**3. Antimonials**

and/or Haber-Weiss reactions (Massimine et al., 2006). At physiologically low levels, ROS can function as second messenger in redox signaling, with H2O2 best providing the specifici‐ ty in its interaction with effectors in signaling processes (Forman et al., 2010). Balancing the generation and elimination of ROS maintains the proper function of redox-sensitive signal‐ ling proteins. However, severe increases of ROS induce oxidative modifications in the cellu‐ lar macromolecules DNA, proteins and lipids, this leading to a disruption of redox homeostasis and irreversible oxidative damage (Trachootham et al., 2008). Depending on the cellular context, the levels of ROS and the redox state of the cells, alterations of the deli‐

cate redox balance can promote cell proliferation and survival or induce cell death.

and Go, 2010).

228 Drug Discovery

To maintain redox homeostasis and eliminate ROS, aerobes are equipped with enzymatic/ nonenzymatic antioxidants and metal sequestering proteins to either prevent or intercept the formation of pro-oxidants. Furthermore, protective mechanisms are put in place to re‐ pair and replace damaged macromolecules. Two central thiol/disulfide couples are involved in controlling the redox state of the cell: glutathione/glutathione disulfide (GSH/GSSG) is the major redox couple that determines the antioxidative capacity of cells, other redox couples include the active site dithiol/disulfide of thioredoxins (Trxred/Trxox) interacting with a differ‐ ent subset of proteins and thus forming a distinct but complementary redox system (Jones

Enzymatic antioxidants can be categorized into primary or secondary antioxidants, the first reacting directly with pro-oxidants (e.g. catalase, superoxide dismutase), the latter are in‐ volved in the regeneration of low molecular weight antioxidant species (Halliwell, 1999). Here, the reduced state of GSSG and Trx-enzymes is restored by the glutathione reductase (GSR) and the Trx reductase using electrons obtained from NADPH. Additionally glutare‐ doxins (Glrx) utilize GSH for the reduction of intracellular disulfides (Fernandes and Holmgren, 2004). While Trx, Trx reductase and Trx peroxidase (peroxiredoxin, Prx) consti‐ tute the Trx-system, the versatile GSH-system includes enzymes required for GSH synthesis and recycling, for its use in metabolism, in defense against ROS-induced damage and in a multitude of detoxification processes. Furthermore, for normal GSH turnover and disposi‐ tion of GSH-conjugated metabolites and xenobiotics, export from the cell is required that is

In spite of the diversity of parasites, all are faced with similar biological problems that are related to their lifestyle. Besides coping with ROS levels generated from intrinsic sources, all have to deal with the oxidative stress imposed by the host´s immune response. Furthermore, parasites are faced with ROS that are produced during the epithelial innate immune re‐ sponse of their vector, by vector-resident gut bacteria (Cirimotich et al., 2011) or during mel‐

Since the redox system plays such a fundamental and indispensable role for parasite surviv‐ al within their host (Massimine et al., 2006), drugs that either promote ROS generation or inhibit cellular antioxidant systems will lead to redox imbalance by pushing ROS levels above a certain threshold level that will ultimately lead to parasite death (Müller et al., 2003). In general, drugs that target vital redox reactions or promote oxidative stress are

carried out by GSH efflux transporters and pumps (Sies, 1999) (Fig. 1).

anotic encapsulation processes (Kumar et al., 2003).

Despite the fact that antimonials were already identified in 1921, they still remain the firstline treatment, although the precise mode of action is not known. But it is generally accepted that pentavalent antimonials (SbV) represent a pro-drug which is converted to trivalent anti‐ monials (SbIII) for antileishmanial activity. Recently it has been indicated that thiols act as re‐ ducing agents in this conversion. Furthermore, the participation of a unique parasite-specific trimeric glutathione transferase TDR1 in the activation of antimonial prodrugs has been sug‐ gested (Fyfe et al., 2012).

Treatment with antimonials requires parenteral administration and is accompanied by toxic side effects such as cardiac arrhythmia and acute pancreatitis (Sundar and Rai, 2002). Some studies have been carried out to investigate the activity mechanism of antimonials which correlates with an interference with the antioxidant defence system of the parasite: Trivalent antimonials decrease the thiol-reducing capacity of *Leishmania* by inducing an efflux of try‐ panothione. In contrast to *Leishmania*, mammalian cells depend on GSH to control their in‐ tracellular thiol-redox status. Here, ROS and oxidized cell components are efficiently reduced by GSH, thereby generating GSSG. The glutathione disulfide form can then be re‐ generated by the GSR (Monostori et al., 2009). In contrast, the redox metabolism of *Leishma‐ nia* relies on a modified GSH-system, *N*1,*N*8-bis(l-γ-glutamyl-l-hemicystinylglycyl) spermidine, also known as trypanothione (Fairlamb et al., 1985). The oxidised form, trypa‐ nothione disulfide, is generated when trypanothione reduces ROS and its reconversion is catalysed by the trypanothione reductase. Antimonials inhibit this enzyme, leading to an ac‐ cumulation of trypanothione disulfide, which subsequently is not accessible for the reduc‐ tion of ROS (Krauth-Siegel and Comini, 2008). The influence of antimonials on the parasite's redox biology has been verified on cellular level by the fact that trivalent antimonials-resist‐ ant parasites display an increased IC50-value for nitric oxide donors such as NaNO2, SNAP, and DETA NONOate compared to antimonial-sensitive strains (Souza et al., 2010; Holzmül‐ ler et al., 2005; Vanaerschot et al., 2010). Whether nitric oxide resistance is due to elevated trypanothione levels or due to another antioxidant mode of action is not yet clear.

**6. Oxidative chemotherapeutic intervention**

*Trypanosoma* infections, caused by the flagellate protozoan *Trypanosoma* are responsible for a high degree of health problems in endemic countries. They can be divided into two types of pathogens: *Trypanosoma cruzi*, the causative agent of Chagas disease, also known as American trypanosomiasis, since it occurs in Latin America and *Trypanosoma brucei ssp*., the causative agent of sleeping sickness, or human African trypanosomiasis, since it is endemic to sub-Saharan Africa. The current medication is known for its toxici‐ ty, poor activity in immune-suppressed patients and long-term treatment combined with high costs. Moreover, vaccines are not foreseeable in the near future. The *T. cruzi* life cy‐ cle includes three fundamental forms characterized by the relative positions of the flagel‐ lum, kinetoplast and nucleus: Trypomastigotes, epimastigotes and amastigotes, the latter one characterized by their proliferation in any nucleated cell (Prata, 2001). On the one hand Chagas' disease is controlled through elimination of its vectors by using insecti‐ cides and on the other side by chemotherapy. Currently, the drugs used are nifurtimox (4[(5-nitrofurfurylidene)amino]-3-methylthiomorpholine-1,1-dioxide), derived from nitro‐ furan, and benznidazole (*N-*benzyl-2-nitroimidazole-1-acetamide), a nitroimidazole deriv‐ ative. Nifurtimox and benznidazole (Fig. 2) are trypanocidal to all forms of the parasite (Rodriques Coura and de Castro, 2002). However, severe side effects and toxicity have been observed (Kirchhoff, 2000). In addition, there are also reports of mutagenesis result‐ ing in DNA damage (Zahoor et al., 1987). An additional aspect that complicates treat‐ ment is the different susceptibility of different parasite strains to the applied chemotherapeutics (Filardi and Brener, 1987). The mode of action of nifurtimox and benznidazole (Fig. 2) is via the formation of free radicals and/or charged metabolites. The nitro group of both drugs is reduced to an amino group by the catalysis of nitro-re‐ ductases, leading to the formation of various free radical intermediates. Cytochrome P450-related nitro-reductases initiate this process by producing a nitro anion radical (Moreno et al., 1982). Subsequently, the radical reacts with oxygen, which regenerates the drug (Mason and Holtzman, 1975). For example, nifurtimox-derived free radicals may undergo redox cycling with O2, thereby producing H2O2 in a reaction catalysed by the SOD (Temperton et al., 1998). Furthermore, in the presence of Fe3+ the highly reac‐

Oxidative Stress in Human Infectious Diseases – Present and Current Knowledge About Its Druggability

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OH is also being formed according to the Haber-Weiss reaction. These free radicals can subsequently bind to cellular macromolecules such as lipids, proteins and DNA, re‐ sulting in severe damage of parasitic cells (Díaz de Toranzo et al., 1988). In contrast, the trypanocidal effect of benznidazole does not depend on ROS but it is likely that reduced metabolites of benznidazole are covalently binding to cellular macromolecules, thereby revealing their trypanocidal effect (Díaz de Toranzo et al., 1988; Maya et al., 2004). Addi‐ tionally, it has been demonstrated that benznidazole inhibits the *T. cruzi* NADH-fuma‐

**of Trypanosoma infections**

tive ‧

rate reductase (Turrens et al., 1996).
