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| SPOTLIGHTS ON NEW PUBLICATIONS |
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| Year : 2016 | Volume
: 9
| Issue : 1 | Page : 59-63 |
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Spotlights on new publications
Sherif M Abaza PhD
Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
| Date of Submission | 11-Apr-2016 |
| Date of Acceptance | 19-Apr-2016 |
| Date of Web Publication | 25-Oct-2016 |
Correspondence Address:
Sherif M Abaza
Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/1687-7942.192998
How to cite this article:
Abaza SM. Spotlights on new publications. Parasitol United J 2016;9:59-63 |
| New drug targets III | |  |
Schistosomiasis
After publications of several reports concerning the appearance of some schistosome strains resistant to praziquantel, Long T and his colleagues from USA and France focused their search on a new antischistosomal drug targeting the immature larval stage: schistosomula. One of these drugs is the anticancer agent ‘polo-like kinase’ (PLK) inhibitor. The human OK PLK1 (huPLK1) enzyme was found to be critical for normal mitotic progression, and its inhibitors are used as chemotherapeutic drugs against cancer in case of PLK1 overexpression in human tumors. S. mansoni PLK1 (SmPLK1) is highly expressed in sporocysts as well as the reproductive organs of the adult worms. A previous study (Long et al., 2010) showed that the huPLK1 inhibitor (BI2536) selectively inhibited SmPLK1 in vitro and produced reduction in the number of oocysts and spermatocytes in female and male reproductive organs, respectively. In the present study, the investigators maintained the S. mansoni life cycle in snails and hamsters and the collected worms were prepared for phenotypic screening. The double stranded RNA (dsRNA) of the fragments regulating the SmPLK1 gene was synthesized, and using reverse transcription-quantitative real time PCR, the investigators coincubated dsRNA with S. mansoni schistosomula to decrease SmPLK1 expression. The results showed that it is critically important for schistosomula survival. Then, both schistosomula and adults were incubated with 11 huPLK1 inhibitors (one of them is benzimidazole thiophene, which was used in a phase I clinical trial for the treatment of solid malignancies). The results revealed that BI2536 showed high efficacy, where phenotypic alterations were observed in both stages at 5 μmol/l after 24 h, whereas five of the huPLK1 inhibitors showed little or no activity. Benzimidazole thiophenes showed much more activity against both stages at 1 μmol/l after 24 h. Therefore, the investigators expanded their experiment to include 38 benzimidazole thiophene inhibitors from GlaxoSmithKline of the Published Kinase Inhibitors Set (PKIS) 1 and 2. Using combined automated and microscopy phenotypic analyses, the investigators identified several PKIS that showed high potency and bioactivity against both schistosomula and adults, and interestingly, they also inhibit huPLK1 and kill cancer cells. The investigators also identified a structure–activity relationship in the inhibitor sets (PKIS 1 and 2) that is shared between schistosomula and adult schistosomes, and they proposed these compounds as novel targets for future development of an antischistosomal drug for praziquantel-resistant strains. Compiled from ‘‘Structure-bioactivity relationship for benzimidazolethiophene inhibitors of polo-like kinase 1 (PLK1), a potential drug target in Schistosoma mansoni. PLoSNegl Trop Dis; 2016 Jan; 10(1): e0004356.’’
Lymphatic filariasis
Elephantiasis, a disfiguring parasitic disease with high morbidity and disability manifestations, is endemic in tropical and subtropical countries. To eradicate filariasis, WHO recommended mass drug administration (MDA) with several reports of strains resistant to diethylcarbamazine, ivermectin, and albendazole (ABZ). This necessitates drug replacement of annual MDA rounds, and thus encourages scientists to search for novel policies to develop new drug targets. Another obstacle in this respect is the difficulties in the in vitro cultivation of different filarial worms and the unavailability of in vivo small experimental animal models. In previous studies (2011, 2013), a British investigator, Elizabeth Bilsland and her colleagues, developed a novel approach to screen protein compounds as potential drug targets using yeast (S. cerevisiae) cultures for the expression of nematode proteins. Based on this approach, the investigators replaced essential yeast genes with filarial or human genes to assess drug efficacy without harmful effects on the host. These yeast strains were labeled with different fluorescent proteins to identify the inhibitory drug that affects the parasite, and not its human ortholog. Using this method, yeast strains expressing different parasitic proteins could be used in screening of new inhibitors to identify compounds with potent inhibitory effects against the expressed parasitic proteins. In the present compilation, and according to certain criteria, the investigators selected 15 B. malayi enzymes and their human orthologs to be cloned. The clones were transferred to yeast strains after deletion of their target genes. The yeast strains were cultivated and monitored for their growth in the presence of B. malayi and human genes to identify enzymes controlling yeast growth. The perfect parasite enzyme that could be utilized as a potential drug target is one that inhibits yeast growth with expression of B. malayi genes, but not with human orthologs. The results showed eight B. malayi enzymes as potential drug targets, which were screened in the library (Open Access Malaria Box; http://www.mmv.org/malariabox) against 400 inhibitors. The investigators were able to identify nine specific inhibitors; five of them showed in vitro antifilarial activity against B. pahangi. The investigators recommended the use of this approach for other antiparasitic drug developments as it is easy to set up, less expensive (use of yeast instead of in vitro parasite cultivation or in vivo animal experiments), and above all distinguishes between compounds affecting only parasite enzymes. They also recommended further studies to standardize the minimum drug concentration with high efficacy on adult filarial worms. Compiled from ‘‘Yeast-based high-throughput screens to identify novel compounds active against Brugia malayi. PloS Negl Trop Dis; 2016 Jan; 10(1): e0004401.’’
Alveolar echinococcosis (AE)
It is an endemic life-threatening disease in China caused by E. multilocularis. Most infected patients are asymptomatic at the early stages due to the lack of pressure symptoms which are common in other types of hydatid cysts. As AE is characterized by its proliferation and diffuse infiltration of the infected tissue, it requires radical removal combined with pre- and post-operative medical treatment. However, its diagnosis is usually late, with most cases becoming inoperable, and in these cases, oral ABZ administration remains the only choice. Due to poor solubility of ABZ in water and hence limited intestinal absorption, this necessitates an increase in the dose uptake for a longer time, favoring severe side effects. Similar results were obtained in studies using other antiparasitic drugs (e.g. praziquantel, nitazoxanide, and mefloquine) in combination with ABZ in the treatment of AE. With the recent advances in nanotechnology and the success of gold or silver nanoparticles, chitosan (CS), and liposomes (L) in drug delivery to the target organ, Maitiseyiti Abulaihaiti and Xin-Yu Peng from China were prompted to conduct their study. Their objective is to investigate ABZ efficacy conjugated with CS and L in the treatment of AE in vivo compared with the oral administration of ABZ alone. After isolation of E. multilocularis metacestodes from naturally infected gerbils, vital metacestodes were suspended to experimentally infect mice by subcutaneous injections. Three ABZ formulations were prepared and the infected mice were selected 3 months post-infection and divided into nine groups (treated by three formulations at three different doses: 37.5, 75, and 150 mg/kg). The treatment was administered three times per week for 12 weeks post-infection. In addition, two groups of control mice were studied as uninfected and infected non-treated groups. The following parameters were used to assess the efficacy of the three ABZ formulations: (1) mean cyst weight, (2) gross morphological alterations, (3) histopathological examination using transmission electron microscopy (TEM), (4) serum level of cytokines (IL-2 and IL-10) as biomarkers for Th1 and Th2 responses, respectively using capture-ELISA, and (5) plasma levels of ABZ-efficient metabolite, ABZ-sulfoxide (ABZ-SO), using high-performance liquid chromatography. The results revealed that ABZ conjugated with CS or L significantly had high ABZ-SO levels, reduced the AE cyst growth, and produced cyst collapse especially at the higher doses (75 and 150 mg/kg) compared with ABZ alone. However, at the highest dose, ABZ-SO increased significantly in the CS group compared with the L group. In addition, both conjugations produced morphological alterations including damage in AE layers (laminated and germinal), with more severe cellular destruction of the germinal layer in mice receiving the CS formulation. Evaluation of Th1 (IL-2) and Th2 (IL-10) immune responses showed correlation between high IL-2 levels and a shift from a Th2-dominant to a Th1-dominant immune response. Finally, the investigators concluded that CS could be a new drug vehicle as it improved ABZ absorption, increased its bioavailability in AE treatment in mice, is easily manufactured as capsules or tablets, and above all, is nontoxic and of low cost. Compiled from ‘‘Efficacy of albendazole-chitosan microsphere-based treatment for alveolar echinococcosis in mice. PLoSNegl Trop Dis; 2015 Sep; 9(9): e0003950.’’
Toxoplasmosis
Being a multi-systemic disease, it also gains its importance from being prevalent all over the world. Although it is asymptomatic in most cases, it yields to several clinical presentations, especially in immunocompromised hosts, resulting in ocular toxoplasmosis and encephalitis. More important is its complications during pregnancy, with its vertical transmission leading to abortion and congenital malformations in newborns. Most cases are treated with pyrimethamine combined with sulfadiazine or clindamycin or azithromycin or atovaquone. However, intolerance to sulfadiazine, with its long-course therapy and gastrointestinal disorders, when associated with the others, necessitates a search for new drug targets. Erica S. Martins-Duarte and colleagues from Brazil and France utilized one of the apicoplast metabolic pathways controlling parasite virulence or survival. Based on this concept, fluoroquinolone (ciprofloxacin; Cipro) and novobiocin were used in the treatment of toxoplasmosis through inhibition of DNA replication (an apicoplast pathway) targeting DNA gyrase. The latter is an enzymatic complex, composed of two subunits (A and B), and their gene products are exclusively targeted to the apicoplast. Both drugs inhibit subunits A and B, with inhibition of apicoplast genome replication leading to parasite death. In a previous study, the same group of investigators (Dubar et al., 2011), synthesized several novel ethyl–ester derivatives of Cipro and used them for in vitro treatment of toxoplasmosis and malignant malaria. The results revealed the significant efficacy of three derivatives (Et-Cipro, Ph-Cipro, and Adam-Cipro) compared with Cipro. These results directed the investigators to study the effects of these three derivatives: (1) on noninfected mice (cytotoxic effects), (2) on T. gondii experimentally infected mice (using Cipro as control), (3) in vitro on T. gondii tachyzoites using immunofluorescence and transmission electron microscopy (TEM), and (4) in vitro on cell culture infected with C. parvum oocysts to test for sporozoite proliferation. The investigators used four doses of the three derivatives (25, 50, 100, and 200 mg/kg/day) to test their cytotoxic effects; three doses (50, 100, and 150 mg/kg/day) to test Cipro and Ph-Cipro effects; and two doses (50 and 100 mg/kg/day) to test Et-Cipro and Adam-Cipro on experimentally infected mice. The results revealed that the three derivatives had no cytotoxic effects on noninfected mice, except for a slight increase in creatinine kinase in animals receiving 200 mg/kg Ph-Cipro. All mice treated with Cipro died by day 10 post-infection infection, whereas the three derivatives increased mice survival acutely infected with T. gondii. In addition, they survived the entire experimental period (60 day) and did not show any parasite encystation in the brain after their sacrifice. In vitro study of the tachyzoites showed an antiproliferative effect after 6–8 h that was sustained after removal from the culture medium (for 18 h). Cipro derivatives inhibited parasite replication early in the first cycle of infection. Adam-Cipro was the most effective even at a low concentration. Using immunofluorescence microscopy, treated parasites showed the formation of large multinucleated parasites, with loss of the typical rosette structure, and drastic effects on tachyzoite division with arrest of endodyogeny. Similarly, a TEM study showed malformation of the inner membrane complex and enlarged apicoplast, which moved posterior to the nucleus during cell division. Lastly, Cipro and its derivatives failed to treat cryptosporidiosis due to apicoplast absence in the apicomplexan Cryptosporidium spp., confirming that these derivatives target alone the apicoplast. Accordingly, the investigators recommended further studies to explore more benefits that could be achieved by conducting similar studies on inhibition of apicoplast genome replication. Compiled from ‘‘Ciprofloxacin derivatives affect parasite cell division and increase the survival of mice infected with Toxoplasma gondii. PLoS One. 2015; 10(5): e0125705.’’
Protozoal diseases
Heat-shock proteins (HSPs) or stress proteins are conserved compounds that are expressed upon exposure to several stress conditions such as temperature increase, oxygen deprivation, pH extremes, and nutrient deprivation. They are categorized according to their molecular weight and/or their location, for example cytosolic (Cy), mitochondrial (Mit), and endoplasmic reticulum (ER). The majority of HSPs are fundamental to cell growth and survival. Among them is HSP90, which deserves considerable attention because of its essential role in the pathogenesis of all the intracellular protozoan diseases caused by T. cruzi, T. gondii, Leishmania, Plasmodium, Babesia More Details, and Cryptosporidium species as well as giardiasis. Accordingly, its inhibitors are expected to be promising new drug targets and candidate vaccines. In humans, there are four HSP90 isoforms, Cy (α and β), ER (Grp94), and Mit (TRAP1), and its inhibitors are used as chemotherapeutic agents against cancer. Therefore, the main objective of the study carried out by Ngonidzashe Faya and colleagues from South Africa was to analyze different HSP90 sequences from human, vector, and parasites using large-scale bioinformatics aiming to investigate their inhibitory sites and to examine the potentiality of these sites for the development of novel drugs targets. In addition to the human isoforms, 100 HSP90 sequences from 22 protozoa, eight helminthes, eight vectors, and one ectoparasite were retrieved and categorized as Cy, ER, and Mit. The investigators also retrieved six HSP70 from human and P. falciparum (two for each category) as controls. Each group was subjected to: (1) multiple sequence analysis, (2) characterization of physicochemical properties (molecular weight, amino acid composition, isoelectric point, and others), (3) identification of phosphorylation sites, and (4) motif analyses. The results showed that protozoan HSP90 proteins are located far from the human ones in the three categories, i.e. they formed their own distant clusters, whereas ectoparasite and helminthes clustered together with the vector and human sequences. Cy HSP90s showed the highest conservative sequence within the 104 studied HSP90s, followed by Mit, and ER was the least conserved HSP90s. They also showed high conservation in their domains, and the most conserved domain proved to be the N-terminal domain, which contains the adenosine triphosphate-binding (ATP) site and which is considered the most important functional site. Certain phosphorylation sites showed high conservation in all the studied categories. Except for human Mit HSP90, the isoelectric point indicated the acidic nature of all HSP90s. Amino acid composition showed a uniform distribution among parasitic Cy HSP90s. High motif conservations were observed in all Cy protozoan HSP90s, except G. lamblia, whereas Mit and ER HSP90s showed motif variations, which are unique in specific protozoa. Mit HSP90s showed conserved and unique motifs in Leishmania, Plasmodium, and Babesia species, while those of ER HSP90s proved unique in Leishmania and Trypanosoma species. The investigators suggested that this unique conservative motif among certain protozoa could be an evidence for the possibility to develop novel selective drug target sites against these diseases. In conclusion, protozoan HSP90s proved to be suitable for further in silico analysis and clinical trials to develop new drug targets for the tropical protozoan diseases with high morbidity and mortality rates and high endemicity in developing countries such as malaria, Babesiosis More Details, visceral leishmaniasis, African trypanosomiasis, and American Chagas disease Compiled from ‘‘Human, vector and parasite Hsp90 proteins: A comparative bioinformatics analysis. FEBS Open Bio; 2015; 5: 916–927.’’
Cryptosporidiosis and giardiasis
Diarrhea associated with protozoan diseases results in high mortality rates in children and immunocompromised patients. In the present compilation, Yukiko Miyamoto and Lars Eckmann from USA reviewed the current therapeutic status, resistance development to therapeutic drugs, and the potential compounds that could be used in the treatment of two common protozoa responsible for the majority of diarrheal diseases: Cryptosporidium spp. and G. lamblia. For the treatment of cryptosporidiosis, nitazoxanide is the only drug approved by the Food and Drug Administration. It is administered at a dose of 100, 200, and 500 mg twice daily for a maximum of 2 weeks for 1–3, 4–11 years old children, and patients older than 11 years of age, respectively. Another drug was paromomycin, but with lower efficacy, while immunotherapy showed good results, especially in immunocompromised patients. The reviewers discussed two important obstacles for the development of new drug targets against cryptosporidiosis. The first is the inability of different culture systems to capture all Cryptosporidium spp. life stages (intracellular and extracellular). Second, small animal models (mice and rats) are not associated with the characteristic diarrheal disease that occurs in humans, whereas large animals (pigs and calves) develop marked severe diarrhea. For the potential drug targets, the reviewers discussed an important point; Cryptosporidium spp. lack genes encoding metabolic pathways and rely on essential core genes for biosynthesis of nucleic acids and the enzymes of these pathways are different from those of the host, making them potential drug targets. Of these enzymes, inosine 5′-monophosphate dehydrogenase (IMPDH), required for guanine salvage pathway, and its specific inhibitors were investigated in several studies and showed promising results in vitro and in vivo. Another example is triacsin C, which inhibits long-chain fatty acyl-coenzyme A (essential for fatty acid metabolism), and the inhibitors of calcium-dependent protein kinase 1 (CDPK1), which exhibited significant efficacy. Cysteine protease inhibitors are another molecular group of potential drug targets, especially those belonging to the papain-like family of cysteine proteases. Lastly, the reviewers screened the library of Open Access Malaria Box (400 inhibitors) and identified 19 compounds with promising activity against cryptosporidiosis.
In contrast to cryptosporidiosis, several drugs are currently used for the treatment of giardiasis, the most common is metronidazole (250 mg in three divided doses/day for 7 days). Other drugs include nitazoxanide, benzimidazoles (ABZ and mebendazole), and paromomycin. Also, in contrast to cryptosporidiosis, its in vitro axenic cultivation is performed in most research laboratories and parasite numbers and viability could be microscopically studied. Moreover, animal models include different rodents, dogs, and cats, whereas mice and gerbils are the most appropriate animal models. However, drug resistance is common in giardiasis and the reviewers postulated drug resistance to the inability of G. lamblia trophozoites to activate nitro prodrugs to toxic radicals by reduction. For potential drug targets in giardiasis, the reviewers discussed four strategies. First, modifications of the existing drugs such as metronidazole, nitazoxanide, and ABZ. They gave examples of several studies with substitution of side chains or placement of the benzene ring or addition of an alkylthiol group or an ethoxy group, and these modifications showed significant potent antigiardial activities. Second, screening libraries known for inhibitors of G. lamblia growth; the reviewers detected several inhibitors; however, being toxic, they are not suitable for clinical trials. Screens in other libraries including compounds suitable for human use detected several compounds that can kill G. lamblia. Auranofin, used in the treatment of rheumatoid arthritis, inhibited G. lamblia growth and proved to be efficient in metronidazole-resistant giardiasis. Fumagillin, carbadox, and tioxidazole are other detected drugs with significant antigiardial efficacy against metronidazole-resistant strains. Orlistat, that is used for the treatment of obesity and is characterized by poor absorption from the intestinal tract, proved to be a potent inhibitor of G. lamblia replication. Miltefosine, used in the treatment of visceral leishmaniasis, showed adequate activity in vitro and in vivo. The third strategy is the use of herbal medicine, natural weed (Oxalis corniculata) and sandalwood (Osyris alba), which showed satisfactory results. Fourthly, G. lamblia molecular targets are less promising efficient drugs compared with Cryptosporidium spp. Arginine deiminase (ADI), carbamate kinase (CK) and fructose 1,6-bisphosphate aldolase (FBPA) are essential enzymes for generating adenosine triphosphate for trophozoite viability, and for parasite metabolism and survival, respectively. The reviewers suggested that future development of potent and selective inhibitors of ADI, CK and FBPA enzymes could be potential novel drug targets against giardiasis. Compiled from ’’Drug development against the major diarrhea-causing parasites of the small intestine, Cryptosporidium and Giardia. Front Microbiol; 2015, 6: 1208.’’
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Conflicts of interest
There are no conflicts of interest.
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