Potential therapeutic targets and the role of technology in developing novel antileishmanial drugs

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Potential therapeutic targets and the role of technology in developing novel antileishmanial drugs Rajalakshmi Rajasekaran and Yi-Ping Phoebe Chen College of Science, Health and Engineering, La Trobe University, Melbourne, VIC, Australia

Leishmaniasis is the most prevalent pathogenic disease in many countries around the world, but there are few drugs available to treat it. Most antileishmanial drugs available are highly toxic, have resistance issues or require hospitalization for their use; therefore, they are not suitable for use in most of the affected countries. Over the past decade, the completion of the genomes of many human pathogens, including that of Leishmania spp., has opened new doors for target identification and validation. Here, we focus on the potential drug targets that can be used for the treatment of leishmaniasis and bring to light how recent technological advances, such as structure-based drug design, structural genomics, and molecular dynamics (MD), can be used to our advantage to develop potent and affordable antileishmanial drugs.

Introduction Leishmaniasis is one of the most neglected tropical diseases in terms of drug discovery. It is a disease caused by protozoan parasites belonging to the genus Leishmania, transmitted via the bite of plebotomine sand flies. Leishmaniasis, in each of its three clinical forms, namely cutanous, mucosal and visceral, remains a serious disease in tropical and subtropical areas of the world. In 2012, the World Health Organization (WHO) reported that leishmaniasis threatened approximately 350 million people in 88 countries around the world [1,2]. This disease generally occurs in underdeveloped countries, where most patients do not avail themselves of a complete course of treatment because of the cost, availability, invasive route of administration, and long treatment duration, which, in turn, increases the chance of drug resistance [3]. This generates the need for new treatment methods and the use of recent technologies to develop new chemotherapeutic agents that are easily available to, and affordable by, the affected population. Leishmania parasites have a dual-form life cycle, occurring as a promastigote flagellar or an amastigote form. Promastigotes are found in the insect vector and are transmitted to the human host. Once inside the host cell, the promastigote differentiates into amastigote and multiplies until the death of the host cell. This Corresponding author: Chen, Y.-P. ([email protected]) 1359-6446/ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2015.04.006

complex life cycle of the parasite provides many targets to explore for drug design and optimization. Unfortunately, because of a lack of commercial interest, few new drugs are being developed or introduced against this deadly disease. Currently, no effective vaccines have been developed and the control of leishmaniasis primarily relies on chemotherapy. The first-line drug, pentavalent antimony, has long been the cornerstone of antileishmanial chemotherapy, but the development of resistance against it has limited its usefulness [4]. Second-line drugs include pentamidine and amphothericin. However, toxicity and emerging resistance prevents the use of pentamidine, whereas amphothericin B has the potential to induce acute toxicity, requiring patient hospitalization. Amphotericin B in its lipid formulation (Ambisome) has proved to be efficient but the high cost is a major drawback. Miltefosine, which is an alkylphosphocholine and originally an anticancer agent [5], was registered for the treatment of visceral leishmaniasis (VL) in India in 2002 and cutaneous leishmaniasis in Colombia in 2005. It has many advantages, including oral efficiency and a short course of treatment. However, its major limitation is its teratogenicity and long half-life, which could favor the development of resistance [6]. A list of all available antileishmanial drugs, their mode of action, and specific adverse effects are detailed in Table 1. Combination treatment for VL has been used to increase the efficiency, reduce costs, and tackle the problem of drug resistance,

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TABLE 1

Current antileishmanial drugs and their mechanism of action

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Drug

Mechanism of action

Adverse effects

Dosage

Administration route

Cost a

Refs

Sodium stibogluconate (Fig. 2g)

Behaves as a prodrug, undergoing biological reduction to a more active/ toxic trivalent form of antimony that exhibits antileishmanial activity

Cardiac arrhythmia and hepatitis, leading to reduction or cessation of treatment

20 mg/kg/day for 30 days

Intravenous or intramuscular

US$70–100 per 100-ml vial

[77]

Meglumine antimonite (Fig. 2h)

Inhibits macromolecular biosynthesis in amastigotes possibly via perturbation of energy metabolism because of inhibition of glycolysis and fatty acid beta-oxidation

Amphotericin B (Fig. 2k)

Selectivity for 24 substituted sterols, mainly ergo sterol, the primary sterol counterpart in mammalian cells, eventually helping to increase drug selectivity toward the microorganism

Thrombophlebitis; occasional serious toxicities, such as myocarditis; severe hypokalaemia; renal dysfunction and even death

1 mg/kg/day for 15 days

Intravenous

US$7.5 per 50-mg vial

[78]

Ambisome (lipid formulation of amphotericin B)

Thought to be drug binding to parasite ergosterol precursors, such as lanosterol, causing disruption of parasite membrane

Fever with rigor and chills; nausea

1 mg/kg/day for 30 days

Intravenous

US$18 per 50-mg vial

[79]

Paromomycin (Fig. 2i)

Binds to 30S ribosomal subunit, interfering with initiation of protein synthesis by fixing the 30S-50S ribosomal complex at start codon of mRNA, leading to accumulation of abnormal initiation complex

Ototoxicity and problems in liver function

15 mg/kg/day for 21 days

Intramuscular

US$15 per course

[80]

Miltefosine (Fig. 2j)

Primary mode of action is uncertain; possible ether remodeling inhibition. Postulated apoptosis and inhibition of cytochrome C oxidase

Teratogenicity (pregnancy must be avoided during treatment and following 2 months)

2.5 mg/day for 28 days

Oral

US$70 for 56 capsules

[6]

a

Prices as quoted in http://www.who.int/leishmaniasis.

which has been a significant challenge [7]. Combining drugs from different classes could result in shorter treatment duration and fewer adverse effects. A combination therapy of miltefosine with amphotericin B or paromomycin is efficient and could be helpful for treating antimony-resistant VL infections in India [3,8]. Studies on combined drug treatments have shown them to be effective, with fewer adverse effects [7]. The combination treatment approach is now being studied more widely, and some combinations are in clinical trials and are showing promising results [9]. However, combination therapies must be used with care. There is a possibility that, if not applied in a controlled and regulated way, the parasite could develop resistance, resulting in a rapid loss of efficacy of not one but two therapeutic options. Therefore, it is important to design suitable experimental studies to determine whether Leishmania parasites are able to develop resistance to the different antileishmanial drug combinations available or in development [10]. 2

Drug discovery for many neglected tropical diseses is carried out using both target-based and phenotypic approaches. Both approaches have their own pros and cons and are being continuously explored by researchers [11]. Phenotypic screening is a powerful method and has been used successfully; this approach does not define any specific molecule or even pathways as a target, but simply selects compounds that are able to eliminate the parasite. The identification of molecular targets from phenotypic approaches can be a way to identify potential new drug targets. Target-based approaches are extensively used in the pharmaceutical industry and involve screening a library of compounds against a protein. The compounds are then optimized for potency against the enzyme, selectivity, and cellular activity [12]. In the case of parasitic diseases, there are few validated molecular targets. This is partly because of the lack of translation from target-based activity to whole-cell assays or in vivo activities [13]. The phenotypic screening of smaller subsets of compounds belonging to a specific

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chemical class could lead to the identification and validation of targets of interest, and have made target-based screens more attractive in the field of trypanosomatid drug discovery [14]. The complete genomes of many Leishmania species, when combined with genomic and proteomic studies, could enhance and speed up the process of target identification and validation. Here, we focus on the potential drug targets that could be used for the treatment of leishmaniasis and how recent technological advancements have had a major role in validating drug targets and developing new thereupeutic agents.

Computation and target-based drug design Identifying a suitable drug target is essential for effective drug development. The various techniques applied in target discovery can be grouped into two broad categories, ‘molecular’ and ‘in vivo’ [15]. The in vivo approach is geared toward target discovery through the study of disease in whole organisms. This approach uses information from clinical studies and animal studies that reveals the physiology, pathology, and epidemiology of the disease. This approach has traditionally been the main target-discovery strategy and still is in diseases such as obesity, atherosclerosis, stroke, behavioral disorders, and neurodegenerative diseases, in which the relevant phenotype can only be detected at the organismal level. By contrast, the molecular approach is geared toward the identification of ‘druggable’ targets using the cellular mechanism underlying the disease phenotype of interest [16]. This approach involves understanding and then targeting the major pathways involved in the disease to control it. Target-based drug design is lengthy and expensive, with several decision gates. With the availability of the complete genome sequence of leishmanial species such as Leishmania major, Leishmania donovani, Leishmania infantum, and Leishmania braziliensis [17], genomic and transcriptomic analyses of the genome have resulted in a better understanding of the biology of the parasites. Similarly, large-scale analyses of metabolites produced during the course of infection, both by the parasite and the vertebrate host, can result in the identification of novel targets [18]. The genome sequence, when conjugated with comparative genomics and proteomics, can provide molecules and pathways that are not present in the host that, in turn, provide new potential target proteins. Some enzymes have also been identified as drug targets despite their presence in the host. This is because they have different enzyme kinetics. Research at a metabalomic level can provide the dynamics associated with the mode of action. Metabalomics not only provides a detailed understanding of the parsite biology addressed by genomics and proteomics, but can also be used to identify metabolic signatures [19]. Understanding how small chemicals interfere with cellular metabolism is a crucial part of modern drug development [20]. The process of target identification uses several filtering techniques, where the sequence, structure, and pathway data are analyzed to come up with potential targets. It is important that the selected drug target is assayable [21]. The identified drug targets can be validated with the use of techniques such as gene knockout, where a knockout parasite is generated by targeted gene replacement. Studying the effect of gene deletion in vitro and in vivo helps develop an understanding of the function and importance of the target gene [22]. Docking with known ligands and high throughput screening (HTS) are other

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techniques that are widely used for target validation (Fig. 1). A brief description of the techniques used in target identification and validation are given in Box 1. Identifying a potential target usually starts with studying the main metabolic pathways, which leads to the identification of possible target proteins. In the case of Leishmania, many such pathways are similar to those in humans and, thus, identifying suitable targets is a challenge. Some of these targets have been identified and proved to be essential for parasite, including the parasite dihydrofolate reductase (DHFR) cyclin-dependent kinases (CDKs) and the well-characterized sterol biosynthesis pathway. The latter is a classic antifungal therapeutic target and has been pursued by several laboratories for antileishmanial and antitrypanosomal chemotherapy, because both Trypanosoma and Leishmania require specific sterols for growth and cell viability. Inhibitors of CRK3-CYC6 and CDK were discovered on a recent target HTS, showing in vitro activity against promastigotes and intramacrophagic amastigotes [23]. Although significant progress in the identification and validation of these targets has been made, no drug focusing on these targets is currently being developed for VL. The TDR targets database (https://www.tdrtargets.org) is a database created as a repository of target-related data by the WHO Special Programme for Research and Training in Tropical Diseases (TDR). This database was created based on genetic, biochemical, and pharmacological data related to tropical disease pathogens in addition to computationally predicted druggability [24]. In silico, the method was used to prioritize pathogen proteins based on whether their properties met criteria considered desirable in a drug target. Prioritization queries that cover sequence-derived information, functional data on expression, metabolic pathways, and assay development feasibility were applied for the prioritization of leishmanial targets from the TDR database. These queries ranked cysteine peptidase C, trypanothione reductase, and DHFR as the top targets for Leishmania [25].

Potential antileishmanial drug targets Target-identification and mechanism-of-action studies have important roles in drug discovery. It is important that the putative target should be either absent in the host or markedly different from the host homolog so that it can be exploited as a drug target. The identification of potential drug targets is typically through the comparison of the parasite and the host enzymes, metabolites, and proteins. These proteins should have significant structural and functional differences compared with their mammalian counterparts to facilitate selective inhibition [26]. There is a significant difference in the sequence identity between proteins from Leishmania and their orthologs in humans, which can be explored to design specific ligands that block the growth of the parasite.

Folate metabolism Folates are essential vitamins that undergo a series of biochemical reactions, from nucleotide biosynthesis to remethylation of homocysteine. One of these is the endogenous folate biosynthesis pathway found in protozoan parasites. Humans do not synthesize folate de novo but instead use a membrane-bound reduced folate carrier to bring the folic acid into the cell. Hence, enzymes related to this metabolism are of interest as drug targets, and the

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L.major genome

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Leishmanial gene expression data

Technologies: Multiple sequence alignment Sequence profiling Gene prediction Active site prediction

Analyzing different leishmanial pathways

Sequence analysis of leishmanial protein

Structural analysis of leishmanial proteins

Technologies: Structure prediction Structure alignment Protein–protein interactions Active site prediction

Gene replacement

Potential antileishmanial drug targets

Gene knockout

Target validation

High-throughput screening/ virtual screening

Reverse docking

Pteridine reductase

N-myristalotransferase

Promising antileishmanial targets

Hypoxanthine-guanine phosphoribosyl transferase

Leishmanolysin

Unlikely antileishmanial targets Drug Discovery Today

FIGURE 1

Target identification and validation in leishmaniasis, showing the different technologies involved in each step.

incorporation of antifolates is believed to provide an ideal therapy for many diseases. The enzymes DHFR and thymidylate synthase (TS) have an important role in folate metabolism and, therefore, are recognized 4

as important targets against leishmaniasis. In most organisms, DHFR and TS exist as separate molecular entities, whereas in Leishmania, these enzymes are a part of a bifunctional DHFR–TS complex. This enzyme catalyzes both the reductive methylation of

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Description of target identification and validation techniques Functional genomics Functional genomics is a branch of genomics that determines gene function and the resulting gene product. Understanding gene function would enable researchers to predict their disease relevance and the suitability of the gene as a drug target. Comparative genomics Comparative genomics is a field in which the genomes of different organisms can be compared to understand the structure and function of the gene of interest. This strategy can be used to identify potential targets, such as essential genes and genes conserved in particular organisms. Gene knockout Gene knockout is a technique in which genes are deleted or disrupted to stop their expression. This works on the assumption that knocking out a target gene will cause the same effect as administering a specific inhibitor of the target protein. Reverse docking In reverse docking, a drug or a small molecule is docked to the binding site of a group of target proteins. Scoring the docked molecules based on their affinity and interactions can identify potential drug targets. HTS HTS is the process of screening a large number of biological modulators against a set of chosen targets. It is an efficient and quick way of eliminating compounds with poor or no effect on the target.

20 -deoxyuridylate and the subsequent reduction of dihydrofolate to yield 20 -deoxythymidylate and tetrahydrofolate at two spatially discrete sites situated on different protein domains [27]. The sequence of the DHFR gene has diverged through evolution, facilitating species-specific inhibition. Studies have shown that it is possible to achieve selective inhibition of the L. major DHFR– TS complex [28]. Unfortunately, the common DHFR inhibitors that are available are not effective against the leishmanial enzyme and appear to be selective for the human enzyme. Inhibitors based on substituted 5-benzyl-2,4-diaminopyrimidines (Fig. 2f) were synthesized and showed good selectivity toward leishmanial DHFR. The structure–activity relation (SAR) study of the 5-benzyl-2,4-diaminopyrimidines class of compounds showed activity against Trypanosoma for several compounds, but were observed to be less active against Leishmania [29]. The absence of a selective inhibitor toward leishmanial DHFR–TS was reported to be the result of pteridine reductase (PTR1), which is capable of performing folate reduction when DHFR is inhibited. PTR1 is a short chain reductase that catalyzes the reduction of folates and pterins. It is widely reported that the failure of the antifolate drug methotrexate is the result of PTR1 synthesis. Given that Leishmania are pteridine auxotrophs, they have a complete and sophisticated pathway for salvaging pteridines from the host and incorporating them into intermediary metabolism. It is the reductase that enables Leishmania to incorporate internalized pterins into its metabolic pathways. Thus, Leishmania differs considerably from the host cells, which can synthesize pterins de novo from GTP in the form of dihydroneopterin and ultimately tetrahydrobiopterin [30]. This enzyme acts as a metabolic bypass for DHFR and, hence, proved to be a valuable candidate for targeted chemotherapy [31]. Studies in animal models indicated that the

loss of folate enzymes resulted in attenuation, which makes it a strong target for chemotherapy [30]. There is still a lack of understanding of the compartmentalization of the folate pathway in Leishmania, which remains a key problem in targeting these enzymes.

Cysteine proteases In protozoa, cysteine proteases (CP) are the most identified and characterized proteinases, and are similar to mammalian cathepsins. In Leishmania infection, CPs are not essential for parasite survival, but do have a role in Leishmania infection. Analysis of the L. major genome revealed three classes of CP, namely A (cathepsin L-like proteases), B (papain like enzymes), and C (cathepsin B-like proteases). Although host homologs exist, leishmanial CPs have distinct structural and biochemical properties, including pH optima and stability. Leishmania CPs have a key role in host immune response, autophagy, and differentiation from promastigote to amastigote. Gene knockout studies have shown that the inhibition of Leishmania CP resulted in replication avoidance. CPBs are thought to have a virulence factor, which could be targeted in the initial phase of the disease [32]. Metacaspases are cysteine peptidases grouped in clan CD, family 14. They are orthologous to caspases and have a major role in apoptosis. In L. donovani, two metacaspases, LdMCA1 and LdMCA2, have been characterized and reported to show 98% homology with each other; they contain a characteristic C terminal proline-rich domain and both are expressed in both promastigotes and amastigotes [33]. Studies indicate that the metacaspases of Leishmania have a major role in cell death path˜oz et al. showed that, ways and in the cell cycle [34]. Castanys-Mun although metacaspases are not essential for leishmanial proliferation and cell death, they act as a growth suppressor [35]. Apart from the discrepancies in the function of metacaspases in Leishmania, conservation of these proteins in these parasites indicates their key role in parasite life or death decisions. Thus, metacaspases should be considered as a potential drug target [36]. From the Leishmania targets mentioned above, the CPs are the most thoroughly studied in terms of inhibitor development of the number of molecules described. Aziridine-2-3-dicarboxylate derivatives (Fig. 2d), vinyl sulfones, hydrazides, and thiosemicarbazones (Fig. 2c) are examples of some classes of compound that inhibit parasitic CPs [37]. Although cost is usually crucial for any (antileishmanial) drug discovery, it is particularly important for this class of compounds because it is a challenge for most developing countries.

Glycolysis Glycolysis is considered to be a promising drug target in Leishmania because it has an essential role in ATP generation. In Leishmania, seven of the glycolytic enzymes are compartmentalized in glycosomes, which makes most of the enzyme structurally and kinetically unique. Most of the enzymes are also phylogenetically distant from those of the mammalian host. Studies using comprehensive 13C-stable isotope-resolved metabolomics have led to a better understanding of carbon metabolism in Leishmania promastigote stages. Leishmania promastigotes catabolize sugars via a typically compartmentalized glycolytic pathway, in which the first five enzymes are located in glycosomes. The ATP and NAD

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BOX 1

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Novel antileishmanial inhibitors

H O

OH

(a)

H

(b)

H

N

N

NH

NH

O N

O

N

O

S

H H

H

O

HO

(c)

H

N

HN

N

O

O

H

N

H N

H

O

H

N

HN H

O

O

Reviews  GENE TO SCREEN

Allopurinol derivative

Adenosine analog

(d)

(e)

H

H H

CI

H

O

O

N

H

H

H

N

N H

N

N

H

O

H

H

H

Purvalnol

Aziridine-2,3-dicarboxylate derivative

Known antileishmanial drugs

H

O

H H

OH O

HO

H

(h)

OH

OH O

O Sb

Sb

O

O

H

O

OH

H

H

O

O

H

N H

H

(i)

H

H

O

H

OH

H

H H

N

Diaminopteridine derivative

H

H

H

H

N

HO

H

N

O

N

N

H

N

(g)

H N

O

HN

H

O N

H

O O

H

O

H

O

Thiosemicarbazones

(f)

OH

O

H

OH

O

HO

O

HO

O

H

O

H

O

HO

O

OH

OH

O O

O

O

O

Sb

H OH

O

Sodium stibogluconate Target: DNA topoisomerase

Melgumine antimoniate Target: Unconfirmed

Paromomycin Target: 16s ribosomal RNA OH

OH

(j) O

HO

O

OH

O

(k)

O

OH

OH

OH

OH

O

O H

P

OH

O O

Miltefosine Target: Cytochrome C oxidase

Amphotericin B Target: sterols

HO

O

OH

Drug Discovery Today

FIGURE 2

Chemical structures of novel antileishmanial inhibitors and drugs. Groups derived from the main compound are ringed in purple.

consumed in these early glycolytic reactions are regenerated, at least in part, by the fermentation of phosphoenolpyruvate (PEP) to succinate. The endproducts of glycolysis and succinate fermentation are further catabolized in a tricarboxylic acid (TCA) cycle [38]. Amastigotes of several Leishmania species were found to exhibit reduced glucose uptake, as compared to rapidly dividing promastigotes, while simultaneously increasing the uptake of amino and fatty acids. Moreover, subsequent genetic studies indicated that Leishmania amastigotes are dependent on hexose uptake and catabolism in vitro and in vivo, despite having reduced capacity to take up sugars [39]. The blocking of a parasite enzyme without causing damage to the glycolysis in the host remains challenging. Several approaches have been considered where the metabolic, structural, and kinetic differences are exploited to design specific inhibitors. Although there were controversies in the inhibition of triose phosphate isomerases, recent studies have proved them to be potential targets. With the availability of the 3D structure of several of the enzymes in Table 2, attempts have been made to develop selective inhibitors targeting these enzymes. Glyceraldehyde3-phosphate dehydrogenase (GAPDH) is a well-studied target, 6

and specific inhibitors have been developed based on its crystal structure. GAPDH is a homotetramer that catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bis-phosphoglycerate using NAD+ as a cofactor. Whereas adenosine acted as a poor inhibitor, several adenosine-based derivatives (Fig. 2a) were demonstrated to be selective against leishmanial GAPDH and showed increased inhibition with low IC50 values [40]. A search of antigens eliciting cellular responses in Leishmania parasites led to the identification of two enzymes from the glycolytic pathway, enolase and aldolase, as potential Th1 stimulatory proteins. Aldolase is a central glycolytic enzyme in carbohydrate metabolism, catalyzing the cleavage of fructose 1,6-bisphosphate into two triose sugars, glyceraldehyde 3-phosphate and dihydroacetone phosphate, whereas enolase catalyzes the reversible dehydration of D-2-phosphoglycerate to PEP in both glycolysis and gluconeogenesis, making it a potential vaccine candidate [41]. In Leishmania, apart from its glycolytic function, enolase also localizes on the surface and binds plasminogen, which contributes to the virulence of the parasite. An anti-enolase antibody was observed to interfere with plasminogen binding, thus making this a candidate vaccine antigen [42,43].

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TABLE 2

Pathway

Target proteinsa

Drug target

PDB structure

Inhibitorsb

Feasibility as targets

Glycolysis

Glucose 6-phosphate isomerase Triosephosphate isomerase GAPDH Pyruvate kinase Aldolase

Yes

Yes

Yes

Yes

2,5-Anhydro-D-mannose and its analogs; adenosine analogs; N-hydroxy-4-phosphono-butanamide

Selective inhibition is a challenge and only high affinity and competitive inhibitors can be used [81]

Yes Yes Yes

Yes Yes Yes

Yes

Yes

Crystal violet; polyamine derivatives; naphthoquinone

Yes

Yes

Absence of these enzymes in mammalian hosts, along with their sensitivity to oxidative stress, makes them a potential target for antileishmanial chemotherapeutics [82]

Thiol metabolism

Trypanothione reductase Trypanothione synthase

CPs

GSK-3 Metacaspases

Yes Yes

Yes No

Aziridine-2,3dicarboxylate; vinyl sulfones; hydrazides; thiosemicarbazones

CPs are essential for the growth, differentiation, and pathogenicity and play a pivotal role in host-parasite interactions, thus making it a potential target with a promising number of inhibitors in-line [83]

Metalloproteases

Leishmanolysin CDKs MAPK

No Possibly Yes

Yes No Yes

Azapurines; thiazoles; paullones; purvalanols

Not all enzymes of this pathway can be targeted; for instance leishmanolysin is a poor target; selective inhibition of CDKs can be achieved and is currently being researched [55]

Purine salvage

HGPRT APRT Adenosine kinase

Unlikely Unlikely No

Yes Yes No

Allopurinol; phthalic anhydride derivatives; phthalimide derivatives

The parasite has multiple salvage routes, which means that targeting individual enzymes is not effective [45]

Folate and/or pteridine metabolism

DHFR TS PTR1

Yes Yes

No Yes

5-Benzyl-2,4-diaminopyrimidines; 8-octyloxy derivatives; 2,4-diaminopteridines

Antifolate therapies against leishmanial DHFR TS have failed because of presence of PTR1, which can reduce pteridebiopterine; It is necessary to design strong inhibitors that can inhibit both enzymes [84]

a b

Widely targeted proteins of the pathways are alone mentioned and some of them have failed to be promising targets. The structures of some of the inhibitors are shown in Fig. 2.

All studies have implied that there are important structural and mechanistic differences between leishmanial enzymes and their human equivalents, which has enabled the development of specific inhibitors that target glycolytic enzymes. It has been stated that, because of conserved glycolytic machinery, single enzymatic inhibition will not be sufficient. Focusing on some of the glycolytic enzymes that have been identified as drug targets will hopefully lead to potent antileishmanial agents.

Purine salvage pathway The purine salvage pathway of parasitic protozoans has been regarded as an attractive chemotherapeutic target. Leishmania species lack de novo synthesis and rely entirely on the purine salvage pathway to meet their purine demands. There is a range of phylogenetic differences between leishmanial parasites and their hosts, and this can be exploited to design specific inhibitors for the parasitic enzymes [44,45]. The leishmanial purine salvage activities include three phosphoribosyltransferases, hypoxanthineguanine phosphoribosyltransferase (HGPRT), xanthine phosphoribosyltransferase (XPRT), and adenine phosphoribosyltransferase (APRT), which catalyze the PRPP-dependent phosphoribosylation of purine bases and adenosine kinase (AK), which phosphorylates

adenosine and a multiplicity of purine interconversion enzymes [46]. Various inhibitors have been targeted on HGPRT because of the difference in substrate specificity compared with the host enzyme. Allopurinol (Fig. 2b) has proved to be an effective inhibitor of HGPRT and, when used with other inhibitors, is more efficient. With the aim of further improving treatment efficacy, a new therapeutic regimen was designed that combines allopurinol (AL) and low-dose meglumine antimoniate (MA). This was shown to be as effective as high-dose MA in the treatment of cutaneous leishmaniasis caused by L. major [47]. The selective metabolism of prodrugs to the nucleotide level is achievable despite the overlap with human enzymes [48]. Virtual screening of purine and piramidine analogs has resulted in several promising lead compounds. Nucleoside hydrolases (NH) of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. NH of L. donovani (LdNH) is the main antigen of the first licensed vaccine against canine VL [49]. Given that NHs are expressed during the early stages of infection, they are excellent candidate targets for pathogen recognition by adaptive immune responses. The identification of the target to the immune response of NH provided a basis for the development of a vaccine against Leishmania [50]. The major issue in targeting this

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Leishmanial enzymes that are used as drug targets and their known inhibitors

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metabolic pathway is that there seems to be alternative salvage pathways in Leishmania that require the targeting of more than one enzyme at a time [45].

Protein kinases

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Protein kinases are promising drug targets for several human and animal diseases. Although the role of protein kinases is well studied in mammalian cells, only limited information is available on protein kinases of protozoans. Studies have shown that protozoan protein kinases differ from mammalian protein kinases, both structurally and functionally. These differences render these kinases as potential drug targets. CDKs have a crucial role in cell division, transcription, apotopsis, and differentiation. Analysis of the genomes of Leishmania species showed that the CDK family is relatively large compared with other unicellular organisms, with 11 cyclins. Compared with two trypanosomatid protozoan parasites, Trypanosoma brucei and Trypanosoma cruzi, Leishmania has an additional cyclin, CYCA. A screen to test 634 chemically diverse antimitotic compounds revealed 27 inhibitors of CRK3, 16 of which also inhibited the growth and replication of L. donovani amastigotes in infected peritoneal macrophages [23]. The chemical inhibition of CRK3 led to aberrant DNA content and abnormal morphology of the cells, which makes it a more potent target [51]. Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation and is an attractive target in trypanosomatids. GSK-3 phosphorylates many regulatory proteins and is involved in transcription and cell cycle regulation. Kinases also provide an important link between cell cycle coordination and apoptosis [52]. This was demonstrated by the inhibition of glycogen synthase-3 short isoform and CRK3. Xingi et al., showed that the indirubins, 6brindirubin-30 -oxime and 6-Br-5-methylindirubin-30 oxime, induced apoptosis in L. donovani promastigotes. This inhibition shows that leishmanial GSK-3 has potential as a drug target [53]. Ongoing studies indicate that GSK-3 inhibitors could be used in the treatment of leishmaniasis with minimal toxicity [54]. Mitogen-activated protein kinases (MAPKs) are important regulators of differentiation and cell proliferation in many eukaryotes. Despite the completion of leishmanial genome sequencing, there is still a lack of understanding of the MAPK signaling pathway in this genus. So far, ten MAPK (LmxMPK–LmxMPK9) and one MAPK activator (MAPKK) (LmxMKK) have been identified and partially characterized. Few studies have been reported on targeting the leishmanial MAPK, but those done so far indicate that LmxMPK is essential for the growth of amastigote and could be explored as a drug target [55].

Thiol metabolism Trypanothione [bis-(glutathionyl) spermidine] is a key molecule against oxidative stress in Leishmania. It is not unique to the parasite, but is crucial in maintaining the cellular redox potential and, thus, is essential for parasite survival. Trypanothione synthase (TryS) is required for the biosynthesis of trypanothione thiol. TryS catalyzes trypanothione synthesis from two molecules of glutathione and spermidine. Trypanothione is then maintained in its reduced form by trypanothione reductase in the presence of NADPH. Reduced trypanothione, in turn, reduces tryparedoxin (TX), followed by the reduction of the TX-recycling enzyme, tryparedoxin peroxidase (TP) [12]. Gene knockout experiments 8

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have shown that TP is essential for survival of the parasites. Recently, TryS was characterized in a nonpathogenic strain of L. donovani and some inhibitors of this enzyme showed leishmanicidal activity, suggesting it to be an attractive and potential drug target [56]. Studies indicated that, despite the strong genetic and chemical validation of TryS, there was a drop in the potency of the inhibitors from the enzyme level to the cellular level, which implies that a longer dosing period is needed. This lack of success might be the result of several factors. The wide active site of the parasite, insufficient uptake, rapid extrusion, and metabolism all have significant roles in determining the in vivo efficacy of a drug. There is also a suggestion that TryS inhibitors combined with other compounds could be used as therapeutic agents.

N-Myristoyltransferase N-Myristoyltransferase (NMT) catalyzes the attachment of the 14-carbon saturated fatty acid, myristate, to the amino-terminal glycine residue of a subset of eukaryotic proteins that function in multiple cellular processes, including vesicular protein trafficking and signal transduction. In L. major, the single-copy NMT gene is constitutively expressed in all parasite stages, with the 421-residue monomeric protein localizing to both cytoplasmic and membrane fractions. NMT has been shown to be essential for survival of the bloodstream form of T. brucei, and in insect stages of L. major [57] and L. donovani [58]. Metabolic labeling studies identified at least ten myristate-labeled proteins in L. major requiring N-terminal myristoylation for translocation to the parasite plasma membrane [57]. However, a comprehensive understanding of the N-myristome and the roles of N-myristoylated proteins in protozoans, particularly infective stages, is still lacking [59]. Previous studies identified myristoyl-CoA–protein N-myristoyl transferase (NMT) as a suitable candidate for drug development against protozoan parasitic infections, including L. major. Recently, to identify inhibitors of protozoan NMTs, Bell et al. screened a diverse subset of the Pfizer corporate collection against Plasmodium falciparum and L. donovani NMTs [60]. Analysis of the screening results showed that SARs for Leishmania NMT varied from those of the other NMTs tested, resulting in the identification of four novel series of Leishmania-selective NMT inhibitors [60]. A detailed understanding of the structure and function of this protein would make it a more suitable target for antileishmanial drug discovery.

Role of technology in antileishmanial drug discovery The field of structural genomics, which relies on the fact that proteins with similar amino acid sequences will have very similar structures, is less than a decade old. Having structural information on the target protein and its complexes with a variety of compounds can provide useful hints for designing compounds that have higher affinity for the target protein [61]. In the case of parasitic protozoans, a large number of proteins that are potential drug targets are selected from a particular pathogen. Projects that focused primarily on pathogenic protozoans, including the Structural Genomics of Pathogenic Protozoa (SGPP; http://www.sgpp. org/) [62] and its continuation, the Medical Structural Genomics of Pathogenic Protozoa (MSGPP; http://www.msgpp.org/), deposited a total of 69 proteins in the Protein Data Bank (PDB), of which coproporphyrinogen III oxidase, adenylate kinase, and uracil-DNA glycosylase from three species of Leishmania were identified using

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MSGPP cocktail crystallography [63]. The application of structural genomics approaches to proteins of pathogenic protozoans has led to determination of the 3D structure of proteins that, in turn, helps in structure-based drug discovery [64]. Structure-based lead discovery is considered to be a boon in the process of drug discovery, where the 3D structure of the protein can be used in the generation of promising lead compounds. A lead compound is a series of molecules that has the potential to progress to a full drug development program [65]. Previously, where thousands of chemical compounds were screened in the laboratory to identify a lead, now, with the massive chemical databases and libraries currently available, computational filtering of molecules is used by many pharmaceutical companies [66]. Virtual screening has emerged as a high-end technology that is gaining increasing use in drug discovery. It is seen as a complementary approach to the experimental HTS and, when coupled with structural biology, promises to increase the number, and enhance the success, of projects in the lead identification stage of the discovery process. This technique involves the computational docking of large databases into the active site of the receptor to identify potential lead structures [67] (Fig. 3). Several inhibitors of CP have been identified through virtual screening, including three compounds that inhibited the proliferation of L. major promastigotes at lower concentration. Although virtual screening has been carried out against a range of leishmanial targets, only a few compounds reached clinical trials, because of the lack of more-refined techniques. The computational docking method has had a major role in the identification of 40 parasite specific inhibitors of leishmanial GAPDH [68]. (a) Screening of chemical libraries

To maximize the chances of finding new drugs and to exploit new potential drug targets emerging from proteomics and genomic studies, we are in need of a more rational approach. One such approach is the use of MD combined with docking. MD is one of the most widely applied techniques to speed up the process of drug discovery. MD simulations can provide the acute details of the motion of particles as a function of time. Thus, these can be used to address the dynamic behavior of the protein [69]. Current MD simulation packages enable the simulation of systems comprising 104–106 atoms and simulation times in the order of psec to Msec. It is also possible to study the effect of solvent molecules on the target protein and acquire important properties, such as interaction energies and entropies [70]. Grover et al. used structure-based screening and MD analysis to discover potent inhibitors of L. donovani spermidine synthase [71]. Enzyme-cofactor docking and refinement/validation with MD simulations of L. major NADH-dependent fumarate reductases provided insight into the binding mode of NADH in each protein [72]. Results currently available make clear that MD will have an important role in understanding the biology of these parasites. Another approach to speeding up drug discovery is to find new uses for existing approved drugs. This is termed ‘drug repositioning’ or ‘drug repurposing’, and traditionally has occurred by serendipity [73]. Many computational strategies for drug repositioning have been published [74]. One way to classify these methods is by categorizing them as either ‘drug based’, where the discovery of repositioning opportunities initiates from the chemical or pharmaceutical perspective, or ‘disease based’, where discovery initiates

(c) Docking and simulation

(b) Analog approach N

3D structure of L. major DHFR TS

N

N

Methotrexate-known inhibitor N

N

O H N OH

571633

567788

77793

554448

62186

552014

587191

369123

580658

299194

569663

69386

570546

50811

14443

73228

569397

366370

O

O

OH

Chemical analogs of methotrexate Docking ligands to the binding site of DHFR TS

Molecular dynamics and simulation studies

Final lead compound 568058

49872

367843

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FIGURE 3

Lead discovery using computational drug design, showing leishmanial dihydrofolate reductase thymidylate synthase (DHFR TS) as the target protein. (a) Screening of the available chemical libraries. (b) Generating structural analogs of the known inhibitor. (c) Docking of the compounds from (a) and (b) to the binding site of the protein followed by further simulation studies. www.drugdiscoverytoday.com 9 Please cite this article in press as: Rajasekaran, R., Chen, Y.P.P. Potential therapeutic targets and the role of technology in developing novel antileishmanial drugs, Drug Discov Today (2015), http://dx.doi.org/10.1016/j.drudis.2015.04.006

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from the perspective of disease management, symptomatology, or pathology [75]. In the neglected and rare disease field, predominantly academic researchers have looked at repositioning compounds that are already approved for other indications. Drug repositioning has been reviewed extensively in the context of finding uses for drugs against major diseases, such as obesity and Parkinson’s disease. Drugs active against leishmaniasis that have arisen via repurposing, include amphotericin B, paromomycin, and miltefosine. Amphotericin B deoxycholate (AmBD) was first licensed as an infusion in 1959 for use against life-threatening fungal infection and has since been used in India for the treatment of VL [76].

Concluding remarks and outlook Leishmaniasis remains endemic in several parts of the world and is a serious health problem in numerous developing countries. In

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the postgenomic era, many potential enzymes and pathways have been identified that are unique to the leishmanial pathogen. With the increase in technological advances, targeted drug discovery can be taken in the right direction with the intelligent use of software and databases along with the biological information available. As outlined here, even with the abundant resources available, developing a pathogen-specific drug is a great challenge because of the major issues of the development of drug resistance and available funding. Pharmaceutical research on natural products is a major strategy for discovering new drugs. During the past decade, although progress has been made in leishmanial research, identifying a potential drug is far from reach. Although many potential targets have been reported with the help of technologies such as comparative genomics and proteomics, a more in-depth study of parasite biochemistry is still necessary.

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