Current Bioactive Compounds 2007, 3, 00-000
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Targeting Amoebiasis: Status and Developments A. Azam1,* and S.M. Agarwal1,2 1 Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi- 110025, India. 2Bioinformatics Centre, School of Information Technology, Jawaharlal Nehru University, New Delhi- 110067, India
Abstract: Amoebiasis, a disease caused by Entamoeba histolytica, remains a major health problem that afflicts several million people worldwide. Moreover, in recent years there has been a rise in the number of reports with amebic brain abscess as well as in developed countries the microorganisms that cause diarrheal diseases are a cause of concern because of their potential to be used as bioterrorist agent. Metronidazole, an antiamoebic agent, is the drug of choice for treating amoebiasis in humans, but it has been shown to be both mutagenic and carcinogenic in bacteria and rodents respectively. The completely safe treatment for this disease does not exist at present and therefore newer and safer agents are required either from synthetic or natural resources. This review covers brief description of the disease, plant secondary metabolites and synthetic compounds that have exhibited moderate to high activity in vitro and in vivo bioassays against E. histolytica. The review also discusses some of the key biochemical targets that are unique and vital for the existence and growth of the parasite which are being further exploited so as to search for therapeutically active antiamoebic agents.
INTRODUCTION Protozoan parasites are among the most common pathogens in the world and are recognized as the causative agent of various tropical diseases in both man and domestic animals. Malaria, amoebiasis, toxoplasmosis, trypanosomiasis, and leishmaniasis are diseases caused by protozoan parasites that affect approximately 25% of the world’s population, mostly in developing countries causing loss of lives and productivity [1-2]. Entamoeba, the human enteric parasite has two species, the potent pathogenic “Entamoeba histolytica” and the non-pathogenic “Entamoeba dispar” [3]. Amoebiasis, a disease caused by ingestion of contaminated food or water containing cysts of E. histolytica, an early-branching protozoan eukaryote remains one of the major threats to public health in most parts of the globe. It infects over 50 million people per annum leading to 50,000 to 100,000 deaths annually [4]. In Bangladesh alone, one in thirty children die of diarrhea or dysentery [5]. Also in Mexico and Brazil a high frequency of incidence of amoebiasis has been reported by various investigators [6-7]. Prevalence of this disease is more common among populations in regions with poor sanitary conditions and contaminated drinking water [8] making it the second leading cause of death among parasitic diseases [4]. Moreover in recent years there has been a rise in the number of reports with amebic brain abscess [9-13] as well as in developed countries the microorganisms that cause diarrheal diseases are a concern because of their potential to be used as bioterrorist agent [5]. During the last 50 years a number of compounds have been isolated and/or synthesized that have been screened in vitro against E. histolytica as a result of which numerous anti-amoebic compounds have been identified. Many of them are being used in medical practice and in certain cases it became necessary to use them in combination of two or more drugs for treatment [4-5, 14]. Chemically they are derivatives of imidazoles, alkaloids, furan and quinolines. The first drug was emetine (1) but severe side effects limited its clinical use [15]. The agents used to cure amoebiasis are classified as tissue amoebicides and luminal amoebicides. Tissue amoebicides such as metronidazole (2), tinidazole (3) and emetine kill amoeba in host tissue and organ, whereas the poorly absorbed luminal amoebicides are active only in the intestinal lumen. The luminal agents currently used are iodoquinol (4), diloxanide furoate (5) and paromomycin (6). Nitroimidazoles derivatives, particularly metronidazole, tinidazole and ornidazole (7) are the main synthetic *Address correspondence to this author at the Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi- 110025, India; E-mail:
[email protected] 1573-4072/07 $50.00+.00
CH3O N
CH3O
N
H
H
C2H5
H
H
O2N
OCH3
NH
CH3
N
CH2CH2OH 2
OCH3
1
I
N O2N
CH3
N
I
N OH
CH2CH2SO2CH2CH3 3
4 Cl
CH3 N
O O
Cl O
O 5
CH2OH OH
O O NH2
OH
CH2OH O
NH2 OH
OH
O O
NH2
NH2
O
OH
OH
NH2 N O2N
6
N
CH3
CH2CH(OH)CH2Cl 7
agents of therapy for invasive amoebiasis [4-5]. The highly selective effect of these drugs is due to reduction of these drugs by nitroreductase enzymes resulting in the formation of highly reactive free radical species [16-17]. In approximately 90% of cases,
© 2007 Bentham Science Publishers Ltd.
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patients presenting with mild to moderate amoebic dysentery respond to nitroimidazole treatment, the drug of choice. Yet in 40 to 60% cases it is required that nitroimidazole administration be followed with paromomycin or the second line agent diloxanide furoate to take care of luminal infection as parasite is found to persist in the intestine [18-20]. Treatment with nitroimidazole too has several side effects that include nausea, vomiting, dry mouth, metallic taste, abdominal pain and headache. In some cases additional side effects viz. dizziness, vertigo, paresthesias and rarely encephalopathy or convulsions have been reported leading to even discontinuation of the drug. Occurrence of neutropenia associated with metronidazole has been shown to be temporary and reversible on discontinuation of the drug [4-5, 14, 21]. Although there is no carcinogenicity or mutagenicity of metranidazole in human beings [14], it has been shown mutagenic in bacteria and carcinogenic in rodents [22-24]. In addition, the drug recipient’s exhibit lower immune response both cell and humoral mediated [25]. Moreover there is possibility of developing drug resistance as demonstrated in other protozoan parasites [26]. Some recent reports also have described the in vitro generation of strains resistant to metronidazole [27-28].
Azam and Agarwal
OH HO
OH OH
O 8 OH
HO
O
OH
O OH
O O
O O
OH OH
HO 9
OH
NATURAL PRODUCTS The first drug used for the treatment of amoebiasis was emetine, isolated from the root and rhizome of Cephaelis ipecacuanha [15]. Literature survey reveals that extracts from traditional preparations are used in popular medicine in various countries like India [29], Phillipines, Europe, South Africa [30-31], Mexico [32-33], Cameroon [34], Sierra leone [35] and Congo [3637]. In the following section the authors discuss the bioactive molecules shown to have significant antiamoebic activity from early ninetees to 2005. FLAVONOIDS The significant in vitro antiamoebic activity of natural flavonoids like kaempferol (8), tiliroside (9) and quercetin (10) on E. histolytica was reported for the first time in 1995 [38-39]. In a systematic manner several flavonoids which differed in the number, nature and allocation of the oxygen bearing subsituents in both the rings (A and B) as well as in the presence or absence of a 2,3double bond, a hydroxy functionally at C-3 and a 4-keto group in ring C were studied [40]. Among the compounds analyzed (-)epicatechin (11), (-)-epigallocatechin (12) and kaempferol were detected to be the most potent flavonoids against Entamoeba. The structure-effect correlations study revealed that the 2,3-double bond and the 4-keto group of ring C is not essential for high antiamoebic activity. Rubus corrifolius used among the Maya people to treat bloody diarrhea was investigated resulting in isolation of seven known compounds including two flavonoids (-)-epicatechin and (+)-Catechin (13) [41]. Further from the roots of Geranium mexicanum HBK, which is an endemic species used in Mexican traditional medicine as purgative, and as a remedy against tonsillitis, whooping cough, urticaria, dysentery and diarrhoea, (-)epicatechin, (-)-Catechin and Tyramine were isolated [42]. However, the activity of the extract was seven times lower than the Rubus corrifolius extract in spite of the presence of tyramine, a compound shown to be antiamoebic for the first time. From the root of Geranium niveum two new A-type proanthocyanidins, epi-afzelechin-(48,2O7)-afzelechin and epi-catechin-(48,2O7)-afzelechin (Geranin A and B) and five known compounds were isolated. Of these the most active antiamoebic agent was Geranin B (14). It was shown that these compounds do not display cytotoxic activity when tested on three different cell lines (MCF-7 breast carcinoma, HT-29 colon adenocarcinoma and A-549 lung carcinoma) [43]. Later on, another two new compounds Geranin C and Geranin D were isolated from
O
OH HO
O OH OH
O 10 R4 R5
R3
O
R6 R1
R2 11: R1 = OH(a), R2=R3=R4=R5= OH, R6 = H 12: R1 = OH(a), R2=R3=R4=R5=R6 = OH 13: R1 = OH(b), R2=R3=R4=R5= OH, R6 = H
OH OH HO
O OH O OH O
OH
OH
HO 14
the root of Geranium niveum belonging to A-type proanthocyanidins [44]. Of these Epi-afzelechin-(48,2O7)-afzelechin(48,2O7)-afzelechin (Geranin D) (15) showed moderate antiprotozoal activity against E. histolytica. It is observed that simple flavonoids like catechin, epigallocatechin, kaempferol to
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OH HO
O
N
N H H
H
OH O
H
OH O
HO
OH
17
OH
OH O
H CH2 N
H3C H N
H3CO
OCH3
O OH
H3C
N
OH
H
N
O
H
CH3
OH
O
15
HO
more complex ones like geranin B and tiliroside have antiamoebic activity but the potency varies significantly in vitro. Moreover mechanism of action of these compounds is generally not known. ALKALOIDS Alkaloids have been well investigated for their pharmacological properties over the years [45]. The in vitro antiplasmodial, antiamoebic, and cytotoxic activities of alkaloids isolated from Strychnos usambarensis have been reported [46-48]. Strychnos usambarensis was used for isolation of number of alkaloids which were then assessed for in vitro activity against E. histolytica. Of these usambarensine (16), usambarine (17) and 18,19-dihydrousambarine oxalate were found active in vitro. Although the mode of action of usambarensine and usambarine is not yet known but it has been proposed that they act as protein inhibitors in view of their structural similarity to tubulosine [49]. Usambarensine is reported to act as DNA intercalator and thus induces apostosis [50]. In another study twenty-four bisbenzylisoquinoline alkaloids were synthesized and in vitro screened for antiplasmodial, antiamoebic, and cytotoxic activities. Only three alkaloids were found active against E. histolytica, aromoline (18), isotrilobine (19), and insularine (20), with none being significantly cytotoxic [51]. Bisbenzylisoquinoline although was demonstrated to have antiamoebic activity but its potency was demonstrated to be lower than of Usambarensine and their mechanism of action remains uninvestigated.
18 O H3C
N
H3CO N
O
H
H
CH3
O OCH3 19
OCH3 H3C
N
OCH3
H
O
H NCH3
O CH2
H3CO
O 20 H N
O
N
N H H
Cl 21
H N
H CH3 N
16
CARBAMIC ACID DERIVATIVE Carbamic acid is an acid that is known by its salts as ammonium carbamate or as its esters (urethane). Recently, antiamoebic and toxicity studies of a carbamic acid derivative, Ethyl 4chlorophenylcarbamate (21) in addition to its therapeutic effect in a hamster model, showed good activity against axenic culture of E. histolytica and significantly reduced the development of amoebic liver abscess in hamsters [52].
FATTY ACID DERIVATIVES Alkylphosphocholines (alkyl-PC) such as hexadecyl-PC (miltefosine) developed originally as antitumour agents have been used successfully for the treatment of visceral leishmaniasis in humans [53]. Of the several alkyl-PCs examined for their possible biological activity against two strains of Entamoeba SFL-3 and HM-1:IMSS, oleyl-PC (22), octadecyl-PC (23), and nonadecenylPC (24) showed highest activity with 50% effective concentrations for 48 h of treatment [54]. While they did not study the mechanism of biological action, they did observe that high concentration of oleyl-PC caused destruction of amoeba surface in the local area. This is similar to the effects of morphological changes observed in case of hexadecyl-PC treated tumor cells. It led them to put forward the view that inhibition of phospholipid biosynthesis and signal
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Current Bioactive Compounds 2007, Vol. 3, No. 1
O R
O
P
Azam and Agarwal
PO3H2
CH3 O
CH2
CH2
N
N
NH
PO3H2
CH3
O
PO3H2
NH
CH3
PO3H2 Br
where R = 22, 23, 24
Br 30
22
29
23 24
transduction can be the possible mechanism of action of these compounds. COUMARINS A coumarinic acid derivative melilotoside (25) was isolated from Teloxys graveolens. This exhibited the most potent activity towards E. histolytica supporting the traditional use of the plant in some gastrointestinal diseases in Mexicans [55].
OH HO
O
HO
O
O
MISCELLANEOUS Diphenyl bisamidines with various substituted groups had been synthesised and tested for antiamoebic activity in Golden hamsters and Wistar rats [57]. Three compounds having subsitution at R1-R 8 (31-33) showed excellent in vivo activity against hepatic infection in the hamster with respect to the standard drugs although they had displayed less pronounced in vitro activity. Under the trade name of Liroldine the compound 31 is registered [58].
OH
R4
OH
R5
25
PO3H2
CH3
OH 4
R7
R2
R3 R3
R2
2
R7
OH
CH3
H3C H3C
OH HO 34
OH
PO3H2
OH
OH 2
R6
The anti-amoebic activity of four compounds maackiain and formononetin and chlorophorin and iroko isolated from plant extracts of Virgilia oroboides and Chlorophora excelsa were evaluated respectively. The anti-amoebic test conducted on trophozoites of E. histolytica established that all four compounds had effect with chlorophorin (34) showing highest activity followed by maackiain (35) and iroko. Out of these chlorophorin was found to be non mutagenic while maackiain showed mutagenicity. None
O
PO3H2 N
PO3H2
R5
31: R1 = F, R2=R3=R4=R5=R6=R7=R8 = H 32: R1 = F, R2=R3=R5=R6=R7=R8 = H, R4 = CH3 33: R1 = NO2, R2=R3=R4=R5=R6=R7=R8 = H
27
O
R4
R8 N
N
OH 2
R1
N
PO3H2
N
PO3H2 26
R1
R8 N
R6
BISPHOSPHONATES Effect of series of bisphosphonates has been studied on the growth of E. histolytica in vitro and in vivo [56]. As many as fortyseven compounds belonging to different classes showed activity against E. histolytica in vitro. The most active compounds were nitrogen-containing bisphosphonates with relatively large aromatic side chains. Several pyridine-derived bisphosphonates were quite active and this activity was shown to correlate with the basicity of the aromatic group, with activity decreasing with increasing pKa values. A series of five compounds described having good IC50 and therapeutic index (TI) values were then selected for in vivo testing in a hamster model of E. histolytica induced liver abscess formation. The compounds chosen were alkyl bisphosphonate (26); N-[methyl(4-phenylbutyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonate (27), a known potent bone resorption drug; the phenoxyethyl analogue of N-Me pamidronate (28); a biphenyl aminomethylene bisphosphonate (29) and the dibromopyridyl aminomethylene bisphosphonate (30).
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Surprisingly, N-Me pamidronate, found most active in vitro, was ineffective in vivo. The maximum decrease in liver abscess formation was reported with compound 27. In contrast, the alkyl bisphosphonate showed a 68% reduction in liver abscess formation but only a 9% reduction in normal liver weight while the biphenyl showed a 36% abscess reduction with an insignificant change in normal liver weight. These were promising initial in vivo results which caused reduction in liver abscess formation and suggest that alkyl bisphosphonate (26) and biphenyl aminomethylene bisphosphonate (29) may represent useful new leads for the development of antiamebic drugs.
O
H
O
O H
28
35
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Current Bioactive Compounds 2007, Vol. 3, No. 1
of the compounds showed cytotoxic effects on any of the cell lines [59]. Based on the chlorophorin’s lower MIC than that of metronidazole, release of acid phosphatase and reduction in the level of alpha amylase, it was suggested that this compound could become a potential antiamoebic drug in future. Furthermore, the mechanism of action was studied and it was demonstrated that like other aromatic phenols, it too destructs trophozoites of E. histolytica, which was dose dependent, and the destruction began with disintegration of nucleus. Oryzalin (36) a member of the dinitoaniline class of drugs is known to be a potent herbicide. The herbicidal action is due to their ability to disrupt microtubules and therefore cell division [60]. These drugs have been effective against parasitic protozoa including Leishmania species [61-62], Trypanosoma brucei, Plasmodium falciparum, Toxoplasma gondii [63] and Cryptosporidum parvum [64]. It has been shown that the growth of both E. histolytica and E. invadevs is inhibited by Oryzalin as it arrests the cell cycle in the mitotic phase [65-66]. H3C
H2C
H2C
N
CH2
O2N
CH2
H O2N
C
S
N
HN
O2N
C
O HN N
C2H5
H S
Cl
N
Cu N
Cl
S
C2H5
OH
O
C
S
SO2NH2
HN
O
CH2OH
NH NO2
39
36
S
N
H
O2N
NOSO3
Cl
S 38
NO2
HO
Cl Pd
N
CH3
Benzyl glucosinolate (37) isolated from the roots of Lepidium virginicum (Brassicaceae) has been shown to be the major antiamoebic compound [67] thus supporting the ancedotal reports of the traditional use of L. virginicum roots in the control of diarrhoea and dysentery.
5
H2C
N
N
H Cl Ru
S
Cl
40
of Mannich bases with thiosemicarbazides of variegated nature [79]. This study was undertaken as pyrazoles and their reduced form are well known nitogen containing heterocyclic compounds known to possess antibacterial, antiinflammatory, antihypersensitive, anticonvulsant, antidepressant properties [80-83]. The results revealed that 3-chloro (41) and 3-bromo substituents on the phenyl ring at position 3 of the pyrazoline ring enhanced the antiamoebic activity as compared to unsubstituted phenyl ring.
OH 37
THIOSEMICARBAZONES Thiosemicarbazones, a very promising class of compounds that show a broad spectrum of therapeutic properties viz. antibacterial, antimalarial, antiviral, antitumor etc, against a range of diseases models [68-69]. Many thiosemicarbazones such as marboran and triapine are already used in medical practice [70]. Over the years we synthesized range of thiosemicarbazone derivatives that exhibited potent antiamoebic activity in vitro [71-78]. Many of the synthesized N-substituted thiosemicarbazone derivatives showed remarkable antiamoebic activity, comparable or superior to metronidazole, the reference drug. Also thiosemicarbazone coordination complexes with different metals like Palladium II, Copper II and Ruthenium II were synthesized. Antiamoebic activity of these compounds was also determined to ascertain their effecttiveness in comparison to their corresponding thiosemicar-bazones. It was observed that a number of these complexes (38, 39, 40) possess noteworthy potencies against E. histolytica in vitro. PYRAZOLINE THIOSEMICARBAZONES In another study we carried out synthesize of 1-N-substituted cyclised pyrazoline analogues of thiosemicarbazones by cyclisation
N
N
N
CH2CH2CH3 CH2CH2CH3
S Cl
41
TRIAZINE Triazines, a class of heterocyclic compounds have been demonstrated to have diverse biological activities [84-88]. Besides, they are proved to be excellent drugs against trypanosomiasis [89] and are also used in cancer chemotherapy [90]. A series of 3,7dimethyl-pyrazolo[3,4-e][1,2,4]triazin-4-yl thiosemicarbazide derivatives were prepared and evaluated in vitro against E. histolytica. Screening led to identification of four compounds that demonstrated better inhibition than metronidazole [91]. The compound (42) that demonstrated the most promising antiamoebic activity is shown. METRONIDAZOLE DERIVATIVES Metronidazole is widely used alone or in combination with other antimicrobial agents to treat protozoan diseases [92]. It is an
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H3C
N N
N
N
N
Azam and Agarwal
CH3
NH C
S
N
H
42
accepted view that on coordination of metal ions the drug action is accelerated as well the efficacy is enhanced [93-94]. The metronidazole complexes of Pd (II), Pt (II), Cu (II), Au (II) and Ru (II) showed better results than metronidazole in vitro [95-96]. The Cu-metronidazole complex (43) displayed highest in vitro activity, which is considerably superior to other metal complexes of metronidazole demonstrating that the incorporation of the copper fragment produced a marked enhancement of the efficacy of metronidazole. 2
OH
parasites that cause some of the most severe diseases. Moreover, biochemical analysis and genome sequencing, both have helped in identifying potential targets: enzymes, transporters and metabolites that are distinct in parasites and their mammalian host [99]. The completion of genome sequencing of E. histolytica is further enabling scientists to understand the parasite better as also relationship between host and parasite. As a consequence one can design even better and safer effective drugs that can be used in future to combat the disease caused by the organism. Furthermore in the present era, homology modeling along with computer aided virtual database screening has become one of the most preferred approaches for discovering newer structural scaffolds that can act as lead compounds [100]. Several drugs currently in therapeutic use, including angiotensin-converting enzyme inhibitors [101] and HIV1 protease inhibitors [102] were identified or optimized using computer assisted modeling. In the following discussion some of the most important and known Entamoeba targets and their inhibitors are summarized. CARBOHYDRATE METABOLISM In E. histolytica, an amitochondriate parasite that resides in the human intestine energy is derived from the fermentation of glucose with CO2, acetate and ethanol as the recognized end products through a process called glycolysis (Fig. 1). Glycolytic pathway is the therapeutic target of clinically used antiamoebic drug metronidazole since unlike the human host which uses oxidative
NO2 N
HO N
N N
O2N
H2O Cu
H2O
Cl Cl
Cu
NO2
N N
N
OH
N NO2 HO
43
OXIME ETHERS Oxime and its ether derivative are commonly used as antiprotozoal, anti-trichonomal, antifungal and antimicrobial agents [97]. 12 oxime ether derivatives of 2-acetylpyridine and 2-acetylfuran series were synthesised and evaluated for their antiamoebic activity [98]. Of these the most promising lead (44) from 2acetylfuran series is shown. CH3 N
C
N
O
CH2CH2
N
O
44
DRUG TARGETS Comparative biochemistry has been the conventional route to drug target identification. Enzymes, metabolites or proteins present in parasites and absent in the mammalian host, or if present then in strikingly different form, have been considered as ideal drug targets. In the recent years, many of the new drug targets are being discovered with much more ease as the genomic sequences of many parasitic organisms is becoming available. This advancement has enabled researchers not only to identify new biochemical pathways and gene families that can be short-listed as potential drug targets but also has significantly increased their basic understanding of
Legend: PPi-PFK: Pyrophosphate dependent Phosphofructokinase; TPI: Triose phosphate isomerase; PPDK: Pyruvate Phosphate Dikinase Fig. (1). Glycolytic pathway indicating metabolic enzymes proposed as target (in bold) in E. histolytica.
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respiration the amoeba are dependent upon fermentation of glucose [103-104]. In the case of Entamoeba, this pathway has been thought to be an ideal target for intervention with the long-term goal of developing antiamoebic drugs. The metabolic intermediates although in the pathway are not unique, however many of the enzymes are either absent in most eukaryotic organism or use unusual cofactors [105-106]. This section of the review presents the currently available data for the different enzymes of glycolytic pathway and the development of selective inhibitors of these enzymes. PHOSPHOFRUCTOKINASE (PFK) In most eukaryotes, an ATP-dependent PFK is present which is a key regulatory enzyme of glycolysis. E. histolytica along with a number of other parasitic protists utilizes an unusual form of phospkofucto-1-kinase (EC 2.7.1.90) in a central step in carbohydrate metabolism. This form of PFK employs inorganic pyrophosphate (PPi) as a phosphoryl donor [107-108]. This PPi dependent PFK catalyses the reversible reaction: Fructose-6-phosphate + PPi Fructose-1, 6-bisphosphate + Pi The critical role played by PFK in energy metabolism together with difference from the human host PFK, makes it an attractive target for discovering antiamoebic drugs. In Entamoeba two genes for PPi-PFK has been described. The larger gene codes for 60 kDa protein and has greater identity to more phylogenetically advanced plant PPi-PFK than it does to bacterial PPi-PFK. While the smaller gene encodes for 48 kDa protein having much lower specific activity as compared to larger enzyme [109-110]. Various bisphosphonates, which are analogues of pyrophosphate with a carbon instead of oxygen atom and synthetic pyrophosphate have been demonstrated to inhibit amoebic PPi-PFK [111]. Among these competitive inhibitors CGP48084 and zoledronate have been demonstrated to exhibit maximum inhibition. Also a homology model of E. histolytica PPi-PFK was constructed and screened against selected bisphosphonates [112]. The study suggested that two of the compounds BA49280E (45) and CGP42446A (46) act as competitive inhibitor of PPi-PFK when tested against E. histolytica, while that of S.mansoni or T.brucei both of which utilize ATP-PFk were not affected.
O
O
P
P
HO HO
HO
CH3 45
OH OH
O
O
P HO HO
P
OH OH
HO
N N 46
TRIOSEPHOSPHATE ISOMERASE (TPI) Triosephosphate isomerase is another relatively well-studied glycolytic enzyme with both its sequence and structure known in a large number of organisms including E. histolytica [113-114]. It was not only the first protein from E. histolytica whose X-ray crystal structure was characterized (PDB code: 1M6J) but also it is the first TIM whose structure has been solved at 1.5 o resolution. It has been proposed to be a target for drug design due to presence of an cysteine residue (Cys 14) at the dimer interface in Entamoeba. Thiol-specific reagents like methylmethane thiosulfonate (47) inactivates EhTIM completely and brings about its dissociation into stable monomers [114], while in contrast HsTIM which has methionine instead of cysteine in the equivalent position is largely or completely insensitive to these reagents [115-117].
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O H3C
S
S
CH3
O 47
ENOLASE Enolase (EC 4.2.1.11), the second last enzyme in glycolytic pathway, catalyses the reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate. Beanon et al. [118] characterized E. histolytica enolase gene, containing 436 amino acid with a Mol. Wt. 47.3 kDa. Although E. histolytica enolase exhibits 60% identity to rat, Drosophila and Sacchromyces Cerevisiae enolase, it was proposed that a 19 amino acid E. histolytica specific region could serve as a target for designing antiparistic drugs [119]. This region corresponds to a coil between strand 3 of beta barrel and helix 9. They also demonstrated high solvent accessibility, high protusion index and a sequence difference from human enolase, suggesting that the region between 254 and 273 might be a putative candidate for antiparasitic drug design. PYRUVATE PHOSPHATE DIKINASE (PPDK) Several glycolytic enzymes in E. histolytica utilize inorganic pyrophosphate as donor of phosphate group instead of ATP, PPDK being one of them. It catalyses the reversible conversion of AMP, phosphoenolpyruvate (PEP) and pyrophosphate (PPi) to ATP, pyruvate and inorganic phosphate (Pi) thereby generating one of the two ATP molecules generated during glycolysis [120]. Thus, PPDK, an inorganic pyrophospahte dependent enzyme substitutes pyruvate kinase present in humans thereby making it relevant as a target for therapeutic studies. The effect of some pyrophosphate analogs like tetrasodium 1-hydroxyl methylidene bisphosphonate, tetrasodium 1-hydroxyl ethylidene bisphosphonate and tetrasodium 1-hydroxynonane bisphosphonate has been tested as inhibitors [121]. Their study demonstrated that tetrasodium 1-hydroxynonane bisphosphonate is the most effective inhibitor. However, the presence of previously unidentified pyruvate kinase (PK) activity in the extracts from E. histolytica trophozoites was demonstrated recently [122]. The existence of such PK activity might overcome inhibition of PPDK and thus may support the parasitic survival under conditions in which PPDK is inhibited. More detailed efforts are likely to be made in coming years by researchers to validate PPDK as a drug target. ALCOHOL DEHYDROGENASE 2 (EHADH2) In E. histolytica, during the last 2 steps of extended glycolytic pathway, acetyl CoA is converted to acetaldehyde followed by its reduction to ethanol [123]. EhADH2, a 97 kDa, NAD and Fe2+ dependent bifunctional enzyme with both alcohol (ADH) and aldehyde (ALDH) dehydrogenase activities catalyses this reaction [123-124]. This enzyme does not have a homologue in man. However, E. histolytica possesses other NADP dependent ADH and ALDH enzymes that could serve a similar function i.e. EhADH1, EhADH3 and EhALDH1 [125-127]. Beside the presence of these enzymes it has been experimentally demonstrated that growth and survival of E. histolytica trophozoites requires EhADH2 expression [128]. Moreover, the conversion of acetaldehyde to ethanol is NADH dependent rather than NADPH [129]. EhADH2 prefers ethanol as substrate whereas EhADH1 enzyme, which is NADP dependent shows marked preference for branched chain alcohols. In addition, EhADH1 enzyme does not utilize acetyl CoA as substrate thereby suggesting that EhADH2 is solely responsible for the conversion of acetyl CoA to acetaldehyde [127]. Based on this data it has been suggested that EhADH2 could also serve as a target for discovering new antiamoebic drugs.
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Current Bioactive Compounds 2007, Vol. 3, No. 1
Azam and Agarwal
Gossypol (48), a polyphenolic compound extracted from cotton seed is a natural racemic mixture composed of equal proportions of the (+)- and (-)- enantiomers inhibits NAD and NADP dependent alcohol dehydrogenases. It has been suggested that gossypol should be active against all sites of contamination as it gets not only concentrated in the liver but also in colon and therefore has been proposed as an ideal antiamoebic lead [130-131]. CHO OH
OH
C H
CH2
CH2 S
CH2
O 51
H2N
NH2 N
CHO
HO
OH
HO H3C
H2C CH
OH
CH3 CH3 CH3
H3C
N
CH3
O
48
O
H N
OH O
In a recent study it has been shown that cycloalkanol compounds, cyclopropyl (CPC) (49) and cyclobutyl (CBC) (50) carbinols have an inhibitory effect on in vivo trophozoites growth and in vitro EhADH2 enzymatic activities [132]. Although the mechanism underlying inhibition is yet to be worked out fully but it has been proposed that EhADH2 converts both cyclic alcohols into corresponding aldehyde and thereafter attacks the active site residue of the glycolytic enzyme and affects its activity. Moreover, it has been speculated that CBC can interfere irreversibly with substrate binding of acetyl CoA and bring about inhibition of this enzyme. OH
OH 49
H
50
CYSTEINE PROTEINASES A large number of extracellular cysteine proteinase, a member of papain superfamily, are observed in E. histolytica [133]. Experimental evidence have been provided with regard to the multiple roles of cysteine proteinase in causing infection and invasion of host tissue making it as another critical target which has the potential to be used for developing inhibitors in order to treat amoebiasis [134]. While they exhibit considerable homology to the cathepsin L-like enzymes, they demonstrate cathepsin B-like substrate specificity [135]. E. histolytica trophozoites when incubated with diazopeptidyl inhibitors the growth decreased by 50% suggesting that cysteine proteinase are important for acquisition of nutrients from a liquid medium [136]. Also Mirelman reported growth inhibitory effects of allicin [S-(2-propenyl) 2-propene-1-sulfonothioate] (51), an active principles of freshly crushed garlic, on E. histolytica due to inactivation of cysteine proteinase [137]. Moreover it has been also shown that hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors like E-64 (52) and Z-Phe-Argflouromethylketone [138]. Recently, the presence of an endogenous chagasin like cysteine proteinase inhibitor in Entamoeba has been reported. Their study suggests that the conserved NPTTG motif contributes to the interaction of the inhibitor protein with active site of target protein. Thus, NPTTG based peptides have been proposed as starting point for the rational development of an antiamoebic drug [139]. CALCIUM BINDING PROTEINS Calcium, an important secondary messenger in many signal transduction pathways, is thought to be involved in the pathogenesis of amebiasis [140]. The involvement of protein kinase C and calmodulin in the secretory activities of E. histolytica has also been demonstrated [141-142]. It is well established that
O
52
regulation of the cytoskeleton is triggered by calcium and calcium binding protein that are crucial for the progression and completion of phagocytosis, growth and host-parasite interaction. Furthermore, the significant effects on amoebic cytolysis when treated with agents that block either calcium function or its release have been documented, demonstrating the pivotal role calcium plays in the pathogenesis of amoebiasis [143-145]. In Entamoeba, a number of calcium binding proteins have been identified [146]. More importantly two distinct calcium binding proteins EhCaBP1 and EhCaBP2, has been characterized and their role has been simultaneously demonstrated [147-151]. Calcium chelators like ethyleneglycol bis (-aminoethyl ether)N,N’-tetraacetate (EGTA) (53) and ethylene diaminetetraacetate (EDTA) (54), putative antagonists of intracellular calcium flux like 8-(N, N-diethylamino) octyl-3,4,5-trimethoxybezoate (TMB-8) (55), slow Na+-Ca+ channel blockers like bepridil (56) and verapamil (57) and calmodulin (CaM) inhibitors like W-7 (N-(6aminohexyl)-chloro-1-naphtalene sulphonamide) and triflouroperazine (TFP) (58), all have been shown to inhibit the growth of Entamoeba [152]. These results indicate that extracellular calcium ions, amebic intracellular calcium flux, calcium channels and a CaM dependent process do contribute to the growth of Entamoeba and therefore inhibiting these process may help in preventing amoebiasis. Also, Carreo et. al [153] demonstrated that cyclosporin A (59) inhibits calcineurin and P-glycoprotein activity and thereby inhibits proliferation of Entamoeba. Also recently it has been shown that when expression of EhCaBP1, a calmodulin like four EF-hand containing 134 amino acid long protein demonstrated to be essential for Entamoeba is blocked the cellular proliferation gets blocked [154]. O HO HO
O
N O
O
O N
OH
OH O
53
CYSTEINE BIOSYNTHESIS Very recently in Entamoeba, it has been documented that most amino acid biosynthetic pathways have been eliminated except serine and cysteine [99]. The existence of biosynthetic pathway for sulfur containing amino acid, cysteine has been demonstrated to be
Targeting Amoebiasis
Current Bioactive Compounds 2007, Vol. 3, No. 1
HOOCH2C
N
HOOCH2C
In mammals cysteine is produced solely from incorporated methionine and cystathionine in a pathway called reverse transsulfuration pathway. In contrast, plants, fungi and some bacteria have so called sulfur assimilation pathway to fix inorganic sulfur into a serine derivative (O-acetyl serine) to synthesize cysteine. It has been also demonstrated previously that Entamoeba possesses the sulfur assimilatory cysteine biosynthetic pathway and is thus capable of procuring cysteine de novo [159-160]. These findings do suggest that enzymes playing important role in cysteine biosynthesis should serve as a critical novel target for developing chemotherapeutic agents.
CH2COOH
CH2CH2 N
CH2COOH
54 H3CO O C
H3CO H3CO
C2H5
CH2 (CH2)7 N
O
C2H5
55
O N
N
56 H3CO
H3C
H3CO
CH C
CH3 H2C
CH3 H2C
H2C
N
H2C
CH2
C N 57
OCH3 OCH3
S N
CF3
(CH3)3 N N CH3 58
H3C H3C
CH3
CH2
O O H3C
REFERENCES
HO
N
N
H3C
O
CH3
CH3
O
[1]
N
N CH3
N
CH3 [2]
O
O H3C
CH3 CH3 H3C
N
N O
O
N
O N CH3
N O
CONCLUSION Amoebiasis spread can be addressed by the provision of adequate sanitation worldwide, but this is an ideal situation and unlikely to happen in foreseeable future. Therefore development of effective chemotherapy is required. Scientists from diverse fields are investigating plants anew with an eye to locate compounds having antiamoebic activities. Thus, a large number of compounds have been identified that demonstrate inhibitory effect on Entamoeba. Besides isolating and synthesizing new compounds even existing compounds need to be subjected to drug target screening followed by animal and human studies to determine their real effectiveness towards whole organism systems including their toxicity in particular and their effects on beneficial normal microbial flora. In spite of so much advancement made in understanding the molecular biology of the parasite, amoebiasis continues to be scientifically very challenging disease in terms of discovering foolproof safe drug. It is a major health problem of developing countries. Tremendous progress has been made in synthetic and natural product chemistry but this effort should be further focused in the direction of developing newer and better screening procedures, models and methods. In absence of these we have not been able to come out with new drugs that can take care of the disease and not limiting physicians by making the same drugs available in their armory to treat the disease. Recent sequencing of Entamoeba genome has raised new hopes. Pharmaceutical companies and scientists both are trying to discover the biochemical pathways, identify critical targets and develop assay method, with the expectation that inhibition of one or combination of these targets by a single or a combination of molecules will lead to discovery and development of newer, safer and better therapeutic agents. ACKNOWLEDGEMENT SM Agarwal acknowledges Senior Research Fellowship from University Grants Commission for Pre-doctoral fellowship.
H3C
CH3
9
CH3 H3C
N
[3]
CH3
[4] [5]
CH3 O
[6]
CH3
59
essential for growth and various cellular activities of amoeba [155156]. Moreover it is thought that the production of this intracellular thiol was retained, as high level of cysteine may compensate for the lack of glutathione and its associated enzyme a major component of oxidative stress resistance in many organisms [157]. Sulphur containing amino acid metabolism varies among organisms [158].
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