Background & objectives: The study was undertaken to explore the locus of interaction of clofazimine and niclosamide which showed substantial growth inhibition property in Leishmania donovani promastigotes. Methods: The uptake of final electron acceptor oxygen and 2, 6-dichlorophenolindophenol (DCPIP) reduction in the electron transport chain were measured by constant volume Warburg respirometer and monitoring absorbance at 600 nm, respectively. Irreversibility of Oa uptake inhibition by clofazimine and niclosamide was determined by dilution of cell suspension followed by centrifugation.
Results: Clofazimine and niclosamide showed their minimum inhibitory concentration (MIC) at 33 and 150 Itg/ml, respectively. Oxygen uptake inhibition by clofazimine and niclosamide was not reversed by removal of the drug by centrifugation. Rotenone, a potent inhibitor of mammalian electron transport chain showed no inhibition on the electron transport chain of L. donovani promastigotes. Cyanide at 1 mM concentration showed partial inhibition in L. donovani promastigotes. Oxygen uptake and DCPIP reduction by L. donovani promastigotes were highly sensitive.to sulphhydryl group inhibitors. Strong inhibition of oxygen uptake (80-100%) by L. donovani promastigotes was achieved by clofazimine, niclosamide and amphotericin B. Amphotericin B failed to inhibit DCPIP reduction by L. donovani promastigotes, whereas DCPIP reduction was inhibited by clofazimine and niclosamide, respectively.
Interpretation & conclusion: DCPIP reduction was mediated by transplasma membrane electron transport as evidenced by its inhibition with membrane impermeable quinone 1, 2-naphthoquinone-4-sulphonic acid (NQSA). Transplasma membrane electron transport requires b-cytochromes and sulphhydryl groups for its function and was inhibited by clofazimine and niclosamide.
Key words Amphotericin B - antileishmanial agent - elofazimine - electron transport - Leishmania donovani - niclosamide plasma membrane
Human leishmaniasis, caused by trypanosomatid protozoa of the genus Leishmania, affects millions of people in the tropical and subtropical regions of the world'. This disease is an increasingly serious public health problem throughout the world, in part because of drug resistant strains of Leishmania spp. Current therapy for the disease is inadequate. The pentavalent antimonials sodium stibogluconate, meglutamine antimoniate and pentamidine, a biguanide compound, are the available drugs of choice for visceral leishmaniasis, and have variable efficacy and side effects2. Amphotericin B, a polyene antibiotic, has been widely used in several forms of dermal, subcutaneous and drug resistant visceral leishmaniasis with considerable success;. The spread of drug resistant strains of L. donovani has led to a resurgence of interest in the development of new anti-leishmanial agents4-6. Characteristic of all leishmanial infections is the intracellular parasitism of the macrophages by amastigotes, the mammalian stage of these parasites which is responsible for all the symptoms and pathology'. The vector stage of leishmania, the promastigotes, can be cultured readily in artificial media and has been used frequently for investigation. Energy metabolism in Leishmania species appears to be significantly different from mammalian cells in several important aspects. In Leishmania species, some of the glycolytic enzymes are grouped into a unique organelle termed `glycosome' from where the end products are transported to the single kinetoplast-mitochondrial complex for terminal oxidation. Moreover L, donovani is also characterised by incomplete oxidation of the input energy source and release of considerable amount of organic acids, particularly succinate and alanine in the medium9. Partial sensitivity to cyanide and presence of plant-like alternative oxidases have been clearly demonstrated in L. donovani promastigotes'. Employing spectroscopic techniques, Martin and Mukkada" had earlier presented compelling evidence for the presence of functional cytochromes in the kinetoplast-mitochondrial complex of L. tropica. Existence of a chloroquine sensitive transplasma membrane redox system, quite different from the mitochondrial electron transport system, was characterized in the eukaryotic microorganism Tetrahymena"2. During random screening of anti leishmanial activity of several anti infective agents, the antitubercular drug clofazimine -3 and anticistodal drug niclosamide'4, were tested for their antileishmanial activity in vitro. Attempts were also made to identify the locus of interaction of these drugs. This is the first report on the anti leishmanial property of niclosamide and clofazimine, but these are poorly soluble in water which limits their activity. Further studies on the synthesis of more water soluble derivatives of these drugs and the evaluation of their antileishmanial property have potential importance in the field of chemotherapy of leishmaniasis.
Material & Methods
Chemicals: All biochemicals unless otherwise mentioned were from Sigma Chemicals, U.S.A. Clofazimine was a gift from S.G. Pharmaceuticals, Baroda. NQSA and iodoacetic acid were purchased from Hi Media, Mumbai.
Organisms and their maintenance: The promastigotes of L. donovani strain MHOM/IN/1978/UR-6, were obtained from the Indian Institute of Chemical Biology, Calcutta. The culture was maintained on modified Ray's blood agar slopes".
Measurement of oxygen uptake by L. donovani cells: Oxygen uptake by whole cell was measured in Warburg constant volume respirometer (from Sambros Pvt. Ltd., Calcutta, India), with air as gas phase at 37C, with 0.2 ml 20 per cent KOH in the centre well and shaking at 80 cycles/min, according to the methods described in Umbreit et al's. For respiration studies, the cells were washed at 1000xg twice in cold phosphate buffered saline (PBS), 140 mM, pH 7.2 and kept at 4C until use. The Warburg flask contained 5 mg cell protein in a total volume of 3 ml. Water insoluble compounds were given as a solution in dimethyl sulphoxide (DMSO; 5 pl/ml PBS). Appropriate vehicle controls (DMSO) were used for each experiment.
Measurement of 2,6-dichlorophenolindophenol (DCPIP) reduction by L. donovani promastigote cells: A reaction mixture of 2.7 ml PBS, 140 mM, pH 7.2 contained 15 Nmoles D-glucose and 0.5 mg cell protein, with or without effectors with proper vehicle control (DMSO). Reaction mixture was incubated for 15 min at 25C. The reduction was started by the addition of 0.3 ml DCPIP + PMS mixture containing 90 nmoles DCPIP and 2.4 pmoles PMS, in 2.7 ml incubation mixture. The absorbance change was monitored for 3 min at 600 nm. DCPIP reduction rate was calculated by E60 2.1 x 104 litre/ mole/cm. Heat inactivated cell suspension was prepared by treating cell suspension at 80C for 60 min and cold inactivated cell suspension by freezing at -20*C for 72 h.
Exposure of L. donovani promastigotes to clofazimine, niclosamide and amphotericin B: The liquid media used for growth experiments was a semisynthetic medium developed by Chaudhuri et all' Cultures of promastigotes in 100 ml conical flasks were incubated for 100 h with added drug supplied as a solution in DMSO (5 pl/ml medium) with proper vehicle control (DMSO). After incubation, the number of elongated motile promastigotes were counted. MIC was the lowest concentration of drug required to stop population growth.
Determination of the reversibility of inhibition in vitro: Oxygen uptake was measured during a period of lh in the presence of either clofazimine or niclosamide with proper vehicle control (DMSO) as described under measurement of oxygen uptake by L. donovani cells. Drug treated cell suspension from the Warburg flask was transferred to a centrifuge tube and diluted five-fold with cold (4'C) PBS followed by centrifugation at 1000xg for 15 min (4'C), and resuspended in PBS containing I per cent bovine serum albumin (BSA). Control cells never exposed to either drug (but centrifuged and resuspended so as to control. for cell loss due to handling) were defined as 0 per cent inhibited; lack of oxygen consumption was defined as 100 per cent inhibition. Protein estimation: The amount of protein was determined by the biuret method". BSA was used as standard. I mg whole-cell protein corresponds to 1.4 x 101 cells.
Results & Discussion
Antileishmanial activity of clofazimine, niclosamide and amphotericin B: Amphotericin B, clofazimine and .niclosamide have growth inhibitory effect (Fig.) on the promastigote form of L. donovani at concentrations of 0.5 pg/ml (5.4 x 10-' M), 33 pg/ml (7 x lOw M) and 150 pug/ml (4.6 x 10-4 M), respectively. Amphotericin B was most potent followed by clofazimine and niclosamide. Vehicle controls using solvent at the same concentrations had no effect on growth. Amphotericin B exerts its action by pore formation through the sterol containing plasma membrane'9. The inhibitory effects of clofazimine and niclosamide on electron transport of bacteria"3 and cestodes", respectively have already been reported. So far no report is available on the mode of action of these drugs on L. donovani promastigotes. Since clofazimine and niclosamide inhibit the reduction of OZ and DCPIP (Table I), it may be concluded that these drugs exert their growth inhibition effect by inhibiting some locus in the transplasma membrane electron transport system. The concentrations required to inhibit 50 per cent glucose stimulated OZ uptake (ICS) for amphotericin B, clofazimine and niclosamide were 2, 24 and 50 pg/ml, respectively. Similarly, the concentrations required to inhibit 50 per cent glucose stimulated DCPIP reduction (ICS) by amphotericin B, clofazimine and niclosamide were found to be 5, 48 and 60 pg/ml, respectively. Incubation of L. donovani promastigotes with these three drugs at MIC caused complete loss of motility, though their cellular integrity was not affected within one hour period. Heat and cold shocked cells failed to reduce DCPIP. The ICS values of 02 uptake and DCPIP reduction for clofazimine, niclosamide and amphotericin B were comparable. Hence, it may be suggested that these drugs inhibit some locus common to both OZ uptake and DCPIP reduction linked chain or pathway. Amphotericin B inhibited OZ uptake and DCPIP reduction by complexing sterols of the plasma membrane2. Binding of amphotericin B with the plasma membrane caused efflux of small moleculesz'. DCPIP reduction inhibition by amphotericin B appears to be either due to efflux of reducing equivalents from the cytoplasm or binding of.redox enzyme with amphotericin B.
The effects of electron transport chain and sulphhydryl group inhibitors on 02 uptake and DCPIP reduction by L. donovani promastigotes are shown in Table 1. Rotenone, an inhibitor of nicotinamide adenine dinucleotide reduced (NADH): ubiquinone reductase (complex 1), had no effect on 02 uptMce even at very high concentration (0.5 mM), whereas at the same concentration 95 per cent inhibition was achieved in mammalian preparation 22. Partial inhibition of DCPIP reduction by rotenone at the same concentration appears to be due to nonspecific interaction. Antimycin A, an inhibitor of reduced ubiquinone: cytochrome C ieductase (complex, III), showed potent inhibition on 02 uptake at a very low concentration, whereas DCPIP reduction was inhibited partially at the same concentration. 2 (n-nonyl)-4 - hydroxyquinoline N - oxide (n-HQNO), an inhibitor of coenzyme Q: cytochrome bct reductase showed high inhibition of both OZ uptake and DCPIP reduction. It is evident from these data that b cytochromes are involved in Oz uptake and DCPIP reduction, but these b cytochromes differ from each other by their inhibitor sensitivity. Cyanide, an inhibitor of cytochrome c oxidase (complex IV) showed only 40 per cent 02 uptake inhibition at a high concentration, whereas DCPIP reduction was completely unaffected by cyanide at the same concentration. From this observation it may be concluded that 60 per cent OZ uptake was dependent on classical cytochrome oxidase and the remaining 40 per cent was dependent on cyanide insensitive cytochrome 023. DCPIP reduction was independent of cyanide sensitive cytochrome system. It is evident from Table I that NQSA is a potent inhibitor of both O, uptake and DCPIP reduction. Since lipophillic oxidant NQSAz4 was impermeable to plasma membrane, it may be concluded that NQSA exerted its action on plasma membrane, presumably by inhibiting the sulphhydryl group25. Specific sulphhydryl group inhibitorsz5 NBDC, HgCl2 and iodoacetate showed also complete inhibition on O,_ uptake and DCPIP reduction'(Table I).
appears from the results in Table II that the action of clofazimine and niclosamide on OZ uptake by L. donovani promastigotes are irreversible in nature. This indicates that these drugs are strongly bound to the plasma membrane due to their lipophilicity.
The experiments described by us demonstrated that clofazimine and niclosamide exerted their effect on transplasma membrane electron transport system of L. donovani promastigotes, and this electron transport is largely dependent on plasma membrane sulphhydryl group. Identification of the inhibition property of clofazimine and niclosamide offers an interesting target against leishmaniasis, which may lead to less toxic treatments than those currently available. An improved understanding of transplasma membrane electron transport may enable identification of novel drug targets in this pathogen.
Financial aid received from the Indian Council of Medical Research, New Delhi is acknowledged.
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Gautam Datta & Tanmoy Bera
Division of Medical Biochemistry, Department of Pharmaceutical Technology Jadavpur University, Calcutta
Accepted May 23, 2000
Reprint request: Dr Tanmoy Bera, Department of Pharmaceutical Technology Jadavpur University, Calcutta 700032
Copyright Indian Council of Medical Research Jul 2000
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