Toward New Antileishmanial Compounds: Molecular Targets for Leishmaniasis Treatment

The leishmaniases are a group of diseases caused by protozoan parasites— Leishmania sp. Leishmaniasis is classified among the 20 neglected diseases by WHO. Although the disease has been known for more than 120 years, the number of drugs used for the treatment is still limited to 5–6. The first-line drugs against leishmaniasis are pentavalent antimonials, which were introduced to the treatment 70 years ago—despite all their side effects. Molecular targets are becoming increas-ingly important for efficacy and selectivity in postgenomic drug research studies. In this chapter, we have discussed potential therapeutic targets of antileishmanial drug discovery such as pteridine reductase (PTR1), trypanothione reductase (TR), N-myristoyltransferase (NMT), trypanothione synthetase (TryS), IU-nucleoside hydrolase, and topoisomerases, enzymes and their inhibitors reported in the literature.


Introduction
Leishmaniasis is a parasitic disease that occurs in the tropic and subtropics regions, and the parts of southern Europe. The disease is classified among neglected tropical diseases (NTDs) [1]. Leishmaniasis is spread by the bite of phlebotomine sand flies that causes the infection with Leishmania parasites. There are three main forms of the disease-cutaneous leishmaniasis (CL) known as the most common form, that causes skin sores; visceral leishmaniasis (VL; kala-azar) is the most severe form, that affects several internal organs; and mucocutaneous leishmaniasis (MCL) that has a chronic and metastatic behavior [2,3].
Although the disease has been known for more than 120 years, the number of drugs used for the treatment is still limited to 5-6. The first-line drugs used against leishmaniasis are pentavalent antimony (Sb V ) compounds namely sodium stibogluconate (Pentostam®) and meglumine antimonate (Glucantime®), which was introduced into treatment more than 70 years ago, despite all their side effects. Neither their mechanism of action nor their chemical structures have been clarified/verified yet in spite of their wide use for a long time. Other drugs used in Leishmania infections are liposomal amphotericin B (L-AmB), miltefosine, paromomycin (aminosidine), and azole-derived antifungals; ketoconazole, itraconazole, and fluconazole. eukaryotes (Figure 2). This physiological pathway, N-myristoylation, plays an important role in the correct cellular localization and biological functions. NMT enzyme was purified and characterized from yeasts for the first time and it is thought to be a target for development of a new class of antifungal drugs [26]. The presence of NMT in L. major was verified in 1997 [27]. Later, NMT enzyme activity was proven essential for viability in Leishmania sp. then, it attracted attention as a potential drug target in kinetoplastid parasites [28]. The validation of this enzyme as a target for antitrypanosomal and antileishmanial drug discovery was not until 2010 (Figure 2) [29,30].
A group of antifungal agents was tested to identify the first NMT inhibitors by Panethymitaki et al. in 2006 [31]. Although some of the tested compounds were found to be NMP inhibitors in a low μM concentration range, their antileishmanial activity has not been reported [31].
In an HTS campaign led by Pfizer, around 150.000 compounds from the Pfizer Global Diverse Representative Set were screened against protozoan NMTs. Following the previous study, the crystal structures of PF-03393842 and PF-03402623 with the enzyme, the initial hits selected in the HTS campaign, were elucidated. Based on this data, a fused hybrid compound 43 was developed as a highly potent L. donovani NMT inhibitor (Ki of 1.6 nM) with good selectivity over the human isoform of the enzyme (Ki 27 nM) (Figure 3) [33]. Although the lack of cell activity of 43 attributed to its poor uptake, the HTS campaign, and hybridization of the hit compounds have resulted in the discovery of a new scaffold [33].
Another HTS assay dedicated to identifying novel Leishmania sp. NMT inhibitors was focused on a set of 1600 pyrazolyl sulfonamide compounds [34]. Interestingly, no correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes was observed. This might be rationalized by the fact that poor cellular uptake considering the basicity of the compounds. The most potent inhibitor of LmNMT (compound 2, Ki of 0.34 nM) exhibited modest activity against L. donovani intracellular amastigotes  (EC50 of 2.4 μM). Yet, advanced studies on compound 2 confirmed the on-target mechanism. Moreover, oral use of compound 2 resulted in a 52% reduction in parasite burden in the mouse model of VL (Figure 3) [34].

Inosine-uridine (IU) nucleoside hydrolase (IU-NH, EC:3.2.2.2)
The nucleoside hydrolase enzyme is an important target for the development of antiparasitic drugs due to its role in the purine salvage pathway. The amino acid sequence and X-ray structure of the enzyme from L. major were revealed in 1999 [39]. IU-NH enzyme establishes a homolog in Leishmania species.
In contrast to these facts, there is no study on IU-NH enzyme inhibitors possessing in vitro/in vivo antileishmanial activity up to our knowledge.

Enzymes Involved in Polyamine metabolism in Leishmania
In Leishmania parasites (and other members of the trypanosomatids), polyamine pathways can be considered as a unique pathway; most enzymes are essential for parasitic survival and infectivity (Figure 4).
Arg is an enzyme that catalyzes the conversion of L-arginine amino acid to L-ornithine and urea.
The expression of the Leishmania amazonensis ARG in a bacterial host was done   Reguera et al. suggest that broad inhibition of ARG activity alone will be insufficient to achieve therapeutically useful control of leishmaniasis, but combined inhibition of ARG with downstream enzymes leading to polyamine synthesis could result in improved therapeutic responses [46]. 3′-methoxy-cinnamoyl-1,3,4thiadiazolium-2-phenylamine, an ARG inhibitory compound, exhibited moderate antileishmanial activity upon amastigotes of L. amazonensis [47].

Spermidine synthase (SpdSyn, SpdS, EC 2.5.1.16)
SpdS catalyzes the conversion of putrescine to spermidine, a crucial polyamine for parasite proliferation. Genetic studies proved that SpdS is an essential gene in L.donovani [66]. Additionally, it was demonstrated that L. donovani amastigotes require SpdS activity to sustain a robust infection in mice; which is required for virulence [67].
Up to our knowledge, the only reported SpdS inhibitor with antileishmanial properties is natural compound hypericin [68].
CGP40215A, a specific AdoMetDC inhibitor, was also reported with the antileishmanial effect that verified the potential of AdoMetDC enzyme inhibition strategy [70]. TryS bifunctionally catalyzes both biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, which is the main regulator in intracellular thiol-redox metabolite for parasitic trypanosomatids. As TryS is absent in humans, targeting this enzyme provides selectivity. Inhibition of TryS results in controlling relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania [71]. Genetic and chemical analyses reveal that TryS is essential for Leishmania infantum [72].
In a computational screening campaign, oxabicyclo[3.3.1]nonanone skeleton was identified not only as a TryS inhibitor but also with TR inhibitory properties. A modest antileishmanial activity was reported for compound PS203 upon L. donovani promastigotes (Figure 5) [73]. In another study, TryS from L. donovani was characterized and inhibition studies with the natural compounds selected from an earlier Micro Source discovery natural product data set were performed [74]. Among the tested natural compounds, conessine and uvaol showed good TryS inhibition (Ki of 3.12 μM and 3.55 μM, respectively) with significant antileishmanial activity on L. donovani promastigotes (IC50 of 13,42 μM and 11,23 μM, respectively) (Figure 5) [74].
About 144 compounds belonging to seven different scaffolds were tested for TyrS inhibitory properties in a study by Benitez et al. One of the most promising inhibitors (IC50 of 0.15 μM) namely MOL2008, an N 5 -substituted paullone derivative was evaluated upon L. infantum promastigotes (EC50 of 12.6 μM) (  One of the main strategies of the host organism to overcome the infection is oxidative stress. TR has been purified from T.cruzi [77], first, and then from Labrus donovani [78]. TR enzyme is responsible for keeping trypanothione in the reduced state that is a variant of glutathione in Leishmania parasites. These enzyme  inhibitors have been investigated in antileishmanial drug discovery as the enzyme is essential for the parasite survival and its absence in the host, in which glutathione reductase (GR) is found, provides selectivity [79]. Although both TR and GR are inhibited by trivalent antimonials, TR is considerably more sensitive [80]. TR enzyme is also a target for anti-Chagas compounds and antimalarials. The main limitation of TR becoming a target in antileishmanial drug discovery is that in order to obtain a considerable effect in parasites' redox state, a minimum of 85% inhibition is required [81]. Additionally, GR should be considered as an off-target for TR inhibitors and the selectivity over TR enzyme of the compounds may be presented. Apart from being an interesting target for antileishmanial drug design, it is also a popular target for antimalarial compounds.
The early discovery of tricyclic inhibitors that are specific for TR over GR led to the design and synthesis of a group of phenothiazine derivatives and their openedring analogs.
A series of bis (2-amino diphenyl sulfides) were designed and synthesized to inhibit TR [84]. Among them, compound 15 was found to be the most active with the IC50 value of 200 nM. Although there was no correlation between TR inhibition and antileishmanial activity, the compounds showed activity upon L. infantum amastigotes (Figure 6) [84]. Sulfonamide and urea derivatives of quinacrine with varying methylene spacer lengths were designed as TR inhibitors and their antiprotozoal activities were evaluated [85]. Compound 2b (TR IC50 of 3.3 μM and GR IC50 of 27.2 μM) was also one of the most active compounds upon L. donovani among with Trypanosoma cruzi and Trypanosoma brucei [85] (Figure 6).
In the pursuit of discovering novel lead heteroaromatic frameworks, harmaline, pyrimidobenzothiazine, and aspidospermine scaffolds were tested against TR inhibition (Ki of 35.1 μM, Ki of 26.9 and Ki of 64.6 μM, respectively) and L. amazonensis promastigote toxicity. Moreover, compounds have not exhibited any GR inhibitory activity [86]. Interestingly, Blackie et al. has introduced ferrocenic 4-aminoquinoline urea compounds with TR inhibitory and antileishmanial properties to the literature [87]. Although compounds inhibited TR in a low μM range with good selectivity over GR and showed antileishmanial activity on L. donovani amastigotes, unfortunately, these compounds were found to be toxic to macrophages (Figure 6) [87].
In an HTS campaign, 100,000 lead-like compounds were evaluated for their TR inhibition. As our focus on antileishmanial compounds, 2 series of compounds namely, nitrogenous heterocycles (triazine and pyrimidine derivatives) and conjugated indole derivatives took our interest in their potential on L. donovani amastigotes (Figure 6) [88].
Imidazo[1,2-b]pyridazin scaffold was designed to inhibit various eukaryotic kinases by Bendjeddou et al. [119]. In this study, some of the compounds were tested against L. amazonensis parasites. The compounds showed antileishmanial activity at rather high concentrations (10 μM) although the compounds have not exhibited any toxicity at cell viability assays regarding concentrations [119].
Because of Leishmania parasite has a life cycle in the mammalian host, inhibition of signal transduction protein kinases for antileishmanial activities was investigated. Polyfluoroalkyl sp 2 -glycolipid compounds were reported with antileishmanial properties by binding p38a-MAPK [120]. Purine derivatives, benzopyrroles, and benzopyrrolidines exhibited CRK3 cyclin-dependent kinase inhibitory properties and showed antileishmanial activity upon Labrus donovani amastigotes [121]. Lastly, a chemical inhibitor of heat shock protein 78 (HSP78), namely Ap5A reported with antileishmanial activity [122].

2.2)
Topoisomerases are enzymes that modulate DNA topology. Firstly, topoisomerase II and then topoisomerase I enzymes were reported in Leishmania species [123,124].
Different classes of TOP inhibitors show activity against L. donovani parasites by the means of DNA TOPI catalytic activity. The most important point is providing selectivity over parasite-human topoisomerase enzymes [125]. Pentostam's one of the proposed modes of action is inhibition of TOPI of L. donovani [126]. Werbovetz et al. tested known TOPII inhibitors, acridine derivatives, against L. chagasi and L. donovani, therefore, it was suggested that TOPII could serve as a useful target for parasite chemotherapy [127].
In another study, compounds bearing 1,5-naphthyridine scaffold were reported [129]. Compound 22 was found to be one of the promising ones with the IC50 value (0.58 ± 0.03 μM) against L. infantum amastigotes similar to the standard drug amphotericin B (0.32 ± 0.05 μM) and selectivity over host murine splenocytes. Additionally, this compound showed remarkable inhibition on leishmanial TopIB [129].

Cysteine synthase (CS, O-acetylserine sulfhydrylase, OASS, EC 2.5.1.47)
Cysteine biosynthesis is a potential target for antileishmanial drug development. The structure of L. major cysteine synthase was revealed in 2012 by Fyfe et al. [145]. Cyclic imide derivatives were identified with a multitarget profile including TOPOI, N-myristoyltransferase, cyclophilin, and CS enzymes using in silico approach and L. amazonensis activity of the compounds were reported [146].

Oligopeptidase B (OPB, EC 3.4.21.83)
It was found out that a high level of serine protease activity was expressed by L. donovani, which was explained by an increase in OPB enzyme activity [147]. The crystal structure of L. major OPB was revealed in 2010 by McLuskey et al. [148]. Epoxy-α-lapachone was shown activity on both promastigote and amastigote forms of L. amazonensis in a study exploring natural compounds as potential antileishmanial agents. Moreover, this activity was associated with serine proteinase inhibitory activity of epoxy-α-lapachone in the same study [149]. Peptidic structure ShPI-I (Kunitz-type protease inhibitor from the sea anemone Stichodactyla helianthus) was shown to be a potent inhibitor of L. amazonensis serine proteases [150].
2.9 Superoxide dismutase (SOD, EC 1.15.1.1) SOD enzyme was found in L. tropica by Meshnick and Eaton and it was suggested that the enzyme may be containing iron (Fe) which causes a difference from its host's enzymes which is linked to a copper or zinc atom [151]. Later, molecular isolation and characterization of Fe containing SOD cDNAs of L. chagasi were reported in 1997 [152] and the 3D structure of Fe-dependent superoxide dismutases (FeSODs) from L. major was reported [153].
In a study, imidazole-containing phthalazine derivatives were found to be potent inhibitors of Fe-SOD with antileishmanial properties. Additionally, the tested compounds were selective toward parasite Fe-SOD over human CuZn-SOD [154]. Arylamine Mannich base derivatives, known to be effective against Trypanosoma cruzi, were exhibited remarkable activity against Leishmania species. The mechanism of action of these compounds was linked to their potent Fe-SOD inhibition [155].
Augustyns's research group design and synthesize various compounds and tested against IAG-NH (inosine-adenosine-guanosine nucleoside hydrolase) from Tabanus vivax. In contrast to promising enzyme activity of the compounds, antileishmanial activity of the compounds hasn't been investigated [41, 173,174].
It was found out that hydroxychromenone and tetrahydrocyclohexanecarboxylic acid fragments could bind to the enzyme in a fragment-based analysis on LdNH using saturation transfer difference (STD) NMR spectroscopy [176].
In a recent study, a natural product from Brazilian flora, flavonoids, and proanthocyanidins, with antileishmanial activity screened against LdNH and described as an inhibitor of LdNH [43,177].
Interestingly, LdNH (NH36) is the main area of interest for human recombinant vaccine-based studies and phase I trial of nucleoside hydrolase NH36 of L. donovani, the main antigen of the Leishmune® vaccine, and the sterol 24-c-methyltransferase (SMT) from L. infantum is in progress [178].

Cysteine proteases
There are two cysteine protease genes from L. major-one is structurally similar to the cathepsin L (CatL) family and the other is similar to the cathepsin B (CatB) family of cysteine proteases. These cysteine protease enzymes were isolated and sequenced by Sakanari et al. [179].

Glyceraldehyde
Although GAPDH enzyme is found in Leishmania sp., it is an attractive target for the development of novel antitrypanosomatid agents rather than antileishmanial compounds.

Dihydroorotate dehydrogenase (DHODH, EC 1.3.5.2)
DHODH enzyme catalyzes the stereoselective oxidation of (S)-dihydroorotate (DHO) to orotate (ORO) in the de novo pyrimidine biosynthetic pathway. The structure of L. major DHODH was revealed by X-ray diffraction analysis [189]. It was reported that natural compounds from Asteraceae species could inhibit LmDHODH by Chibli et al., though the antileishmanial effect of the compounds has not been evaluated [190].
DDD806905, a known TbMetRS inhibitor, tested against LdMetRS and showed antileishmanial effect upon Leishmania axenic amastigote yet, it has not shown efficacy in an animal model of leishmaniasis due to high protein binding as well as sequestration of this dibasic compound into acidic compartments [192]. Researchers have characterized a new series of LdMetRS inhibitors bearing 4,6-diamino-substituted pyrazolopyrimidine core that target a previously undefined, allosteric binding site in the enzyme recently [193].

Phosphodiesterases (PDE, EC 3.1.4.17)
Phosphodiesterases control the cellular concentration of the second messengers cAMP and cGMP that are key regulators of several physiological processes.
A correlation between cAMP concentration in Leishmania cells and proliferation and transformation is demonstrated. By the addition of phosphodiesterase inhibitors to the culture medium, the intracellular level of cAMP was increased [194].
Crystal structure of the L. major phosphodiesterase LmjPDEB1, one of the five PDE encoding genes, was reported in 2007 [195].

Uridinediphosphate-glucose pyrophosphorylase (UGPase, EC 2.7.7.9)
UGPase enzyme catalyzes the reaction of UTP and glucose-1-phosphate to 3-UDP-glucose and PPi in the presence of Mg 2 in vivo. It was reported that protozoan UGP differed from its mammalian counterparts which might provide selectivity [202]. L. major UGPase three-dimensional structure was reported but there has not been any reported in vitro/in vivo inhibitor of the enzyme yet although virtual screening campaigns have been applied to the enzyme [203].

2.19
Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) The levels of dUTP are kept low by the action of dUTPase, a ubiquitous enzyme that catalyzes the hydrolysis of dUTP to PPi and dUMP, a substrate for thymidylate synthase (TS) [204]. The purification and characterization of L. major dUTPase were reported alongside its crystal structure [205,206].
Deoxyuridine derivatives were shown to inhibit L. major, and human dUTPase enzymes exhibited moderate activity against L. donovani [207].

γ-Glutamylcysteine synthetase (Gcs, EC 6.3.2.2)
Gcs is an essential protein of the trypanothione biosynthesis pathway, which catalyzes ATP-dependent ligation of L-cysteine to L-glutamate. Characterization of L. donovani Gcs was reported to the literature in 2016 [208]. Agnihotri et al. identified carbamate, urea, and purine derivatives as Gcs inhibitors using in silico tools, then antileishmanial effect of the compounds was reported in vitro [209].

Cyclophilin (Cyp, Peptidylprolyl isomerase, EC 5.2.1.8)
Cyclophilins are a ubiquitous class of proteins with peptidylprolyl cis-trans isomerase activity. The structure of cyclophilin from L. donovani bound to cyclosporin was reported in 2009 [210]. Interestingly, a recent study showed that cyclosporin A, cyclophilin A modulator, does not express any significant inhibitory effect on intracellular L. donovani amastigotes, therefore, further studies are needed to validate this enzyme [211].