Mitochondrial Inhibitor Research Articles

Celastrol alleviates atopic dermatitis by regulating Ezrin-mediated mitochondrial fission and fusion.

Celastrol, a bioactive molecule extracted from the plant Tripterygium wilfordii Hook F., possesses anti-inflammatory, anti-obesity and anti-tumour properties. Despite its efficacy in improving erythema and scaling in psoriatic mice, the specific therapeutic mechanism of celastrol in atopic dermatitis (AD) remains unknown. This study aims to examine the role and mechanism of celastrol in AD using TNF-α-stimulated HaCaT cells and DNCB-induced Balb/c mice as invitro and invivo AD models, respectively. Celastrol was found to inhibit the increased epidermal thickness, reduce spleen and lymph node weights, attenuate inflammatory cell infiltration and mast cell degranulation and decrease thymic stromal lymphopoietin (TSLP) as well as various inflammatory factors (IL-4, IL-13, TNF-α, IL-5, IL-31, IL-33, IgE, TSLP, IL-17, IL-23, IL-1β, CCL11 and CCL17) in AD mice. Additionally, celastrol inhibited Ezrin phosphorylation at Thr567, restored mitochondrial network structure, promoted translocation of Drp1 to the cytoplasm and reduced TNF-α-induced cellular reactive oxygen species (ROS), mitochondrial ROS (mtROS) and mitochondrial membrane potential (MMP) production. Interestingly, Mdivi-1 (a mitochondrial fission inhibitor) and Ezrin-specific siRNAs lowered inflammatory factor levels and restored mitochondrial reticular formation, as well as ROS, mtROS and MMP production. Co-immunoprecipitation revealed that Ezrin interacted with Drp1. Knocking down Ezrin reduced mitochondrial fission protein Drp1 phosphorylation and Fis1 expression while increasing the expression of fusion proteins Mfn1 and Mfn2. The regulation of mitochondrial fission and fusion by Ezrin was confirmed. Overall, celastrol may alleviate AD by regulating Ezrin-mediated mitochondrial fission and fusion, which may become a novel therapeutic reagent for alleviating AD.

Effect of Tianma Gouteng Decoction on PINK1/Parkin pathway in oxidative stress in vascular smooth muscle cell induced by AngⅡ

This study explored the effect of Tianma Gouteng Decoction on oxidative stress induced by angiotensin Ⅱ(AngⅡ) in vascular smooth muscle cell(VSMC) and its molecular mechanism. Primary rat VSMC were cultured using tissue block method, and VSMC were identified by α-actin immunofluorescence staining. AngⅡ at a concentration of 1×10~(-6) mol·L~(-1) was used as the stimulating factor, and Sprague Dawley(SD) rats were orally administered with Tianma Gouteng Decoction to prepare drug serum. Rat VSMC were divided into normal group, model group, Chinese medicine group, and inhibitor(3-methyladenine, 3-MA) group. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation activity. Bromodeoxyuridine(BrdU) flow cytometry was used to detect cell cycle. Transwell assay was used to detect cell migration ability. Enzyme-linked immunosorbent assay(ELISA) was used to detect the activity of superoxide dismutase(SOD), catalase(CAT), and malondialdehyde(MDA) in VSMC. The intracellular reactive oxygen species(ROS) fluorescence intensity was detected using DCFH-DA fluorescent probe. Western blot was used to detect the expression of PTEN-induced putative kinase 1(PINK1), Parkin, p62, and microtubule-associated protein 1A/1B-light chain 3(LC3-Ⅱ) proteins in VSMC. The results showed that Tianma Gouteng Decoction-containing serum at a concentration of 8% could significantly inhibit VSMC growth after 48 hours of intervention. Compared with the normal group, the model group showed significantly increased cell proliferation activity and migration, significantly decreased levels of SOD and CAT, significantly increased levels of MDA, significantly enhanced ROS fluorescence intensity, significantly decreased expression of PINK1, Parkin, and LC3-Ⅱ proteins, and significantly increased expression of p62 protein. Compared with the model group, the Chinese medicine group showed significantly reduced cell proliferation activity and migration, significantly increased levels of SOD and CAT, significantly decreased levels of MDA, significantly weakened ROS fluorescence intensity, significantly increased expression of PINK1, Parkin, and LC3-Ⅱ proteins, and significantly decreased expression of p62 protein. Compared with the Chinese medicine group, the addition of the mitochondrial autophagy inhibitor 3-MA could block the intervention of Tianma Gouteng Decoction-containing serum on VSMC proliferation, migration, mitochondrial autophagy, and oxidative stress levels, with statistically significant differences. In summary, Tianma Gouteng Decoction has good antioxidant activity and can inhibit cell proliferation and migration. Its mechanism of action may be related to the activation of the mitochondrial autophagy PINK1/Parkin signaling pathway.

MitoNEET preserves muscle insulin sensitivity during iron overload by regulating mitochondrial iron, reactive oxygen species and fission.

Iron overload (IO) is known to contribute to metabolic dysfunctions such as type 2 diabetes and insulin resistance. Using L6 skeletal muscle cells overexpressing the CDGSH iron-sulfur domain-containing protein 1 (CISD1, also known as mitoNEET) (mitoN) protein, we examined the potential role of MitoN in preventing IO-induced insulin resistance. In L6 control cells, IO resulted in insulin resistance which could be prevented by MitoN as demonstrated by western blot of p-Akt and Akt biosensor cells. Mechanistically, IO increased; mitochondrial iron accumulation, mitochondrial reactive oxygen species (ROS), Fis1-dependent mitochondrial fission, mitophagy, FUN14 domain-containing protein 1 (FUNDC1) expression, and decreased Parkin. MitoN overexpression was able to reduce increases in mitochondrial iron accumulation, mitochondrial ROS, mitochondrial fission, mitophagy and FUNDC1 upregulation due to IO. MitoN did not have any effect on the IO-induced downregulation of Parkin. MitoN alone also upregulated peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) protein levels, a master regulator of mitochondrial biogenesis. The use of mitochondrial antioxidant, Skq1, or fission inhibitor, Mdivi-1, prevented IO-induced insulin resistance implying both mitochondrial ROS and fission play a causal role in the development of insulin resistance. Taken together, MitoN is able to confer protection against IO-induced insulin resistance in L6 skeletal muscle cells through regulation of mitochondrial iron content, mitochondrial ROS, and mitochondrial fission.

Deuterium MRI of serine metabolism in mouse models of glioblastoma.

Serine is a major source of one-carbon units needed for the synthesis of nucleotides and the production of intramitochondrial nicotinamide adenine dinucleotide phosphate (NADPH), and it plays an important role in cancer cell proliferation. The aim of this study was to develop a deuterium (2H) MRS imaging method for imaging tumor serine metabolism. Sequential (2H) spectra and spectroscopic images were used to monitor the metabolism of [2,3,3-2H3]serine in patient-derived glioblastoma cells in vitro and in tumors obtained by their orthotopic implantation in mouse brain. [14,14-2H2] 5,10-methylene-tetrahydrofolate, [2H]glycine, [2H]formate, and labeled water were detected in cell suspensions and water labeling in spectroscopic images of tumors. Studies in cells and tumors with variable mitochondrial content and inhibitor studies in cells demonstrated that most of the labeled serine was metabolized in the mitochondria. Water labeling in the cell suspensions was correlated with formate labeling; therefore, water labeling observed in tumors could be used to provide a surrogate measure of flux in the pathway of one-carbon metabolism in vivo. The method has the potential to be used clinically to select patients for treatment with inhibitors of one-carbon metabolism and subsequently to detect their early responses to such treatment.

Construction of mitochondrial-targeting nano-prodrug for enhanced Rhein delivery and treatment for osteoarthritis in vitro

Rhein, a natural anthraquinone compound derived from traditional Chinese medicine, exhibits potent anti-inflammatory properties via modulating the level of Reactive oxygen or nitrogen species (RONS). Nevertheless, its limited solubility in water, brief duration of plasma presence, as well as its significant systemic toxicity, pose obstacles to its in vivo usage, necessitating the creation of a reliable drug delivery platform to circumvent these difficulties. In this study, an esterase-responsive and mitochondria-targeted nano-prodrug was synthesized by conjugating Rhein with the polyethylene glycol (PEG)-modified triphenyl phosphonium (TPP) molecule, forming TPP-PEG-RH, which could spontaneously self-assemble into RPT NPs when dispersed in aqueous media. The TPP outer layer of these nanoparticles enhances their pharmacokinetic profile, facilitates efficient delivery to mitochondria, and promotes cellular uptake, thereby enabling enhanced accumulation in mitochondria and improved therapeutic effects in vitro. The decline in RONS was observed in IL-1β-stimulated chondrocyte after RPT NPs treating. RPT NPs also exert excellent anti-inflammatory (IL-1β, TNF-α, IL-6 and MMP-13) and antioxidative effects (Cat and Sod) via the Nrf2 signalling pathway, upregulation of cartilage related genes (Col2a1 and Acan). Moreover, RPT NPs shows protection of mitochondrial membrane potential and inhibition of chondrocyte apoptosis. Moreover, These findings suggest that the mitochondria-targeted polymer-Rhein conjugate may offer a therapeutic solution for patients suffering from chronic joint disorders, by attenuating the progression of osteoarthritis (OA).

Mitigating the Functional Deficit after Neurotoxic Motoneuronal Loss by an Inhibitor of Mitochondrial Fission.

Amyotrophic lateral sclerosis (ALS) is an extremely complex neurodegenerative disease involving different cell types, but motoneuronal loss represents its main pathological feature. Moreover, compensatory plastic changes taking place in parallel to neurodegeneration are likely to affect the timing of ALS onset and progression and, interestingly, they might represent a promising target for disease-modifying treatments. Therefore, a simplified animal model mimicking motoneuronal loss without the other pathological aspects of ALS has been established by means of intramuscular injection of cholera toxin-B saporin (CTB-Sap), which is a targeted neurotoxin able to kill motoneurons by retrograde suicide transport. Previous studies employing the mouse CTB-Sap model have proven that spontaneous motor recovery is possible after a subtotal removal of a spinal motoneuronal pool. Although these kinds of plastic changes are not enough to counteract the functional effects of the progressive motoneuron degeneration, it would nevertheless represent a promising target for treatments aiming to postpone ALS onset and/or delay disease progression. Herein, the mouse CTB-Sap model has been used to test the efficacy of mitochondrial division inhibitor 1 (Mdivi-1) as a tool to counteract the CTB-Sap toxicity and/or to promote neuroplasticity. The homeostasis of mitochondrial fission/fusion dynamics is indeed important for cell integrity, and it could be affected during neurodegeneration. Lesioned mice were treated with Mdivi-1 and then examined by a series of behavioral test and histological analyses. The results have shown that the drug may be capable of reducing functional deficits after the lesion and promoting synaptic plasticity and neuroprotection, thus representing a putative translational approach for motoneuron disorders.

A metabolic redox relay supports ER proinsulin export in pancreatic islet β cells.

ER stress and proinsulin misfolding are heralded as contributing factors to β cell dysfunction in type 2 diabetes, yet how ER function becomes compromised is not well understood. Recent data identify altered ER redox homeostasis as a critical mechanism that contributes to insulin granule loss in diabetes. Hyperoxidation of the ER delays proinsulin export and limits the proinsulin supply available for insulin granule formation. In this report, we identified glucose metabolism as a critical determinant in the redox homeostasis of the ER. Using multiple β cell models, we showed that loss of mitochondrial function or inhibition of cellular metabolism elicited ER hyperoxidation and delayed ER proinsulin export. Our data further demonstrated that β cell ER redox homeostasis was supported by the metabolic supply of reductive redox donors. We showed that limiting NADPH and thioredoxin flux delayed ER proinsulin export, whereas thioredoxin-interacting protein suppression restored ER redox and proinsulin trafficking. Taken together, we propose that β cell ER redox homeostasis is buffered by cellular redox donor cycles, which are maintained through active glucose metabolism.

Mitochondrial division inhibitor (mdivi-1) induces extracellular matrix (ECM)-detachment of viable breast cancer cells by a DRP1-independent mechanism

Increasing evidence supports the hypothesis that cancer progression is under mitochondrial control. Mitochondrial fission plays a pivotal role in the maintenance of cancer cell homeostasis. The inhibition of DRP1, the main regulator of mitochondrial fission, with the mitochondrial division inhibitor (mdivi-1) had been associated with cancer cell sensitivity to chemotherapeutics and decrease proliferation. Here, using breast cancer cells we find that mdivi-1 induces the detachment of the cells, leading to a bulk of floating cells that conserved their viability. Despite a decrease in their proliferative and clonogenic capabilities, these floating cells maintain the capacity to re-adhere upon re-seeding and retain their migratory and invasive potential. Interestingly, the cell detachment induced by mdivi-1 is independent of DRP1 but relies on inhibition of mitochondrial complex I. Furthermore, mdivi-1 induces cell detachment rely on glucose and the pentose phosphate pathway. Our data evidence a novel DRP1-independent effect of mdivi-1 in the attachment of cancer cells. The generation of floating viable cells restricts the use of mdivi-1 as a therapeutic agent and demonstrates that mdivi-1 effect on cancer cells are more complex than anticipated.

Abstract PO-034: Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs

Abstract Cancer cells need metabolic adaptations to survive in hostile environment such as low nutrient and drug treatment conditions. DLBCLs (Diffuse large B cell lymphoma) originate from a metabolic competitive/challenging environment of germinal center (GC) when B cells undergo positive selections against antigens. However, it is largely unknown whether and how metabolic flexibility (the ability to use different nutrients) and plasticity (the ability to process the same nutrient differently) contribute to DLBCLs. Our previous study identified that SIRT3, the only mitochondrial protein deacetylase, is critical to survival and proliferation of DLBCLs by promoting glutaminolysis. Our mitochondrial localized SIRT3 inhibitor, YC8-02, has pan-toxicity to all DLBCL cells tested regardless of different subtypes with somatic mutations. Here, we observed glucose as an alternative nutrient source to compensate in the TCA cycle in SIRT3 deficient cells. DLBCLs exhibited resistance to SIRT3 inhibition in high glucose condition, while SIRT3 inhibited DLBCLs highly depend on glucose metabolism. The combination of YC8-02 with glucose metabolism inhibitors induces synergistic killing to DLBCLs. As glucose is the main carbon source of cells, it is unknown how DLBCLs manage the flexibility of using glucose for different purposes. Using in vivo metabolic imaging (MALDI), we observed that, in contrast to normal GC B cells, lymphomas specifically use the glucose for de novo nucleotide synthesis. SIRT3 inhibition decreased nucleotide levels in DLBCL cells in vitro and in vivo. The metabolic flux tracing showed that DLBCL cells requires SIRT3 activity to activate a glucose/serine/one-carbon metabolism pathway for de novo nucleotide biosynthesis, and Inhibition of SIRT3 decreased the glucose and serine being used for nucleotides in DLBCLs. Serine as another potent source for nucleotides can rescue DLBCLs from SIRT3 inhibition. Furthermore, we validated that overexpression of enzymes, like MTHFD2, in one carbon metabolism pathway can rescue the proliferation in SIRT3 deficient cells. High nucleotides levels in medium also help DLBCLs survival in YC8-02 treatments. To overcome the metabolic flexibility on environmental nucleotides, we used inhibitors of nucleoside transporters (ENT1/2), dipyridamole and draflazine, which can synergize with YC8-02 to kill DLBCLs in vitro and eradicate systemic lymphomas in vivo. In summary, our results illustrate that DLBCLs with active SIRT3 exhibit metabolic plasticity by repurposing glucose to nucleotide biosynthesis, while lymphoma cells can also reply on metabolic flexibility to manage nutrient usage and gain extracellular nucleotides for survival under SIRT3 inhibition. Our combination treatment SIRT3 and ENT inhibitors can be a promising therapy for DLBCLs by restraining their metabolic plasticity and flexibility. Citation Format: Meng Li, Noel R Park, Chris Chin, Coraline Mlynarczyk, Hening Lin, Shawn Davidson, Ari M Melnick. Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-034.

Dependence on mitochondrial respiration of malignant T cells reveals a new therapeutic target for angioimmunoblastic T-cell lymphoma

Cancer metabolic reprogramming has been recognized as one of the cancer hallmarks that promote cell proliferation, survival, as well as therapeutic resistance. Up-to-date regulation of metabolism in T-cell lymphoma is poorly understood. In particular, for human angioimmunoblastic T-cell lymphoma (AITL) the metabolic profile is not known. Metabolic intervention could help identify new treatment options for this cancer with very poor outcomes and no effective medication. Transcriptomic analysis of AITL tumor cells, identified that these cells use preferentially mitochondrial metabolism. By using our preclinical AITL mouse model, mimicking closely human AITL features, we confirmed that T follicular helper (Tfh) tumor cells exhibit a strong enrichment of mitochondrial metabolic signatures. Consistent with these results, disruption of mitochondrial metabolism using metformin or a mitochondrial complex I inhibitor such as IACS improved the survival of AITL lymphoma-bearing mice. Additionally, we confirmed a selective elimination of the malignant human AITL T cells in patient biopsies upon mitochondrial respiration inhibition. Moreover, we confirmed that diabetic patients suffering from T-cell lymphoma, treated with metformin survived longer as compared to patients receiving alternative treatments. Taking together, our findings suggest that targeting the mitochondrial metabolic pathway could be a clinically efficient approach to inhibit aggressive cancers such as peripheral T-cell lymphoma.

Study on the mechanism of glucocorticoid receptor mitochondrial translocation and glucocorticoid-induced apoptosis in macrophages

Objective Our research aimed to investigate the therapeutic effects of Tubastatin-A, a glucocorticoid receptor (GR) mitochondrial translocation inhibitor, and mitoquinone (MitoQ), an antioxidant, on attenuating dexamethasone (DEX)-induced macrophage apoptosis. Methods We treated RAW264.7 macrophages with different combinations of DEX and either Tubastatin-A or MitoQ. Parameters such as mitochondrial GR translocation, mitochondrial reactive oxygen species levels, mitochondrial membrane potential, mitochondrial permeability transition pore opening, cytochrome C efflux to the cytosol, and apoptosis were subsequently evaluated in the different treatment groups via qRT–PCR, western blotting, and immunofluorescence assays. Results DEX intervention increased the translocation of GRs into the mitochondria, while reducing the expression of the mitochondrial gene MT-CO1 and the activity of mitochondrial respiratory chain complex IV in macrophages. In addition, DEX administration increased mtROS levels, mitochondrial permeability transition pore opening, and mitochondrial cytochrome C release in macrophages, which promoted their apoptosis. We found that Tubastatin-A inhibited mitochondrial GR translocation and reversed the DEX-induced increase in GR levels within the mitochondria. Furthermore, Tubastatin-A mitigated various mitochondrial changes induced by DEX, including reducing the efflux of mitochondrial cytochrome C and inhibiting macrophage apoptosis. Similarly, MitoQ exerted its effects on macrophage apoptosis by reducing mtROS levels through the mitochondrial pathway. Conclusions The DEX-mediated translocation of GR into mitochondria disrupts the mitochondrial function of macrophages, which induces their apoptosis. By inhibiting mitochondrial translocation of GR and reducing mtROS levels, Tubastatin-A and MitoQ can effectively attenuate macrophage apoptosis, which has clinical implications for reducing the notable side effects associated with glucocorticoid use.

STL Inhibited Angiogenesis of DPSCs Through Depressing Mitochondrial Respiration by Enhancing RNF217.

Angiogenesis is the determining factor during dental pulp regeneration. Six-twelve leukemia (STL) is identified as a key regulatory factor on the biological function of dental pulp stem cells (DPSCs) under hypoxic conditions, but its effect on angiogenesis is unclear. Co-culture of DPSCs and human umbilical vein endothelial cells (HUVECs) is used to detect tubule formation ability in vitro and the angiogenesis ability in vivo. RNA-seq and bioinformatic analyses are performed to screen differentially expressed genes. Seahorse Cell Mito Stress Test is proceeded to exam mitochondrial respiration. STL decreased tubule formation and mitochondrial respiration of DPSCs in vitro and restrained the number of blood vessels and the expression of VEGF in new formed tissue in vivo. Furthermore, pretreating STL-depleted DPSCs with rotenone, a mitochondrial respiration inhibitor, counteracted the promoting effect of STL knockdown on tubule formation. Then, RNA-seq and bioinformatic analyses identified some angiogenesis relevant genes and pathways in STL-depleted DPSCs. And STL enhanced expression of mRNA-ring finger protein 217 (RNF217), which inhibited the tubule formation and mitochondrial respiration of DPSCs. STL inhibited the angiogenesis of DPSCs through depressing mitochondrial respiration by enhancing RNF217, indicating that STL is a potential target for angiogenesis of DPSCs.

Deoxynivalenol Induces Drp-1-Mediated Mitochondrial Dysfunction via Elevating Oxidative Stress.

Mitochondrial dysfunction is often linked to neurotoxicity and neurological diseases and stems from oxidative stress, yet effective therapies are lacking. Deoxynivalenol (DON or vomitoxin) is one of the most common and hazardous type-B trichothecene mycotoxins, which contaminates crops used for food and animal feed. Despite the abundance of preliminary reports, comprehensive investigations are scarce to explore the relationship between these fungal metabolites and neurodegenerative disorders. The present study aimed to elucidate the precise role of DON in mitochondrial dynamics and cell death in neuronal cells. Excessive mitochondrial fission is associated with the pathology of several neurodegenerative diseases. Human SH-SY5Y cells were treated with different concentrations of DON (250-1000 ng/mL). Post 24 and 48 h DON treatment, the indexes were measured as follows: generation of reactive oxygen species (ROS), ATP levels, mitochondrial membrane potential, calcium levels, and cytotoxicity in SH-SY5Y cells. The results showed that cytotoxicity, intracellular calcium levels, and ROS in the DON-treated group increased, while the ATP levels and mitochondrial membrane potential decreased in a dose-dependent manner. With increasing DON concentrations, the expression levels of P-Drp-1, mitochondrial fission proteins Mff, and Fis-1 were elevated with reduced activities of MFN1, MFN2, and OPA1, further resulting in an increased expression of autophagic marker LC3 and beclin-1. The reciprocal relationship between mitochondrial damage and ROS generation is evident as ROS can instigate structural and functional deficiencies within the mitochondria. Consequently, the impaired mitochondria facilitate the release of ROS, thereby intensifying the cycle of damage and exacerbating the overall process. Using specific hydroxyl, superoxide inhibitors, and calcium chelators, our study confirmed that ROS and Ca2+-mediated signaling pathways played essential roles in DON-induced Drp1 phosphorylation. Therefore, ROS and mitochondrial fission inhibitors could provide critical research tools for drug development in mycotoxin-induced neurodegenerative diseases.

Nutrient-sensitizing drug repurposing screen identifies lomerizine as a mitochondrial metabolism inhibitor of chronic myeloid leukemia.

In chronic myeloid leukemia (CML), the persistence of leukemic stem cells (LSCs) after treatment with tyrosine kinase inhibitors (TKIs), such as imatinib, can lead to disease relapse. It is known that therapy-resistant LSCs rely on oxidative phosphorylation (OXPHOS) for their survival and that targeting mitochondrial respiration sensitizes CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy or have shown adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. We performed a high-throughput drug repurposing screen designed to identify mitochondrial metabolism inhibitors in myeloid leukemia cells. This identified lomerizine, a US Food and Drug Administration (FDA)-approved voltage-gated Ca2+ channel blocker now used for the treatment of migraines, as one of the top hits. Transcriptome analysis revealed increased expression of voltage-gated CACNA1D and receptor-activated TRPC6 Ca2+ channels in CML LSCs (CD34+CD38-) compared with normal counterparts. This correlated with increased endoplasmic reticulum (ER) mass and increased ER and mitochondrial Ca2+ content in CML stem/progenitor cells. We demonstrate that lomerizine-mediated inhibition of Ca2+ uptake leads to ER and mitochondrial Ca2+ depletion, with similar effects seen after CACNA1D and TRPC6 knockdown. Through stable isotope-assisted metabolomics and functional assays, we observe that lomerizine treatment inhibits mitochondrial isocitrate dehydrogenase activity and mitochondrial oxidative metabolism and selectively sensitizes CML LSCs to imatinib treatment. In addition, combination treatment with imatinib and lomerizine reduced CML tumor burden, targeted CML LSCs, and extended survival in xenotransplantation model of human CML, suggesting this as a potential therapeutic strategy to prevent disease relapse in patients.

Mitochondrial dysfunction results in enhanced adrenal androgen production in H295R cells

The role of mitochondria in steroidogenesis is well established. However, the specific effects of mitochondrial dysfunction on androgen synthesis are not fully understood. In this study, we investigate the effects of various mitochondrial and metabolic inhibitors in H295R adrenal cells and perform a comprehensive analysis of steroid and metabolite profiling. We report that mitochondrial complex I inhibition by rotenone shifts cells toward anaerobic metabolism with a concomitant hyperandrogenic phenotype characterized by rapid stimulation of dehydroepiandrosterone (DHEA, 2 h) and slower accumulation of androstenedione and testosterone (24 h). Screening of metabolic inhibitors confirmed DHEA stimulation, which included mitochondrial complex III and mitochondrial pyruvate carrier inhibition. Metabolomic studies revealed truncated tricarboxylic acid cycle with an inverse correlation between citric acid and DHEA production as a common metabolic marker of hyperandrogenic inhibitors. The current study sheds light on a direct interplay between energy metabolism and androgen biosynthesis that could be further explored to identify novel molecular targets for efficient treatment of androgen excess disorders.

A chemical modification of a peroxisome proliferator-activated receptor pan agonist produced a shift to a new dual alpha/gamma partial agonist endowed with mitochondrial pyruvate carrier inhibition and antidiabetic properties

New analogs of the PPAR pan agonist AL29-26 encompassed ligand (S)-7 showing potent activation of PPARα and -γ subtypes as a partial agonist. In vitro experiments and docking studies in the presence of PPAR antagonists were performed to help interpretation of biological data and investigate the main interactions at the binding sites. Further in vitro experiments showed that (S)-7 induced anti-steatotic effects and enhancement of the glucose uptake. This latter effect could be partially ascribed to a significant inhibition of the mitochondrial pyruvate carrier demonstrating that (S)-7 also acted through insulin-independent mechanisms. In vivo experiments showed that this compound reduced blood glucose and lipid levels in a diabetic mice model displaying no toxicity on bone, kidney, and liver. To our knowledge, this is the first example of dual PPARα/γ partial agonist showing these combined effects representing, therefore, the potential lead of new drugs for treatment of dyslipidemic type 2 diabetes.

Phase 2 study to evaluate the novel mitochondrial PRX3 inhibitor, RSO-021, as an intrapleural monotherapy and in combination with IV paclitaxel in patients with malignant pleural effusion due to mesothelioma or another advanced solid tumor.

TPS8124 Background: Tumor cells generate elevated levels of reactive oxygen species (ROS), leading to increased expression and activity of critical ROS scavenging pathways. Peroxiredoxin 3 (PRX3) is the principal peroxide scavenging enzyme in mitochondria. RSO–021 is a novel small molecule PRX3 inhibitor that inactivates PRX3 through direct covalent adduction of active site cysteine residues, in turn inducing oxidative stress that is incompatible with tumor cell survival. Preclinical studies of RSO-021 show that it selectively inhibits mitochondrial PRX3 in vitro and is highly effective in a variety of tumor models, including mesothelioma and many solid tumors. RSO-021 has completed phase 1 evaluation in patients with advanced mesothelioma or solid tumors with predominant pleural disease and malignant pleural effusions. The phase 1 data supported weekly intrapleural dosing of RSO-021 at the 90 mg dose level, with acceptable safety and early signals of efficacy. Some patients were able to receive >30 weeks of treatment. Pre- and post-dose primary prophylaxis for local inflammatory reactions to intrapleural injection was instituted during dose escalation, using corticosteroids, NSAIDS and paracetamol. It also became clear that RSO-021 should not be administered to patients with a dry pleural tap, due to risk of extravasation. Methods: The objectives of this open-label, multi-center, 4-arm phase 2 study (MITOPE) are to confirm the efficacy, safety, tolerability, and pharmacology of weekly intrapleural RSO-021 administration. Exploratory endpoints include target engagement as well as biomarkers of response and inflammation. The study explores 2 different RSO-021 doses (90 mg/wk and 45 mg/wk) per FDA Project Optimus, both as monotherapy and in combination with IV paclitaxel 175 mg/m2 every 3 weeks at until progression or limiting toxicity. Each arm at each dose has a 2-stage Simon design (12-21 patients) requiring 1 response to trigger stage 2 (null/target response rate 3%/20% and type I/type II error 0.05/0.8) for a maximum of 168 patients in the trial. Three of the 4 study arms are investigating the antitumor activity of RSO-021 monotherapy (arms 1-3) and the fourth is assessing the paclitaxel combination. Eligibility (by arm) requires: newly diagnosed treatment-naïve mesothelioma with pleural effusion; mesothelioma with pleural effusion that progressed on ≥1 standard of care (SoC) regimen; solid tumor with predominant pleural/lung disease and effusion that progressed on ≥1 SoC regimen; and breast, epithelial ovarian or non-small cell lung cancer with predominant pleural/lung disease and effusion that progressed on ≥1 SoC regimen. The first patient was treated in 4Q2023 and the trial is actively recruiting to all 4 arms at 9 UK sites. Additional sites are planned. Clinical trial information: NCT05278975 .

Inhibition of OGG1 ameliorates pulmonary fibrosis via preventing M2 macrophage polarization and activating PINK1-mediated mitophagy

Background8-Oxoguanine DNA glycosylase (OGG1), a well-known DNA repair enzyme, has been demonstrated to promote lung fibrosis, while the specific regulatory mechanism of OGG1 during pulmonary fibrosis remains unclarified.MethodsA bleomycin (BLM)-induced mouse pulmonary fibrosis model was established, and TH5487 (the small molecule OGG1 inhibitor) and Mitochondrial division inhibitor 1 (Mdivi-1) were used for administration. Histopathological injury of the lung tissues was assessed. The profibrotic factors and oxidative stress-related factors were examined using the commercial kits. Western blot was used to examine protein expression and immunofluorescence analysis was conducted to assess macrophages polarization and autophagy. The conditional medium from M2 macrophages was harvested and added to HFL-1 cells for culture to simulate the immune microenvironment around fibroblasts during pulmonary fibrosis. Subsequently, the loss- and gain-of function experiments were conducted to further confirm the molecular mechanism of OGG1/PINK1.ResultsIn BLM-induced pulmonary fibrosis, OGG1 was upregulated while PINK1/Parkin was downregulated. Macrophages were activated and polarized to M2 phenotype. TH5487 administration effectively mitigated pulmonary fibrosis, M2 macrophage polarization, oxidative stress and mitochondrial dysfunction while promoted PINK1/Parkin-mediated mitophagy in lung tissues of BLM-induced mice, which was partly hindered by Mdivi-1. PINK1 overexpression restricted M2 macrophages-induced oxidative stress, mitochondrial dysfunction and mitophagy inactivation in lung fibroblast cells, and OGG1 knockdown could promote PINK1/Parkin expression and alleviate M2 macrophages-induced mitochondrial dysfunction in HFL-1 cells.ConclusionOGG1 inhibition protects against pulmonary fibrosis, which is partly via activating PINK1/Parkin-mediated mitophagy and retarding M2 macrophage polarization, providing a therapeutic target for pulmonary fibrosis.

Heat stress upregulates arachidonic acid to trigger autophagy in sertoli cells via dysfunctional mitochondrial respiratory chain function

As a key factor in determining testis size and sperm number, sertoli cells (SCs) play a crucial role in male infertility. Heat stress (HS) reduces SCs counts, negatively impacting nutrient transport and supply to germ cells, and leading to spermatogenesis failure in humans and animals. However, how HS affects the number of SCs remains unclear. We hypothesized that changes in SC metabolism contribute to the adverse effects of HS. In this study, we first observed an upregulation of arachidonic acid (AA), an unsaturated fatty acid after HS exposure by LC-MS/MS metabolome detection. By increasing ROS levels, expression of KEAP1 and NRF2 proteins as well as LC3 and LAMP2, 100 µM AA induced autophagy in SCs by activating oxidative stress (OS). We observed adverse effects of AA on mitochondria under HS with a decrease of mitochondrial number and an increase of mitochondrial membrane potential (MMP). We also found that AA alternated the oxygen transport and absorption function of mitochondria by increasing glycolysis flux and decreasing oxygen consumption rate as well as the expression of mitochondrial electron transport chain (ETC) proteins Complex I, II, V. However, pretreatment with 5 mM NAC (ROS inhibitor) and 2 µM Rotenone (mitochondrial ETC inhibitor) reversed the autophagy induced by AA. In summary, AA modulates autophagy in SCs during HS by disrupting mitochondrial ETC function, inferring that the release of AA is a switch-like response, and providing insight into the underlying mechanism of high temperatures causing male infertility.

Fmo induction as a tool to screen for pro-longevity drugs.

Dietary restriction (DR) and hypoxia (low oxygen) extend lifespan in Caenorhabditis elegans through the induction of a convergent downstream longevity gene, fmo-2. Flavin-containing monooxygenases (FMOs) are highly conserved xenobiotic-metabolizing enzymes with a clear role in promoting longevity in nematodes and a plausible similar role in mammals. This makes them an attractive potential target of small molecule drugs to stimulate the health-promoting effects of longevity pathways. Here, we utilize an fmo-2 fluorescent transcriptional reporter in C. elegans to screen a set of 80 compounds previously shown to improve stress resistance in mouse fibroblasts. Our data show that 19 compounds significantly induce fmo-2, and 10 of the compounds induce fmo-2 more than twofold. Interestingly, 9 of the 10 high fmo-2 inducers also extend lifespan in C. elegans. Two of these drugs, mitochondrial respiration chain complex inhibitors, interact with the hypoxia pathway to induce fmo-2, whereas two dopamine receptor type 2 (DRD2) antagonists interact with the DR pathway to induce fmo-2, indicating that dopamine signaling is involved in DR-mediated fmo-2 induction. Together, our data identify nine drugs that each (1) increase stress resistance in mouse fibroblasts, (2) induce fmo-2 in C. elegans, and (3) extend nematode lifespan, some through known longevity pathways. These results define fmo-2 induction as a viable approach to identifying and understanding mechanisms of putative longevity compounds.