1、Role of Reactive Oxygen Species (ROS) in Therapeutics and DrugResistance in Cancer and BacteriaAllimuthu T. Dharmaraja*Department of Genetics and Genome Sciences and Comprehensive Cancer Center, School of Medicine, Case Western ReserveUniversity, Cleveland, Ohio 44106, United StatesABSTRACT: Evading
2、 persistent drug resistance in cancer and bacteria isquintessential to restore health in humans, and impels intervention strategies. Adistinct property of the cancer phenotype is enhanced glucose metabolism andoxidative stress. Reactive oxygen species (ROS) are metabolic byproducts ofaerobic respira
3、tion and are responsible for maintaining redox homeostasis incells. Redox balance and oxidative stress are orchestrated by antioxidantenzymes, reduced thiols and NADP(H) cofactors, which is critical for cancercells survival and progression. Similarly, Escherichia coli (E. coli) and life-threatening
4、infectious pathogens such as Staphylococcus aureus (SA) andMycobacterium tuberculosis (Mtb) are appreciably sensitive to changes in theintracellular oxidative environment. Thus, small molecules that modulateantioxidant levels and/or enhance intracellular ROS could disturb the cellularoxidative envir
5、onment and induce cell death, and hence could serve as noveltherapeutics. Presented here are a collection of approaches that involve ROSmodulation in cells as a strategy to target cancer and bacteria.INTRODUCTIONChemical genetics is one of the successful strategies widelyexplored for the identicatio
6、n of drug candidates and theirmechanism of action in cells.1Phenotypic screening of drugcandidates exploits the genetic dierences in cancerous cellsand has identied many successful FDA approved drugs,including afatinib, imatinib, and trastuzumab, and others, suchas neratinib, that are in advanced cl
7、inical development.However, sustaining the ecacy of marketed drugs has beena challenge due to the prevailing drug resistance in cancer,which often necessitates intervention strategies.2Duringaerobic respiration and cellular metabolism, oxygen isconverted into water and carbon dioxide, respectively,
8、toproduce energy in the form of adenosine triphosphate (ATP)in cells.3In these processes, oxygen is partially reduced toreactive radical and nonradical oxygen species. In cells,superoxide (O2) is generated by 1 etransfer to O2eitherfrom the electron transport chain, ETC, or by NADPH oxidase(NOX) enz
9、yme. O2is known to damage ironsulfur clusterproteins (FeS), which leads to release of Fe(II) into theextracellular matrix and, as an eect, inactivation of the functionof FeS cluster proteins (Schemes 1a and 2).4,5This O2species undergoes a dismutation to hydrogen peroxide (H2O2)in a buer or catalyze
10、d by a family of enzymes called superoxidedismutases (SOD, Scheme 2). H2O2is reactive toward a varietyof functional groups in biomolecules; for example, the thiol ofthe cysteine-containing proteins is oxidized to form sulfenicacids (Scheme 1b). This, in turn, can undergo subsequentoxidations by addi
11、tional equivalents of H2O2to form sulnicand sulfonic acids that could permanently inactivate the proteinfunction.58Next, hydroxyl radical (OH), one of the highly reactiveROS, is generated by a metal (Fe(II) or Cu(I) mediatedreduction of H2O2through the Fenton reaction (Schemes 1and 2). Hydroxyl radi
12、cal directly reacts with DNA, which isoften irreversible, causing oxidative damage and eventuallyleading to mutations in the DNA sequence.5,7The reactive sitesin DNA are the sugar backbone and nucleobases. In ribose, C-4 tertiary radical is generated and subsequent uncontrolledreactions lead to DNA
13、degradation or mutations. Among thenucleobases, guanine is a highly reactive base that forms 8-oxoguanine during its reaction with OH. Thymine, anothernucleobase, undergoes an addition reaction with OH, formingthymine radical species and inducing mutations in DNA.Proteins are known to be oxidized by
14、 OH, mainly thiolcontaining amino acids including methionine, and theseresidues are oxidized to their corresponding sulfoxides (Scheme1b). Protein carbonyls are generated by a reaction of OHwithamino acids such as lysine, arginine, proline, and histidine at thecarboxylic acid functional group, and o
15、xidation of histidineresidues in proteins produces 2-oxo-histidines.6,8Formation of hypochlorous acid (HOCl) from H2O2in cellsis catalyzed by the enzyme myeloperoxidase (MPO, Scheme 2).This species, HOCl, is reactive toward biomolecules and foundto oxidize cysteine residues to cysteine sulfenic acid
16、 andtyrosine residues to dityrosines in proteins. HOCl is muchmore reactive (3 107M1s1) than H2O2(0.9 M1s1), asobserved in oxidizing glutathione. Hydroxyl radical, OH, is thesource of a generation of hydroperoxyl or organoperoxyReceived: August 16, 2016Published: January 30, 2017Perspectivepubs.acs.
17、org/jmc XXXX American Chemical Society A DOI: 10.1021/acs.jmedchem.6b01243J. Med. Chem. XXXX, XXX, XXXXXXradicals, which can damage lipids by oxidation and peroxidation(Scheme 1b).6These radical and nonradical oxygen speciescould indiscriminately react with biomacromolecules in cells toinduce oxidat
18、ive damage (Scheme 1b).9Enhanced ROS-mediated oxidative damage builds up stress in cells,appropriately called as oxidative stress. Overall, excess ROSproduction can induce a plethora of damaging eects to cellularcomponents (Figure 1). Immune cells have directed thedamaging capacity of ROS to microor
19、ganisms duringpathogenic conditions, where ROS are aberrantly produced asan immune response to combat pathogens.10Apart fromdamaging eects to cellular components, ROS are recognized asa key factor in many cellular signaling events, including cellproliferation and survival, which necessitates balance
20、 in ROSproduction and maintenance of redox homeostasis by cellularmachinery.6,11Hence, to attenuate the oxidative stress-mediated damage to biomacromolecules and to regulateredox homeostasis, cellular machinery has evolved stringentantioxidant systems, such as antioxidant thiols and enzymes.12For ex
21、ample, the pool of O2produced in electron transportchain and cellular metabolic events is quenched by the enzymesuperoxide dismutase (SOD), where O2is converted intoH2O2. The subsequent quenching sequence involves anotherenzyme catalase, which transforms H2O2to water.5,8Although abasal level of ROS
22、is crucial for maintaining redox homeostasisand cellular functioning, enhanced ROS levels were implicatedin many neurodegenerative disorders, carcinogenesis, andaging.5,10,13Dening the precise role and safe level ofintracellular ROS is challenging due to mixed eects of ROSin cells such as protective
23、 roles in pathogenic conditions anddeleterious eects to the host cellular components at higherlevels. The benecial level of ROS to cells remains enigmatic;however, a signicant interest is spreading across the chemicalbiology and medicinal chemistry community on ROS basedtherapeutic strategies.ROS in
24、ducing small molecules by mechanisms of (1)inhibiting antioxidant systems, (2) transferring electrons toO2and producing ROS, or (3) a combination of the above-mentioned processes (1 and 2) in cells were reported toselectively kill cancer phenotypes.2Similarly, ROS basedtherapeutics are in developmen
25、t as antibacterial agents againstdrug resistant Gram-negative and Gram-positive bacterialstrains. A number of reviews on the chemistry of ROS andtheir biological roles have been published.2,4,5,1012,1418In thisPerspective, an eort has been made to collective literature ofsome of the ROS-based therap
26、eutic strategies to target cancerand bacteria, and their acquired drug resistance.REDOX HOMEOSTASIS REGULATED BY ENZYMESIN CELLSOxidative damaging eects of ROS to biomacromolecules, suchas DNA, protein, and lipids, through nonspecic rapid reactionsplaced on ROS the imprint of toxicity to cells. Henc
27、e, ROS areshowcased as harmful species, and that fact was hyped with thediscovery of overexpressed cellular antioxidant enzymesincluding superoxide dismutase and catalase. Until the 1990s,NADPH oxidase in phagocytes was the only known ROSgenerator in phagocytic conditions, which is activated inrespo
28、nse to growth factors, cytokines, and inammation.19Later, many biologically important enzymes were identied thatgenerate ROS during their primary functions.20A series of 7enzymes of the NADPH oxidase family were identied asgenerating ROS in various tissues and not only in phagocytes.Cytosolic NADPH
29、is used as a substrate (electron source), andelectrons are transferred to molecular oxygen in the NADPHoxidase catalyzed ROS production.21Ligandreceptor inter-action with NOX mediates ROS generation, and such ligandsinclude platelet derived growth factors, chemokines, and tumorScheme 1. (A) General
30、Scheme for ROS Production; (B). Damaging Eects of ROS to BiomacromoleculesScheme 2. ROS Generation Induced by Cellular EnzymesaaaETC: Electron transport chain; NOX: NADPH oxidase; SOD:Superoxide dismutase; MPO: Myeloperoxidase.Figure 1. Schematic representation of ROS generation and their level-depe
31、ndent eects in cancer and bacteria.Journal of Medicinal Chemistry PerspectiveDOI: 10.1021/acs.jmedchem.6b01243J. Med. Chem. XXXX, XXX, XXXXXXBnecrosis factors.22,23The mitochondrial respiratory chain is oneof the major sources of ROS including O2, which is thenconverted into H2O2. During respiration
32、, molecular oxygen isthe nal electron acceptor in the ETC, where it is reduced towater; however, escape of electrons in the ETC leads to partialreduction of O2(14%) to produce O2and H2O2.24Mammalian targets of rapamycin (mTOR), p53, and B-celllymphoma 2 (BCL-2) family members are other factorsrespon
33、sible for mitochondrial ROS production.22,25Xanthineoxidase (XO) is a metalloavoprotein constitutively expressedin cells. XO converts hypoxanthine and xanthine to urate, andin the process, O2is reduced into O2.26Cytochrome P450(CYP) enzymes are avoenzymes with an iron-containing haemcenter. CYPs are
34、 known for oxygen-dependent metabolism ofcholesterol, steroids, and small molecules. Here, the oxygenbound iron haem center in CYPs is known to produce O2and H2O2. ROS are also generated during a metabolism ofarachidonic acid by lipoxygenases and cyclooxygenases invarious cell types (Figure 2).27Tak
35、en together, intracellularROS is produced by specic enzymes in various parts of cells ina tightly controlled manner for cellular functions.Although a cluster of known enzymes is responsible forintracellular ROS production, until recently, superoxidedismutase and catalase enzymes were the only recogn
36、izedantioxidant systems in cells for balancing the level of ROS tomaintain redox homeostasis. Glutathione (GSH), cysteine(Cys), vitamin C (ascorbic acid), and vitamin E (-tocopherol)are other nonenzymatic antioxidant molecules that mitigate theexcess level of ROS produced in cells. Later, glutathion
37、eperoxidase (GPx), thioredoxin reductase, and peroxiredoxinwere some of the other antioxidant enzymatic systemsuncovered as handling ROS levels in cells.22,28,29GSH is alow molecular thiol and a major antioxidant found in cells. GSHserves as a substrate for GPx in annihilating lipid peroxidationand
38、is oxidized to disulde (GSSG), which is reduced back toGSH by another enzyme, glutaredoxin, to restore GSH levels incells. Disulde bonds formed by oxidation of thiols in proteins,peptides, and glutathione are either during a reaction with ROSor in the process of attenuating ROS. The reduced forms ar
39、eeciently reinstated through reduction of the disulde bond bythioredoxin reductase systems using NADPH as a cofactor(Figure 2).30With timely activation of these stringentantioxidant systems, cells are able to tightly regulate ROSlevels and maintain redox homeostasis.ROS PARADOX IN CANCERMitochondria
40、l genetic mutations have been one of the primarycauses of the increase in cellular ROS. Upon mutagenesis inmitochondrial DNA, the electron transport sequence in ETC istracked, which leads to accumulation of electrons in themitochondrial membrane, and a reaction of these electrons withmolecular oxyge
41、n produces O2.31Diusible H2O2can beproduced by a dismutation of O2by SOD, which can enterinto the nucleus and cause DNA damage to induce geneticmutations. These overall genomic instabilities, caused byenhanced ROS levels and oxidative stress, trigger cancermetastasis and progression.32However, due t
42、o the needs of cellproliferation, cancer cells adapt to signicantly enhanced levelsof intracellular ROS and reach nearly the toxic threshold limit.Balancing the ROS overproduction has been handled byenhanced levels of GSH and other antioxidant enzymes in cells.Thioredoxin reductase-1 (TR-1) is one o
43、f the antioxidantselenoproteins that is activated upon oxidative stress responseto maintain the reducing environment in cells. Many malignanttumors were characterized by overexpression of TR-1, and theinhibition of TR-1 function was recognized as to abrogatecancer progression. Treatment of cancer us
44、ing antioxidants wasconsidered as a potential strategy, since ROS is contemplatedto promote cancer.33,34However, clinical trials of treatment ofcancers with antioxidants such as N-acetylcysteine, ebselen,edaravone, vitamin A, vitamin C, vitamin E, and -carotenefailed and were found to aggravate canc
45、er progression over longtreatment regimens (Figure 3).30Therefore, ROS-mediatedmutations could promote cancer, paradoxically, enhancingintracellular levels of ROS beyond or to the toxic threshold, byusing external sources of ROS or by inhibition of an antioxidantsystem, which could surmount cellular
46、 antioxidant defenses andkill cancer cells, hence oering an opportunity to develop ROSbased therapeutics for cancer treatment.ROS-BASED THERAPEUTICS IN CANCERIn cancer cells, growth and proliferation are encouraged with acondition of a modest rise in intracellular ROS; on the otherhand, apoptosis is
47、 induced at higher levels of ROS. Despitesustaining high levels of intracellular ROS, cancer cells are moresensitive to enhanced intracellular ROS than the non-transformed cells. Thus, utilization of ROS inducing smallmolecules to target cancer has been considered as a potentialstrategy. Piperlongum
48、ine (PL, 1, Figure 4) is an alkaloid naturalproduct isolated from a pepper plant (piper longum L).35Figure 2. Maintenance of redox homeostasis in cells by enzymes.Journal of Medicinal Chemistry PerspectiveDOI: 10.1021/acs.jmedchem.6b01243J. Med. Chem. XXXX, XXX, XXXXXXCCompound 1 was identied as one
49、 of the eective anticanceragents that selectively kills cancer cells by enhancing the levelsROS through inhibition cellular machinery responsible forsuppressing ROS and oxidative stress in cells.35,36Cancer-selective cytotoxicity induced by 1 was unambiguouslycharacterized in both in vitro and in vivo models. Proteinsresponsible for modulating oxidative stress in cells, such asglutathione-S-transferase pi 1 (GSTP1) and carbonyl reductase1 (CBR1), were identied as binding targets of 1 using anunbiased ta
