Characterizing OXPHOS inhibitor-mediated alleviation of hypoxia using high-throughput live cell-imaging.

BACKGROUND: Hypoxia is a common feature of many solid tumors and causes radiotherapy and immunotherapy resistance. Pharmacological inhibition of oxidative phosphorylation (OXPHOS) has emerged as a therapeutic strategy to reduce hypoxia. However, the OXPHOS inhibitors tested in clinical trials caused only moderate responses in hypoxia alleviation or trials were terminated due to dose-limiting toxicities. To improve the therapeutic benefit, FDA approved OXPHOS inhibitors (e.g. atovaquone) were conjugated to triphenylphosphonium (TPP+) to preferentially target cancer cell's mitochondria. In this study, we evaluated the hypoxia reducing effects of several mitochondria-targeted OXPHOS inhibitors and compared them to non-mitochondria-targeted OXPHOS inhibitors using newly developed spheroid models for diffusion-limited hypoxia. METHODS: B16OVA murine melanoma cells and MC38 murine colon cancer cells expressing a HIF-Responsive Element (HRE)-induced Green Fluorescent Protein (GFP) with an oxygen-dependent degradation domain (HRE-eGFP-ODD) were generated to assess diffusion-limited hypoxia dynamics in spheroids. Spheroids were treated with IACS-010759, atovaquone, metformin, tamoxifen or with mitochondria-targeted atovaquone (Mito-ATO), PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) or mitochondria-targeted tamoxifen (MitoTam). Hypoxia dynamics were followed and quantified over time using the IncuCyte Zoom Live Cell-Imaging system. RESULTS: Hypoxic cores developed in B16OVA.HRE and MC38.HRE spheroids within 24 h hours after seeding. Treatment with IACS-010759, metformin, atovaquone, Mito-PEG-ATO and MitoTam showed a dose-dependent reduction of hypoxia in both B16OVA.HRE and MC38.HRE spheroids. Mito-ATO only alleviated hypoxia in MC38.HRE spheroids while tamoxifen was not able to reduce hypoxia in any of the spheroid models. The mitochondria-targeted OXPHOS inhibitors demonstrated stronger anti-hypoxic effects compared to the non-mito-targeted OXPHOS inhibitors. CONCLUSIONS: We successfully developed a high-throughput spheroid model in which hypoxia dynamics can be quantified over time. Using this model, we showed that the mitochondria-targeted OXPHOS inhibitors Mito-ATO, Mito-PEG-ATO and MitoTam reduce hypoxia in tumor cells in a dose-dependent manner, potentially sensitizing hypoxic tumor cells for radiotherapy.

Mitigating gut microbial degradation of levodopa and enhancing brain dopamine: Implications in Parkinson's disease.

Parkinson's disease is managed using levodopa; however, as Parkinson's disease progresses, patients require increased doses of levodopa, which can cause undesirable side effects. Additionally, the oral bioavailability of levodopa decreases in Parkinson's disease patients due to the increased metabolism of levodopa to dopamine by gut bacteria, Enterococcus faecalis, resulting in decreased neuronal uptake and dopamine formation. Parkinson's disease patients have varying levels of these bacteria. Thus, decreasing bacterial metabolism is a promising therapeutic approach to enhance the bioavailability of levodopa in the brain. In this work, we show that Mito-ortho-HNK, formed by modification of a naturally occurring molecule, honokiol, conjugated to a triphenylphosphonium moiety, mitigates the metabolism of levodopa-alone or combined with carbidopa-to dopamine. Mito-ortho-HNK suppresses the growth of E. faecalis, decreases dopamine levels in the gut, and increases dopamine levels in the brain. Mitigating the gut bacterial metabolism of levodopa as shown here could enhance its efficacy.

Mito-metformin protects against mitochondrial dysfunction and dopaminergic neuronal degeneration by activating upstream PKD1 signaling in cell culture and MitoPark animal models of Parkinson's disease.

Impaired mitochondrial function and biogenesis have strongly been implicated in the pathogenesis of Parkinson's disease (PD). Thus, identifying the key signaling mechanisms regulating mitochondrial biogenesis is crucial to developing new treatment strategies for PD. We previously reported that protein kinase D1 (PKD1) activation protects against neuronal cell death in PD models by regulating mitochondrial biogenesis. To further harness the translational drug discovery potential of targeting PKD1-mediated neuroprotective signaling, we synthesized mito-metformin (Mito-Met), a mitochondria-targeted analog derived from conjugating the anti-diabetic drug metformin with a triphenylphosphonium functional group, and then evaluated the preclinical efficacy of Mito-Met in cell culture and MitoPark animal models of PD. Mito-Met (100-300 nM) significantly activated PKD1 phosphorylation, as well as downstream Akt and AMPKα phosphorylation, more potently than metformin, in N27 dopaminergic neuronal cells. Furthermore, treatment with Mito-Met upregulated the mRNA and protein expression of mitochondrial transcription factor A (TFAM) implying that Mito-Met can promote mitochondrial biogenesis. Interestingly, Mito-Met significantly increased mitochondrial bioenergetics capacity in N27 dopaminergic cells. Mito-Met also reduced mitochondrial fragmentation induced by the Parkinsonian neurotoxicant MPP+ in N27 cells and protected against MPP+-induced TH-positive neurite loss in primary neurons. More importantly, Mito-Met treatment (10 mg/kg, oral gavage for 8 week) significantly improved motor deficits and reduced striatal dopamine depletion in MitoPark mice. Taken together, our results demonstrate that Mito-Met possesses profound neuroprotective effects in both in vitro and in vivo models of PD, suggesting that pharmacological activation of PKD1 signaling could be a novel neuroprotective translational strategy in PD and other related neurocognitive diseases.

Gut microbiome, short-chain fatty acids, alpha-synuclein, neuroinflammation, and ROS/RNS: Relevance to Parkinson's disease and therapeutic implications.

In this review, we explore how short-chain fatty acids (SCFAs) produced by the gut microbiome affect Parkinson's disease (PD) through their modulatory interactions with alpha-synuclein, neuroinflammation, and oxidative stress mediated by reactive oxygen and nitrogen species (ROS/RNS). In particular, SCFAs-such as acetate, propionate, and butyrate-are involved in gut-brain communication and can modulate alpha-synuclein aggregation, a hallmark of PD. The gut microbiome of patients with PD has lower levels of SCFAs than healthy individuals. Probiotics may be a potential strategy to restore SCFAs and alleviate PD symptoms, but the underlying mechanisms are not fully understood. Also in this review, we discuss how alpha-synuclein, present in the guts and brains of patients with PD, may induce neuroinflammation and oxidative stress via ROS/RNS. Alpha-synuclein is considered an early biomarker for PD and may link the gut-brain axis to the disease pathogenesis. Therefore, elucidating the role of SCFAs in the gut microbiome and their impact on alpha-synuclein-induced neuroinflammation in microglia and on ROS/RNS is crucial in PD pathogenesis and treatment.

OXPHOS-targeting drugs in oncology: new perspectives.

INTRODUCTION: Drugs targeting mitochondria are emerging as promising antitumor therapeutics in preclinical models. However, a few of these drugs have shown clinical toxicity. Developing mitochondria-targeted modified natural compounds and US FDA-approved drugs with increased therapeutic index in cancer is discussed as an alternative strategy. AREAS COVERED: Triphenylphosphonium cation (TPP+)-based drugs selectively accumulate in the mitochondria of cancer cells due to their increased negative membrane potential, target the oxidative phosphorylation proteins, inhibit mitochondrial respiration, and inhibit tumor proliferation. TPP+-based drugs exert minimal toxic side effects in rodents and humans. These drugs can sensitize radiation and immunotherapies. EXPERT OPINION: TPP+-based drugs targeting the tumor mitochondrial electron transport chain are a new class of oxidative phosphorylation inhibitors with varying antiproliferative and antimetastatic potencies. Some of these TPP+-based agents, which are synthesized from naturally occurring molecules and FDA-approved drugs, have been tested in mice and did not show notable toxicity, including neurotoxicity, when used at doses under the maximally tolerated dose. Thus, more effort should be directed toward the clinical translation of TPP+-based OXPHOS-inhibiting drugs in cancer prevention and treatment.

Mitochondria, which are the cell's powerhouse of energy, are functional in cancer cells. Inhibition of cancer cell respiration is associated with inhibition of cancer cell proliferation. Therefore, mitochondria have become a promising target for developing antitumor drugs to treat cancer. Several classes of drug molecules selectively target cancer cell mitochondria and inhibit mitochondrial respiration or oxidative phosphorylation (OXPHOS). A new class of OXPHOS-targeting drugs is emerging as a potential cancer therapeutic. One of the OXPHOS inhibitor drugs, IACS-010759, developed by investigators at MD Anderson Cancer Center, was tested in patients with acute myeloid leukemia. Patients who were administered the drug developed peripheral neuropathy and other complications (lactic acidosis), resulting in dose reduction. At lower doses, this drug was not effective. Subsequently, the clinical trial was terminated. The investigators then showed the same type of neurotoxicity using a mouse model. These findings were recently published. Thus, there is an urgent need to develop new OXPHOS inhibitors that do not have neurotoxicity in mice or humans.In this opinion article, we make a case that there are other triphenylphosphonium cation (TPP⁺)-based mitochondrial OXPHOS inhibitors (inhibiting both complex I and complex III) that are structural modifications of naturally occurring molecules or US FDA-approved drugs. These mitochondria-targeted drugs (MTDs) are as potent as IACS-010759 in cells and in preclinical models. Several TPP⁺-based MTDs have been tested in mice and did not exert neurotoxicity. TPP⁺-containing MTDs such as mitochondria-targeted coenzyme Q10 (MitoQ) have been tested in patients with Parkinson's disease, with no evidence of peripheral neuropathy or other toxicity (e.g., lactic acidosis). Other US FDA-approved drugs (metformin and atovaquone [ATO] or papaverine) are in clinical trials alone or in combination with other standard-of-care treatments (e.g., radiation therapy). We recommend that TPP⁺-based drugs that have been tested in preclinical models or in humans should undergo clinical trials in patients with cancer.

Redox-crippled MitoQ potently inhibits breast cancer and glioma cell proliferation: A negative control for verifying the antioxidant mechanism of MitoQ in cancer and other oxidative pathologies.

Mitochondria-targeted coenzyme Q10 (Mito-ubiquinone, Mito-quinone mesylate, or MitoQ) was shown to be an effective antimetastatic drug in patients with triple-negative breast cancer. MitoQ, sold as a nutritional supplement, prevents breast cancer recurrence. It potently inhibited tumor growth and tumor cell proliferation in preclinical xenograft models and in vitro breast cancer cells. The proposed mechanism of action involves the inhibition of reactive oxygen species by MitoQ via a redox-cycling mechanism between the oxidized form, MitoQ, and the fully reduced form, MitoQH2 (also called Mito-ubiquinol). To fully corroborate this antioxidant mechanism, we substituted the hydroquinone group (-OH) with the methoxy group (-OCH3). Unlike MitoQ, the modified form, dimethoxy MitoQ (DM-MitoQ), lacks redox-cycling between the quinone and hydroquinone forms. DM-MitoQ was not converted to MitoQ in MDA-MB-231 cells. We tested the antiproliferative effects of both MitoQ and DM-MitoQ in human breast cancer (MDA-MB-231), brain-homing cancer (MDA-MB-231BR), and glioma (U87MG) cells. Surprisingly, DM-MitoQ was slightly more potent than MitoQ (IC50 = 0.26 µM versus 0.38 µM) at inhibiting proliferation of these cells. Both MitoQ and DM-MitoQ potently inhibited mitochondrial complex I-dependent oxygen consumption (IC50 = 0.52 µM and 0.17 µM, respectively). This study also suggests that DM-MitoQ, which is a more hydrophobic analog of MitoQ (logP: 10.1 and 8.7) devoid of antioxidant function and reactive oxygen species scavenging ability, can inhibit cancer cell proliferation. We conclude that inhibition of mitochondrial oxidative phosphorylation by MitoQ is responsible for inhibition of breast cancer and glioma proliferation and metastasis. Blunting the antioxidant effect using the redox-crippled DM-MitoQ can serve as a useful negative control in corroborating the involvement of free radical-mediated processes (e.g., ferroptosis, protein oxidation/nitration) using MitoQ in other oxidative pathologies.

Antiproliferative effects of mitochondria-targeted N-acetylcysteine and analogs in cancer cells.

N-acetylcysteine (NAC) has been used as an antioxidant drug in tumor cells and preclinical mice tumor xenografts, and it improves adaptive immunotherapy in melanoma. NAC is not readily bioavailable and is used in high concentrations. The effects of NAC have been attributed to its antioxidant and redox signaling role in mitochondria. New thiol-containing molecules targeted to mitochondria are needed. Here, mitochondria-targeted NAC with a 10-carbon alkyl side chain attached to a triphenylphosphonium group (Mito10-NAC) that is functionally similar to NAC was synthesized and studied. Mito10-NAC has a free sulfhydryl group and is more hydrophobic than NAC. Mito10-NAC is nearly 2000-fold more effective than NAC in inhibiting several cancer cells, including pancreatic cancer cells. Methylation of NAC and Mito10-NAC also inhibited cancer cell proliferation. Mito10-NAC inhibits mitochondrial complex I-induced respiration and, in combination with monocarboxylate transporter 1 inhibitor, synergistically decreased pancreatic cancer cell proliferation. Results suggest that the antiproliferative effects of NAC and Mito10-NAC are unlikely to be related to their antioxidant mechanism (i.e., scavenging of reactive oxygen species) or to the sulfhydryl group-dependent redox modulatory effects.

Polyphenolic Boronates Inhibit Tumor Cell Proliferation: Potential Mitigators of Oxidants in the Tumor Microenvironment.

Boronate-based compounds have been used in brain cancer therapy, either as prodrugs or in combination with other modalities. Boronates containing pro-luminescent and fluorescent probes have been used in mouse models of cancer. In this study, we synthesized and developed polyphenolic boronates and mitochondria-targeted polyphenolic phytochemicals (e.g., magnolol [MGN] and honokiol [HNK]) and tested their antiproliferative effects in brain cancer cells. Results show that mitochondria-targeted (Mito) polyphenolic boronates (Mito-MGN-B and Mito-HNK-B) were slightly more potent than Mito-MGN and Mito-HNK in inhibiting proliferation of the U87MG cell line. Similar proliferation results also were observed in other cancer cell lines, such as MiaPaCa-2, A549 and UACC-62. Independent in vitro experiments indicated that reactive nitrogen species (e.g., peroxynitrite) and reactive oxygen species (e.g., hydrogen peroxide) stoichiometrically react with polyphenolic boronates and Mito-polphenolic boronates, forming polyphenols and Mito-polyphenols as major products. Previous reports suggest that both Mito-MGN and Mito-HNK activate cytotoxic T cells and inhibit immunosuppressive immune cells. We propose that Mito-polyphenolic boronate-based prodrugs may be used to inhibit tumor proliferation and mitigate oxidant formation in the tumor microenvironment, thereby generating Mito-polyphenols in situ, as well as showing activity in the tumor microenvironment.

NAC, NAC, Knockin' on Heaven's door: Interpreting the mechanism of action of N-acetylcysteine in tumor and immune cells.

N-acetylcysteine (NAC) has been used as a direct scavenger of reactive oxygen species (hydrogen peroxide, in particular) and an antioxidant in cancer biology and immuno-oncology. NAC is the antioxidant drug most frequently employed in studies using tumor cells, immune cells, and preclinical mouse xenografts. Most studies use redox-active fluorescent probes such as dichlorodihydrofluorescein, hydroethidine, mitochondria-targeted hydroethidine, and proprietary kit-based probes (i.e., CellROX Green and CellROX Red) for intracellular detection of superoxide or hydrogen peroxide. Inhibition of fluorescence by NAC was used as a key experimental observation to support the formation of reactive oxygen species and redox mechanisms proposed for ferroptosis, tumor metastasis, and redox signaling in the tumor microenvironment. Reactive oxygen species such as superoxide and hydrogen peroxide stimulate or abrogate tumor cells and immune cells depending on multiple factors. Understanding the mechanism of antioxidants is crucial for interpretation of the results. Because neither NAC nor the fluorescent probes indicated above react directly with hydrogen peroxide, it is critically important to reinterpret the results to advance our understanding of the mechanism of action of NAC and shed additional mechanistic insight on redox-regulated signaling in tumor biology. To this end, this review is focused on how NAC could affect multiple pathways in cancer cells, including iron signaling, ferroptosis, and the glutathione-dependent antioxidant and redox signaling mechanism, and how NAC could inhibit oxidation of the fluorescent probes through multiple mechanisms.

The Chemistry of HNO: Mechanisms and Reaction Kinetics.

Azanone (HNO, also known as nitroxyl) is the protonated form of the product of one-electron reduction of nitric oxide (•NO), and an elusive electrophilic reactive nitrogen species of increasing pharmacological significance. Over the past 20 years, the interest in the biological chemistry of HNO has increased significantly due to the numerous beneficial pharmacological effects of its donors. Increased availability of various HNO donors was accompanied by great progress in the understanding of HNO chemistry and chemical biology. This review is focused on the chemistry of HNO, with emphasis on reaction kinetics and mechanisms in aqueous solutions.

Therapeutic Targeting of Tumor Cells and Tumor Immune Microenvironment Vulnerabilities.

Therapeutic targeting of tumor vulnerabilities is emerging as a key area of research. This review is focused on exploiting the vulnerabilities of tumor cells and the immune cells in the tumor immune microenvironment (TIME), including tumor hypoxia, tumor acidity, the bidirectional proton-coupled monocarboxylate transporters (MCTs) of lactate, mitochondrial oxidative phosphorylation (OXPHOS), and redox enzymes in the tricarboxylic acid cycle. Cancer cells use glucose for energy even under normoxic conditions. Although cancer cells predominantly rely on glycolysis, many have fully functional mitochondria, suggesting that mitochondria are a vulnerable target organelle in cancer cells. Thus, one key distinction between cancer and normal cell metabolism is metabolic reprogramming. Mitochondria-targeted small molecule inhibitors of OXPHOS inhibit tumor proliferation and growth. Another hallmark of cancer is extracellular acidification due lactate accumulation. Emerging results show that lactate acts as a fuel for mitochondrial metabolism and supports tumor proliferation and growth. Metabolic reprogramming occurs in glycolysis-deficient tumor phenotypes and in kinase-targeted, drug-resistant cancers overexpressing OXPHOS genes. Glycolytic cancer cells located away from the vasculature overexpress MCT4 transporter to prevent overacidification by exporting lactate, and the oxidative cancer cells located near the vasculature express MCT1 transporter to provide energy through incorporation of lactate into the tricarboxylic acid cycle. MCTs are, therefore, a vulnerable target in cancer metabolism. MCT inhibitors exert synthetic lethality in combination with metformin, a weak inhibitor of OXPHOS, in cancer cells. Simultaneously targeting multiple vulnerabilities within mitochondria shows synergistic antiproliferative and antitumor effects. Developing tumor-selective, small molecule inhibitors of OXPHOS with a high therapeutic index is critical to fully exploiting the mitochondrial vulnerabilities. We and others developed small-molecule inhibitors containing triphenylphosphonium cation that potently inhibit OXPHOS in tumor cells and tissues. Factors affecting tumor cell vulnerabilities also impact immune cells in the TIME. Glycolytic tumor cells supply lactate to the tumor-suppressing regulatory T cells overexpressing MCTs. Therapeutic opportunities for targeting vulnerabilities in tumor cells and the TIME, as well as the implications on cancer health disparities and cancer treatment, are addressed.

Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo.

Multiple roles of reactive oxygen species (ROS) and their consequences for health and disease are emerging throughout biological sciences. This development has led researchers unfamiliar with the complexities of ROS and their reactions to employ commercial kits and probes to measure ROS and oxidative damage inappropriately, treating ROS (a generic abbreviation) as if it were a discrete molecular entity. Unfortunately, the application and interpretation of these measurements are fraught with challenges and limitations. This can lead to misleading claims entering the literature and impeding progress, despite a well-established body of knowledge on how best to assess individual ROS, their reactions, role as signalling molecules and the oxidative damage that they can cause. In this consensus statement we illuminate problems that can arise with many commonly used approaches for measurement of ROS and oxidative damage, and propose guidelines for best practice. We hope that these strategies will be useful to those who find their research requiring assessment of ROS, oxidative damage and redox signalling in cells and in vivo.

Chemoprevention of Lung Cancer with a Combination of Mitochondria-Targeted Compounds.

Combined treatment targeting mitochondria may improve the efficacy of lung cancer chemoprevention. Here, mitochondria-targeted honokiol (Mito-HNK), an inhibitor of mitochondrial complex I and STAT3 phosphorylation, and mitochondria-targeted lonidamine (Mito-LND), an inhibitor of mitochondrial complexes I/II and AKT/mTOR/p70S6K signaling, were evaluated for their combinational chemopreventive efficacy on mouse lung carcinogenesis. All chemopreventive treatments began one-week post-carcinogen treatment and continued daily for 24 weeks. No evidence of toxicity (including liver toxicity) was detected by monitoring serum levels of alanine aminotransferase and aspartate aminotransferase enzymes. Mito-HNK or Mito-LND treatment alone reduced tumor load by 56% and 48%, respectively, whereas the combination of Mito-HNK and Mito-LND reduced tumor load by 83%. To understand the potential mechanism(s) of action for the observed combinatorial effects, single-cell RNA sequencing was performed using mouse tumors treated with Mito-HNK, Mito-LND, and their combination. In lung tumor cells, Mito-HNK treatment blocked the expression of genes involved in mitochondrial complex ǀ, oxidative phosphorylation, glycolysis, and STAT3 signaling. Mito-LND inhibited the expression of genes for mitochondrial complexes I/II, oxidative phosphorylation, and AKT/mTOR/p70S6K signaling in lung tumor cells. In addition to these changes, a combination of Mito-HNK with Mito-LND decreased arginine and proline metabolism, N-glycan biosynthesis, and tryptophan metabolism in lung tumor cells. Our results demonstrate that Mito-LND enhanced the antitumor efficacy of Mito-HNK, where both compounds inhibited common targets (oxidative phosphorylation) as well as unique targets for each agent (STAT3 and mTOR signaling). Therefore, the combination of Mito-HNK with Mito-LND may present an effective strategy for lung cancer chemoprevention.

Prevention of Tumor Growth and Dissemination by In Situ Vaccination with Mitochondria-Targeted Atovaquone.

Atovaquone, an FDA-approved drug for malaria, is known to inhibit mitochondrial electron transport. A recently synthesized mitochondria-targeted atovaquone increased mitochondrial accumulation and antitumor activity in vitro. Using an in situ vaccination approach, local injection of mitochondria-targeted atovaquone into primary tumors triggered potent T cell immune responses locally and in distant tumor sites. Mitochondria-targeted atovaquone treatment led to significant reductions of both granulocytic myeloid-derived suppressor cells and regulatory T cells in the tumor microenvironment. Mitochondria-targeted atovaquone treatment blocks the expression of genes involved in oxidative phosphorylation and glycolysis in granulocytic-myeloid-derived suppressor cells and regulatory T cells, which may lead to death of granulocytic-myeloid-derived suppressor cells and regulatory T cells. Mitochondria-targeted atovaquone inhibits expression of genes for mitochondrial complex components, oxidative phosphorylation, and glycolysis in both granulocytic-myeloid-derived suppressor cells and regulatory T cells. The resulting decreases in intratumoral granulocytic-myeloid-derived suppressor cells and regulatory T cells could facilitate the observed increase in tumor-infiltrating CD4+ T cells. Mitochondria-targeted atovaquone also improves the anti-tumor activity of PD-1 blockade immunotherapy. The results implicate granulocytic-myeloid-derived suppressor cells and regulatory T cells as novel targets of mitochondria-targeted atovaquone that facilitate its antitumor efficacy.

Exploiting the tumor immune microenvironment and immunometabolism using mitochondria-targeted drugs: Challenges and opportunities in racial disparity and cancer outcome research.

Black and Hispanic cancer patients have a higher incidence of cancer mortality. Many factors (e.g., socioeconomic differences, insufficient access to healthcare) contribute to racial disparity. Emerging research implicates biological disparity in cancer outcomes. Studies show distinct differences in the tumor immune microenvironment (TIME) in Black cancer patients. Studies also have linked altered mitochondrial metabolism to changes in immune cell activation in TIME. Recent publications revealed a novel immunomodulatory role for triphenylphosphonium-based mitochondrial-targeted drugs (MTDs). These are synthetically modified, naturally occurring molecules (e.g., honokiol, magnolol, metformin) or FDA-approved small molecule drugs (e.g., atovaquone, hydroxyurea). Modifications involve conjugating the parent molecule via an alkyl linker chain to a triphenylphosphonium moiety. These modified molecules (e.g., Mito-honokiol, Mito-magnolol, Mito-metformin, Mito-atovaquone, Mito-hydroxyurea) accumulate in tumor cell mitochondria more effectively than in normal cells and inhibit mitochondrial respiration, induce reactive oxygen species, activate AMPK and redox transcription factors, and inhibit cancer cell proliferation. Besides these intrinsic effects of MTDs in redox signaling and proliferation in tumors, MTDs induced extrinsic effects in the TIME of mouse xenografts. MTD treatment inhibited tumor-suppressive immune cells, myeloid-derived suppressor cells, and regulatory T cells, and activated T cells and antitumor immune effects. One key biological disparity in Black cancer patients was related to altered mitochondrial oxidative metabolism; MTDs targeting vulnerabilities in tumor cells and the TIME may help us understand this biological disparity. Clinical trials should include an appropriate number of Black and Hispanic cancer patients and should validate the intratumoral, antihypoxic effects of MTDs with imaging.

Combining PEGylated mito-atovaquone with MCT and Krebs cycle redox inhibitors as a potential strategy to abrogate tumor cell proliferation.

Glycolytic and mitochondrial oxidative metabolism, which are two major energy sources in tumors, are potential targets in cancer treatment. Metabolic reprogramming from glycolysis to mitochondrial oxidative metabolism and vice versa is an adaptive strategy with which tumor cells obtain energy to survive and thrive under the compromised conditions of glycolysis and mitochondrial respiration. Developing highly potent, nontoxic, and tumor-selective oxidative phosphorylation (OXPHOS) inhibitors may help advance therapeutic targeting of mitochondrial drugs in cancer. The FDA-approved antimalarial drug atovaquone (ATO), a mitochondrial complex III inhibitor, was repurposed in cancer treatment. Here, we developed a new class of PEGylated mitochondria-targeted ATO (Mito-(PEG)n-ATO). Depending on the PEGylation chain length (n), Mito-PEG-ATO analogs inhibit both mitochondrial complex I- and complex III-induced oxygen consumption in human pancreatic (MiaPaCa-2) and brain (U87MG) cancer cells. Mito-PEG5-ATO is one of the most potent antiproliferative mitochondria-targeted compounds (IC50 = 38 nM) in MiaPaCa-2 cells, and is more effective than other inhibitors of OXPHOS in MiaPaCa-2 and U87MG cells. Furthermore, we show that the combined use of the most potent OXPHOS-targeted inhibitors (Mito-PEG5-ATO) and inhibitors of monocarboxylate transporters (MCT-1 and MCT-4), Krebs cycle redox metabolism, or glutaminolysis will synergistically abrogate tumor cell proliferation. Potential clinical benefits of these combinatorial therapies are discussed.

Functionalized polyanhydride nanoparticles for improved treatment of mitochondrial dysfunction.

Parkinson's disease (PD) is a devastating neurodegenerative disease affecting a large proportion of older adults. Exposure to pesticides like rotenone is a leading cause for PD. To reduce disease progression and prolong life expectancy, it is important to target disease mechanisms that contribute to dopaminergic neuronal atrophy, including mitochondrial dysfunction. Achieving targeted mitochondrial delivery is difficult for many therapeutics by themselves, necessitating higher therapeutic doses that could lead to toxicity. To minimize this adverse effect, targeted nano-carriers such as polyanhydride nanoparticles (NPs) can protect therapeutics from degradation and provide sustained release, enabling fewer administrations and lower therapeutic dose. This work expands upon the use of the polyanhydride NP platform for targeted drug delivery by functionalizing the polymer with a derivative of triphenylphosphonium called (3-carboxypropyl) triphenylphosphonium (CPTP) using a novel method that enables longer CPTP persistence on the NPs. The extent to which neurons internalized both nonfunctionalized and functionalized NPs was tested. Next, the efficacy of these nanoformulations in treating rotenone-induced mitochondrial dysfunction in the same cell line was evaluated using a novel neuroprotective drug, mito-metformin. CPTP functionalization significantly improved NP internalization by neuronal cells. This was correlated with significant protection by CPTP-functionalized, mito-metformin encapsulated NPs against rotenone-induced mitochondrial dysfunction. However, nonfunctionalized, mito-metformin encapsulated NPs and soluble mito-metformin administered at the same dose did not significantly protect cells from rotenone-induced toxicity. These results indicate that the targeted NP platform can provide enhanced dose-sparing and potentially reduce the occurrence of systemic side-effects for PD therapeutics.

Reactive oxygen species, proinflammatory and immunosuppressive mediators induced in COVID-19: overlapping biology with cancer.

This review analyzes the published literature linking the different mechanisms focused on oxidative stress and inflammation that contribute to COVID-19 disease severity. The objective is to bring together potential proinflammatory mechanisms of COVID-19 pathogenesis and address mitigation strategies using naturally occurring compounds and FDA-approved drugs. Outstanding questions addressed include the following: What is the mechanistic basis for linking enhanced vulnerability in COVID-19 to increased oxidative damage and proinflammatory mediators (e.g., cytokines), especially in high-risk people? Can we repurpose anti-inflammatory and immunomodulatory agents to mitigate inflammation in COVID-19 patients? How does 2-deoxy-d-glucose function as an anti-COVID drug? COVID-19, cancer biology, and immunotherapy share many mechanistic similarities. Repurposing drugs that already have been FDA-approved for mitigating inflammation and immunosuppression in cancer may be a way to counteract disease severity, progression, and chronic inflammation in COVID-19. What are the long-term effects of reactive oxygen species-inducing immune cells and sustained inflammation in so-called long-haulers (long COVID) after recovery from COVID-19? Can we use mitochondria-targeted agents prophylactically to prevent inflammation and boost immunity in long-haulers? Addressing the oxidative chemical biology of COVID-19 and the mechanistic commonalities with cancer may provide new insights potentially leading to appropriate clinical trials and new treatments.

Impaired Mitochondrial Bioenergetics Function in Pediatric Chronic Overlapping Pain Conditions with Functional Gastrointestinal Disorders.

Background: Fatigue is often the primary complaint of children with functional gastrointestinal disorders (FGDI) and other chronic overlapping pain disorders (COPC). The basis for this symptom remains unknown. We evaluated mitochondrial function in the white blood cells of these patients. Methods: This prospective Children's Wisconsin IRB approved study recruited subjects aging 10-18 years from pediatric neurogastroenterology clinics and healthy comparison subjects (HC). Environmental and oxidative stressors can damage the mitochondrial respiratory chain. The known low-grade inflammation in COPC could, therefore, impact the respiratory chain and theoretically account for the disabling fatigue so often voiced by patients. Mitochondrial energy generation can be easily measured in peripheral mononuclear cells (PMC) as a general marker by the Seahorse XF96 Extracellular Flux Analyzer. We measured 5 parameters of oxygen consumption using this methodology: basal respiration (BR), ATP linked oxygen consumption (ATP-LC), maximal oxygen consumption rate (max R), spare respiratory capacity (SRC), and extracellular acidification rate (ECAR), which reflect non-electron chain energy generation through glycolysis. In health, we expect high ATP linked respiration, high reserve capacity, low proton leak, and low non-mitochondrial respiration. In disease, the proton leak typically increases, ATP demand increases, and there is decreased reserve capacity with increased non-mitochondrial respiration. Findings and clinical data were compared to healthy control subjects using a Mann-Whitney test for skewed variables, Fisher's exact test for dichotomous variables, and regression tree for association with functional outcome (functional disability inventory, FDI). Results: 19 HC and 31 COPC showed no statistically significant difference in age. FGID, orthostatic intolerance, migraine, sleep disturbance, and chronic fatigue were present in the majority of COPC subjects. BR, ECAR, and ATP-LC rates were lower in the COPC group. The low BR and ATP-LC suggest that mitochondria are stressed with decreased ability to produce ATP. Tree analysis selected SRC as the best predictor of functional disability: patients with SRC >150 had a greater FDI (more disability) compared to patients with SRC <=150, p-value = 0.021. Conclusion: Subjects with COPC have reduced mitochondrial capacity to produce ATP. Predisposing genetic factors or reversible acquired changes may be responsible. A higher SRC best predicts disability. Since a higher SRC is typically associated with more mitochondrial reserve, the SRC may indicate an underutilized available energy supply related to inactivity, or a "brake" on mitochondrial function. Prospective longitudinal studies can likely discern whether these findings represent deconditioning, true mitochondrial dysfunction, or both.