Leishmaniasis: Current Status of Available Drugs and New Potential Drug Targets Nisha Singh1, Manish Kumar1,2, Rakesh Kumar Singh1*

Asian Pacific Journal of Tropical Medicine (2012)485-497 485

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Document heading doi: Leishmaniasis: Current status of available drugs and new potential drug targets Nisha Singh1, Manish Kumar1,2, Rakesh Kumar Singh1*

1Molecular Immunology Laboratory, Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi- 221 005, India

2Department of Chemistry, Bose Institute, APC Road, Kolkata -700009, India

ARTICLE INFO ABSTRACT

Article history: Leishmania The control of infection relies primarily on chemotherapy till date. Resistance Received 15 February 2012 to pentavalent antimonials, which have been the recommended drugs to treat cutaneous and Received in revised form 15 March 2012 visceral leishmaniasis, is now widespread in Indian subcontinents. New drug formulations Accepted 15 April 2012 like amphotericin B, its lipid formulations, and miltefosine have shown great efficacy to treat Available online 20 June 2012 leishmaniasis but their high cost and therapeutic complications limit their usefulness. In addition, irregular and inappropriate uses of these second line drugs in endemic regions like state of Bihar, India threaten resistance development in the parasite. In context to the limited drug options Keywords: and unavailability of either preventive or prophylactic candidates, there is a pressing need to develop true antileishmanial drugs to reduce the disease burden of this debilitating endemic Leishmaniasis disease. Notwithstanding significant progress of leishmanial research during last few decades, Drug resistance identification and characterization of novel drugs and drug targets are far from satisfactory. This Drug targets review will initially describe current drug regimens and later will provide an overview on few Drugs important biochemical and enzymatic machineries that could be utilized as putative drug targets for generation of true antileishmanial drugs.

survive and multiply within phagolysosomal compartment 1. Introduction of macrophages. Leishmaniasis has traditionally been classified into three major clinical forms: visceral (VL), The leishmaniases are a wide spectrum of vector born cutaneous (CL), and mucocutaneous leishmaniasis (MCL), disease with great epidemiological and clinical diversity. It which differ in immunopathologies andLeishmania degree of morbiditydonovani 20 and mortality. Most VL caused by is caused by more than species of protozoan Leishmania parasite that Leishmania is fatal if untreated, whereas CL caused by belongs to family kinetoplastida and genus Phlebotamine. The major Leishmania mexicana Leishmania braziliensis 30 , , , and disease is spread by more thanLeutzomia species of Leishmania panamensis [1] , frequently self cures within 3-18 sand fly in old world andLeishmania in new world . The digenetic life cycle of consists of motile, months, leaving disfiguring scars[2]. flagellated, extracellular promastigotes form in the gut of Over more than 90% cases of VL ensue in five countries: sand fly vector that infects mammalian host and transform India, Bangladesh, Nepal, Sudan and Brazil and 90% of into nonmotile, nonflagellated amastigotes form, which CL cases occur in seven countries: Afghanistan, Algeria, Brazil, Iran, Peru, Saudi Arabia and Syria[3]. However, these numbers only reflects the reported cases and there is gross * : Corresponding author: Rakesh Kumar Singh, Molecular Immunology Laboratory, under reporting of cases in endemic areas that hide the [4] Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi- actual severity of the disease . Although, spread of disease 221 005, India. in endemic and non-endemic regions is multi-factorial Tel: +91-54/2-6702477 E-mail: [email protected] but lack of effective control measures for both, parasite and Foundation prject : The financial supports received from Department of Biotechnology, its vector are main factors. The poor knowledge about the New Delhi (BT/PR11177/MED/29/99/2008) is greatly acknowledged. This article is disease and lack of effective health policies are the primary dedicated to all those poor people who lost their lives during drug trials in various parts of the world. hurdles in the elimination of leishmaniasis from every Nisha Singh et al./Asian Pacific Journal of Tropical Medicine (2012)485-497 486 V [19] corner of the world is far from Leishmania reality. parasites to Sb . It has also been reported that this Soon after realization that causes this disease, reduction takes place primarily in macrophage rather generic pentavalent antimonials have been the cornerstone than parasite[20]. The supporting evidences that come from of leishmanial chemotherapy in disease endemic countries organisms like bacteria and yeast, where the metal reduction especially in Indian subcontinent[5]. In addition, branded is mediated by host specific enzymes suggests that this [21] sodium stibogluconate and meglumine antimoniate are conversion is host specific . Leishmania the alternatives of generic antimonials. The other second The routes of antimonials entrance into line drugs like amphotericin B, its liposomal formulations and macrophages are not known. However, parasitic and miltefosine are being used in the treatment with more aquaglyceroporin, aquaporin 1 transporter is supposed efficacies and dramatic potential for curing leishmaniasis to be responsible for the transport of antimonialsV into however, they are comparably costlier than the generic amastigotes[22]. In addition, the transport of Sb via [6] antimony . Other drugs like paromomycin and pentamidine phosphate transporters is based on Vthe fact that pentavalent have shown some usefulness and could be a potential arsenate, a metal related to Sb , is ableV to enterIII the [21] supplement in the drugs regimen but their use and parasiteLeishmania. Both form of antimonials Sb and Sb , kills availability in disease endemic regions is limited[6-8]. species by DNA fragmentation, suggesting Identification and characterization of cellular targets and the role of apoptosis, 毬-oxidation of fatty acid and answering the problem of drug resistance in leishmaniasis adenosine diphosphate phosphorylation. However, the has always been the main thrust of protozoan research exact mechanisms of action are still unexplored[23-25]. In worldwide. The recent advancements in innovative animal addition, the antimonials inhibit glycolysis and metabolic models and parasites with reporter gene constructs have pathways and increases efflux of intracellular thiols by a ATP ABC provided rapid andin highvivo throughin vitro output drug screening binding cassette ( ) transporter, multi drug resistant methods in both, and [9,10]. Unfortunately protein A (MRPA)[26]. Pentamonials are also known to no vaccine candidates either prophylactic or preventive inhibit trypanothion reductases, an enzyme responsible for are under animal or clinical trials[11-13]. Thus it becomes protection from host reactive oxygen and nitrogen species to significantly important to search an effective drug/s and/or parasites[27]. a prophylactic vaccine/s for an early and effective control The widespread misuse of drug, as it was easily available of leishmaniasis. This review gives an overview of 1) drugs over the counters in endemic regions; along with loss that are being used and/or are still in phase of clinical trials, of drug activation by parasites are the major causes of in vitro V 2 3 ) mechanisms of drug action and resistance, ) and finally acquiredLeishmania resistance. The studies on Sb resistant elaborates biochemical machineries and enzymes as new axenic amastigotes and promastigotesV III indicate [10] potential drug targets. their diminished ability to reduce Sb to Sb III . A study Leishmaniaon amastigote and promastigote forms of Sb resistant 2. Current scenario of available drugs , have shown reduction in accumulation of metals due to either reduced uptake or increased efflux[28]. HSP70 2.1. Pentavalent antimonials Overexpression of a heat shock protein ( ) gene has been found to be associated with the antimonial resistance[29]. The transporters of ABC family, MRPA and 1 PRP1 Pentavalent antimonials, the generic sodium stibogluconateetc pentamidine resistant protein ( ) that act as efflux pump (pentosam) and branded meglumine antimoniate , are for antimonials,are also linked to antimony resistance[15,30]. being used in the treatment of leishmaniasis over more Further, various genes identified in antimonial unresponsive than five decades and still they are the first line drugs of clinical isolates suggests the multifactorial mechanism of choice where resistance is not reported[14]. The growing resistance[31-34]. incidence of resistance has raised serious concern for its use [15] 2.2. Amphotericin B (AmB) in diseaseV endemic area . The pentavalent antimoniate (Sb ) considered as a pro-drug,III which is further converted to trivalent antimonite (Sb ), an active form of Vthe drug It is a polyene antifungal drug widely used to treat albeit to the parasite is also susceptible to Sb [16]. The systemic fungal infections[35]. In endemic areas of Bihar reduction of pentavalent to trivalent compound takes place where antimonials resistance is common, AmB is the either in macrophages or in the parasite however, it is still a drug of choice[36]. AmB shows high affinity for ergosterol, dilemma[17]. Parasite mediated reduction has been found to the predominant sterol of fungal and leishmanial cell be associated with the loss of reductase activity of parasite, membrane. Despite its high efficiency, AmB is also toxic which may also lead to drug resistance. This is supported and its side effect has been reported[37,38]. Adverse effects V Leishmania donovani by the observation that Sb resistant of plain AmB have been circumvented with its three clinical amastigotes lose their reductase activity. The recent finding formulations in which deoxycholate have been replaced TDR 1 A B of a parasite thiol dependent reductasesV III ( ) enzyme, by other lipids. These formulations are liposomal m that catalyze the conversion of Sb to Sb using glutathione (L-AmB: Ambiosome), AmB colloidal dispersion (ABCD: as a reductant also supports this possibility[18]. In addition, Amphocil) and AmB lipid complex (ABL: Abelcit). These 2 ACR2 A B arsenate reductaseLeishmania ( ) a new antimoniate reductase lipid formulations of m retain their antifungal activity characterized in sp. increases sensitivity of and show very high efficacy to cure this deadly disease and Nisha Singh et al./Asian Pacific Journal of Tropical Medicine (2012)485-497 487 are less toxic. In VL cases, liposomal AmB has been proved The activity of miltefosine is due to intracellular as an efficient drug with more than 95% efficacy but high accumulation of drug, which is regulated by two cost limits its use to common man suffering from this deadly transporters, LdMT and its 毬-subunit LdRos3, a P-type ATP disease. ase, belonging to aminophospholipid Leishmaniatranslocase The antileishmanial activity of AmB and its lipid family[52]. In studies on resistant mutant of , i.e.formulation is due Leishmania to its interaction of both sterols it is observed that due to decreased influx, accumulation ergosterol of and cholesterol of host of miltefosine inside the parasite is greatly hampered[52]. A B macrophages. Since cholesterol Leisis complexedhmania donovani by m , Although, the exact mode of antileishmanial action is still it markedly inhibits binding unclear but itLeishmania has been founddonovani that it causes apoptosis like promastigotes to macrophage[39]. Further, at higher processes in as observed in amastigote concentration (<0.1 M), it induces the formation of aqueous but how it happens, still unknown[53]. Miltefosine also pores in leishmanial promastigotes cell membrane that reduces the lipid content in promastigotes membrane and result in osmotic changes leading to the cell lysis[40]. Inspite enhances the phosphatidylethanolamine content suggesting of excellent efficacy the administration of AmB is also a partial inhibition of phosphatidylethanolamine-N- associated with the toxicity and emergence of parasitic methyltransferase that leads to decreased parasite A B [54] resistance. The damaging effect of m in2 +kidney tubular proliferation . cell+ is mainly due to increased salt and Ca concentration, The clinical resistance is not yet reported but being an H permeability across the aqueous2+ pores that lead to oral agent its improper use in endemic countries like sustained collapse of pH and Ca gradient across the India increases the probability of resistance and spread membrane, a mechanismin vitro responsible for apoptosis in of resistance parasites where prevalence of infection is eukaryotic Leishmania cells. The studies demonstrate that significantly high. This is now being observed that few resistant Leishmania lacks donovani ergosterol, the main target of patients after successful treatment with miltefosine relapsed AmB[41]. In AmB resistant strain, after 9-12 months. However, more studies are required parasitic cell membrane lacks C-24 alkylated sterols to understand whether these are relapse or reinfection or S that might be due to inactivation of enzyme -adenosyl resistance. Broadly, decreased drugLeishmania efflux is the main reason methionine transferase which is responsible for alkylation at for miltefosine resistance in , though many C-24 position in ergosterol moieties leading to resistance[42]. mechanisms are reported for this decreased intracellular Another study has shown that resistance to AmB was drug concentration. It has been reported that single point 64 4 512 2 L MT LDR 3 found to be associated Leishmania with genetarentolae TarII . and tarII . mutation at d and os in experimental leishmaniasis amplification in mutant cell lines[43]. may lead to resistance. In addition to LdMT, over expression Till date clinical resistance against AmB is not reported but of multidrug resistant MDR1 gene which encodes a the relative nonspecific mode of action of AmB at the level glycoprotein is also responsible for drug resistance[55]. of membrane may be a factor for its infrequent resistance. Furthermore, miltefosine resistance is also correlated with It has been shown in a study that success of AmB treatment lipid content in parasite membrane. It has been observed greatly depends on patient immunity status and indicate that the amount of unsaturated phospholipid alkyl chains that successive relapse could enhance emergence of AmB was lower in miltefosine resistance parasites[56]. [44,45] resistant isolates . These finding warrants the possibility 2.4. Paromomycin of resistance against the most successful drug. 2.3. Miltefosine Paromomycin is chemically an aminoglycosidic antibiotic and has both antileishmanial and antibacterial activity. Miltefosine is originally developed as anticancerous agent, Paromomycin cures both, VL and CL (more effectively) but which is an alkylphosphocholine (hexadecylphosphocholine) limited availability restricts its use in endemic regions[57,58]. moiety[46]. It is the first oral drug used for the treatment A controlled study on the efficacy of topical paromomycin of VL and was considered a major breakthrough in anti- sulfate and methylbenzethonium chloride in CL has leishmanial chemotherapy[47,48]. Its phase I/II/III trials shown total elimination of parasite within the first 10 days provoked a storm of protection against VL that was followed of treatment[59]. The Indian and Sudanese trials further by phase IV trial, which also proved its relevance in demonstrate its high efficacy and excellent tolerability, outpatient setting in those areas where VL is endemic[49,50]. which have also been comparable to AmB[60,61]. A phase II The combination of miltefosine and ambisome has also study in Tunisia and France has shown that WR279, 396, been evaluated, and has been found effective with good a formulation of paromomycin and gentamicin was found tolerability but unfortunately side effects raise questions safe and highly effective in treatment of CL. It offers great against extreme efficacy of this combinatio[51]. The main potential as a new, simple, easily applicable and expensive adherence of the drug is compromised by its long terminal topical therapy for CL[62]. residence, time and teratogenicity. Miltefosine has a median The mechanism modus operandiof paromomycin Candida action krusei is largely 152 long half-life of approximately hours, which could unclear. Its inLeishmania supports encourage development of clinical resistance. Further, cytochrome C inhibition but in requires further its teratogenic and abortifacient nature limits its use in elucidation. Recently it has been shown that cationic pregnancy. paromomycin binds to the negatively charged leishmanial Nisha Singh et al./Asian Pacific Journal of Tropical Medicine (2012)485-497 488 2.6. Pentamidine glycocalyx suggesting mitochondria as a primary target[63]. In addition, paromomycin inhibits translocation and Pentamidine is an aromatic diamine used to cure recycling of ribosomal subunitsLeishmania and donovani hence protein synthesis. Paromomycin in promotes leishmaniasis as a second line drug. Its isothionate and association of 50S and 30S subunits of both, cytoplasmic methansulphonate salts are mainly usedV for the treatment and mitochondrial ribosomes and stops their recycling of VL. It was initially used to treat Sb refractory patients in [64] India but its declining efficacy and high resistance risk has that eventually inhibits protein synthesis .e tFurther al exploration came from the study of Hirokoma that led to its closure in India. Some combinational strategies proves that paromomycin interacts with both 30S and 50S have also been tried with this drug. A study on antimony subunits without inhibiting the association of translation unresponsive patient revealed that combination of low initiation factor-3 (IF3) to the 30S ribosomal subunit[65]. dosage of pentamidine and allopurinol as compare to the full dosages of pentamidine are more effective and less toxic Due to its limited use resistance is not yet reported in in with and ultimate cure of 73% and 58%, respectively[74]. outpatient treatment but resistance has been reported vitro Leishmania donovani Leishmania tropica However, its efficacy is questionable in comparison to other in and [63,66]. drugs. A comparative study on pentamidine andLeishmania meglumine However, rapid emergence against paromomycin due to its CL braziliensisantimoniate (glucantime) against due to aminoglycosidic nature cannot be over ruled. in Peru shows that glucantime was more effective [75] 2.5. Sitamaquine than pentamidine . However, Leishmania it has been panamensis found more CL effectiveLeishmania to curbguyanensis caused by and [76,77]. Sitamaquine, chemically 8-aminoquinoline, is the only Although, its precise mode of action is Leishmanianot known, drug that was developed for treatment of VL. It was originally donovaniit is reported that the drug enters inside developed in collaboration with GlaxoSmithKline and promastigote through arginine and polyamine Wlater Reed Army Institute[49]. The advantage of this drug [78,79] transporters . In a biochemicalLeishmania study it wasdonovani found II is its oral administration. The phase trial of sitamaquine that Leishmaniain pentamidine amazonensis resistant, in India demonstrated its efficacy against VL and was well and promastigote clones, drug tolerated[67]. However, despite efficacy few side effects like resistivity is due to decreased uptake followed by increased vomiting, dyspepsia, cyanosis, nephritic syndrome and efflux of drugs. There is alteration in polyamine carrier that glomerulonephritis were also observed. The consequences of might be responsible for the alteration in surface protein Kenyan phase II trial were different from Indian trial[68]. The nature and content leading to decreased influx of drug. Kenyan trial showed somewhat equal efficacy but observed Furthermore, this drug gets accumulated in mitochondria side effects were abdominal pain, headache and kidney and enhances efficacy of mitochondrial respiratory chain dysfunctioning. complex II inhibitors suggesting its leishmanicidal activities Sitamaquine at high concentration affects parasite motility, due to decreased mitochondrial membrane potential. It is [69] also reported that it inhibit mitochondrial topoisomerase morphology and growth . Mechanism of its action involves [80] electrostatic interaction between phospholipid anionic polar II . Pentamidine is highly toxic;etc causes hypoglycemia, nephrotoxicity and hypotension . Pentamidine resistance head groups and positively charged sitamaquine and then mechanism is not well understood, but intracellular ABC with phospholipid acyl chains leading to drug insertion protein PRP1 can confer resistance to pentamidine in within biological membranes[70]. After binding to the Leishmania Leishmania intracellular stage of [81]. membrane, sitamaquine accumulate in cytosolic Despite the significant progress that has been made acidic compartments, acidocalcisome. However, correlation [71] during last few years in chemotherapies for leishmaniasis, between its action and accumulation is not clear . There is all drugs of this regimen have some limitations like price, transient affinity between sitamaquine and membranes and feasibility, safety, efficacy, toxicity, side effects and also energy dependent efflux was demonstrated, suggesting probability of growing resistance. The most important [70] the presence of an uncharacterized transporter . Although, consideration of present therapeutics is gradual increase in resistance against this in drug vitro has not been reported Leishmania yet in resistance to antimonials in particular and others in general, clinicaldonovani practices but, resistance against predominantly in Indian subcontinents. In recent years, promastigote has been reported by selecting drug clinical trials of combination therapies are operational[6]. pressure of sitamaquine at 160 毺m concentration[72]. In Combination of two or more drugs could reduce treatment one of the Leishmaniastudy, conducted major on cutaneous leishmaniasis duration and drug doses and consequently drug toxicity caused by on BALB/c mice, sitamaquine but probabilities of resistance development against current dihydrochloride did not reduce the parasite burden and available drug regimen cannot be denied. The main lesion progression was continued. The lack of its efficacy combination drugs currently under consideration are LAmB and activity seriously restricted further clinical trials[73]. (Liposomal amphotericin B) and miltefosine, LAmB and However, with available status of knowledge, more studies paromomycin, LAmB and antimonials and paromomycin are required to understand its efficacy, mode of action as and antimonials in India and also being evaluated in other well as toxicity. part of the world (www.clinicaltrials.gov). There is no vaccine candidate available, and current recent progress Nisha Singh et al./Asian Pacific Journal of Tropical Medicine (2012)485-497 489 3.1. Enzymes of polyamine biosynthesis on leishmanial research does not guaranty about future availability of candidate leishmanial antigens/immunogens. Hence, identification of novel drug targets and development The putrescine, spermidine and spermine, like polyamine of true antileishmanial agents should be the priority area of play important role in growth and differentiation of parasite research. from promastigote to amastigote stage[82]. Polyamines not only involves in parasite growth and differentiation but also 3. New potential drug targets down regulates lipid peroxidation generated by oxidants compounds and makes the environment compatible for survival[83]. There is reduced polyamine metabolism Leishmaniasis is the only tropical disease, which is during initial phase of parasite adaptation in varying being treated by non-leishmanial drugs. Moreover, the environment from vector to host[84]. Parasites overexpress exact mechanisms of action of these drugs are not clearly arginase, ornithine decarboxylase, s-adenosylmethionine understood. The recent research funding from various DC WHO/TDR decarboxylase (Adomet ) and spermidine synthase, the organizations like , Tropical Medicineetc Research Centre, USA and European Commission , to disease enzymes involved in polyamine metabolism. However, endemic countries like India and Sudan, only encourage polyamine pool remains unchanged or marginally affected clinical trials and diagnostic evaluation studies. Recent during their growth and metabolism that imply existence [85] publications in leishmanial research reveals that focus of regulatory mechanisms . Any of these regulatory is being made only on drug trials/combination therapy of mechanisms offer a greater possibility for a future drug available non-leishmanial drugs, evaluation of diagnostic target.Leishmania and prognostic capability of available tools, and very little In , arginine is converted to L-ornithine emphasis is being paid on other aspects by leishmanial by enzyme arginase. L-ornithine is further converted to biologist and researchers. In the past several decades very putrescine through decarboxylation by enzyme ornithine little emphasis has been made on novel control strategies in decarboxylase, which in turn converted to spermidine and terms of new drug targets and vaccine candidates. spermine, the substancesLeishmania responsible for cell growth and Search of new potential drug targets mainly focus on proliferation of as well as Th2 type response biochemical and metabolic pathways essential for parasite that is responsible for pathogen survival in mammalian survival (Figure 1). The target enzymes of these pathways host[86]. Being first step of polyamine metabolism, targeting should have significant structural and functional difference arginase pathway will be highly beneficial. Inhibitors of from its mammalian counterparts for selective inhibition polyamine biosynthetic pathway have shown antileishmanial of target sites. Further, strategies to target more than one activity. Adomet DC inhibitor cures animal leishmaniasis enzyme of a metabolic pathway simultaneously may prove but have not been tested on humans and seeks further more usefulness and effectiveness. experimental studies[87]. However, failure of alpha- difluoromethylornithine, a polyamine inhibitor used to treat trypanosomiasis, to cure leishmaniasis proves some differences between two closely relatedLeishmania parasites. Therefore, more efforts are needed to discover specific [88] polyamineLm inhibitors . The polyamine transporters ( POT1) that transport both putrescine and spermidine are also good targets that regulate the intracellular polyamine level. Hence, development of inhibitors to stop polyamine biosynthesis and transportation may be quite useful as novel antileishmanial therapies. 3.2. Peptidases

Peptidases are increasingly being seen as potential drug Figure 1. Leishmania targets[89]. Therapeutically, peptidase inhibitors have An overveiw of potential drug targets in species. HIV The coloured boxes represent the potential drug targets. ODC: been successfully introduced to treat , hypertension, 154 ornithin decarboxylase, SpdS: spermidine synthase, TR: trypanothion pancreatitis and multiple myeloma. Leishmania A total of major peptidases reductase, TDPX: trypanodoxin peroxidase, DHFR: dihydrofolate were found to be present in the genome, rudutase, DH: dihydrofolate, TH: tetrahydrofolate, DHFR-TS: with example of serine, cysteine, aspartic, threonine and dihydrofolate rudutase thymidylate-synthase, M-THF: methylene tetrahydrofolate, dTMP: deoxy thymidine monophosphate, dUMP: metallopeptidases.Leishmania Twomajor aspartic peptidases were found deoxy uridine mono phosphate, MAPKKK: mitogen activated proteins in the genome sequence, one with kinase kinase kinase. MAPKK: map kinase kinase, MAPK: map sequence similarity to presenlin1 (PS1), which is a multipass kinase, CDKs: cyclin dependent kinase, NT: nucleotide transporter, membrane peptidase and the other with intramembrane FT: folate transporter. MT: mitochondria, ER: endoplasmic reticulam. signal peptide peptidase (SPP)[90]. PS1 is potentially involved

in autophagy while SPP cleaves the transmembrane domain Nisha Singh et al./Asian Pacific Journal of Tropical Medicine (2012)485-497 490 of signal peptidase that may be vital drug target[91]. in mammalian counterpart, so it could be used as effective Twenty-oneLeishmania threoninemajor peptidases have been found in drug target. the genome, though all are classified as Moreover, inside glycosome first seven steps of proteasome subunit[91]. The proteasome is a multisubunit, glycolysis occur whereas rest three occurs in cytosol. multicatalytic peptidase responsible for degradation Enzymes hexokinase (HKK), phosphofructokinases (PFK) of ubiquitinated proteins in the cytosol[92]. Similar to are autocatalytic enzymes and their hexose phosphate plasmodialLeishmania and trypanosomatids proteasome, the proteasome intermediates accumulation are lethal for parasite, thus of is a potential therapeutic target, as the use of in trypanosome unique mechanism has been evolved to specific inhibitorsLeis hashmania shown maxicana the proteasome to be necessary escape this lethality. The first seven steps of glycolysis have ATP for growth ofin vitro promastigotes and no net gain of and rest steps occur in cytosol and that amastigotes [93]. result in ATP production. This unique compartmentalization HXK PFK The cysteine peptidase of theLeishmania papain family are regulates and autocatalysis, which depend on extensively studied peptidase of , comprising ATP[99]. These glycolytic enzymes are essential for parasite the cathepsin-L like peptidases CPA and CPB, and the survival and enzymes of glycosomes may be targeted due to B CPC PTS1 PTS2 cathepsin- like peptidase , which are Leishmanialysosomal. presence of unique targeting signal sequences, & CPA CPC N C Neithermaxicana nor are essential for survival of present either on -terminal or -terminal end of these in the host but, CPB has been found to be a enzyme. If the targeting sequences are disrupted may lead virulence factor and may be targeted in initial phase of to mistargeting and degradation of essential enzymes of disease[94]. In addition, the use of an inhibitor specific to glycolysis that creates lethality[100]. Thus unique organization cathepsin-L like cysteine peptidase, K11777, has shown that of glycolytic pathway and evolutionary distance from these peptidases are necessary for the growth of parasites mammalian host posing leishmanial glycolytic intermediate that indicates potential of the peptidase as a drug target[95]. enzymes could be potential drug targets, however due to Among several serine peptidases of protozoan, subtilisin- conserved glycolytic machinery single enzymatic inhibition like serine peptidase, a part of the secretary endosomal will not be sufficient to marshal with parasite survival or system, which participate in processing of secreted death. proteins may be very useful as drug target. PlasIt hasmodium been 3.4. Enzymes of thiol metabolic shown that the subtilisin- like peptidases of [96] are potential drug targets . Investigation of theLeishmania effect of Leishmania serine peptidase inhibitors on the survival of The parasite survives and proliferates in has shown that TPCK(N-tosyl-l-lysyl-chloromethylketone) hazardous environment of macrophage in mammalian and benzamidine both reducesLe ishmaniaviability amazonensisand induce host. This remains a puzzle that how parasite escape morphological changes in the from reactive oxygen and reactive nitrogen species that is promastigotes, suggesting serine peptidases could be useful generated by host as immune effectors mechanisms against potential drug targets[97]. parasite. Recent reports indicate that parasite has developed 3.3. Enzymes of glycosomal machinery unique defense mechanism to marshal respiratory burst activities of macrophages. Cascades of three antioxidant Leishmania enzyme of trypanothion metabolism are required to like other trypanosomatids depends solely counteract mammalian antioxidant glutathione metabolism. on their host for carbon source to fulfill its energy Trypanosomatids contain trypanothion [T(SH)2], a dithiol requirements. The amastigotes uptake blood glucose from instead of a glutathione as a main reductant and replaced mammalian blood stream and other essential component the ubiquitous GSH/glutathione reductase redox couple with like fatty acids, amino acids receives from phagolysosome of its T(SH)2 /trypanothion reductase(TR) system[101,102]. Why macrophages. In trypanosomatids glycosomes, peroxisome T(SH)2 is evolved inside parasitic system remain an enigma. like organelle, are found. These glycosomes plays important TR is a flavoenzyme that reduces thioredoxin and role in many metabolic activities like glycolysis, oxidation tryparedoxin and some short chain protein like dithiol. of fatty acid,etc lipid biosynthesis, and purine salvage These reactions delivers reducing equivalent to peroxidase pathways . Due to the result of these metabolic activities for detoxification of toxic radicals produced during superoxide radicals are generated as side products in large hydroperoxide and deoxyribonucleotide synthesis. amount. To protect glycosomal enzymes from superoxide Trypanothion enable metabolic pathways by maintaining low SOD radical toxicityLeishmania Fe-superoxide dismutaseLeishmania (Fe chagasi's ) are molecular mass thiols trypanothione and monoglutathionyl evolved in species. In two i.e.spermidine, glutathiol and ovathiol in their reduced state different types of FeSOD, Lcfesodb1 and Lcfesodb2 have important for the uninterrupted progression of metabolic [103] been characterized, which are differentially expressed in pathwaysTrypanosoma. brucie Leishmania infantum stationary promastigotes, amastigotes and early logarithmic In and it is promastigotes stage, respectively. These are responsible reported that T(SH)2 is capable of reducing NO and Fe into for survival and protection from lethal superoxide radicals a harmless stable dinitrosyl iron complex with 600 time within glycosomes[98]. More importantly, FeSod is absent more affinity than mammalian GSH reductase system, that Nisha Singh et al./Asian Pacific Journal of Tropical Medicine (2012)485-497 491

NO MAPK protects parasites from potentially lethal nitric oxide ( ) s are not only important to amastigotes but also in molecule. The absence of this pathway in mammalian host forvitro promastigotes. The direct evidenceLeishmania came major from the and trypansomatids sensitivity towards oxidative stress, study on overexpression of MAPK trypanothione reductase and enzymes of trypanothione shows stage specific phosphotransferase activity and [113] metabolism are an attractive drug targets for antileishmanial Leishmania accumulation in axenicLeishmania amastigotes not in promastigotes . [104] MAPK infantumdrug designing . Homology modeling of Identification of specific sequences of TR , and mammalian glutathione reductase show and theirLeishmania targeting offers great probability for an effective remarkable difference in their three dimensional and drug. MAPK inhibitors should have same effect catalytic active sites. Hence specific inhibitors designed on amastigote survival as in case of gene deletion. Hence against TR may be an ideal drug that will stop parasite efforts are required to identify leishmanial MAPK related growth without altering host glutathion reductase (GR) targets. These kinases may be potential drug target not activity. only for the development of novel drug regimen but also for 3.5. Cyclin dependent kinases therapeutic immunomodulation. 3.7. Enzymes of sterol biosynthesis

Cyclin dependent kinases (cdks) play crucial role in cell Leishmania division cycle, transcription, apoptosis and differentiation. In species the main endogenous sterols are 10 The Leishmania genomic analysis hasLeishmania reported major orthologous cyclin ergosterol and stigmasterol, which differs from mammalian in species. In one additional counterpart cholesterol, therefore can be used as potent mitotic like cyclin, CYCA is also found[105,106]. Cdk requires drug targets. Ergosterol has two important functions: first, active cyclin for its own activation while few cdks requires it is a structural component of cell membrane and second, activation by the phosphorylation for their functional it might play hormonal role. AmB sounds strongly against activity. This phosphorylation could be achieved by cdc2 leishmaniasis because of sterol based mechanism of action, activating kinases, at their conserved threonine residues but AmB resistance for leishmaniasis dictates researchers (t-loop)[107]. Cdk related kinase3 (CRK3 gene) encodes cdc2 to find an alternative drug of AmB deliberately. Azasterols, L related protein kinase with activity matchingLeishmania to eukaryotic a known class of s-adenosyl- -methionine, show H1 CRK3 G2 M 24 histone Leishmania. is active mexicana at / phase of cell antileishmanial activity and inhibits -methyltransferase, cycle. In , disruption of CRK3 leads to which is a vital enzyme in ergosterol biosynthesis[114]. change in cell ploidy though it was avoided when extra copy Other sterolsLeishmania like azol and triazole are also effective CRK3 CRK3 14 14 of was expressed from episome ensuring that is against , whichLeishmania inhibits 毩-methylsterol - essential[108]. The chemical inhibitors of CRK3 impair the demethylase. However, has potential to survive parasite viability within macrophage, thus validating CRK3 in altered sterol profile, and also have ability to utilize as potential drug target. The most potent inhibitor of CRK3 and metabolize host sterol[85]. This consideration must belongs to indirubin class, which providesLeishmania pharmacophores donovani be accounted during novel drug development. The most for further drug development[105]. In , it appropriate way will be the inhibitors of sterol biosynthesis was recently shown that glycogen synthase kinase (LdGSK3) with other metabolic pathway combined together. Along with is also involved in cell cycle control and apoptosis based on the combinational therapy, to find some another potential indirubin test[109] exploiting the LdGSK3 as potential drug sites in sterol biosynthetic pathway that can be used as target in combination with CRK3. Likewise, other cdk may attractive drug targets. also be explored as possible targets. 3.8. Dihydrofolate reductase (DHFR) 3.6. Mitogen activates proteins kinases (MAPK) DHFR is a key enzyme in folate metabolism, linked to the In mammals MAPK play important role in all aspects of production of thymidine[115,116]. DHFR reduces dihydrofolate immune response from initiation of innate immunity to to tetrahydrofolate using NADPH as cofactor. Therefore, activation of adaptive immunity. In addition, they also inhibition of DHFR prevents biosynthesis of thymidine DNA regulate cell differentiation, proliferation and apoptosis. and as a consequence,Leishmania major biosynthesis. Trypanosoma Fortunately, cruzi this MAPK receiver molecules, receive external stimuli and enzyme from and have signals, and through cascades of intermediates regulate been crystallized and the structural data may be exploited transcriptional, proliferativeLeishmania mexicanaand differentiation status to observe structural difference between parasite and [110] 15 MAPK DHFR of a cell . In Leishmania mutant, have human enzymes that may help to design selective been identified[110,111]. lacking MAPK inhibitors[117,118]. An approach to discover novel parasite gene has shown their significance in transformation and DHFR inhibitors using database mining has also been made cellular growth. The MAPK gene deleted promastigotes to search the Cambridge structural database but DHFR as after differentiation into amastigote lose proliferative drug target requires further attention[119,120]. In addition, capacity, also peritoneal macrophages were able to cope enzyme dihydrofolate reductase-thymidylate (DHFR-TS) with infection, which their importance for amastigotes[112]. that catalyzes conversion of dihydrofolate from methyhylene Nisha Singh et al./Asian Pacific Journal of Tropical Medicine (2012)485-497 492 tetrahydrofolate(M-THF) and thymidine, has been shown Molecules that can target to metacaspase biosynthetic to be related to parasite survival and parasite lacking this machinery and induces their early expression might prove as enzyme are not able to survive in animals[121]. Inspite of efficient antileishmanial agents. In addition, since they are these advantages several potential resistance mechanisms required for chromosomal segregation and parasite survival, to DHFR have been discovered including over-expression of they can also be directly targeted[132]. However, more the enzyme DHFR-TS and enzyme ptrI[122,123]. The enzyme studies are required to understand the complete function of ptr1 predominantly involved in reduction of biopterin to leishmanial metacaspases. dihydrobiopterin and tetrahydrobiopterin but also capable 3.11. Leishmanial antigens that modulates host immune of reducing dihydrofolate to tetrahydrofolate. Hence, a functions combined strategy to target both DHFR and ptrI in will be more effective as a true antileishmanial drug. Leishmania survival and proliferation within 3.9. Topoisomerases parasitophorous vacuole primarily depends on its strategy to evade macrophage effector molecules and diverting its DNA topoisomerases are ubiquitous enzymes needed to immune response. Macrophage arsenal is full of its potent remove torsional stress in DNA by introducing transient armatures such as reactive nitrogen and oxygen species, protein-bridge DNA breaks either on one (type I) or lysozymes, and other microbicidal molecule, production both (type II) DNA strands. Topoisomerases are major of variousTh1 cytokines for parasitic inhibition[133]. On targets in cancer and bacterial chemotherapy[124,125]. In parasitic infection the chase begin, macrophage attack on parallel protozoan parasites are not distinct, they require parasite with its reactive nitrogen and oxygen molecules topoisomerase specially topoisomerase II due to the as a result of induction of respiratory burst activity, while presence of complex intercatenated network of thousands parasite has to defend itself for its survival, it has to of minicircles as well as maxicircles in kinetoplastids produce something that will counterattack and suppress mitochondria. Topoisomerase II has been reported to be this activity or develop a strategy to skip from this attack. Leishmania overexpressed and shows increased activity in arsenite survival strategy includes: (1) its glycoprotein Leishmania donovani resistance [126]. The topological (gp63: a metalloproteinase) facilitates binding of metacyclic problem associated with mitochondrial DNA presents promastigotes to macrophages via CR3 receptor without potential sites for topoisomerase II activity. Antibacterial eliciting oxidative burst in macrophages, (2) It inactivates and anticancerous drugs like novobiocin, etoposide, and complement components C3, C5, C9 by phosphorylation, fluoroquinolones can be used to target topoisomerase II in (3) The parasitic superoxide dismutase scavenges reactive order to inactivation of genetic integrity and cell survival[127]. oxygen intermediates, (4) It suppresses iNOS expression of More efforts are required towards topoisomerase targeted host macrophages, and (5) It induces disease exacerbating drug interaction and development of anti-topoisomerase Th2 cytokines production such as TGF-毬 and IL-10 and Leishmania viz. chemicals against drug resistant parasites. suppresses the production of Th1 cytokines IL-4, IL- etc Leishmania 12 [134]. Further, parasite also alters various 3.10. Metacaspases signaling cascades by induction of PTPase activity of host leading to down regulation or inactivation of various Metacaspases are orthologous to caspases that play signaling cascade intermediates like NF-毷毬, AP1, STAT1 etc crucial role in apoptosis but imperfectly understood in that ultimately lead to the down regulation RNI, ROS, protozoa. It has been found that metacaspases may be Nramp1 and other immune responses of macrophage[135]. possible candidates to induce programmed cell death One of the other survival strategies is the down regulation Leishmania donovani in trypasomatids[128]. In two or dysfunctioning of ion transporters recruited on the metacaspases: LdMCA1 and LdMCA2 are reported showing cell membrane of phagolysosomes. The divalent cationic 2+ 2+ 2+ 98% homology with each other and contain characteristic transporters creates Fe , Mn , Zn deprived environment C terminal proline rich domain and both are expressed in inside the phagosomes by pumping them out. These ions promastigotes and amastigotes form[129]. A metacaspase from specifically iron is required for various crucial processes Leishmania major Lm ( jMCA) has found to be essential for like antioxidant defense (Fe-Sod), mitochondrial respiration the proper segregation of the nucleus and kinetoplast[130]. and DNA replication that are required for parasitic growth Leishmania major Leishmania Metacaspase gene of is actively expressed and survival[136]. The parasite effectively down in amastigotes and procyclic promastigotes but surprisingly regulates or blocks the activity of these transporters but the at a lower level in metacyclic promastigotes. Metacaspases mechanisms are not known. However, a divalent cations Leishmania in , on treatment with H2O2 trigger process of transporter natural resistance associated with macrophage programmed cell death of parasites. It has also been found protein1 (NRAMP1) now referred as SLC11A1 that belongs to that parasites, which over express metacaspases are more a divalent cations transporter solute carrier family, play an sensitive to H2O2 induced programmed cell death[131]. important role in host resistance against diverse pathogenic Nisha Singh et al./Asian Pacific Journal of Tropical Medicine (2012)485-497 493 Salmonella Mycobacterium organism such as , and priority research areas to combat disease progression Leishmania by maintaining cations deficient environment worldwide. inside the phagolysomes[137]. NRAMP1 is exclusively expressed on professional phagocytes and pumps out cations, Conflict of interest statement required for parasitic growth and survival, from phagosomal milieu to cytosolic compartment of macrophage (Figure Leishmania 2). The parasite expresses iron transporter ZIP We declare that we have no conflict of interest. 2+ family one (LIT1) transporter, which transport Fe inside [138] parasite . It seems that some parasitic proteins, soluble, Acknowledgements surface or excretory-secretory, modulate Nramp1 pump function during leishmanial pathogenesis. These leishmanial antigens might work as potential drug targets but they are The financial supports received from Department of needed to be identified and characterized for development Biotechnology, New Delhi (BT/PR11177/MED/29/99/2008) is of drug and vaccine candidates. greatly acknowledged. This article is dedicated to all those poor people who lost their lives during drug trials in various parts of the world.

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