treatment. Our work reveals a suitable in vivo ferroptosis-inducing tool to explore the mechanisms underlying ferroptosis and the relevance of ferroptosis to pathogenesis of human diseases.

ANP32E contributes to gastric cancer progression via NUF2 upregulation.

Acidic nuclear phosphoprotein 32 family member E (ANP32E) is a histone chaperone that removes H2A.Z from chromatin. ANP32E is implicated in numerous cellular processes, including cell proliferation, apoptosis and cell differentiation. Increasing evidence suggests that dysregulation of ANP32E expression is strongly associated with carcinogenesis. However, the relationship between ANP32E in the development of gastric cancer (GC) is unknown. The present study aimed to explore the potential role of ANP32E in the development of GC using gain­of­function, loss­of­function, CCK­8, colony formation, apoptosis, reverse transcription­quantitative PCR, immunoblotting and luciferase reporter assay. The results of the present study demonstrated that ANP32E expression levels were significantly increased in GC tissues. ANP32E knockdown markedly inhibited GC cell proliferation and colony formation and significantly induced GC cell apoptosis, whereas overexpression of ANP32E significantly induced GC cell malignancy. Furthermore, the results demonstrated that there was a positive association between ANP32E and NUF2 component of NDC80 kinetochore complex (NUF2) expression levels. By assessing NUF2 expression levels, it was demonstrated that ANP32E promoted tumor cell proliferation and inhibited cell apoptosis by increasing NUF2 expression levels in GC cell lines. In conclusion, the present study indicated that ANP32E may function as an efficient oncogene, which promotes tumorigenesis of GC cells by inducing NUF2 expression.

Ferroptosis Inhibitory Aromatic Abietane Diterpenoids from Ajuga decumbens and Structural Revision of Two 3,4-Epoxy Group-Containing Abietanes.

Two new 3,4-epoxy group-containing abietane diterpenoids (1 and 2), together with five known diterpenoids (3-7), were isolated from Ajuga decumbens. Their structures were elucidated by spectroscopic data analysis, NMR calculations, and X-ray diffraction experiments. The structures of two known abietane diterpenoids were revised based on NMR calculations and X-ray diffraction data. Notably, compound 4 specifically inhibited RSL3-induced ferroptosis with an EC50 of 56 nM by antioxidation. Moreover, 4 significantly decreased RSL3-induced lipid and cytosolic ROS accumulation and ferroptosis marker gene PTGS2 mRNA expression. This work reports the most potent natural inhibitor against ferroptosis found so far.

Discovery of novel diphenylbutene derivative ferroptosis inhibitors as neuroprotective agents.

Ferroptosis is a regulated and iron-dependent cell death. Ferroptosis inhibitors are promising for treating many neurological diseases. Herein, with phenotypic assays, we discovered a new diphenylbutene derivative ferroptosis inhibitor, DPT. Based on this hit, we synthesized fourteen new diphenylbutene derivatives, evaluated their ferroptosis inhibitory activities in HT22 mouse hippocampal neuronal cells, and found that three compounds exhibited improved inhibitory activities compared with DPT. Among these active compounds, compound 3f displayed the most potent anti-ferroptosis activity (EC50 = 1.7 µM). Further studies demonstrated that 3f is a specific ferroptosis inhibitor. And we revealed that different from the classic ferroptosis inhibitors, 3f blocked ferroptosis by increasing FSP1 protein level. Moreover, 3f can penetrate blood-brain barrier (BBB). In a rat model of ischemic stroke, 3f effectively mitigated cerebral ischemic injury. Therefore, we are confirmed that 3f, as a novel ferroptosis inhibitor with a new scaffold, is promising for further development as an agent against neurological diseases.

miR­486­5p suppresses gastric cancer cell growth and migration through downregulation of fibroblast growth factor 9.

Non­coding RNAs serve essential roles in regulating mRNA and protein expression and dysregulation of non­coding RNAs participates in a variety of types of cancer. microRNAs (miRNAs/miRs), which are 21­24 nucleotides non­coding RNAs, have been shown to be important for the development of gastric cancer (GC). However, the role of miR­486­5p in GC remains to be elucidated. The present study found that miR­486­5p was downregulated in GC tissues. Comparing with gastric normal cells GES­1, GC cells, including MKN­45, AGS, HGC27 and MKN74, had reduced abundance of miR­486­5p transcript. CCK8 and colony formation assays demonstrated that GC cell growth and proliferation were enhanced by miR­486­5p inhibitors and were suppressed by miR­486­5p mimics. miR­486­5p also suppressed cell cycle process and migration and promoted apoptosis in GC cells, as verified by propidium iodide (PI) staining, Transwell assay and PI/Annexin V staining. miR­486­5p downregulated fibroblast growth factor 9 (FGF9) through combining to its 3'untranslated region. Overexpression of FGF9 accelerated the growth and proliferation of GC cells. The expression of miR­486­5p was negatively associated with FGF9 mRNA expression in GC samples. These results revealed that miR­486­5p was a tumor suppressor in GC. Downregulation of FGF9 contributed to the role of miR­486­5p in GC.

A new ferroptosis inhibitor, isolated from Ajuga nipponensis, protects neuronal cells via activating NRF2-antioxidant response elements (AREs) pathway.

Ferroptosis is a new form of cell death, and inhibition of ferroptosis is a promising strategy to treat neurological diseases. In this work, sixteen compounds were isolated from Ajuga nipponensis and assayed for anti-ferroptosis activity in HT22 mouse hippocampal neuronal cells. Ajudecunoid C (1, ADC), a new neoclerodane diterpenoid, showed significant inhibitory activity against erastin and RSL3-induced ferroptosis with EC50 values of 4.1 ± 1.0 and 3.6 ± 0.3 µM, respectively. Experimental results demonstrated that ADC effectively prevented ferroptosis through scavenging free radical and activating NRF2-antioxidant response elements (AREs) pathway. This study reveals that ADC, as a new ferroptosis inhibitor, is a promising lead compound for the development of drugs against ferroptosis-related neurological diseases.

Discovery of phloroglucinols from Hypericum japonicum as ferroptosis inhibitors.

Ferroptosis is a new type of cell death, which involves central neuronal system. Inhibition of ferroptosis is a promising strategy to prevent and treat neurological diseases. Thirteen phloroglucinols (1-13) were obtained from the whole plants of Hypericum japonicum. Of them, compounds 1-3 are new ones. Their structures were elucidated by extensive analysis of spectroscopic data and X-ray diffraction. All the isolates were evaluated for their inhibitory effect on RSL3-induced ferroptosis. Two new compounds 2-3 showed significant inhibitory effect with EC50 of 0.48 ± 0.14 µM and 0.94 ± 0.14 µM, respectively. DPPH free radical scavenging abilities of all compounds were assessed to evaluate their antioxidant effect. This work first reports the anti-ferroptosis activity of phloroglucinols.

Inhibiting Ferroptosis through Disrupting the NCOA4-FTH1 Interaction: A New Mechanism of Action.

Ferroptosis is an iron-dependent form of oxidative cell death, and the inhibition of ferroptosis is a promising strategy with which to prevent and treat neurological diseases. Herein we report a new ferroptosis inhibitor 9a with a novel mechanism of action. It is demonstrated that nuclear receptor coactivator 4 (NCOA4), a cargo receptor for ferritinophagy, is the target of 9a. Compound 9a blocks ferroptosis by reducing the amount of bioavailable intracellular ferrous iron through disrupting the NCOA4-FTH1 protein-protein interaction. Further studies indicate that 9a directly binds to recombinant protein NCOA4383-522 and effectively blocks the NCOA4383-522-FTH1 interaction. In a rat model of ischemic stroke, 9a significantly ameliorates the ischemic-refusion injury. With the first ligand 9a, this work reveals that NCOA4 is a promising drug target. Additionally, 9a is the first NCOA4-FTH1 interaction inhibitor. This work paves a new road to the development of ferroptosis inhibitors against neurological diseases.

Mechanisms of Modulation of Ferroptosis and Its Role in Central Nervous System Diseases.

Ferroptosis is a new form of programmed cell death characterized by intracellular iron-dependent accumulation of lipid peroxide and primarily associated with iron metabolism, glutathione-dependent pathway, and coenzyme Q10-dependent pathway. Recent studies demonstrate that ferroptosis is associated with central nervous system (CNS) diseases, such as stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. This review summarizes the key regulatory mechanisms of ferroptosis and its role in CNS diseases. These updates may provide novel perspective for the development of therapeutical agents against CNS diseases.

Indoleamine 2,3-Dioxygenase 1 (IDO1) Promotes Cardiac Hypertrophy via a PI3K-AKT-mTOR-Dependent Mechanism.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme for tryptophan metabolism, involved in immune cell differentiation/maturation and cancer biology. IDO1 is also expressed in cardiomyocytes, but its roles in the cardiovascular system are not fully understood. Here, we reported the functions of IDO1 during cardiac hypertrophy. Quantitative real-time PCR and Western blot experiments demonstrated the upregulation of IDO1 mRNA and protein levels in human and hypertrophic mouse hearts, as well as in angiotensin II (Ang II)-induced hypertrophic rat cardiomyocytes. IDO1 activity and metabolite product kynurenine were upregulated in rodent hypertrophic hearts and cardiomyocytes. Inhibition of IDO1 activity with PF-06840003 reduced Ang II-induced cardiac hypertrophy and rescued cardiac function in mice. siRNA-mediated knockdown of Ido1 repressed Ang II-induced growth in cardiomyocyte size and overexpression of hypertrophy-associated genes atrial natriuretic peptide (Anp or Nppa), brain natriuretic peptide (Bnp or Nppb), ß-myosin heavy chain (ß-Mhc or Myh7). By contrast, adenovirus-mediated rat Ido1 overexpression in cardiomyocytes promoted hypertrophic growth induced by Ang II. Mechanism analysis showed that IDO1 overexpression was associated with PI3K-AKT-mTOR signaling to activate the ribosomal protein S6 kinase 1 (S6K1), which promoted protein synthesis in Ang II-induced hypertrophy of rat cardiomyocytes. Finally, we provided evidence that inhibition of PI3K with pictilisib, AKT with perifosine, or mTOR with rapamycin, blocked the effects of IDO1 on protein synthesis and cardiomyocyte hypertrophy in Ang II-treated cells. Collectively, our findings identify that IDO1 promotes cardiomyocyte hypertrophy partially via PI3K-AKT-mTOR-S6K1 signaling.

Synthesis and evaluation of andrographolide derivatives as potent anti-osteoporosis agents in vitro and in vivo.

In this work, we found that 14-deoxy-11,12-didehydroandrographolide (2), a derivative of andrographolide (AP, 1), had greatly reduced cytotoxicity compared with AP and exhibited moderate anti-osteoclastogenesis activity. Thirty compounds were synthesized by introducing anti-osteoporosis chemotypes at C-19 of 2. Six of them exhibited stronger inhibition of osteoclastogenesis than AP. Of note, compound 12g displayed the most potent activity with IC50 value of 0.35 µM. The expression levels of osteoclast-specific genes such as TRAcP, CTSK, NFATc1, and MMP-9 were also decreased by 12g treatment. Furthermore, Western blot and immunofluorescence analyses demonstrated that compound 12g inhibited osteoclast differentiation through downregulation of RANKL-induced NF-κB signaling pathway. In an ovariectomized (OVX) female mice model, compound 12g significantly ameliorated bone loss. Therefore, compound 12g exhibited promising in vivo efficacy and low toxicity, indicating its therapeutic potential for the treatment of osteoporosis.

JMJD1A Represses the Development of Cardiomyocyte Hypertrophy by Regulating the Expression of Catalase.

The histone demethylase JMJD family is involved in various physiological and pathological functions. However, the roles of JMJD1A in the cardiovascular system remain unknown. Here, we studied the function of JMJD1A in cardiac hypertrophy. The mRNA and protein levels of JMJD1A were significantly downregulated in the hearts of human patients with hypertrophic cardiomyopathy and the hearts of C57BL/6 mice underwent cardiac hypertrophy induced by transverse aortic constriction (TAC) surgery or isoproterenol (ISO) infusion. In neonatal rat cardiomyocytes (NRCMs), siRNA-mediated JMJD1A knockdown facilitated ISO or angiotensin II-induced increase in cardiomyocyte size, protein synthesis, and expression of hypertrophic fetal genes, including atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), and Myh7. By contrast, overexpression of JMJD1A with adenovirus repressed the development of ISO-induced cardiomyocyte hypertrophy. We observed that JMJD1A reduced the production of total cellular and mitochondrial levels of reactive oxygen species (ROS), which was critically involved in the effects of JMJD1A because either N-acetylcysteine or MitoTEMPO treatment blocked the effects of JMJD1A deficiency on cardiomyocyte hypertrophy. Mechanism study demonstrated that JMJD1A promoted the expression and activity of Catalase under basal condition or oxidative stress. siRNA-mediated loss of Catalase blocked the protection of JMJD1A overexpression against ISO-induced cardiomyocyte hypertrophy. These findings demonstrated that JMJD1A loss promoted cardiomyocyte hypertrophy in a Catalase and ROS-dependent manner.

Structure-Activity Relationship Analysis on Antioxidant and Anticancer Actions of Theaflavins on Human Colon Cancer Cells.

The roles of natural products as effective cancer prevention and therapeutic agents have been documented by various studies in recent years, but the action mechanisms and structure-activity relationship need more elucidation. The present study showed that theaflavins (theaflavin and its derivatives, TFs) from black tea caused an inhibitory effect on the proliferation of human colon adenocarcinoma cancer SW480 cells and human colon cancer SW620 cells [half maximal inhibitory concentration (IC50) < 32.0 µM] by the induction of cell cycle arrest but exerted lower toxicity against normal cells with a high safety index (1.89-6.26). Moreover, TFs triggered a decrease in reactive oxygen species in SW480 cells as a result of their excellent radical-scavenging ability (e.g., the IC50 value of TF4 to ABTS• + was 1.91 ± 0.21 µM). More importantly, the structure-activity relationship analysis of TFs exhibited that the galloyl group was an important factor to affect these activities. Taken together, we revealed that the TFs could act as substitutes for natural antioxidants and promising anticancer agents with beneficial influence on human health and then anticipated that this study may provide useful information on the development of therapeutic natural products.