|SPOTLIGHTS ON NEW PUBLICATIONS
|Year : 2016 | Volume
| Issue : 2 | Page : 112-117
Spotlights on new publications
Sherif M Abaza
Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
|Date of Submission||26-Nov-2016|
|Date of Acceptance||09-Dec-2016|
|Date of Web Publication||25-Apr-2017|
Sherif M Abaza
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 2016;9:112-7
| New drug targets IV|| |
Malaria: From the Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, UK, Sebastian Kirchner and his colleagues published a review article aiming to highlight the recent progress in genome technology in Plasmodium spp. with valuable advanced knowledge. The reviewers claimed that this will guide scientists and researchers to discover and expect future emergence of drug resistance as well as to search for development of new drug targets. According to their review, to eradicate malarial infections, research should be focused in three main directions: human genomics to increase human resistance to Plasmodium invasion to RBCs; vector genomics (mainly A. gambiae mosquitoes) to decrease malarial transmission; and parasite genomics to discover the evolution of selection pressures against drugs and vaccines. Analyses of several epidemiological studies on different human-infected populations have resolved issues related to the first point of focus. The recent progress of African projects to identify the specific gene (toll-11), protecting African mosquitoes against falciparum malaria, also succeeded in solving the second issue. For the third issue, several strains of P. falciparum, P. vivax, P. knowlesi and other species affecting primates have been sequenced. It was found that the Plasmodium genome has ∼5000 genes that are encoded by 14 linear chromosomes. Therefore, the challenge is in the ability of the Plasmodium genome to evolve and the occurrence of single-nucleotide polymorphisms (SNPs) in certain gene exons in response to drug effects, resulting in the emergence of drug-resistant strains. The reviewers presented an interesting figure that showed major advances in different fields related to different Plasmodium spp. omics during the period 2002–2016 that dealt with landmark keys of parasite development and pathogenesis.
One of those postulated mechanisms explaining how gene expression is regulated during the Plasmodium life cycle stages is epigenetic control: i.e., changes in gene expression without change in nucleotide sequence through post-translational modifications (PTMs) of histones. In contrast to mammals, unicellular eukaryotes have more activation than silencing marks. Therefore, histone PTMs play an important role in Plasmodium gene regulation, leading to continuous dynamic changes across the intraerythrocytic development cycle. In other words, there are several genes in Plasmodium genomics, some are identified and few of them are still under detailed investigation that encodes proteins controlling addition or removal of histone PTM marks. Accordingly, the reviewers presented some of these histone modifiers as antimalarial drug targets, such as histone lysine deacetylase (HDAC) and bromodomain protein 1 (BDP1), leading to increased gametocyte conversion and failure to invade new erythrocytes, respectively. In addition, investigating the transcriptions and translations that occurred during the intraerythrocytic development cycle to increase merozoite egress and invasion would guide investigators in discovering mechanism(s) of epigenetic regulation of gene expression in Plasmodium genomics. This might give a thrust to the development of new antimalarial drugs.
Next, the molecular mechanisms behind Plasmodium evasion of the host adaptive immune response through P. falciparum erythrocyte membrane protein 1 (PfEMP1) were discussed. The PfEMP1 is encoded by 60 var genes family (i.e. genes controlling variant surface antigens). The reviewers listed several mechanisms for P. falciparum var gene regulation, and focused on the essential role of the 5′ upstream promoter families. Using these molecules, var genes are divided into five classes (upsA to upsE), which were found to correlate significantly with malaria severity, especially upsC var. The importance of upsC to maintain chromosome-internal var genes in their silent state was observed in severe cases of malignant malaria. The implication of another gene family; Plasmodium helical interspersed subtelomeric protein, was discussed. This gene family is also responsible for regulation of immune evasion due to its ability to bind to the PfEMP1 intracellular acidic terminal segment, leading to the formation of Plasmodium cytoadherence complex. In addition, other mechanisms of immune evasion were also discussed such as genes encoding repetitive interspersed family proteins which are incriminated in malignant malaria in African children with blood group A. The reviewers suggested that knowledge gained from these studies could guide interesting scientists to discover new drug targets that might decrease or ameliorate malaria severity.
The last issue that was discussed in the present review is artemisinin (ART) drug resistance. The reviewers analyzed the data obtained from clinical studies related to ART treatment outcomes. Although the role of kelch13 gene mutation in ART resistance was established and kelch13 SNPs are now used to map the spread of resistance in Africa and Southeast Asia, there are still large numbers of SNPs that could be incriminated in ART resistance. This evidence proposes that further studies be conducted to gain more knowledge on ART mode(s) of action.
In conclusion, the reviewers recommended further studies in the field of Plasmodium lipidomics to discover new areas of Plasmodium membrane composition and organization; a new channel for new anti-malarias. Research in histone modifiers as well as in the var gene family was also strongly recommended. Compiled from a review article. “Recent advances in malaria genomics and epigenomics.” Genome Med 2016; 8(1): 92.
Toxoplasmosis is a major disease caused by the apicomplexan protozoa, Toxoplasma gondii, which affects immunocompromised patients and causes major complications including ocular toxoplasmosis and encephalitis. In 2008, Kentaro Kato and colleagues showed that P. falciparum expressed a protein kinase (PfPK2), a homolog of human calcium calmodulin-dependent protein kinase (CaMK), which had an essential role in the invasion of P. falciparum in human RBCs. Recently, K. Kato et al. worked on another apicomplexan protozoa, T. gondii, aiming to identify a CaMK-like protein in its genome. This might pave the way to understand the mechanism underlying tachyzoites motility, migration, and host cell invasion, which are mediated by increase in cytosolic calcium concentration. The latter leads to secretion of adhesion molecules from their micronemes, and these macromolecules, known as glideosome complex, are phosphorylated during cell invasion to control gliding and motility. The T. gondii glideosome complex includes myosin A, myosin light chain (TgMLC1), T. gondii glideosome-associated proteins (TgGAPs) 40, 45, 50, 70, and 80, aldolase 1, and actin 1 (TgACT1). In their previous article (K. Kato et al. 2008), the investigators also observed that P. falciparum PK1, which is localized in the merozoites periphery, phosphorylates myosin A and PfGAP45 before invasion. The investigators hypothesized that if the T. gondii genome includes a homology of PfPK2, it will have a role in T. gondii tachyzoites motility and invasion, and it might be a step in the development of new chemotherapeutic drugs.
T. gondii (strain RH) tachyzoites were cultivated and maintained in cell culture, followed by PCR amplification, and its CaMK-related kinase (TgCaMKrk) was identified and sequenced. It was found that it has 57% identity and 88% similarity with that previously described for P. falciparum (PfPK2), and it was assigned in GenBank as number AB699221. Then, the TgCaMKrk kinase domain was purified using a wheat germ cell-free protein synthesis system to exhibit its autophosphorylation activity. The results confirmed its possession of protein kinase activity, which has the capability to phosphorylate itself. In contrast to the previous results obtained for PfPK2, the autophosphorylation activity of TgCaMKrk was not affected by the presence or absence of calcium and/or calmodulin. These results indicated that use of neither calcium nor calmodulin antagonists would affect the protein kinase of TgCaMKrk, and subsequently the invasion of T. gondii tachyzoites. However, the investigators found that TgCaMKrk could phosphorylate only one of the glideosome components (GAP45) in vitro. Moreover, after using several rabbit antibodies to localize TgCaMKrk in T. gondii tachyzoites, results suggested its localization at the apical end; however, the investigators recommended further studies for precise localization. In conclusion, parasite’s protein kinases play an important role in its life cycle, and search for their inhibitors offers a good step in the development of new drugs. The investigators recommended further studies in TgCaMKrk with special emphasis on its regulatory mechanism(s) and its impact on the T. gondii life cycle. Compiled from a research article “Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog.” Parasit Vectors 2016; 9:405.
Leishmaniasis: In their search to discover new drugs for leishmaniasis, Patrícia ST Veras and Juliana P Bezerra-de-Menezes from Brazil, a very high endemic country for visceral and cutaneous leishmaniasis, published a review article to reveal the probability of proteomic approach to identify new proteins essential for Leishmania intracellular survival and virulence. They hypothesized that proteomics (the study of large-scale parasite proteins) is a recent advanced tool to understand protein changes in Leishmania amastigotes and promastigotes in response to host and vector cells, respectively. Leishmania proteomic studies are used to identify and characterize the molecular pathways expressed in both Leishmania spp. and their hosts (mammalian and sandfly vectors).
First, the reviewers discussed the mechanisms used to modulate parasite protein expressions during life cycle differentiation (amastigotes↔promastigotes). Several morphological and biochemical adaptive processes are essentially required for this transformation, including adaptation to temperature and pH changes as well as to mammalian host immune response or vector cytotoxic environment. Identification of Leishmania proteins expressed for adaptation followed by identification of their inhibitors could be the first step in drug target development to stop Leishmania intracellular survival. Reviewing the studies conducted in this respect, several proteins were identified. It was found that the majority of specific proteins identified in vector promastigotes and/or cultivated amastigotes belonged to five functional groups of proteins expressed in stress (heat shock proteins 60 and 70), for cytoskeleton and cell membrane, energy metabolism and phosphorylation; as well as multiplication, and amino acid metabolism. However, two specific proteins were highly expressed in amastigotes: isocitrate dehydrogenase (IDH) and triosephosphate isomerase (TIM). The first was found to be essential for glutamate synthesis and for the formation of α-ketoglutarate, which was required for survival at 37°C; hence its expression in amastigotes was higher than in promastigotes. Meanwhile, TIM has an essential role in glycolysis to generate ATP within mammalian cells. A third study showed that enzymes specified in fatty acid β-oxidation and gluconeogenesis were upregulated in amastigotes, while glycolytic enzymes and flagellar proteins were highly expressed in promastigotes. In addition, the reviewers showed the importance of phosphoproteomics (large-scale study of phosphoproteins). In a single study recently conducted, proteins involved in stress and RNA/protein turnover and metabolism, as well as in RNA signaling, were identified, and the investigators stressed on these proteins as new drug targets because of their importance and critical role in Leishmania differentiation. In another recent study aiming to compare between differentially expressed proteins in amastigotes and promastigotes, the investigators used amastigotes differentiated within human monocyte-derived macrophages (not axenic amastigotes as in previous studies). The results also showed significant increase in proteins involved in glucose transport and decline of those involved in cell motility and cytoskeleton in intracellular amastigotes.
Second, the reviewers discussed the mechanisms adopted by host macrophages in response to Leishmania-expressed proteins, aiming to identify in vitro markers of susceptibility and resistance to leishmaniasis. Several proteins, predominantly expressed in infected macrophages, were involved in cellular metabolism, signaling and detoxification, as well as in cell immune response. The presence of 14 proteins only in Leishmania major and a unique protein (phospholipase D1; PLD1) in Leishmania amazonensis-infected cells was an interesting observation. The reviewers listed these 15 proteins which the investigators had analyzed using biological network modeling tool. They found that 14 out of the 15 proteins were organized as proteins for cell development, while PLD1 was involved in the formation of large parasitophorous vacuoles that characterize macrophages of leishmaniasis amazonensis. Another interesting observation was the upregulation of myosin light chain in L. major-infected macrophages and its correlation with small parasitophorous vacuoles. In addition, the reviewers focused on the role played by four additional proteins to modulate host immune response and were upregulated in Leishmania-infected macrophages: hypoxia-inducible factor 1-alpha (HIF-1α), TNF receptor-associated protein 1 (TRAP1), serpin, and PYD and CARD domain-containing protein (PYCARD). The first two were highly expressed in leishmaniasis major; HIF-1α increased production of NO and TNF-α as immune response mediators under hypoxic conditions, and TRAP1 maintained cellular viability under oxidative stress. On the other hand, serpin and PYCARD showed reduced expression in leishmaniasis amazonensis, with their roles in inflammatory cascade and apoptosis. Another study showed that mitochondrial antiviral signaling protein (MAVS) was significantly increased in infected mammalian cells after leishmaniasis. The investigators claimed that MAVS protein was significantly correlated with increased production of proinflammatory cytokines and IFN type I. Furthermore, a phosphoproteomic study showed the response of Leishmania amastigotes to reduced arginine levels in the host cell by increased expression of Leishmania arginine transporter. It was shown that reduced arginine levels in infected cells is mediated by the host immune response through mitogen-activated protein kinase 2 (MPK2)-dependent signaling cascade.
Third, the reviewers discussed proteins expressed in skin lesions in cutaneous leishmaniasis (CL), as well as expressed in the serum of infected patients with visceral leishmaniasis (VL). In a proteomic study conducted to analyze proteins extracted from cutaneous lesions and normal skin, the investigators identified 13 proteins in both lesions and normal samples. Nine of them were upregulated, and four were downregulated in CL in comparison with normal skin samples. The upregulated proteins were found to be involved in apoptosis (caspase 9), immune response (T cell receptor-β), and biosynthesis (transcription factor IIIB; BRF1). The reviewers focused on the role played by the significant increase in the apoptotic process that was positively correlated with the size of the cutaneous lesions. Similarly, very few proteomic studies were conducted to identify the proteins that were highly expressed in VL; however, these proteins were suggested to be used as diagnostic or prognostic or genetic markers rather than drug targets.
In conclusion, there are very few proteins that were identified in Leishmania amastigotes and involved in metabolic pathways to be developed as drug targets. In addition, most of these identified proteins are frequently modulated, and none of their molecular targets could be used to develop new therapeutic drugs. The reviewers recommended further studies to gain more proteomic data regarding these proteins or others and their pathways and signaling for survival and virulence of Leishmania amastigotes and promastigotes. Compiled from a review article. “Using proteomics to understand how Leishmania parasites survive inside the host and establish infection.” Int J Mol Sci 2016 Aug; 17(8):1270.
Filariasis: Nowadays, lymphatic filariasis, onchocerciasis and loaiasis are considered neglected tropical diseases. The use of mass drug administration (MDA) differs from area to another; i.e., MDA with diethylcarbamazine (DEC) + albendazole succeeded in the treatment of lymphatic filariasis in India, South America, and Southeast Asia, while it failed in certain areas of Africa where patients are coinfected with onchocerciasis, causing severe ocular and systemic manifestations. Meanwhile, MDA of ivermectin as the sole drug of choice for onchocerciasis is not suitable in certain areas of Africa where loaiasis is endemic, and ivermectin causes severe neurological manifestations resulting in coma and death. Therefore, development of new drugs is urgently required for these filarial infections. On the other hand, insect growth regulators (IGRs) are widely used in veterinary medicine to interfere in the process of insect larval molt and embryogenesis via a steroid hormone (ecdysone), which is produced by the prothoracic gland to trigger larva molting and metamorphosis. In other words, ecdysteroids are essential for larval development, and ecdysone receptors (EcRs) are thought to be involved in the developmental pathways of invertebrates, as they are absent in vertebrates. Amruta S. Mhashilkar et al., from the USA, assumed the use of EcRs as potential chemotherapeutic targets in filarial nematode infections due to several evident data: (1) filarial nematodes are considered ecdysozoan parasites, in which EcRs are the master regulators of their larval development; (2) EcRs are detected in several parasitic nematodes such as Ascaris suum, Brugia malayi, and O. volvulus; (3) the levels of ecdysteroid vary during larval development − where levels are high the third and fourth molts; (4) all parasitic nematodes have the ability to catabolize ecdysone; (5) ecdysteroids showed developmental properties on some filarial nematodes, such as promotion of microfilaria release in B. pahangi and stimulation of embryogenesis in D. immitis; and (6) EcR homologs were identified in genomic studies of some filarial parasites. To achieve their objective to discover new therapeutic drug targets against filarial infections, the investigators performed four studies. First, they infected gerbils with B. malayi infective larvae, followed by intraperitoneal administration of 20-hydroxyecdysone (20E), given daily for 150 days (time required for development of the third-stage larva into a fertile adult). The results showed no microfilariae in 20E-treated gerbils, while mature microfilariae were observed in the control animals. Second, to search for agonists for B. malayi EcR (BmaEcR), the investigators used a complicated methodology utilizing stable mammalian cell lines and three different constructed plasmids of BmaEcR. A screening strategy was used to test the activity of 40 library-selected compounds belonging to diacyl-hydrazine, tetrahydroxy-quinoline, and steroidal ecdysone analog families against BmaEcR. Third, using docking studies, the investigators conducted another virtual screening to verify the model performance of 25 selected compounds (the investigators mentioned the inclusion criteria for their selection). The results showed an excellent correlation between the results of both library and virtual screening. The EC50 values of the identified compounds (with high activity against BmaEcR) were determined at different concentrations to test for their cytotoxic properties. Based on their results, the investigators found only two compounds (ponasterone A and muristerone A) and they conducted their last and concluding study. They cultivated B. malayi gravid females in cell line cultures in the presence of 20E, ponasterone A, and muristerone A. The results showed that these two compounds resulted in a significant increase in aborted eggs, embryos, and microfilariae in comparison with 20E. Compiled from a research article. “Identification of ecdysone hormone receptor agonists as a therapeutic approach for treating filarial infections”. PLoSNegl Trop Dis 2016 Jun; 10(6): e0004772.
Diroﬁlariasis is a cardiopulmonary disease caused by D. immitis (heartworm) that infects dogs and cats and occasionally man. Until now, there is only a single drug class (macrocyclic lactones; MLs) for treatment of dirofilariasis. Recent studies confirmed the failure of MLs to prevent heartworm infection in dogs and cats and its possibility to be transmitted to man, as well as the resistance to MLs in the treatment of human onchocerciasis. Drug industry is based on targeting a vital system or organ for motility, feeding, and reproduction, which is the neuromuscular system. Canadian investigators (Thangadurai Mani et al.) with Spanish collaboration considered a new drug that targets ion channels (ICs) and their receptors in the neuromuscular system of the filarial nematodes. The investigators hypothesized that possible characterization of polymorphism in D. immitis genes encoding ICs and receptors might be an essential step in the development of new drug targets and/or identification of anticipated drug resistance. On the other hand, ICs are categorized according to either the ion type (cations or anions) or gate type (voltage or ligand gated). In the latter, ICs pass through gate using voltage change or are associated with either an amino acid (e.g. glutamate and γ-amino butyric acid; GABA) or a biogenic amine (e.g. serotonin, tyramine, or dopamine), and the cysteine-loop (cys-loop) superfamily is the most important IC. Therefore, their main objective is to identify possible single nucleotide polymorphic (SNP) sites in D. immitis IC genes through comparative genomic approaches. To identify all putative IC genes in the D. immitis genome, a complexed complementary approach was followed by searching for nucleotide sequences of all ICs genes in all nematodes in available databases (NCBI, Wormbase, Broad Institute and NEMBASE4). A total of 1249 nucleotide sequences were detected and blasted in the nuclear genome of eight different D. immitis strains (either MLs susceptible or with loss of efficacy). The four ML-susceptible isolates (122 worms from 17 dogs) were obtained from different countries (USA, Spain, Grenada, and Italy). Isolates with ML loss of efficacy were obtained from four infected dogs from four different areas in the USA, and from each dog ∼8000 microfilaria were collected. Worms from each country and microfilaria from each dog were pooled (eight strains) for genomic sequencing. According to blasting of sequences of ICs genes on the eight D. immitis genomic sequencing, 224 genes encoding 126 unique genes encoding ICs and receptors were named and classified, and 1762 single nucleotide polymorphic (SNP) sites were identified.
The results revealed that the D. immitis genome contains 81 genes encoding 42 cys-loop ICs genes (with 410 SNPs), 73 genes encoding 43 potassium ICs (with 284 SNPs), 26 genes encoding 9 calcium ICs (with 321 SNPs), 24 genes encoding 9 chloride ICs (with 265 SNPs), and 8 genes encoding 7 sodium ICs, while the remaining 12 genes were found to encode other types of ICs (16 genes). The investigators presented their SNP results in each type of IC and showed the differences between the collected isolates of MLs susceptible or with loss of efficacy (i.e. which SNP is specific or related to which type of isolate). Similar rates of polymorphism (70–75%) were detected in the susceptible isolates of the pooled population from USA, Spain, and Grenada, while the lower rate (43%) detected in the Italy isolate was attributed to the fact that the sample was obtained from a single Italian dog, while the other samples were obtained from multiple dog infections. On the basis of this result, the investigators concluded low genetic variability among ML-susceptible isolates. The lowest rate of SNPs was found in genes encoding potassium ICs (0.09%), while a high polymorphic rate (0.48%) was detected in the genes encoding chloride ICs. Meanwhile, the majority of SNPs (85.5%) were detected in the intron regions of the genes that encode ICs, but a few of them were detected in the ML-susceptible pooled population; only 14.5% SNPs were located in the exon region of the gene, and a few of them were detected in MLs with loss of efficacy population. These results, along with those obtained from previous studies, pushed the investigators to conclude the presence of specific gene mutations at certain codons (intron or exon regions) that are responsible for drug selection and resistance, which requires further studies with more field isolates. The investigators also highlighted SNPs specific to MLs-susceptible or with loss of efficacy population to provide basic information for future studies which search for new drug development and require additional data to ensure drug activity against all allelic variants of the drug target. The investigators also strongly recommended further studies in glutamate-gated and GABA-gated chloride channels as new drug targets for dirofilariasis. As regards voltage-gated ICs, the investigators discussed their role as antihelminthic drug targets and claimed that only two drugs, emodepside and praziquantel, have been developed until now to target genes encoding voltage potassium and calcium IC. Meanwhile, the investigators found high polymorphism (in both intron and exon regions) in these genes with high prediction of resistance to new drugs targeting these genes. Compiled from a research article. “Polymorphism in ion channel genes of Dirofilaria immitis: Relevant knowledge for future anthelmintic drug design.” Inter J Parasitol: Drugs and Drug Resistance 2016; 6:343-355.
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