**6. Target based approaches**

#### **6.1 Targeting thioredoxin glutathione reductase**

The redox system of *Schistosoma* parasites depends on the enzyme thioredoxin glutathione reductase (TGR), This enzyme is critical to the redox homeostasis of schistosomes as it acts in the detoxification of reactive oxygen species present in the host. Inhibitors of this enzyme have been sought and assessed for antischistosomal potential [88–91]. The silencing of *S. mansoni* TGR (*Sm*TGR) expression with RNAi led to parasite death within 4 d in an *in vitro* study [89]. Though **PZQ** does not inhibit this enzyme, two previously studied antischistomals, potassium antimonyl tartrate (**69**, **Figure 11**) and oltipraz (**70**), were found to be effective *Sm*TGR inhibitors.

Auranofin (**71**), a gold complex widely used to treat rheumatoid arthritis, strongly inhibited the enzyme (IC50 < 10 nM) and effected good WBRs (~60%) in infected mice [89]. Further work has established that treatment with **71** causes cysteine-goldcysteine bridges to form in *Sm*TGR, and that this process may be catalyzed by the selenocysteine present in the enzyme [92].

Early HTS efforts in this vein revealed the oxadiazole 2-oxide scaffold as a promising lead for novel *Sm*TGR inhibitors [81]. Treatment with furoxan derivative **72** at 10 μM caused 100% parasite death in adult *S. mansoni, S. japonicum* and *S. haematobium* within 24 h in *in vitro* studies, and was highly effective *in vivo* (>88% WBR at 10 mg/kg dosage) [93]. The parasite's phenotypic response to treatment with **72** resembled the effects of RNAi silencing of *SmTGR* expression [89]. The addition of a nitric oxide (NO) scavenger to the system slowed the schistosomal activity of **72** considerably, indicating that **72**'s release of NO in the presence of *SmTGR*

**Figure 11.** *Inhibitors of S. mansoni thioredoxin glutathione reductase (SmTGR) (***69***–***78***).*

contributes to its potency [93]. Further structure-activity relationship (SAR) work established the 3-cyano-1,2,5-oxadiazole-2-oxide moiety as the pharmacophore of interest [94]. Testing several aryl-substituted furoxans against *S. japonicum* yielded several active compounds, but no correlations between antischistosomal activity and either TGR inhibition or NO release rate [95].

HTS efforts to find other *Sm*TGR inhibitors yielded a set of eight hits with IC50 values under 10 μM [96]. Four of these, **73**–**76**, showed consistent antischistosomal activity against *S. mansoni*, *S. japonicum*, and *S. haematobium*, rapidly killing at least half the adult worms present at a 10 μM dose [96].

A secondary "doorstop pocket" binding site in *Sm*TGR has recently been identified; binding to this site appears to preclude NADPH binding elsewhere in the enzyme [97]. Piperazine derivatives **77** and **78** were predicted to bind tightly to this pocket in binding studies, and in fact proved to be good *Sm*TGR inhibitors with antischistosomal activity against adult worms *in vitro* [97].

#### **6.2 Targeting kinases**

Kinases play critical roles in regulating vital functions like cell proliferation, differentiation, apoptosis, and migration in various organisms. The use of proteinkinase-targeting drugs against *S. mansoni* and *S. japonicum* has been reviewed recently [98–100]. *S. mansoni* has 357 kinases; 351 of those are transcribed in adults with 268 being protein kinases (PKs) [99]. Phenotypic screening of a set of 114 approved oncology drugs against *S. mansoni* NTS revealed several kinase inhibitors with good activity against both NTS and adult *S. mansoni* (IC50 < 10 μM) *in vitro* [101]. Six of those compounds (**Figure 12**)— trametinib (**79**), bosutinib (**80**), ponatinib (**81**), afatinib (**82**), sunitinib (**83**), and vandetanib (**84**)—were then assessed for *in vivo* activity. In a murine model, only **79** and **84** showed *in vivo* efficacy, with WBR values of 63.6% and 48.1%, respectively [101].

Protein tyrosine kinases (PTKs) are involved in angiogenesis, reproduction, cell proliferation, and many other processes [102]. Many PTK inhibitors (or "tyrphostins", for tyrosine phosphorylation inhibitors [103]) are able to inhibit multiple PTKs, including receptor tyrosine kinases (RTKs) like growth factor receptors, insulin receptors, (IR) and Venus kinase receptors (VKR). Among the RTK inhibitors that have demonstrated

**Figure 12.** *Anti-schistosomiasis kinase inhibitors (***79***–***86***).* antischistosomal activity is BIBF1120 (**85**), which inhibits fibroblast growth factor receptors in *S. mansoni* (*Sm*FGFR-A and -B) and which, in *in vitro* testing, caused unpairing of coupled worms at 5 μM and complete worm death within 48 h at 10 μM [104]. Another is tyrphostin AG1024 (**86**), which inhibits both insulin receptors and VKRs in *S. mansoni*, induces death in both schistosomula and adult worms at 10 μM [105].

Other kinases that have been studied as antischistosomal targets include mitogenactivated protein kinases (MAPKs) [106, 107], Polo-like kinases (PLKs) [108], Ablkinase [109], and *Sm*TAO and *Sm*STK25, two protein kinases discovered in a recent large-scale RNAi screen to be critical to worm survival [110].

#### **6.3 Targeting hemozoin formation**

Like other blood-feeding parasites, *S. mansoni* must free themselves of toxic free heme, and do so by polymerizing heme to crystalline hemozoin [111, 112]. Inhibiting the parasites' heme polymerization, then, presents another anti-schistosomiasis strategy; this is considered to be the antischistosomal mode of action for several antimalarials [113, 114]. However, recent work showing that some hemozoin in the *Schistosoma* gut is actually consumed to yield free iron for egg development indicates that there is more to learn about hemozoin formation in this parasite [115].

A series of pyrido[1,2-*a*]benzimidazoles, some of which with demonstrated inhibition of heme polymerization in *P. falciparum*, were screened against *S. mansoni* [116]. A majority of the compounds tested (48 of 57) showed good activity against NTS, with 19 of those demonstrating IC50 values below 3 μM against adult worms. However, the correlation between hemozoin inhibition and antischistosomal activity was weak (R<sup>2</sup> < 0.05 for both NTS and adults).

Further investigation of this scaffold led to analogs **87** and **88**, with IC50's under 0.4 μM against adult *S. mansoni* and moderately good WBR effects in infected mice (62.2% and 69.1%, respectively) [117], and to the chiral 1-phenylethylamine derivative **89**, which combined excellent WBR activity (89.6%) at 50 mg/kg with some toxicity concerns (**Figure 13**) [118].

#### **Figure 13.**

*Antischistosomals targeting hemozoin formation (***87***–***89***), cysteine proteases (***90***–***92***), tubulin (***93***), and histone deacetylase (***94***–***95***).*

#### **6.4 Targeting cysteine proteases**

Cysteine proteases are integral to metabolism, nutrition and immune invasion in several parasites, including *Trypanosoma cruzi, Trypanosoma brucei, and S. mansoni* [119, 120]. In particular, *S. mansoni* cathepsin B1 (*Sm*CB1) inhibitors have been assessed for anti-schistosomiasis activity. A series of thiosemicarbazone and thiazoles were assessed for *Sm*CB1 inhibitory activity and screened for phenotypic effect on *S. mansoni* schistosomula and adult worms [121]. The best *Sm*CB1 inhibitor found, thiosemicarbazole **90** (IC50 = 1.5 ± 0.4 μM), displayed no activity against the parasite *in vitro*, while thiazole **91**, which showed no *Sm*CB1 inhibition, was the most active compound against schistosomula, and the only one active against adult worms, in the set [121]. However, a series of peptidomimetic vinyl sulfones including K11777 (**92**) has demonstrated both excellent *Sm*CB1 inhibitory efficacy (IC50 = 2.09 ± 0.08 nM for **92**) and good activity against schistosomula *in vitro* [122, 123].

### **6.5 Targeting tubulin**

Tubulin, and tubulin-containing cellular components like microtubules, which are essential for cell division and many other functions of the eukaryotic cell, have long been considered druggable targets in *S. mansoni* [124, 125]. In 1977, colchicine and vinblastine were shown to inhibit red blood cell ingestion and microtubule formation in the parasite [126]. However, the cytotoxicity of these natural products preclude their wider application as anti-schistosomiasis agents.

Phenotypic screening of a library of tubulin-binding compounds led to the further exploration of the phenylpyrimidine scaffold as a source of new leads [127]. Further development resulted in thiophene-substituted phenylpyrimidines such as **93**, which reduced worm movement by over 90% at 5 μM but lacked the mammalian cell cytotoxicity of other tubulin-targeting compounds [127].

#### **6.6 Targeting histone deacetylase**

Histone deacetylase (HDAC) inhibitors, developed for epigenetic cancer chemotherapy [128], have shown effectiveness against *S. mansoni* at all stages [129–131]. In target validation studies, reducing expression of *S. mansoni* HDAC8 (*Sm*HDAC8) leads to decreased worm and egg counts in infected mice [132]. A series of hydroxamic acid *Sm*HDAC8 inhibitors has been developed [133, 134]; the most potent of these, dibenzofuran **94**, strongly inhibited *Sm*HDAC8 (IC50 = 270 nM) and killed >98% of *S. mansoni* schistosomula at 10 μM, but its poor solubility foiled efforts to test its *in vivo* activity [134]. Triazole hydroxamic acids such as **95** were found to have similar *in vitro* activity [135]. Related enzyme studied as *S. mansoni* drug targets have included *Sm*HDAC6 [136], histone methyltransferase EZH2 [137], and some sirtuins (particularly *Sm*Sirt1 and *Sm*Sirt2) [138, 139].

#### **6.7 Other targets**

Other *S. mansoni* targets being investigated for new antischistosomal drugs include phosphodiesterase-4 [140–142], dihydroorotate dehydrogenase [143], 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [144, 145], farnesyl transferase [146], carbonic anhydrase [147], NAD+ catabolizing enzyme [148], cytochrome P450 (CYP3050A1) [149] and aldose reductase [9, 150, 151].
