**10. Druggability of oxidative stress systems in helminths**

(Freinbicherler et al., 2011). Moreover, one 1

play a yet unrecognized role in combating 1

Grx GST

Pr-SSG

GS-X

Pr-SH

cytosolic compartment

for GSH synthesis (γ-glutamyl cycle).

Knöckel et al., 2012; Butzloff et al., 2012).

The fact that vitamin B6 is linked to the defense against 1

GST

H2O+O2

GSSG GR

NADP+

GPx H2O2 LOOH

X

tion of ‧

234 Drug Discovery

ly this role of 1

Figure 1:

GS-X/GSSG transporter

GSSG GS-X

extracellular space

O2 molecule can be either synthesised by the reac‐

O2 in the malaria parasite *P. falciparum*. Very recent‐

AA γ-Glu-AA

Gly

Cys-Gly γ-GT DPD

ADP

Cys

γ-GCS

γ-Glu-Cys

GSH-S

ATP

O2 in plants and fungi (Tambasco-Stu‐

Gly Cys AA transporter

γ-Glu-AA

5-Oxoprolin

5-OP

AA γ-GCT

Glu

ATP

O2.

OH and O2-. or two O2-. with two hydrogen ions (Khan and Kasha, 1994). In order to de‐

O2 detoxification has been verified in the malaria parasite (Wrenger et al., 2005;

toxify these ROS, *Plasmodium* has developed – as outlined above - multiple antioxidant defence systems. However - excluding the membrane located lipophilic tocopherol (vitamin E) (Wang and Quinn, 1999) - none of the above mentioned defense systems are capable to detoxify 1

dart et al., 2005; Ehrenshaft et al., 1999), suggests that the vitamin B6 biosynthesis might also

**GSH**

ADP

NADPH

**Figure 1.** Schematic illustration of the glutathione (GSH) system. GSH homeostasis involves intra- and extracellular mechanisms. GSH is synthesized from amino acids (AA) by the action of γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthase (GSH-S), both requiring ATP. As antioxidant, GSH participates in the reduction of peroxides, cata‐ lysed by glutathione peroxidase (GPx), in the reduction of protein-disulfides, catalysed by glutaredoxins (Grx) and in conjugation reactions with electrophils (eg. xenobiotics, X), catalysed by glutathione transferases (GSTs). The gluta‐ thione conjugates (GS-X) and GSSG are transported out of the cell via GS-X/GSSG pumps. The NADPH-dependent GSH reductase (GR) is responsible for the intracellular recycling of GSH, while extracellular GSH gets sequentially hydro‐ lysed by γ-glutamyl transpeptidase (γ-GT) and dipeptidase (DPD), with glutamate, cysteine and glycine being recycled

A number of drugs have been identified that act as inhibitors of the hemozoin formation by binding to heme. This leads to an accumulation of free heme, causes high levels of oxidative stress and ends in the death of the parasite (Meunier et al., 2010). Quinolinecontaining derivatives such as amopyroquine, amodiaquine, tebuquine, halofantrine, py‐ ronaridine, quinine, mepacrine, epiquinine, quinidine, bisquinoline chloroquine (see

GSH

GSH transporter

> Helminths are parasitic worms that encompass nematodes (roundworms), cestodes and trematodes (flatworms) and affect humans in all areas of the world, with more than onethird of humans harbouring these parasites that cause chronic, debilitating morbidity. Fur‐ thermore, co-endemicity and polyparasitism increase the burden of millions (Hotez et al., 2008). In the absence of vaccines, control relies on pharmacotherapy and pharmacoprophy‐ laxis to easy symptoms and reduce transmission. Helminthosis are treated with a limited number of anthelmintics by chemotherapy of symptomatic individuals or, more general, by control programmes that rely on mass drug administration (MDA) and require annual or bi‐ annual treatment of at-risk populations over prolonged period of time (Prichard et al., 2012). A major problem, however, is the development of resistance or tolerance by the parasites to these common antiparasitic drugs (Vercruysse et al., 2011). It is therefore essential to under‐ stand the underlying mechanisms of drug resistance and find new drugs to circumvent it.

> Praziquantel has been used for over 20 years to treat a variety of human trematode infec‐ tions. Its precise mechanism of action has not been fully elucidated, however, there is exper‐ imental evidence that praziquantel acts by increasing the permeability of cell membranes towards calcium ions and/or by interfering with adenosine uptake (Jeziorski and Greenberg, 2006; Angelucci et al., 2007). Furthermore, it has been suggested that praziquantel reduces GSH concentrations, making the parasite more susceptible to the host immune response (Ribeiro et al., 1998). Interestingly, exposure to sub-lethal concentrations of praziquantel shows that schistosomes undergo a transcriptomic response similar to that observed during oxidative stress (Aragon et al., 2009).

active against immature stages of schistosomes. Although a number of potential drug tar‐ gets have been proposed, the mode of action remains ambiguous (O´Neill et al., 2010). It is thought that the primary activator of the drug is an iron source. Therefore, interaction with heme in the worm gut has been suggested, leading to the formation of an unstable species that generates ROS and thus kills the worm (Utzinger et al., 2001). Since artemisinins are

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

http://dx.doi.org/10.5772/53758

237

Schistosomes seem to be poorly adapted to cope with oxidative stress. This is surprising, since they have to deal with host-immune and self-generated ROS and, furthermore, with ROS generated during the consumption of host haemoglobin (Huang et al., 2012).The highly restricted antioxidant network has been widely accepted as an excellent drug target for schistosomes and other platyhelminths, since it is unique and differs significantly from the human host. Interestingly, the parasites have merged the Trx- and GSH-system using a hy‐ brid enzyme, the thioredoxin-glutathione reductase (TGR) (Salinas et al., 2004, Huang et al., 2012). Using RNA interference, the TGR was found to be essential for parasite survival (Kuntz et al., 2007). TGR was indicated to be the main target of schistosomicidal drugs used in the past (antimonyl potassium tartrate and oltipraz) and of the anti-arthritic drug aurano‐ fin (Fig. 2), with a significant worm reduction observed in infected mice (Kuntz et al., 2007; Angelucci et al., 2009). A quantitative high-throughput screen identified highly potent lead compounds against the Schistosoma TGR (Simeonov et al., 2008), with low inhibitory con‐ stants being found with derivatives of phosphinic amides, isoxazolones and the oxadia‐

Preventive chemotherapy is the mainstay in the control of human soil-transmitted helmin‐ thiasis (STH). STH is primarily caused by the nematodes *Ancylostoma duodenale* and *Necator americanus* (hookworms), *Ascaris lumbricoides* (roundworm) and *Trichuris trichiura* (whip‐ worm) that parasitize the human gastrointestinal tract. Four anthelminthics that exhibit a broad spectrum of activity are currently recommended by the World Health Organization: The benzimidazoles albendazole and mebendazole, the synthetic phenylimidazolthiazole le‐ vamisole and the pyrimidine derivative pyrantel pamoate. While benzimidazoles bind to free β-tubulin, leading to tubule capping and degradation (Beech et al., 2011), the choliner‐ gic agonist levamisole activates ligand-gated acetylcholine receptors (Lewis et al., 1980) and the pyrimidine derivative pyrantel pamoate induces persistent activation of nicotinic acetyl‐ choline receptors (Utzinger and Keiser, 2004). The GABA agonist piperazine, the nicotinic acetylcholine receptor agonist tribendimidine are further drugs used in STH. Currently nei‐ ther drug class used to control or treat STH, has been implicated as influencing the redox biology of parasites. Instead, most of the currently used or proposed drugs (Olliaro et al., 2011) of gastro-intestinal nematodes affect ion channel function of the neuromuscular syn‐ apses. These neuroactive drugs cause paralysis of the worm and result in its rapid expulsion

Filarial parasites are classified according to the habitat of the adult worms in the vertebral host, with the cutaneous (*Loa loa* and *Onchocerca volvulus*) and lymphatic (*Wuchereria bancrof‐ ti, Brugia malayi* and *Brugia timori*) groups being the most clinically significant. Chemothera‐ peutic approaches to control parasite transmission and to treat onchocerciasis rely on the

critically important for malaria chemotherapy, they are not available for MDA.

zole-2-oxide chemotype (Furoxan) (Fig. 2) (Huang et al., 2012).

or killing.

**Figure 2.** Molecular structures of chemotherapeutics which are used to treat infectious disease by generating directly or indirectly high levels reactive oxygen species.

Reliance on a single drug for mass treatment is risky. Therefore, anti-schistosomiasis drug development is on the way to identify new compounds with different modes of action. Re‐ cently it was demonstrated that artemisinin-based compounds (e.g. artemether, figure 2) are active against immature stages of schistosomes. Although a number of potential drug tar‐ gets have been proposed, the mode of action remains ambiguous (O´Neill et al., 2010). It is thought that the primary activator of the drug is an iron source. Therefore, interaction with heme in the worm gut has been suggested, leading to the formation of an unstable species that generates ROS and thus kills the worm (Utzinger et al., 2001). Since artemisinins are critically important for malaria chemotherapy, they are not available for MDA.

Schistosomes seem to be poorly adapted to cope with oxidative stress. This is surprising, since they have to deal with host-immune and self-generated ROS and, furthermore, with ROS generated during the consumption of host haemoglobin (Huang et al., 2012).The highly restricted antioxidant network has been widely accepted as an excellent drug target for schistosomes and other platyhelminths, since it is unique and differs significantly from the human host. Interestingly, the parasites have merged the Trx- and GSH-system using a hy‐ brid enzyme, the thioredoxin-glutathione reductase (TGR) (Salinas et al., 2004, Huang et al., 2012). Using RNA interference, the TGR was found to be essential for parasite survival (Kuntz et al., 2007). TGR was indicated to be the main target of schistosomicidal drugs used in the past (antimonyl potassium tartrate and oltipraz) and of the anti-arthritic drug aurano‐ fin (Fig. 2), with a significant worm reduction observed in infected mice (Kuntz et al., 2007; Angelucci et al., 2009). A quantitative high-throughput screen identified highly potent lead compounds against the Schistosoma TGR (Simeonov et al., 2008), with low inhibitory con‐ stants being found with derivatives of phosphinic amides, isoxazolones and the oxadia‐ zole-2-oxide chemotype (Furoxan) (Fig. 2) (Huang et al., 2012).

Preventive chemotherapy is the mainstay in the control of human soil-transmitted helmin‐ thiasis (STH). STH is primarily caused by the nematodes *Ancylostoma duodenale* and *Necator americanus* (hookworms), *Ascaris lumbricoides* (roundworm) and *Trichuris trichiura* (whip‐ worm) that parasitize the human gastrointestinal tract. Four anthelminthics that exhibit a broad spectrum of activity are currently recommended by the World Health Organization: The benzimidazoles albendazole and mebendazole, the synthetic phenylimidazolthiazole le‐ vamisole and the pyrimidine derivative pyrantel pamoate. While benzimidazoles bind to free β-tubulin, leading to tubule capping and degradation (Beech et al., 2011), the choliner‐ gic agonist levamisole activates ligand-gated acetylcholine receptors (Lewis et al., 1980) and the pyrimidine derivative pyrantel pamoate induces persistent activation of nicotinic acetyl‐ choline receptors (Utzinger and Keiser, 2004). The GABA agonist piperazine, the nicotinic acetylcholine receptor agonist tribendimidine are further drugs used in STH. Currently nei‐ ther drug class used to control or treat STH, has been implicated as influencing the redox biology of parasites. Instead, most of the currently used or proposed drugs (Olliaro et al., 2011) of gastro-intestinal nematodes affect ion channel function of the neuromuscular syn‐ apses. These neuroactive drugs cause paralysis of the worm and result in its rapid expulsion or killing.

Filarial parasites are classified according to the habitat of the adult worms in the vertebral host, with the cutaneous (*Loa loa* and *Onchocerca volvulus*) and lymphatic (*Wuchereria bancrof‐ ti, Brugia malayi* and *Brugia timori*) groups being the most clinically significant. Chemothera‐ peutic approaches to control parasite transmission and to treat onchocerciasis rely on the

**Figure 2.** Molecular structures of chemotherapeutics which are used to treat infectious disease by generating directly

Reliance on a single drug for mass treatment is risky. Therefore, anti-schistosomiasis drug development is on the way to identify new compounds with different modes of action. Re‐ cently it was demonstrated that artemisinin-based compounds (e.g. artemether, figure 2) are

or indirectly high levels reactive oxygen species.

236 Drug Discovery

macrocyclic lactone ivermectin, an effective and safe microfilaricide (Basáňez et al., 2008). Ivermectin is an agonist of ligand-gated Cl<sup>−</sup> channels, with particular activity against gluta‐ mate-gated Cl<sup>−</sup> channels of invertebrates (Martin et al., 1997). While ivermectin is less effec‐ tive against adult worms, it causes reproductive quiescence and disappearance of microfilaria from skin or blood. Interestingly, cultured microfilariae are unaffected by iver‐ mectin at concentrations found in treated patients (Bennett et al., 1993), making interference of the drug with protective mechanisms employed against the human immune response fea‐ sible (Geary et al., 2010). The development of ivermectin-resistant strains of *Caenorhabditis elegans* has shown that resistance to low levels of ivermectin is associated with an increased expression of drug efflux pumps and an increase in GSH-synthesis and -conjugation is ob‐ served. Since the overall levels of glutathione decrease, increased drug conjugation and re‐ moval from the cells is suggested (James and Davey, 2009). In a recent study, ivermectin has been identified as a cytotoxic agent to leukemia cells and a previously unknown indirect in‐ fluence of ivermectin on the intracellular redox balance was demonstrated. Mechanistically, ivermectin induced chloride influx, membrane hyperpolarization, and generated ROS, the latter being functionally important for ivermectin-induced cell death (Shrameen et al., 2010).

the model nematode *C. elegans*, GSH-synthesis and a large variety of primary and secondary antioxidant enzymes and GSH-dependent detoxification enzymes are tightly regulated by the sole NF-E2-related (Nrf) transcription factor SKN-1 (An and Blackwell, 2003). Inhibiton of SKN-1 would thus target the expression of a multitude of enzymatic antioxidants and de‐ toxification enzymes rather than affecting only one single protein or protein class, resulting in the downregulation of xenobiotic detoxification and in an enormous increase of oxidative stress. Since SKN-1 is also essential for embryonic development, this would be an additional bonus. Nematode-specific structural differences are observed that make SKN-1 an excellent

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

http://dx.doi.org/10.5772/53758

239

The current bottle-neck for the treatment of parasitic diseases with chemotherapeutics is the increasing drug resistance which forces the continuous discovery and development of new antiparasitic drugs. There is an urgent need for novel chemotherapeutic targets. New drugs should be generated to specifically target the parasite with minimal (or no) toxicity to the human host. Therefore, good drug targets should be distinctly different from processes in the host, or ideally be absent in the latter. Targeting the peculiarities - which are absent in the host - is proposed as such a strategy. In this sense, the parasite-specific biosyntheses rep‐ resent ideal drug targets; similar to the already exploited antifolate interference with the parasite's dihydrofolate (vitamin B9) biosynthesis. There are a variety of reports about reac‐ tive compounds that have antiparasitic activity; however, not all of these are therapeutically viable drug-like molecules due to various limitations such as toxicity, low bioavailability, rapid inactivation under *in vivo* conditions and development of resistance. Recently studies on drug synergism raised special attention, which can open new avenues to improve the ef‐ ficacy of antiparasitic drugs in combination with others. Since parasites such as *Plasmodium, Trypanosoma* or helminths are highly susceptible to oxidative stress - as outlined within this chapter - the identification of new lead compounds that target the parasite's redox systems

candidate for the development of specific nematocidal drugs (Choe et al., 2012).

by inducing oxidative stress, will be an efficient approach to discover novel drugs.

In this chapter we have tried to give an outline of the present situation of redox-active anti‐ parasitic molecules that target human infectious diseases. In future the mechanisms, evolu‐ tionarily developed by the parasite to circumvent the crucial presence of ROS, will open new avenues for the development of novel antiparasitic drugs that combat resistant human

The authors would like to thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for financial support (Project No. 2009/54325-2 to CW). The support of the DFG

(Deutsche Forschungsgemeinschaft, grant LI 793/5-0 to EL) is acknowledged.

**11. Conclusion**

pathogens effectively.

**Acknowledgement**

Diethylcarbamazine (DEC) is still the mainstay for the treatment of lymphatic filariasis and first choice of therapy of loiasis. Surprisingly, its molecular mechanism of action is still not completely understood. Since pharmacologically relevant concentrations of DEC do not have an effect on microfilariae in culture, its mode of action must involve both the worm and its host. A possible involvement of host arachidonate- and NO-dependent pathways was observed (McGarry et al., 2005). Currently no verification of an influence on the redox biology of helminths is available.

It has been postulated that antioxidant enzymes, that defend against host-generated ROS, are of particular importance for long-lived tissue-dwelling parasites that are involved in chronic infections. Here, surface or secreted antioxidant enzymes are of great importance since they can directly neutralize ROS that pose real danger, thereby protecting surface membranes against peroxides. Secreted filarial antioxidant enzymes include SOD, GPx and Prx (Henkle-Dührsen and Kampkötter, 2001). Additionally to their antioxidant role, the Prx have recently been shown to contribute to the development of Th2-responses by altering the function of macrophages (Donnelly et al., 2008). Interestingly, GSH-dependent proteins have been observed that are capable of modifying the local environment via modulation of the immune response. Here the secretory GSTs from *O. volvulus* combine several features that make them excellent drug target: they are accessible since they are located directly at the parasite–host interface, they detoxify and/or transport various electrophilic compounds and secondary products of lipid peroxidation and they are involved in the synthesis of po‐ tential immunmodulators. Significant structural differences to the host homologues are ob‐ served in the xenobiotic binding site; this may support the structure-based design of specific inhibitors (Sommer et al., 2003; Perbandt et al., 2008; Liebau et al., 2008).

As outlined above, GSH-dependent detoxification pathways defend against current drugs and also play a role in mediating resistance to anthelmintics. The antioxidant pathways also provide the parasite with a means to protect against ROS-attack by its host and/or vector. In the model nematode *C. elegans*, GSH-synthesis and a large variety of primary and secondary antioxidant enzymes and GSH-dependent detoxification enzymes are tightly regulated by the sole NF-E2-related (Nrf) transcription factor SKN-1 (An and Blackwell, 2003). Inhibiton of SKN-1 would thus target the expression of a multitude of enzymatic antioxidants and de‐ toxification enzymes rather than affecting only one single protein or protein class, resulting in the downregulation of xenobiotic detoxification and in an enormous increase of oxidative stress. Since SKN-1 is also essential for embryonic development, this would be an additional bonus. Nematode-specific structural differences are observed that make SKN-1 an excellent candidate for the development of specific nematocidal drugs (Choe et al., 2012).
