|SPOTLIGHTS ON NEW DRUG TARGETS
|Year : 2015 | Volume
| Issue : 2 | Page : 130-133
Spotlights on new publications
Sherif M Abaza
Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
|Date of Submission||22-Sep-2015|
|Date of Acceptance||10-Oct-2015|
|Date of Web Publication||27-Jan-2016|
Sherif M Abaza
MD, Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Abaza SM. Spotlights on new publications. Parasitol United J 2015;8:130-3
| New drug targets II|| |
In Brazil, there are several forms of leishmaniasis; the most severe are visceral leishmaniasis caused by L. (L.) infantum chagasi and cutaneous leishmaniasis caused by L. (V.) braziliensis and L. (L.) amazonensis. The latter two species lead to localized lesions with mucocutaneous disfigurements and leishmaniasis with variable clinical presentations in the form of localized or diffuse cutaneous lesions, respectively. Leishmaniasis is considered as an opportunistic parasite, especially in AIDS patients. Three elements are critical in controlling leishmaniasis: (a) Treatment of infected cases; (b) Vector control; and (c) Sacrifice of infected dogs (the main reservoir host). Almost all available drugs induce high toxicity and undesirable side effects; besides, the long duration of administration leads to the emergence of drug-resistant strains. Therefore, it becomes necessary to search for new drug targets with high therapeutic efficacy and low toxicity. In the present issue, recent reviews and research articles handling new drug targets in leishmaniasis will be compiled and discussed.
Several reasons forced a group of investigators from Brazil to review and discuss aspects on the treatment of leishmaniasis, one of the important parasitic diseases endemic in Brazil. Some of these reasons are that the available drugs have high toxic side effects, long duration of administration, and incomplete cure. In their review, Juliana PB de Mendez and colleagues (Veras PST as corresponding author) discussed the available drugs (pentavalent antimonials, amphotericin B, pentamidine, and paromomycin) and their adverse effects. They also presented other drugs with low antileishmanial efficacy (rifampicin, tamoxifen, doxycycline, monomycine, trimethoprim, and nifurtimox), and other drugs that have species-specific activity, such as miltefosine. In addition, they described different clinical studies that dealt with combined drug therapy, use of nanoparticles and liposomal formulations (e.g. ambisome), and use of different drug carriers (e.g. carbon nanotubes). In their presented clinical studies, amphotericin B was the most frequently used drug in nanotechnology studies. After their fruitful analytical introduction, they claimed the importance of molecular techniques in transcriptomics and proteomics studies to identify new drug targets for the treatment of leishmaniasis.
They indicated that, to target a parasite, it is better to interfere with a critical pathway than use a specific enzyme or individual molecule. Accordingly, high-throughput analyses of the parasite critical pathways (large-scale data of the transcriptomics and proteomics studies) should lead to the discovery of new drug targets. This will be assisted with the high development of genomic tools such as microarrays that allow the identification of several stage-specific critical genes highly expressed during the development and differentiation of Leishmania spp. In addition, immunoproteomics offers a new era for T-cell epitope evaluation to be used as diagnostic and chemotherapeutic markers. Moreover, they documented the importance to understand the molecular mechanisms underlying drug resistance using comparative proteomics.
In this context, the reviewers presented their studies (the last was published in 2013) and they succeeded in identifying proteins with potential chemotherapeutic effects in infected macrophages. They selected one of them (hypoxia-inducible factor-1α) that is found to be one of the client proteins for heat shock protein 90 (HSP90). HSP90 proved to have essential roles in Leishmania cell cycle control. The reviewers summarized HSP90 inhibitors such as geldanamycin, tanespimycin (17-AAG), and radicicol and their use in other parasitic infections such as malaria, trypanosomiasis, and toxoplasmosis with promising results. They also presented results of their recently conducted experiments on HSP90 inhibitors. They found that 17-AAG (a) inhibited Leishmania growth, (b) reduced macrophage infection, (c) was able to kill both amastigotes and promastigotes and (d) proved to be more powerful than that of host macrophage in killing intracellular amastigotes. The reviewers additionally found that 17-AAG (a) acts as an anti-inflammatory drug with inhibition of superoxide and nitric oxide production and reduction of proinflammatory cytokines and chemokines, as well as (b) induces morphological autophagic features. Therefore, they concluded that HSP90 inhibition attacked several parasite regulatory proteins and pathways.
Advances in the development of new treatment for leishmaniasis. Biomed Res Int. 2015; 2015:815023
Another review published recently discussed a different aspect in the treatment of leishmaniasis. Miguel A Chαvez-Fumagalli et al. also from Brazil, focused on the creation of new delivery systems using amphotericin B (AmpB) that would decrease its toxicity without affecting its efficacy. Their aim was to improve the delivery system for an existing effective drug against leishmaniasis, which would be much less expensive and time consuming than the development of a new drug target. They chose AmpB because it proved effective against the different Leishmania spp. endemic in Brazil presenting all varieties of the disease (cutaneous, visceral, and mucocutaneous).
Because the existing liposomal formulations to deliver AmpB to infected macrophages, such as AmBisome, amphocil, and abelcet, are costly, the reviewers focused on studies that utilized materials used in nanotechnology as drug delivery systems, such as PLA (polyethylene glycol-polylactide), PE-PEG (1,2-distearoyl-glycerol-3-phosphoethanolamine-N-methoxy polyethylene glycol), PLGA (polylactic-co-glycolic acid), DMSA (dimer captosuccinic acid) individually or combined (PLGA-DMSA and PLGA-PEG). These studies revealed that the efficacy of nano-AmpB delivery systems was greater than that of sole AmpB therapy. The development of oil-in-water microemulsion system improved AmpB solubility up to 1000-fold. Another formulation was developed (lipopolymerosome) and it showed less AmpB toxicity and high antileishmanial efficacy through upregulation of Th1 cytokines and inducible nitric oxide synthase, as well as downregulation of Th2 cytokines. In addition, chitosan and chondroitin sulfate attracted scientists' attention as drug delivery systems after their successful use to deliver vaccine candidates. The reviewers discussed the results obtained from a recent study, which used both substances conjugated with AmpB (individually and combined) against extracellular promastigotes and intracellular amastigotes in comparison with free AmpB therapy. A synergistic interaction in combined conjugated therapy was detected with low toxic effects. This combined conjugated AmpB therapy was also evaluated in another two studies, and the results showed its high uptake in the liver and spleen of murine models. The delivery system also showed anti-inflammatory activity that may reduce the nephrotoxicity caused by free AmpB. The reviewers finally concluded that chitosan and chondroitin sulfate nanoparticles are the best delivery system nowadays for AmpB due to their lower costs.
New delivery systems for amphotericin B applied to the improvement of leishmaniasis treatment. Rev Soc Bras Med Trop; 2015, 48 (3): 235-242
To overcome the undesirable side effects of several drugs used in the treatment of leishmaniasis, Carolina BG Teles and colleagues from Brazil directed their efforts toward herbal medicine. They selected Combretum leprosum fruit, as it contains combretastatin A-4, which is a prodrug designed as anticancer therapy due to its vasculature damage causing central necrosis. The investigators found in a previous study that lupane-triterpene (LT), one of this fruit components, had antileishmanial activity against promastigotes of L. amazonensis, the causative species of diffuse cutaneous leishmaniasis, which is endemic in the Amazon region. In another previous study, the investigators had shown that LT had potent antiproliferative effects against L. braziliensis and L. guyanensis promastigotes. Meanwhile, they hypothesized that LT inhibits DNA topoisomerases. The latter are ubiquitous enzymes that have a major role in the DNA duplex, and they are suggested as potential antiprotozoan drug targets, especially against trypanosomatids. Therefore, the investigators aimed to evaluate the efficacy of LT against L. amazonensis amastigotes and their development in peritoneal mouse macrophages, and to test the link between LT and DNA topoisomerases using bioinformatic analyses.
To achieve their objectives, mice peritoneal macrophages were infected with L. amazonensis promastigotes in vitro, and incubated with different concentrations of LT for different time intervals. Replication and survival of intracellular amastigotes were evaluated during 96 h of incubation against control cultures and cultures with glucantime as control drug. This was followed by determination of the survival index and transmission electron microscopic study. The molecular structure of LT was searched in the available bioinformatics of L. braziliensis and L. donovani and the investigators applied a flexible docking protocol, which was based on a previously described protocol applied to DNA topoisomerase crystal structure of L. donovani. The results showed that LT had no cytotoxic effects on macrophage viability at the concentration range of 6.5-109 μmol/l. After 96 h incubation, LT showed significantly reduced macrophage infection in comparison with control and drug control cultures. Transmission electron microscopic study confirmed their results in the form of vacuolar cytosol, mitochondrial swelling, and shedding of amastigote membranes. Finally, the investigators found that LT had high-affinity binding with DNA topoisomerase suggesting its use as inhibitor. The investigators discussed their results with respect to other compounds isolated from herbs, and recommended bioinformatic analyses to identify other potential drug targets against leishmaniasis.
A lupane-triterpene isolated from Combretum leprosum Mart. fruit extracts that interferes with the intracellular development of Leishmania (L.) amazonensis in vitro. BMC Complement Altern Med; 2015; 15:165
Almost all protozoa obtain their purines, required for DNA synthesis and replication, from exogenous precursors through purine salvage pathways. In such pathways, nucleosides are either hydrolyzed or undergo phosphorolysis leading to purine release. Inhibition of such pathways would prevent purine salvage by the parasite to build up its DNA. Immucillins were reported as synthetic inhibitors of nucleoside hydrolases causing death of some protozoa such as L. major, T. brucei, and P. falciparum. Aiming to identify new potential drugs against leishmaniasis, a team of investigators from Brazil, USA, and New Zealand ( Freitas EO et al.) evaluated the efficacy of different immucillins (eight chemical compounds) against promastigotes of L. infantum chagasi and L. amazonensis in vitro, as well as on L. infantum chagasi intracellular amastigotes in macrophages obtained from infected BALB/c mice. In addition, they tested the effect of each compound on macrophage viability, and conducted an electron microscopic study to determine the morphological changes. All growth assays were determined in a time-dependent and dose-dependent manner. To test the effect of immucillins on the activity of nucleoside hydrolase (NH36), the investigators prepared and purified the gene encoding NH3 of L. donovani, and they found that it is not species specific, and so they used it as nucleoside source to be added in each growth assay.
Six immucillin compounds were found to inhibit promastigote growth of both Leishmania spp., whereas three of them were potent against intracellular amastigote in the infected macrophages. Moreover, two of them inhibited NH36 activity, whereas the third was found to be a poor NH36 inhibitor. The strongest effect of these compounds was detected at 72 and 96 h, as confirmed by the ultrastructural study. Interestingly, each compound showed different morphological alterations in the form of global cellular alterations, kinetoplast swellings, and intense cytoplasmic vacuolization with enlarged vesicles. No macrophage cytotoxicity was detected in the treatment of these three compounds against L. infantum chagasi amastigotes. Finally, the investigators recommended the use of one of these compounds (forodesine) in a clinical human trial as it is available in the form of oral tablets and had already passed safety trials in humans.
Immucillins impair Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis multiplication in vitro. PLoS One; 2015, 10(4): e0124183
During the last decade, few publications have reported the use of β-amino alkanols in the treatment of leishmaniasis; however, their mechanism of action is still unknown. This encouraged María Á Abengózar and colleagues from Spain and Chile to evaluate the efficacy of 15 β-amino alkanols against L. donovani promastigotes and intracellular amastigotes in vitro and to study their actions as potential drugs. First, the investigators selected the compounds according to the presence of free hydroxyl group and their selectivity index values. They tested these compounds in cell cultures (THP-1 cell line) to detect any cytotoxic effects, which was determined using colorimetric measurement of MTT (a chemical substance added to the culture indicating cytotoxic effects on its reduction). Inhibition of MTT reduction by the tested alkanols was also determined by in vitro incubation with L. donovani promastigotes and intracellular amastigotes to select the compounds that could be used as potential drug targets. To elucidate the mode of action of the potential compounds, the investigators used the following methods: (a) Ultrastructural study using electron microscopy and confocal microscopy imaging to detect any morphological alterations; (b) Evaluation of promastigotes plasma membrane permeabilization using vital dye Sytox Green; (c) Measurement of free cytoplasmic ATP in promastigotes using cell titer Glo-luminescence assay; (d) Determination of oxygen consumption rates in promastigotes; and (e) Analysis of DNA content of promastigotes incubated with or without the tested compounds using flow cytometry.
Results showed that only three compounds (no. 5, 8, and 9) have therapeutic activity against the L. donovani promastigotes and intracellular amastigotes with no cytotoxic effects on cell cultures. Confocal and ultrastructural results of treated promastigotes revealed swollen mitochondrion, severe permeabilization with cytoplasmic loss, and vacuolization around the flagellar pocket for compounds 5, 8, and 9, respectively. Similar alterations were observed in treated intracellular amastigotes. Interpretation of their results suggested that compound 5 acts through mitochondrial dysfunction, whereas membrane permeabilization was postulated as mechanism of action of compound 8. Moreover, the investigators attributed interference in membrane traffic as a mechanism of action in case of compound 9. The investigators recommended compound 5 as potential drug target because it causes diminished intracellular ATP content without plasma membrane permeabilization (better than compound 8) and it also inhibits complex II of the respiratory chain as determined by measurement of oxygen consumption rates. Furthermore, it is easy to be encapsulated and delivered to the infected target. Finally, they initiated another protocol for future development of compound 5 as new drug target in the treatment of leishmaniasis.
Mechanisms of action of substituted β-amino alkanols on Leishmania donovani. Antimicrob Agents Chemother; 2015, 59(2): 1211-1218
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