Role of Reactive Oxygen Species (ROS) in ... - ACS Publications

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Role of Reactive Oxygen Species (ROS) in Therapeutics and Drug Resistance in Cancer and Bacteria Allimuthu T. Dharmaraja* Department of Genetics and Genome Sciences and Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States ABSTRACT: Evading persistent drug resistance in cancer and bacteria is quintessential to restore health in humans, and impels intervention strategies. A distinct property of the cancer phenotype is enhanced glucose metabolism and oxidative stress. Reactive oxygen species (ROS) are metabolic byproducts of aerobic respiration and are responsible for maintaining redox homeostasis in cells. Redox balance and oxidative stress are orchestrated by antioxidant enzymes, reduced thiols and NADP(H) cofactors, which is critical for cancer cells survival and progression. Similarly, Escherichia coli (E. coli) and lifethreatening infectious pathogens such as Staphylococcus aureus (SA) and Mycobacterium tuberculosis (Mtb) are appreciably sensitive to changes in the intracellular oxidative environment. Thus, small molecules that modulate antioxidant levels and/or enhance intracellular ROS could disturb the cellular oxidative environment and induce cell death, and hence could serve as novel therapeutics. Presented here are a collection of approaches that involve ROS modulation in cells as a strategy to target cancer and bacteria.

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Next, hydroxyl radical (OH•), one of the highly reactive ROS, is generated by a metal (Fe(II) or Cu(I)) mediated reduction of H2O2 through the Fenton reaction (Schemes 1 and 2). Hydroxyl radical directly reacts with DNA, which is often irreversible, causing oxidative damage and eventually leading to mutations in the DNA sequence.5,7 The reactive sites in DNA are the sugar backbone and nucleobases. In ribose, C4′ tertiary radical is generated and subsequent uncontrolled reactions lead to DNA degradation or mutations. Among the nucleobases, guanine is a highly reactive base that forms 8oxoguanine during its reaction with OH•. Thymine, another nucleobase, undergoes an addition reaction with OH•, forming thymine radical species and inducing mutations in DNA. Proteins are known to be oxidized by OH•, mainly thiol containing amino acids including methionine, and these residues are oxidized to their corresponding sulfoxides (Scheme 1b). Protein carbonyls are generated by a reaction of OH• with amino acids such as lysine, arginine, proline, and histidine at the carboxylic acid functional group, and oxidation of histidine residues in proteins produces 2-oxo-histidines.6,8 Formation of hypochlorous acid (HOCl) from H2O2 in cells is catalyzed by the enzyme myeloperoxidase (MPO, Scheme 2). This species, HOCl, is reactive toward biomolecules and found to oxidize cysteine residues to cysteine sulfenic acid and tyrosine residues to dityrosines in proteins. HOCl is much more reactive (3 × 107 M−1 s−1) than H2O2 (0.9 M−1 s−1), as observed in oxidizing glutathione. Hydroxyl radical, OH•, is the source of a generation of hydroperoxyl or organoperoxy

INTRODUCTION Chemical genetics is one of the successful strategies widely explored for the identification of drug candidates and their mechanism of action in cells.1 Phenotypic screening of drug candidates exploits the genetic differences in cancerous cells and has identified many successful FDA approved drugs, including afatinib, imatinib, and trastuzumab, and others, such as neratinib, that are in advanced clinical development. However, sustaining the efficacy of marketed drugs has been a challenge due to the prevailing drug resistance in cancer, which often necessitates intervention strategies.2 During aerobic respiration and cellular metabolism, oxygen is converted into water and carbon dioxide, respectively, to produce energy in the form of adenosine triphosphate (ATP) in cells.3 In these processes, oxygen is partially reduced to reactive radical and nonradical oxygen species. In cells, superoxide (O2•−) is generated by 1 e− transfer to O2 either from the electron transport chain, ETC, or by NADPH oxidase (NOX) enzyme. O2•− is known to damage iron−sulfur cluster proteins (Fe−S), which leads to release of Fe(II) into the extracellular matrix and, as an effect, inactivation of the function of Fe−S cluster proteins (Schemes 1a and 2).4,5 This O2•− species undergoes a dismutation to hydrogen peroxide (H2O2) in a buffer or catalyzed by a family of enzymes called superoxide dismutases (SOD, Scheme 2). H2O2 is reactive toward a variety of functional groups in biomolecules; for example, the thiol of the cysteine-containing proteins is oxidized to form sulfenic acids (Scheme 1b). This, in turn, can undergo subsequent oxidations by additional equivalents of H2O2 to form sulfinic and sulfonic acids that could permanently inactivate the protein function.5−8 © 2017 American Chemical Society

Received: August 16, 2016 Published: January 30, 2017 3221

DOI: 10.1021/acs.jmedchem.6b01243 J. Med. Chem. 2017, 60, 3221−3240

Journal of Medicinal Chemistry

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Scheme 1. (A) General Scheme for ROS Production; (B). Damaging Effects of ROS to Biomacromolecules

For example, the pool of O2•− produced in electron transport chain and cellular metabolic events is quenched by the enzyme superoxide dismutase (SOD), where O2•− is converted into H2O2. The subsequent quenching sequence involves another enzyme catalase, which transforms H2O2 to water.5,8 Although a basal level of ROS is crucial for maintaining redox homeostasis and cellular functioning, enhanced ROS levels were implicated in many neurodegenerative disorders, carcinogenesis, and aging.5,10,13 Defining the precise role and safe level of intracellular ROS is challenging due to mixed effects of ROS in cells such as protective roles in pathogenic conditions and deleterious effects to the host cellular components at higher levels. The beneficial level of ROS to cells remains enigmatic; however, a significant interest is spreading across the chemical biology and medicinal chemistry community on ROS based therapeutic strategies. ROS inducing small molecules by mechanisms of (1) inhibiting antioxidant systems, (2) transferring electrons to O2 and producing ROS, or (3) a combination of the abovementioned processes (1 and 2) in cells were reported to selectively kill cancer phenotypes.2 Similarly, ROS based therapeutics are in development as antibacterial agents against drug resistant Gram-negative and Gram-positive bacterial strains. A number of reviews on the chemistry of ROS and their biological roles have been published.2,4,5,10−12,14−18 In this Perspective, an effort has been made to collective literature of some of the ROS-based therapeutic strategies to target cancer and bacteria, and their acquired drug resistance.

Scheme 2. ROS Generation Induced by Cellular Enzymesaa

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ETC: Electron transport chain; NOX: NADPH oxidase; SOD: Superoxide dismutase; MPO: Myeloperoxidase.

radicals, which can damage lipids by oxidation and peroxidation (Scheme 1b).6 These radical and nonradical oxygen species could indiscriminately react with biomacromolecules in cells to induce oxidative damage (Scheme 1b).9 Enhanced ROSmediated oxidative damage builds up stress in cells, appropriately called as oxidative stress. Overall, excess ROS production can induce a plethora of damaging effects to cellular components (Figure 1). Immune cells have directed the

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REDOX HOMEOSTASIS REGULATED BY ENZYMES IN CELLS Oxidative damaging effects of ROS to biomacromolecules, such as DNA, protein, and lipids, through nonspecific rapid reactions placed on ROS the imprint of toxicity to cells. Hence, ROS are showcased as harmful species, and that fact was hyped with the discovery of overexpressed cellular antioxidant enzymes including superoxide dismutase and catalase. Until the 1990s, NADPH oxidase in phagocytes was the only known ROS generator in phagocytic conditions, which is activated in response to growth factors, cytokines, and inflammation.19 Later, many biologically important enzymes were identified that generate ROS during their primary functions.20 A series of 7 enzymes of the NADPH oxidase family were identified as generating ROS in various tissues and not only in phagocytes. Cytosolic NADPH is used as a substrate (electron source), and electrons are transferred to molecular oxygen in the NADPH oxidase catalyzed ROS production.21 Ligand−receptor interaction with NOX mediates ROS generation, and such ligands include platelet derived growth factors, chemokines, and tumor

Figure 1. Schematic representation of ROS generation and their leveldependent effects in cancer and bacteria.

damaging capacity of ROS to microorganisms during pathogenic conditions, where ROS are aberrantly produced as an immune response to combat pathogens.10 Apart from damaging effects to cellular components, ROS are recognized as a key factor in many cellular signaling events, including cell proliferation and survival, which necessitates balance in ROS production and maintenance of redox homeostasis by cellular machinery.6,11 Hence, to attenuate the oxidative stressmediated damage to biomacromolecules and to regulate redox homeostasis, cellular machinery has evolved stringent antioxidant systems, such as antioxidant thiols and enzymes.12 3222



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Figure 2. Maintenance of redox homeostasis in cells by enzymes.

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necrosis factors.22,23 The mitochondrial respiratory chain is one of the major sources of ROS including O2•−, which is then converted into H2O2. During respiration, molecular oxygen is the final electron acceptor in the ETC, where it is reduced to water; however, escape of electrons in the ETC leads to partial reduction of O2 (1−4%) to produce O2•− and H2O2.24 Mammalian targets of rapamycin (mTOR), p53, and B-cell lymphoma 2 (BCL-2) family members are other factors responsible for mitochondrial ROS production.22,25 Xanthine oxidase (XO) is a metalloflavoprotein constitutively expressed in cells. XO converts hypoxanthine and xanthine to urate, and in the process, O2 is reduced into O2•−.26 Cytochrome P450 (CYP) enzymes are flavoenzymes with an iron-containing haem center. CYPs are known for oxygen-dependent metabolism of cholesterol, steroids, and small molecules. Here, the oxygen bound iron haem center in CYPs is known to produce O2•− and H2O2. ROS are also generated during a metabolism of arachidonic acid by lipoxygenases and cyclooxygenases in various cell types (Figure 2).27 Taken together, intracellular ROS is produced by specific enzymes in various parts of cells in a tightly controlled manner for cellular functions. Although a cluster of known enzymes is responsible for intracellular ROS production, until recently, superoxide dismutase and catalase enzymes were the only recognized antioxidant systems in cells for balancing the level of ROS to maintain redox homeostasis. Glutathione (GSH), cysteine (Cys), vitamin C (ascorbic acid), and vitamin E (α-tocopherol) are other nonenzymatic antioxidant molecules that mitigate the excess level of ROS produced in cells. Later, glutathione peroxidase (GPx), thioredoxin reductase, and peroxiredoxin were some of the other antioxidant enzymatic systems uncovered as handling ROS levels in cells.22,28,29 GSH is a low molecular thiol and a major antioxidant found in cells. GSH serves as a substrate for GPx in annihilating lipid peroxidation and is oxidized to disulfide (GSSG), which is reduced back to GSH by another enzyme, glutaredoxin, to restore GSH levels in cells. Disulfide bonds formed by oxidation of thiols in proteins, peptides, and glutathione are either during a reaction with ROS or in the process of attenuating ROS. The reduced forms are efficiently reinstated through reduction of the disulfide bond by thioredoxin reductase systems using NADPH as a cofactor (Figure 2).30 With timely activation of these stringent antioxidant systems, cells are able to tightly regulate ROS levels and maintain redox homeostasis.

ROS PARADOX IN CANCER Mitochondrial genetic mutations have been one of the primary causes of the increase in cellular ROS. Upon mutagenesis in mitochondrial DNA, the electron transport sequence in ETC is trafficked, which leads to accumulation of electrons in the mitochondrial membrane, and a reaction of these electrons with molecular oxygen produces O2•−.31 Diffusible H2O2 can be produced by a dismutation of O2•− by SOD, which can enter into the nucleus and cause DNA damage to induce genetic mutations. These overall genomic instabilities, caused by enhanced ROS levels and oxidative stress, trigger cancer metastasis and progression.32 However, due to the needs of cell proliferation, cancer cells adapt to significantly enhanced levels of intracellular ROS and reach nearly the toxic threshold limit. Balancing the ROS overproduction has been handled by enhanced levels of GSH and other antioxidant enzymes in cells. Thioredoxin reductase-1 (TR-1) is one of the antioxidant selenoproteins that is activated upon oxidative stress response to maintain the reducing environment in cells. Many malignant tumors were characterized by overexpression of TR-1, and the inhibition of TR-1 function was recognized as to abrogate cancer progression. Treatment of cancer using antioxidants was considered as a potential strategy, since ROS is contemplated to promote cancer.33,34 However, clinical trials of treatment of cancers with antioxidants such as N-acetylcysteine, ebselen, edaravone, vitamin A, vitamin C, vitamin E, and β-carotene failed and were found to aggravate cancer progression over long treatment regimens (Figure 3).30 Therefore, ROS-mediated mutations could promote cancer, paradoxically, enhancing intracellular levels of ROS beyond or to the toxic threshold, by using external sources of ROS or by inhibition of an antioxidant system, which could surmount cellular antioxidant defenses and kill cancer cells, hence offering an opportunity to develop ROS based therapeutics for cancer treatment.

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ROS-BASED THERAPEUTICS IN CANCER In cancer cells, growth and proliferation are encouraged with a condition of a modest rise in intracellular ROS; on the other hand, apoptosis is induced at higher levels of ROS. Despite sustaining high levels of intracellular ROS, cancer cells are more sensitive to enhanced intracellular ROS than the nontransformed cells. Thus, utilization of ROS inducing small molecules to target cancer has been considered as a potential strategy. Piperlongumine (PL, 1, Figure 4) is an alkaloid natural product isolated from a pepper plant (piper longum L).35 3223



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Compound 1 was identified as one of the effective anticancer agents that selectively kills cancer cells by enhancing the levels ROS through inhibition cellular machinery responsible for suppressing ROS and oxidative stress in cells.35,36 Cancerselective cytotoxicity induced by 1 was unambiguously characterized in both in vitro and in vivo models. Proteins responsible for modulating oxidative stress in cells, such as glutathione-S-transferase pi 1 (GSTP1) and carbonyl reductase 1 (CBR1), were identified as binding targets of 1 using an unbiased target identification approach, i.e., stable isotope labeled amino acids in cell culture (SILAC) and quantitative proteomics. Additionally, studies on cancer phenotypes and cells engineered with mutations of proteins responsible for suppressing oxidative stress (such as GSTP1 and CBR1) exhibited sensitivity to 1, and reversal in the sensitivity was observed upon cotreatment with antioxidants, thus demonstrating the involvement of redox modulation in cells by 1 for its action.36 Following that, a report on the structure−activity relationships of 1 had predicted a dual function in cells such as ROS generation and a depletion of small molecule cellular antioxidant thiol, GSH.37 Some of the derivatives of 1 were found to aberrantly generate ROS better than 1; however, they were not cytotoxic enough to induce cell death in cancer cells at comparable concentrations of 1. Another molecule, Pl-Fph (2, Figure 4), was identified as highly toxic to cancer cells; however, 2 did not elevate intracellular ROS in HeLa and H1703 cells. An ROS-independent, irreversible glutathionylation was identified as a mechanism of action for the cytotoxicity of 1-like molecules.37 Hence, a mechanism of antioxidant GSH depletion and intracellular ROS induction was proposed for the cytotoxic effects of 1 and its analogs to cancer cells.37 Quinones are one of the major sources of ROS in cells and are known to undergo bioreduction in cells. Subsequent reactions with molecular oxygen could produce ROS. Known quinone based anticancer agents, associated with ROSmediated cell-killing along with other mechanisms of action,

Figure 3. Structures of cellular and synthetic antioxidants that are known to mitigate intracellular ROS.

Figure 4. Structures of pipelongumine (1) and Pl-FPh (2), sites of modification for studying the structural activity relationship of the PL scaffold, and their roles.

Figure 5. Quinone based synthetic and natural products that mediate their anticancer effects, in part through modulation of intracellular ROS. 3224



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Figure 6. Antioxidant targeting, and thiol-reactive and ROS-generating small molecule anticancer agents and other anticancer agents that are used in combination with ROS inducing small molecules.

are mitomycin C (3, DNA alkylation),38 geldanamycin (4, heat shock protein 90 (HSP90) inhibitor),39 streptonigrin (5, topoisomerase inhibitor), doxorubicin (6, DNA intercalator and topoisomerase inhibitor), and mitoxantrone (7, topoisomerase inhibitor) (Figure 5).40 Deoxynyboquinone (DNQ, 8) is a potent quinone based anticancer agent and a natural product analog of SCH 538415 (9, Figure 5).41 Earlier, a mechanism of cytochrome C release was identified for 8 to induce apoptosis in cancer cells.41 Later, Joseph S. Bair et al. developed a synthetic route to 8 and studied the compound’s ability to enhance intracellular ROS in cancer cells and associated mechanisms for cell killing. Compound 8 was found to be highly potent at nanomolar concentrations against a panel of cancer cell lines, including human melanoma (SKMEL-5), breast cancer (MCF-7), leukemia (HL-60), and doxorubicin-resistant leukemia (HL-60/ADR). Experiments performed under hypoxic conditions resulted in a diminished potency of 8, thus proving the importance of oxygen for its action. N-Acetylcysteine, when cotreated with 8 and elesclomol (10, a known, copper-mediated superoxide generator and an ROS-based experimental therapeutic, is in clinical trials)42 independently, has suppressed the potency of both 8 and 10 and hence supports a mechanistic action similar to that of 10 (ROS-mediated cell death). Additional evidence for a ROS based mechanism for 8 was obtained from global transcriptional profiling of U-937 cells treated with 8. A number of

repressed and elevated transcripts responsible for mediating and/or suppressing oxidative stress were identified. Heme oxygenase-1 (HO-1), one of the antioxidant proteins in the Nrf2 (Nuclear factor E2-related factor 2) pathway that is activated upon oxidative stress response in cells, was the topranked transcript identified from their experiment, and heat shock protein-70 (HSP-70) and metallothioneins were other major proteins listed. Further, Western blotting analysis of cells treated with various concentrations of 8 showed a concentration dependent enhancement of HO-1 and HSP-70 levels. An addition of N-acetylcysteine suppressed HO-1 and HSP-70 levels, supporting the mechanism of oxidative stress-mediated pathways as targets for 8. NADP(H) quinone oxidoreductase 1 (NQO1) is a 2-electron reductase, overexpressed in many solid tumors for the detoxification of quinones and reducible xenobiotics, such as nitroaromatics and azo dyes.43 Cancer cells such as breast cancer,44 colon cancer, nonsmall cell lung carcinoma, pancreatic cancer, and ovarian cancer are characterized by overexpressed NQO1 levels.45 Nrf2, a transcription factor, controls the expression levels of NQO1 in cells. Normally, Nrf2 is bound to KEAP1 (Kelch ECH associating protein 1) as an inactivated protein and is released via a covalent modification of KEAP1 in the cytoplasm upon exposure to electrophilic or oxidative stress conditions. One of the downstream effects of enhanced levels of Nrf2 leads to overexpressed NQO1 and inducing carcinogenesis. NQO1 3225



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substrates, such as streptonigrin (5) and β-lapachone (11, Figure 5), were known to efficiently suppress tumors, and 11 was good at initial clinical trials of cancer chemotherapy.46 Under comparable conditions, 8 was established as an excellent NQO1 substrate with better efficiency than the putative NQO1 substrates (5 and 11). A recovery of cell killing was observed for 8 when cotreated with NQO1 inhibitors ES936 (12, Figure 5) and dicoumarol (13, Figure 6) in cell viability assays.47 Upon treatment of 8 with NQO1 expressed cancer cells, large amounts of intracellular ROS were generated, and cotreatment with antioxidants reduced the potency of DNQ, further strengthening an ROS-dependent mechanism of 8 with this phenotype, which is a promising success in redox therapeutics.48 Irisferin A (14) is a quinone based natural product derivative of resveratrol (15, Figure 5), identified as a potent inhibitor of cancer cells, including A549 and HCT116. A study of the structure−activity relationships of 14 (Figure 5) has confirmed the importance of the quinone core for the compound’s activity.49 The quinone core of 14 (irisquinone (16), Figure 5) was found to rapidly enhance intracellular ROS, which resulted in disruption of the mitochondrial membrane and activation of apoptosis.49 Cell viability studies against a panel of cancer cell lines demonstrated a higher inhibitory potency for 14 than that for known ROS based anticancer agent 1.49 Glutathione (GSH) is a reduced thiol-containing tripeptide crucial for the maintenance of cellular redox homeostasis, survival, and proliferation. Cancer cells are equipped with high levels of GSH, which protects cells against oxidative attack. Hence, inhibition of γ-glutamylcysteine synthetase, a GSH biosynthetic enzyme, was considered as a viable target for developing anticancer agents. Buthionine-(S, R)-sulfoximine (BSO, 17 Figure 6), a specific inhibitor of γ-glutamylcysteine synthetase, was found to induce ROS accumulation by GSH biosynthesis inhibition and eventually inhibits the proliferation of cancer cells.50 Since then, 17 has been successfully used for sensitizing cancer cells to many anticancer agents with appreciable enhancement in their selectivity and potency.51,52 Clinically, glioma is treated with a combination of Cisplatin (18) and Temozolomide (19, Figure 6), a DNA alkylating agent.53 However, drug resistance to 18 in glioma cells restricts the use of these drugs. Drug-resistant glioma cells, which are resistant to 18, were shown to have high levels of GSH. Seizing on this opportunity by treating this drug-resistant glioma cells with 17 sensitized these cells to 18 and 19, proving GSH biosynthesis to be a key regulator of cancer cell survival and a worthwhile target to reverse drug resistance.53 The small molecule NPD926 (20, Figure 6), which contains a chloroacetamide functionality, from the library of RIKEN NPDepo, was found to kill a variety of cancer cells, including breast, lung, colon, pancreas, prostate, liver, and leukemia, with IC50 (minimum concentration for 50% growth inhibition) ranging from submicromolar to low micromolar concentrations.54 Further studies on understanding the mechanism of action have revealed an accumulation of intracellular ROS in cancer cells. Compound 20 was also found to be a substrate of GSTP, a pi-class of glutathione S-transferase, which has mediated conjugation of GSH with 20, leading to depletion of intracellular GSH levels. Further, cell death was potentiated by an addition of known GSH biosynthesis inhibitors 15 and erastin (21).54 Human colorectal cancer has been characterized with activated KRAS and BRAF mutations with upregulated levels

of GLUT1 (glucose transporter 1).55 GLUT1 is involved in vitamin C transport across the cell membrane, where oxidized vitamin C, dehydroascorbate (DHA, 22, Figure 6), is transported by GLUT1 and GLUT3; then, 22 is reduced intracellularly by a system consisting of thioredoxin, glutathione, and NADPH cofactor.56,57 GLUT1 overexpression was found to enhance the uptake of 22 selectively in oncogene mutant cells. Increased uptake of 22 leads to depletion of GSH and NADPH, as they are involved in reducing 22 to vitamin C. Disturbance in the level of intracellular GSH leads to accumulation of ROS. Enhancement of ROS upon treatment with vitamin C leads to inhibition of glyceraldehyde 3phosphate dehydrogenase (GADPH) by oxidizing the catalytic cysteine residue, activation of poly ADP-ribose polymerase (PARP), and DNA damage; all these downstream effects collectively enhance the cytotoxic effects of vitamin C to colorectal cancers.9 Hence, compounds that deplete GSH and reduce cellular components could induce ROS accumulation and eventually become toxic to cancer cells. Along with GSH depletion, a simultaneous ROS generation could multiply the effects of oxidative stress in cancer cells. In this respect, a dual stimuli responsive hybrid of cinnamaldehyde (23, an ROS generator, Figure 6) and quinone methide (GSH depleting agent) protected with boronate ester, QCA (24, Figure 6) was reported.58 This hybrid, 24, was indeed found to deplete cellular glutathione and enhance intracellular ROS, and this was found to activate apoptosis-inducing signals like caspase activation to initiate apoptosis. Simultaneous GSH depletion and ROS production have enhanced the vulnerability of cancer cells to oxidative attack. In mouse xenograft models, this hybrid, 24, showed promising results by decreasing the tumor size and demonstrated a synergistic antitumor activity with camptothecin (25, Figure 6).58 The naphthoquinone epoxide core is found in many natural products and biologically active compounds; for example, Vitamin K3 epoxide (26, Figure 5) was found to enhance intracellular ROS in human glioma cells, which induced oxidative stress and cell death.59 These epoxides are known to react with biologically relevant nucleophiles such as reduced thiols (GSH and cysteine) and produce dihydroquinone-adduct. The resulting dihydroquinone-adduct can undergo tautomerism to produce hydroquinone, which could react with molecular oxygen to produce O2•− and other ROS. A library of naphthoquinone epoxides was synthesized and characterized for the compounds ability to react with GSH to generate ROS. Steric and electronic effects of substituents on naphthoquinone epoxide suggested that the electron withdrawing group (NNQEp, 27, Figure 5) has enhanced the reactivity of epoxide with GSH and dictated their ROS generation potential.60 Thiol depletion and ROS generation together have nearly correlated with the potency of the compounds to inhibit human leukemia (THP1) cell proliferation.60 Thus, depletion of intracellular antioxidant thiols and subsequent ROS generation using organic small molecules proves to have the potential to inhibit cancer cell proliferation. Antioxidant enzymatic systems in cells are vital for cell survival and proliferation, and they are upregulated in cancerous cells to combat ROS overproduction. Hence, suppressing or inhibiting antioxidant enzymes could result in amplified intracellular ROS levels and oxidative stress mediated biomolecules damage, which eventually leads to cell death. The thioredoxin (Trx) and thioredoxin reductase (TrxR) system is one of the crucial compartments in mammalian cells for 3226



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alleviating lipid hydroperoxides in cells. Moreover, GPX4 uses GSH as the main cofactor for its function. Hence, both GSH depletion by 21 and GPX4 inhibition by 32 could lead to overproduction of ROS and lipid peroxidation products, which eventually induces ferroptotic cell death.73−76 This discovery is exciting in terms of identifying small molecule inhibitors of GPX4, and GSH depleting molecules to augment intracellular ROS together could offer selective toxicity to cancer cells. A high-throughput screening of a library of small molecules has identified many highly efficient enhancers of ROS (BRD5459 (33), BRD56491 (34),77 and BRD9092 (35)); however, they were found nontoxic to osteosarcoma cancer cells (Figure 6). Therefore, ROS induction itself may not be sufficient to induce cell death in cancer; however, upon cotreatment with 17, a glutathione biosynthesis inhibitor potentiated GSH depletion further and sensitized the cancer cells to ROS enhancers, suggesting the potential utility of these compounds in combination therapies.77 Due to limited toxicity and dose levels of drug molecules, this combination regimen is promising and being explored. Combination therapy could reduce adverse effects and drug resistance; hence, a significant number of combination trials are being performed in the area of oncology.78 Compound 1 was shown to potentiate cancer cell death along with tumor necrosis factor-related apoptosisinducing ligand (TRAIL), a cytokine overexpressed in immune cells and neutrophils, and is in a clinical trial for cancer treatment. Safingol (36, Figure 6) is a sphingolipid protein kinase inhibitor and induces autophagy and necrosis.79 Investigation of a combination of 18 and 36 is in a clinical trial for the treatment of later stage solid tumors.79 ROSmediated cell-killing was identified as a mechanism for 36 when cotreated with drugs including doxorubicin (6), gemcitabine (37, Figure 6), and vincristine (38) (Figure 6).80 Apart from a natural product mimic and electrophilic and quinone based organic small molecules, multiple strategies are being developed to generate ROS in cells to study their chemotherapeutic actions, including that of a hematoporphyrin photosensitizer, that generates intracellular ROS upon exposure to light.81 A radical-mediated DNA damage by radiation therapy is another area advancing in combination therapy with novel drug molecules, which can significantly reduce the risk of chemoand radiotoxicity.82−85 Photodynamic therapy (PDT) is a noninvasive photoregulated technology that is successfully used in treating solid tumors. The major mechanism by which PDT operates is through the generation of reactive radical species, such as singlet oxygen and other ROS, which could damage DNA and other important biomolecules.82,86 As a combination of chemo- and photoregulated therapeutics, a PEGylated polyelectrolyte photosensitizer conjugated with doxorubicin prodrug, which could be activated by ROS for intracellular ROS generation by irradiation, was reported. This conjugate can be self-activated upon irradiation and release 6 on demand. This was found to greatly improve the therapeutic index of 6, hence proving a viable strategy to reduce the off-target effects mediated by 6.87 However, the challenge with conventional PDT is the limited tissue penetration of visible light, which makes PDT an unsuitable technology for the treatment of deep tissue tumors such as hypoxia cancer. A development in overcoming this difficulty is upconversion nanoparticle (UNCPs) phototransducers. These are lanthanide coated nanoparticles which can convert low energy near-infrared (NIR) radiation into high energy UV light.88 Based on this technology, a NaYF4 coated UNCPs, with merocyanine 450

maintaining intracellular pathways involving redox homeostasis. NADPH-dependent disulfide reduction of Trx by TrxR leads to dithiol reduced Trx, which is involved in many redox reactions in protein disulfides responsible for events of cell proliferation, differentiation, and cell death.61,62 Trx-TrxR inhibition was considered one of the attractive goals in cancer chemotherapy because many malignant tumors are characterized with multiple-fold higher levels of Trx-TrxR and their active function is crucial for cancer cell survival, proliferation, and drug resistance. In this respect, a number of competitive, noncompetitive, reversible, and irreversible inhibitors of the Trx system were developed.61,62 Px-12 (1-methylpropyl 2-imidazolyl disulfide, 28, Figure 6) is in clinical trials and is an irreversible Trx inhibitor.63 A covalent modification of the noncatalytic cysteine residue is believed to be the mechanism of action of Trx inhibition by 28.63 Another TrxR inhibitor in preclinical trials is ethaselen (29, Figure 6), an organoselenium compound which selectively binds to the selenocysteinecysteine redox pair in TrxR to impart inhibitory activity.64 Compound 29 was found to suppress the tumor growth significantly on human lung cancer cell lines.64 Although a number of Trx inhibitors are available, novel, highly selective, and effective Trx inhibitors are still in pursuit. Parthenolide (30, Figure 6) is a plant natural product with sesquiterpene lactone and an epoxide ring.65 Compound 30 was found to be a highly potent anticancer agent, and the compound’s activity was translated by activation of p53 and modulation of NFkB pathways and BCl2 members, including augmented intracellular ROS. Recently, a molecular mechanism of TrxR inhibition and subsequent ROS generation in cancer cells by 30 was identified.65 H2O2, an ROS and also a precursor for hydroxyl radical formation in cells, is eradicated by antioxidant peroxiredoxin enzyme, PrxI. Cancer cells survival and resistance to chemo- and radiotherapy were augmented by elevated levels of PrxI in many cancer cells,66−71 hence posing as an ideal drug target for anticancer therapeutics. AMRI-59 (31, Figure 6) was identified as a potent inhibitor of PrxI from a high throughput screening of 25000 molecules.72 The hit molecule was found to enhance intracellular accumulation of ROS to activate apoptosis-inducing pathways such as ASK1-JNK/p38 MAPKs and mitochondria-mediated apoptosis, preferentially in cancer cells.72 These approaches encourage the development of novel strategies to target TrxR and PrxI antioxidant systems for cancer therapeutics. Recently, a nonapoptotic regulated cell death induced upon accumulation of ROS, iron, and lipid peroxides through the loss of glutathione peroxidase 4 (GPX4), a lipid peroxide reducing enzyme, is reported as ferroptosis. Erastin (21, Figure 6) and Ras-selective lethal small molecule RSL3 (32, Figure 6) were reported to induce ferroptotic cell death.73,74 Detailed experimental observations have led to the conclusion that the cell death is neither apoptotic nor necrotic but is iron dependent. Compound 21 was found to generate ROS in sensitive cancer cell lines, and there was a significant reduction in the activity of 21 upon treatment with iron chelators, thus confirming the importance of iron for its activity. Further mechanistic studies revealed that depleted GSH levels were observed upon treatment with 21, and the addition of GSH biosynthesis inhibitor BSO, 17, has augmented the induction of ferroptosis. However, 32 does not deplete the GSH levels in cells upon treatment, and the mechanistic target was identified as glutathione peroxidase 4 (GPX4) enzyme, an oxidative lipid damage repair system and the only enzyme responsible for 3227



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to counteract oxidative damages. Abrogation of Nrf2 levels was found to reverse the oxaliplatin (42) resistance in gastric carcinoma by reducing the levels of DDHs.107 Thus, utilization of intracellular ROS generating small molecules might be an alternative to Nrf2 gene knockouts to reverse drug resistance in cancer. Although the effect of ROS-based drugs in regulating the drug resistance is mysterious, cotreatment of anticancer agents with molecules that mediate their effects through a ROS based mechanism has significantly sensitized the cells to drugs by reversing the resistance.51,53,78,97−100,108−110 Hence, strategies to develop ROS inducing small molecules could be a major helping hand in treating cancers and drug resistance in future chemotherapeutics.

and zinc-phthalocyanine photosensitizers, were synthesized and found to be successful in treating deep tissue tumors with improved therapeutic index.89 Subsequently, a thulium (Tm3+) doped triphenylphosphine functionalized on the surface of TiO2 coated UNCPs was developed for a delivery aimed at mitochondria. This was found to activate TiO2 by emitting UV light with 980 nm laser excitation to produce reactive oxygen species, in particular, O2•−.90,91 Thus, advancement in PDT along the line of ROS-based methodologies has shown promise in targeting cancers. Copper-based metal complexes are efficient cancer chemotherapeutics entering into phase I clinical trials, and they function through radical mediated ROS production and DNA damage.92−95 Gentamycin (39, Figure 9) is an aminoglycoside antibiotic used in treating Gram-negative bacterial infections. This was found to enhance intracellular ROS during its action in eukaryotes. The property was used to sensitize NCI-H460 lung cancer cell lines via a ROS based mechanism to anticancer agents such as camptothecin (25), vinblastine (40), and digitoxin (41).96 Taken together, small molecules that induce intracellular ROS by modulating enzymes involved in redox homeostasis and electrophilic molecules capable of altering the redox equilibrium are making an important contribution to the development of chemotherapeutics. These ROS generating compounds are attractive due to their synergistic behavior and potential as an adjuvant to sensitize cancer cells to other drugs when used in combination. Nanocarriers and nanoparticles utilized in photodynamic therapy, in combination with chemotherapeutics acting through ROS generation are appealing due to their enhanced efficiency and reduced offtarget effects. These intriguing results create a lot of interest in the area of ROS-based chemotherapeutics development.

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DAMAGING EFFECTS OF ROS AND DEFENSE ANTIOXIDANT SYSTEMS IN BACTERIA Aerobic organisms are continuously exposed to an oxidative environment and are prone to undergo oxidative damage by ROS. As an evolution, cells have developed a strong antioxidant response to these ROS, mainly by small molecule cellular thiols such as cysteine (Cys) and glutathione (GSH).12 These reduced thiols are important for the maintenance of cellular redox homeostasis, which works through neutralization of ROS. During the redox homeostasis, reduced thiols such as GSH undergo a dimerization to form a glutathione dimer (GS-SG), which is reduced in the cells by reducing enzymes such as glutathione reductase. Apart from small molecule reduced thiols, antioxidant proteins make a significant contribution to keeping up the cellular redox state. Among the bacterial species, E. coli is one of the well-studied models of bacteria through genetic engineering mutations, in their evolution to microenvironmental changes. Also, E. coli is one of the mechanistically well studied bacterial species for their sensitivity to enhanced ROS levels and diminished antioxidant capacity. Due to the spin restrictions, an addition of electrons in triplet oxygen would lead to a formation of one electron reduced species O2•− as the first ROS from O2.4,111 A family of metal cofactored superoxide dismutase is found in cells as antioxidant systems to control the level of O2•− produced in cells (Scheme 3). Some examples are manganese

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DRUG RESISTANCE IN CANCER Although new drugs and novel mechanisms for treating cancer are on the rise, one of the major concerns in cancer therapeutics is the development of multidrug resistance in cancer cells to various commercially used chemotherapeutics.97 Cancers have become resistant by various mechanisms, including altered drug targets, activated drug efflux pumps, suppressing apoptosis inducing factors, and enhanced DNA repairs.98−100 At low concentrations, ROS act as a signaling molecule in activating apoptosis and regulate the Pglucoprotein (P-gp), a drug efflux pump coded by multidrug resistance genes.100,101 Overexpressed levels of P-gp were found at low concentrations of H2O2 (1 μM) treatment; however, high concentrations of H2O2 (10 μM) treatment have downregulated the expression of P-gp in cells,102 hence producing a concentration dependent contribution of ROS in P-gp mediated drug resistance.103 Dihydrodiol dehydratases (DDHs) come under the class of the aldo-keto reductase family of enzymes, which functions in converting polycyclic aromatic hydrocarbons into the corresponding ortho-quinones, and ROS is produced as a byproduct.104 DDHs regulate the level of intracellular ROS and contribute to an enhanced cancer chemoresistance. Suppression of intracellular ROS by overexpressed DDHs was found to increase the drug resistance in ovarian cancer to 18.105,106 Conversely, the ROS level was found to be elevated upon DDHs knockout by siRNA and lead to downsizing the drug resistance to 18.106 Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the antioxidant system, activated upon ROS-mediated stress in cells

Scheme 3. Antioxidant System Maintaining ROS Homeostasisa

a

ETC: Electron transport chain; NOX: NADPH oxidase; SOD: Superoxide dismutase; Cat: Catalase; GPO: Glutathione peroxidase; MPO: Myeloperoxidase; GSH: Glutathione; AA: Amino acids.

superoxide dismutase (MnSOD), copper superoxide dismutase (CuSOD), and iron superoxide dismutase (FeSOD). These SODs are found to quench O2•− by dismutation into H2O2 and O2 (Scheme 3). Next, H2O2 is converted to O2 and water, mainly by catalase and glutathione peroxidase enzymes.12 Sensor proteins such as OxyR, hydroperoxidase I (KatG), and peroxiredoxin (AhpC) are expressed to control the level of 3228



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by eliminating oneself.116 ROS and reactive nitrogen species117 are generated immediately after the recognition of any invading foreign bodies such as viruses and bacteria (Scheme 4). NOX

H2O2 generated in cells. OxyR is an enzyme activated upon exposure to H2O2 via oxidation of a cysteine-199 residue in OxyR and induces more than 20 antioxidants to scavenge H2O2.112,113 Other antioxidants KatG and AhpC were also found to act through oxidation of the cysteine residues.12 Upon detection of any excess H2O2 production in cells, these enzymes activate an array of antioxidant systems to control the intracellular H2O2 levels (Scheme 3). Further, H2O2 can react with a trace amount of metal ions to generate OH•. Reduced thiols, such as cysteine and glutathione, are present in excess in cells to trap the OH• to protect the cellular components from the oxidative damaging effects (Scheme 3).12 Like GSH in eukaryotes, bacteria are evolved with low molecular weight thiol as protecting agents during electrophilic and oxidative stress conditions. Mycothiol (MSH), composed of a linker of Nacetylcysteine-amide bonded to a D-glucosamine α-myoinositol, is the major antioxidant low molecular weight thiol in actinomycetes.114 Bacilithiol (BSH) is the primary low molecular weight thiol along with coenzyme A (Figure 7) in

Scheme 4. Phagocytosis of Bacteria during Immune Response in Cells

proteins are the main source inducing ROS production in macrophages for phagocytic killing of invading pathogens. Nitric oxide synthase118 is an enzyme responsible for generating nitric oxide (NO) during the immune response, where Larginine is converted into citrulline for the production of NO by NOS catalysis.119,120 Mycobacterium tuberculosis (Mtb) causes human tuberculosis, an ancient disease, and is one of the major leading causes of infectious deaths globally.121,122 During phagocytosis, Mtb evades the host defense mechanisms to prolong their existence in humans. Activation of proinflammatory cytokines by ROS in macrophages during phagocytosis is important in eliminating the bacteria. Mtb has found ways to downregulate the inflammatory response and ROS produced in macrophages.123−125 Indefinite persistence is already a challenge posed by Mtb to humans. In addition, development of multidrug and extensive-drug resistance in Mtb is a mountain to climb in terms of clearing out this pathogen from human life. Staphylococcus aureus (SA) is a nosocomial pathogen posing a serious threat to human health by evolving resistance to all commercially used antibiotics, including methicillin (43) (MRSA) and vancomycin (44) (VRSA) (Figure 9).126 Colonization of SA is promoted through the interaction of many virulence factors expressed by SA with fibronectin, collagen, laminin, vitronectin, and thrombospondin.127 Other factors that SA deploys in combating host defense include staphylokinase, protease, nucleases, lipases, and hyaluronate lyase expression in SA (promote tissue invasion), and secretion of coagulase, protein A, fatty acid metabolizing enzymes, and leucocidin.128 S. aureus produces a number of virulence factors which help to evade phagocytosis in macrophages. Phagocytosis is a process initiated for bacterial elimination, but virulence factors drive an inevitable bacterial dissemination upon lysis of phagosomes. These evolutions of sophisticated antioxidant and repair systems kept oxidative damage in check in microorganisms. ROS is an integral component of host defense response, and during situations where oxidative stress can be overcome, infection results. Hence, novel strategies to eliminate these human health challenging pathogens are highly encouraged; due to the capacity of ROS to severely damage cellular components at elevated levels, exposing bacteria to ROS using reliable ROS sources might be effective against bacterial infections.

Figure 7. Structures of antioxidant thiols in bacteria.

Gram-positive bacteria such as bacillus and staphylococcus. Here, the structure of BSH constitutes N-acetylcysteine-Dglucosamine and L-malate, resembling the structure of MSH just with a difference in the linker of myoinositol in MSH by Lmalate (Figure 7).115

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ROS DURING IMMUNE RESPONSE Due to the aforementioned damaging effects of ROS in biomolecules, under pathogenic conditions, ROS are generated deliberately by the immune cells including macrophages and neutrophils.5,12,14 The immune system can be divided into two types: (1) innate immune system, formed by multicellular organisms and (2) acquired immune system, mostly found in vertebrates. T-cells and B-cells play important roles in acquired immunity by the antigen−antibody reaction, whereas the innate immune system contains macrophages, neutrophils, and dendritic cells, which are primary prophylactic systems working

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ENHANCEMENT OF ROS Mimicking biological systems and enhancing the level of ROS in cells by systems biology or chemical approaches have been considered as potential strategies to target bacterial infections. ROS is produced invariably in all aerobic organisms; however, they have evolved antioxidant systems to counteract oxidative stress.12 In addition, bacteria evolved stringent virulence systems could combat the host protective mechanisms for 3229



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their survival and dissemination. Therefore, enhancing the level of endogenous ROS in support of macrophage-mediated host defense could be lethal by affecting bacterial redox homeostasis.129 Either impairing antioxidant systems or enhancing the endogenous levels of ROS in bacteria could be useful strategies to increase oxidative damage for bacterial growth inhibition. Impairing Antioxidant Systems. Bacteria with impaired antioxidant systems are more sensitive to ROS and antibiotics. In E. coli, mutations were induced to deplete SOD levels on the genes of antioxidant MnSOD and FeSOD enzymes. These SOD-deficient mutants were found to be highly sensitive when subjected to treatment with paraquat (45, Figure 10), a O2•− generating small organic molecule, during a reaction with oxygen.130 Conversely, when the bacteria were cultured in anaerobic conditions, no effect on the growth was observed, thus proving a vital role for SOD in protecting bacteria against enhanced oxidative attack by ROS.130 Catalase is an enzyme expressed in cells as a major antioxidant for the scavenging of H2O2. A detailed study of the effect of H2O2 on the growth of catalase deficient mutants of E. coli was reported. When compared with wild-type E. coli bacterium, catalase null mutants were identified to be 50−60-fold more sensitive to H2O2.131 Catalase is one of the most important antioxidant systems present in cells for the protection from harmful effects of H2O2.131 RecA is a gene expressed as a response to DNA damage in cells. RecA mutants of E. coli were found to be 6−7fold more sensitive to H2O2 than the wild type E. coli.131 Not just Gram-negative bacteria but also Staphylococcus aureus, a Gram-positive bacterium, has been shown to be sensitive to ROS when its antioxidant system is impaired. S. aureus is an infective pathogen associated with community and hospital-acquired skin infections, which has been known to affect around a million people each year. Calcium binding protein, calprotectin, is a major cytoplasmic protein in neutrophils and functions as a signaling agent during inflammation.132,133 It has been reported that depletion of manganese (Mn2+), a trace metal ion found in bacterial antioxidant superoxide dismutase (Mn-SOD)134 and Zinc (Zn2+) through chelation by calprotectin in SA, could alleviate the bacterial growth. Equivalently, calprotectin deficient mice were diagnosed with heavy bacterial infection, inflammation, and high Mn2+ levels in comparison to wild-type mice.42 Therefore, depletion of nutrient metal ions, which are important for antioxidant enzymes function in SA, could sensitize bacteria to immune response and elimination by phagocytosis. Carotenoids serve as antioxidants in SA, and are important for survival and developing resistance to neutrophil-mediated killing by the host immune system.135−138 Inhibition of carotenoid biosynthesis has been found to sensitize the pathogen to oxidants mediated killing when compared to wild-type SA.135 Staphyloxanthin is a pigment found in S. aureus, and it is an important virulence factor identified to promote resistance against ROS and the host-immune system (Figure 8). Staphyloxanthin (46) quenches ROS such as O2•−, H2O2, and HOCl generated by the innate immune systems.139 Thus, inhibition of biosynthesis of 46 could increase the levels of endogenous ROS that could be lethal to the bacterium. As the biosynthesis of 46 is a close resemblance to cholesterol biosynthesis, a series of cholesterol biosynthetic inhibitors were evaluated for the inhibition of 46. BPH-652 (47) was found to inhibit 46 at nanomolar concentrations (inhibitory concentration, Ki = 1.5 nm). An increased susceptibility of S. aureus

Figure 8. Structures of 44 and its inhibitor 45.

was observed when 47 was cotreated with H2O2 (Figure 8).139 Thus, impairing the antioxidant system in Gram-positive and Gram-negative bacteria was found to sensitize bacteria to ROS and antibiotics. Enhancement of Endogenous ROS Levels. Another way of interfering or even inhibiting bacterial growth is by increasing endogenous ROS, thereby increasing the oxidative damage. Flavins and flavoproteins in their reduced forms react with molecular O2 to produce ROS such as O2•− and H2O2 by a sequential 1e− reduction process (Scheme 5).140 Flavoproteins such as flavodoxin, flavoredoxin, flavin dehydrogenase (acyl-CoA dehydrogenase) DT-diaphorase, and flavin monooxygenase are known to react with molecular oxygen to generate ROS.140 Each enzyme has a different rate of reactivity with oxygen for ROS generation. The stability of the flavin radical determines the type of ROS generated. Unstable radical species react faster with oxygen, and O2•− is found to be the major ROS generated, whereas more stable flavin radical is found to generate H2O2 as a major ROS.141 In E. coli, NADH is another cofactor undergoing oxidation to generate ROS in cells catalyzed by NADH-oxidase enzyme. An emerging research field in biomedical engineering is systems biology, where complex interactions of cell components and their functions are studied. The systems biologybased approach for studying complex enzymatic interactions and the metabolic products has led to advances in biotechnology and microbiology.142 Using this approach, genome-scale metabolic models were created for predicting the increase in intracellular ROS production in bacteria. In this metabolic pathway, 133 reactions that involve ROS generation were identified and, in addition, 266 ROS producing reactions were also found. For example, SOD for H2O2 production was included for the in-silico analysis. Specific gene deletion analyses provided information about pathways involving ROS enhancement in the bacterium. To validate the findings experimentally, genetic mutations (incorporation or deletion of 21 identified genes) were successfully carried out and the analysis of metabolic byproduct showed a predictable enhancement of ROS in E. coli. Next, bacterial mutants deficient of antioxidants were found to be highly sensitive when treated with oxidants such as H2O2, sodium hypochlorite (NaOCl), and Menadione (48, Figure 10), whereas the wild-type bacterium E. coli was resistant.129 A significantly enhanced sensitivity of mutants to H2O2 suggested the utility of enhancement of ROS as a method to target bacterial infections.142 Traditional antibiotics have largely independent mechanisms for their bactericidal activities, and if bacteria develop resistance to one antibiotic, they typically remain sensitive to others. For example, β-lactam antibiotics (such as ampicillin (49, Figure 9), 3230



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Scheme 5. ROS Generation from Oxidation of Flavins during Reaction with O2

Figure 9. Quinolone, β-lactam, aminoglycoside, and glycopeptide antibiotics.

Figure 10. Structures of antibacterial agents that induce intracellular ROS enhancement and oxidative stress in bacteria.

along with their individual targets for bactericidal activity.144 Although this concept was disputed by a few independent reports,118,145,146 subsequently, several reports have provided evidence and reiterated the contribution of ROS and oxidative stress in antibiotic-mediated bacterial cell killing.147−151 Induction of redox stress upon treatment with antibiotics was confirmed using multiple approaches, including intracellular H2O2 sensors and a number of ROS-specific fluorescent

amoxicillin (50, Figure 9), etc.) are known to inhibit bacterial cell wall biosynthesis; quinolone antibiotics (norfloxacin (51, Figure 9), ciprofloxacin (52, Figure 9), etc.) are DNA gyrase inhibitors; and, amino acid biosynthesis in bacteria is inhibited by aminoglycoside antibiotics (Kanamycin (53, Figure 9), gentamycin (39), etc.) (Figure 9).143 However, a common mechanism of bactericidal activity for traditional antibiotics was proposed as an enhancement intracellular ROS in bacteria 3231



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probes.148 Further, metabolic profiling of antibiotic-treated E. coli has shown accumulation of multiple metabolites indicative of oxidative stress build up and biomolecules damage.149 In addition to the therapeutic role, ROS were used as an adjuvant to potentiate the antibacterial activity of the commercial antibiotics.142 Therefore, the involvement of ROS in antibiotics mediated bacterial cell killing is established with ample evidence. Bacteria are effectively killed by antibiotics; however, they have evolved drug-resistance by altering their respective drug targets during prolonged exposure to a sublethal level of antibiotics.152,153 When E. coli was treated with a nontoxic dose of 49, the bacteria not only became resistant to 49 but also were found resistant to 51 and 53. A mechanism of mutations in multiple genes induced by radical generation upon treatment with the sublethal level of antibiotics has been annotated for multidrug resistance in bacteria.152,153 To understand the role of ROS in E. coli, a design of a nitroreductase activated ROS generator (NTR-ROS, 54, Figure 10) capable of generating ROS intracellularly was reported.154 A detailed investigation has confirmed the generation of ROS only inside bacteria upon activation by nitroreductase, which is a suitable scaffold for testing the effects of enhancement of intracellular ROS. ROS generated by this scaffold in E. coli itself were found to be not toxic enough to kill E. coli at elevated concentrations; however, it could serve as an adjuvant to aminoglycoside antibiotics such 39 and 53.154 Thus, enhancing intracellular ROS can serve as an adjuvant to commercial antibiotics to alleviate bacterial infection. Using the systems biology approach, the toxicity of OH• generated from silver ion in silver nitrate (AgNO3, 55) to E. coli was studied.155 Generation of OH• in Ag+ treated bacteria was detected using a hydroxyphenyl fluorescein (HPF) based fluorescence assay, and was further confirmed by quenching of this fluorescence using thiourea, a standard quencher of OH•.155 A series of mutants such as SOD-A, an O2•− overproducing mutant and iron import gene mutant, were incubated with Ag+, and the growth profile was monitored. A significant inhibition of bacterial growth suggested the utility of ROS as antibacterial agents. In addition, Ag+ was also found to enhance the killing efficacy of the bacterial antibiotics in both in vitro and in vivo models.155 Fosfomycin (56, Figure 10), a small molecule epoxide, is an FDA approved drug commercially used for treating urinary tract infections.156 The mechanism of action of 56 is through irreversible inhibition of a peptidoglycan biosynthetic enzyme, pyruvyl transferase, MurA.156 Compound 56 was found to be highly potent in inhibiting bacterial growth, including methicillin-resistant Staphylococcus aureus (MRSA) in in vitro and in vivo models, where it acts synergistically with phagocytes in killing MRSA by enhancing intracellular ROS. In particular, 56 induced hydroxyl radical production in SA to efficiently inhibit the growth of MRSA.157 Compound 56, also used in combination with many commercially used antibiotics, successfully treats patients with severe MRSA infections.158,159 However, thiol-reactive electrophilic molecule antibiotics, such as 56, are very well handled by antioxidant low molecular weight thiols in bacteria in the mechanism of detoxification.160 BSH is a major antioxidant thiol found in SA, and is found to be important in developing resistance and survival for SA. With the help of FosB, an enzyme prototype for bacillithiol Stransferase enzymes, BSH detoxifies 56. BSH null SA was highly sensitive to 56 treatment, similar to FosB mutants.161 Substituted dinitrobenzenesulfonamide (sulfur dioxide donor, 57, Figure 10), a prodrug of sulfur dioxide, was found to react

with thiols for its activation and release of SO2 in MRSA.162 Upon treatment of MRSA with 57, a depletion in intracellular thiol and an enhanced level of oxidative species were detected in comparison with untreated MRSA. A combination of thiol depletion and oxidative species production by 56 have profoundly inhibited the growth of MRSA and Enterococcus faecalis (a Gram-positive infectious pathogen).162 Redox-active quinone based small molecule 58 (Figure 10) with a scaffold derived from jadomycin and DNQ natural products, was found to be highly potent in inhibiting a panel of patients derived MRSA strains, better than commercially used antibiotic vancomycin. 163 The mechanism identified for bacterial inhibition, in part, acts through the generation of O2•− and other ROS.163 Hence, targeting low molecular weight thiol to deplete antioxidant capacity and subsequently enhancing intracellular oxidative species is an encouraging strategy for inhibiting growth and deterring drug resistance in bacteria. Mycobacterium tuberculosis (Mtb) is a causative agent of tuberculosis and is one of the highly infectious pathogens. Onethird of the world population is infected with Mtb, as estimated by the World Health Organization (WHO).164 Vitamin C is a known antioxidant and one of the essential nutrients for mammals.165 Vitamin C is shown to reduce the ferric ion (Fe3+) and increase the level of free ferrous ions (Fe2+) in the extracellular medium. The free Fe(2+) can react with O2 to generate O2•−, or with H2O2 to generate highly reactive •OH by the Haber−Weiss process and the Fenton reaction, respectively (Scheme 3).166 Vitamin C (4 mM) was shown to enhance ROS levels and found to be lethal to Mtb.167 The addition of deferoxamine (59, Figure 10), a Fe2+ chelator along with vitamin C, showed no effect on bacterial growth, providing evidence for Fe2+ mediated ROS generation for bacterial inhibition.167 Due to a thick waxy cell wall, Mtb is considered to be one of the difficult pathogens to treat and is shown to be sensitive to enhanced oxidative stress.167 Artemisinin (60, Figure 10) is an endoperoxide-containing sesquiterpene natural product, commercially used to treat malaria, and an interaction of peroxide with cellular Fe2+ leads to the formation of oxygencentered free radicals.168 Subsequent reaction of radicals with oxygen and other biomolecules leads to enhanced oxidative stress and cell death. Artemisinin itself is not toxic enough to kill Mtb; however, a conjugate of artemisinin with mycobactin (61, a chelating siderophore (Figure 10)) was found highly potent in killing multidrug resistant and extensively drug resistant Mtb.169 A mechanism proposed for the inhibition of Mtb by 61 was through oxygen-based radicals generation selectively in Mtb.169 A library of anthraquinone-based small molecules has been developed, which are capable of reacting with oxygen in a buffer to generate ROS. Hydroquinol can directly react with molecular O2 to generate ROS; however, substituted hydroquinols are highly unstable. Dihydroquinones, an equivalent of hydroquinols, were synthesized; these molecules are capable of undergoing keto−enol tautomerism to produce hydroquinol in a buffer, which can subsequently react with O2 to generate ROS.170 ROS generated by this scaffold, including O2•−, H2O2, and hydroxyl radical, were thoroughly established with appropriate functional assays. When treating Mtb (H37Rv) with a library of ROS generators, a complete growth inhibition at low micromolar concentration was observed.171 The lead compound ATD-3169 (62, Figure 10) was found to retain its potency in inhibiting a panel of multidrug resistant and extensively drug resistant Mtb strains.172 This compound was 3232



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Figure 11. ROS-inducible anticancer and antibacterial agents.

to 63 when cultured in antioxidant supplemented medium.174,175 Thus, the development of ROS-based antibacterial agents might have therapeutic potential against a wide range of Gram-positive and Gram-negative bacteria and also could serve as an adjuvant to enhance the efficacy of commercially used antibiotics. While aerobic bacteria become resistant to antibiotics, they have been shown to be sensitive to ROS-based therapeutics and drug cotreatments. Drug-resistance in anaerobic bacteria to antibiotics such as clindamycin, β-lactam, and quinolone by multiple mechanisms including decreased drug uptake, as well as overexpression of drug efflux pumps and horizontal gene transfer, is posing challenges to antibacterial drug discovery.176 The antimicrobial agent metronidazole (64, Figure 10) has been used clinically for over five decades for the treatment of anaerobic bacterial infections.177 The mechanism of 64 was not completely understood, but it is dosed as a prodrug, which undergoes a bioreduction in the bacterial cytoplasm to produce nitroso free radical, a short-lived species, which can oxidatively damage biomolecules.177 Activated 64 binds to bacterial DNA nonspecifically and induces oxidative DNA damage by single and double strand breaks, which eventually leads to cell death.178 Although 64 is a preferred frontline drug for the treatment of anaerobic bacterial infections, drug-resistance to 64 by multiple mechanisms has been recorded.179 Anaerobic bacteria are known to have enriched electron-transport protein and negative redox potential. Perhaps small molecules capable of undergoing intracellular bioreduction to produce radical intermediates could serve as efficient therapeutics for drugresistant anaerobic bacteria.

found to disturb cellular redox homeostasis maintained by MSH, which was measured using a highly specific redox biosensor of intrabacterial mycothiol redox potential (EMSH; Mrx1-roGFP2, the biosensor is covalently linked to mycoredoxin, a Mtb mycothiol specific oxidoreductase).173 As an involvement of MSH in protecting cells from ROS-mediated toxicity, MSH mutant Mtb (MTbΔMSHA) was generated; when treated with 62, the mutant strain was found to be much more sensitive by a factor of 10 to the toxicity exerted by 62 when compared to wild-type Mtb. Microarray analysis of 62 treated Mtb revealed most of the genes involved in ROS detoxification pathways were upregulated, such as DNA repair, cell wall lipid biosynthesis, iron homeostasis, sulfur metabolism, redox-active proteins, citric acid cycle proteins, and transcriptional regulators. KatG, an antioxidant enzymatic system in Mtb, was highly upregulated (35-fold) after treatment with 62. A clear link between intracellular ROS enhancement by 62 to disturb the redox balance in Mtb was established. Evidence of upregulation of the antioxidant genes pool upon the treatment with an ROS generator is confirmative of Mtb being sensitive to enhanced oxidative conditions.172 The ability of this ROS generator to induce bactericidal effects in drug-tolerant Mtb is highly commendable and is meriting more efforts toward developing ROS based small molecule therapeutics to combat Mtb infections. Clofazimine (63, Figure 10) is a commercial drug used for the treatment of leprosy, a chronic infection caused by Mycobacterium leprae.174 The mechanism of action exerted by 63 is proposed to generate intracellular ROS and induce oxidative stress.174,175 A bioreduction of 63 by NADHoxidoreductase, which is present in the ETC, leads to reduced 63. A concomitant reaction with O2 produces ROS. As support of ROS generation, the bacterium was found to be less sensitive 3233



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ROS AS A TOOL TO INDUCE ANTICANCER AND ANTIBACTERIAL PRODRUGS Versatile therapeutic functions of ROS in treating cancer and bacterial infections have shown promise in drug discovery. In addition, ROS has been used as a tool to induce anticancer and antibacterial prodrugs, selectively in cellular phenotypes carrying higher levels of intracellular ROS. Numerous strategies have been reported that developed cell/organelle-specific chemical probes to sense intracellular ROS localization and levels.180 Based on this, prodrugs that are specifically activated in certain cells/organelles have been developed to exert their effects on a target. Clinically used cancer chemotherapeutics such as nitrogen mustards exhibit their toxicity by DNA crosslinking; however, limited selectivity toward cancer cells leads to off-target effects. The protected forms of active chemotherapeutic nitrogen mustards, with a cleavable linker such as boronic esters (65, Figure 11), which are sensitive to high levels of intracellular ROS, have been reported to induce cancer selective cell death.181,182 This strategy has been extended further to design H2O2 inducible anticancer agents including amino-ferrocene based anticancer prodrugs183 (66, Figure 11) and matrix metalloproteinase inhibitor prodrugs184 (67, Figure 11). Leinamycin (68, Figure 11) is a natural product activated by cellular thiol for its antitumor action. Leinamycin E1 (69, Figure 11), a biosynthetic intermediate of 68, was found to be activated by enhanced intracellular ROS and was selectively toxic to cancer cells.185 Advancing on this route, a theranostic prodrug (70, Figure 11) capable of diagnosing and releasing anticancer agent 5′-deoxy-5-fluorouridine, upon activation in mitochondria (where high levels of H2O2 are detected), has been reported.186 Furthermore, several ROS-inducible cargos have been developed to deliver anticancer agents in a site-directed fashion.187 Isoniazid (INH, 71, Figure 11) is a front-line drug for the treatment of Mtb infections.188 Compound 71, in the form of a prodrug, undergoes an oxidative activation by a Mtbcatalase-peroxidase system to produce a reactive intermediate, which forms an adduct (INH-NAD) with NADPH.188 The formation of INH-NAD and its subsequent inhibitory function of Mtb-InhA (enoyl-acyl carrier protein reductase, a cell wall mycolic acid biosynthetic enzyme) is reported to be the primary action of this drug in killing Mtb. A cotreatment of 71 with a constant flux of enzymatically generated H2O2 using a glucose/glucose oxidase system was found to enhance the rate of formation of INH-NAD-adduct and found to profoundly inhibit InhA.189 Nitric oxide (NO), a diffusible radical reactive species, is generated in cells to serve diverse cellular functions in human immune, nervous, and vascular systems.190 Overproduction of NO is known to induce nitrosative stress, and a reaction with O2•− would produce peroxynitrite, which is one of the highly reactive species that induce oxidative stress and DNA damage in cells.191 NO has shown a promising therapeutic record against various diseases, including cancer, ischemia-reperfusion injury, and neurodegeneration. Prodrugs of NO have been shown to enhance the potency and therapeutic index of NO.190,192 A H2O2 activated NO-donor, BORO/NO (72, Figure 11) was developed to permeate bacteria and release NO in intracellular milieu, a useful tool to study the enhanced effects of NO in bacteria.193 Taken together, intracellular ROS or external ROS sources could potentially be used to deliver existing anticancer and antibacterial agents in the form of a prodrug; this might

significantly enhance the drugs’ therapeutic indexes and reduce off-target effects.

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CONCLUSIONS Numerous strategies have been developed to combat cancer and bacterial infections; however, promotion of resistance in cancer and bacteria to drugs with a specific mechanism of action warrants novel therapeutic strategies with distinct actions from traditional ones. Enhancement of intracellular ROS by endogenous antioxidant inhibition, modulating the functions of proteins responsible for maintaining redox homeostasis, and using sources of small molecule ROS generators have shown a lot of success in the therapeutic arena. Combination strategy is another route taken to regulate drug resistance. Combinations of ROS inducers and commercial drugs have shown a lot of promise in selectively killing cancer and bacteria with greatly enhanced inhibitory potential. Hence, augmenting ROS in cancer cells and drug-resistant cancer cells could enhance oxidative stress and lead to enhanced vulnerability to chemotherapeutics. Although some evidence has pointed to induction of drug resistance mediated by low intracellular ROS, the involvement of ROS in controlling the drug resistance, in both bacteria and cancer, seems possible. Conceivably, it is only a concentration dependent phenomenon. One of the problems that needs addressing is to appreciate the influence of leveldependent effects of ROS in drug resistance and as a therapeutic in the biological system. A devoted effort in the design and synthesis of organic molecules with the potential of enhancing intracellular ROS in a temporally controlled fashion could help understand the role of this species in a biological context. A controlled generation of intracellular ROS using organic small molecules could alleviate the off-target effects in cells and can be a potential future therapeutic, which warrants a considerable effort.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The author declares no competing financial interest. Biography Allimuthu T. Dharmaraja received his B.Sc., from Government Arts College, Salem, India, in 2000 and his Master’s degree from Bharathidasan University, India, in 2005. He then joined Indian Institute of Science Education and Research, Pune, India, for a Ph.D. degree in Chemistry. In his doctoral studies, he worked on the design, synthesis, and evaluation of organic small molecule based reactive oxygen species generators as anticancer and antibacterial agents. Since 2015, he has been a postdoctoral research scholar at Department of Genetics and Genome Sciences and Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, USA, where he is working on the synthesis of electrophilic small molecule libraries using high throughput synthesis, evaluating their potential in selectively killing cancer cells and understanding the mechanism of action.

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ABBREVIATIONS USED ROS, Reactive oxygen species; Fe−S, Iron−sulfur cluster protein; SOD, Superoxide dismutase; MPO, Myeloperoxidase; Gpx, Glutathione peroxidase; TR1, Thioredoxin reductase-1; GSTP1, Glutathione-S-transferase pi 1; CBR1, Carbonyl reductase 1; SILAC, Stable isotope labeled amino acids in cell 3234



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culture; NQO1, NADPH quinone oxidoreductase 1; NRF2, Nuclear factor E2-related factor 2; KEAP1, Kelch ECH associating protein 1; GLUT1, Glucose transporter 1; GADPH, Glyceraldehyde 3-phosphate dehydrogenase; PARP, Poly ADP ribose polymerase; Trx, Thioredoxin; TrxR, Thioredoxin reductase; PrxI, Peroxiredoxin; GPX4, Glutathione peroxidase 4; TRAIL, Tumor necrosis factor-related apoptosisinducing ligand; P-gp, P-glucoprotein; DDHs, Dihydrodiol dehydratases

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