Thioredoxin reductase and glutathione synthesis in Plasmodium falciparum

The malaria parasite Plasmodium falciparum is still a major threat to human health in the non-industrialised world mainly due to the increasing incidence of drug resistance. Therefore, there is an urgent need to identify and validate new potential drug targets in the parasite's metabolism that are suitable for the design of new anti-malarial drugs. It is known that infection with P. falciparum leads to increased oxidative stress in red blood cells, implying that the parasite requires efficient antioxidant and redox systems to prevent damage caused by reactive oxygen species. In recent years, it has been shown that P. falciparum possess functional thioredoxin and glutathione systems. Using genetic and chemical tools, it was demonstrated that thioredoxin reductase, the first step of the thioredoxin redox cycle, and gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting step of glutathione synthesis, are essential for parasite survival. Indeed, the mRNA levels of gamma-GCS are elevated in parasites that are oxidatively stressed, indicating that glutathione plays an important antioxidant role in P. falciparum. In addition to this antioxidant function, glutathione is important for detoxification processes and is possibly involved in the development of resistance against drugs such as chloroquine.     

Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis

African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.     

Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling

Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the general failure of vector eradication programs, drugs are the main line of defence, but the newest drugs are being tracked by the emergence of resistance in parasites, sharing ominous parallels with multidrug resistance in bacterial pathogens. Any of a number of mechanisms will elicit a drug resistance phenotype in parasites, including: active efflux, reduced uptake, target modification, drug modification, drug sequestration, by-pass shunting, or substrate competition. The role of ABC transporters in parasitic multidrug resistance mechanisms is being subjected to more scrutiny, due in part to the established roles of certain ABC transporters in human diseases, and also to an increasing portfolio of ABC transporters from parasite genome sequencing projects. For example, over 100 ABC transporters have been identified in the Escherichia coli genome, but to date only about 65 in all parasitic genomes. Long established laboratory investigations are now being assisted by molecular biology, bioinformatics, and computational modelling, and it is in these areas that the role of ABC transporters in parasitic multidrug resistance mechanisms may be defined and put in perspective with that of other proteins. We discuss ABC transporters in parasites, and conclude with an example of molecular modelling that identifies a new interaction between the structural domains of a parasite P-glycoprotein.     

Beyond Schuh: early studies on the oxidation of LDL and other lipoproteins and its role in atherosclerosis

Abstract

The malaria parasite Plasmodium falciparum is still a major threat to human health in the non-industrialised world mainly due to the increasing incidence of drug resistance. Therefore, there is an urgent need to identify and validate new potential drug targets in the parasite's metabolism that are suitable for the design of new anti-malarial drugs. It is known that infection with P. falciparum leads to increased oxidative stress in red blood cells, implying that the parasite requires efficient antioxidant and redox systems to prevent damage caused by reactive oxygen species. In recent years, it has been shown that P. falciparum possess functional thioredoxin and glutathione systems. Using genetic and chemical tools, it was demonstrated that thioredoxin reductase, the first step of the thioredoxin redox cycle, and γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting step of glutathione synthesis, are essential for parasite survival. Indeed, the mRNA levels of γ-GCS are elevated in parasites that are oxidatively stressed, indicating that glutathione plays an important antioxidant role in P. falciparum. In addition to this antioxidant function, glutathione is important for detoxification processes and is possibly involved in the development of resistance against drugs such as chloroquine.     

Rational deployment of antimalarial drugs in Africa: should first-line combination drugs be reserved for paediatric malaria cases?

Artemisinin-based combination therapy is exerting novel selective pressure upon populations of Plasmodium falciparum across Africa. Levels of resistance to non-artemisinin partner drugs differ among parasite populations, and so the artemisinins are not uniformly protected from developing resistance, already present in South East Asia. Here, we consider strategies for prolonging the period of high level efficacy of combination therapy for two particular endemicities common in Africa. Under high intensity transmission, two alternating first-line combinations, ideally with antagonistic selective effects on the parasite genome, are advocated for paediatric malaria cases. This leaves second-line and other therapies for adult cases, and for intermittent preventive therapy. The drug portfolio would be selected to protect the 'premier' combination regimen from selection for resistance, while maximising impact on severe disease and mortality in children. In endemic areas subject to low, seasonal transmission of Plasmodium falciparum, such a strategy may deliver little benefit, as children represent a minority of cases. Nevertheless, the deployment of other drug-based interventions in low transmission and highly seasonal areas, such as mass drug administration aimed to interrupt malaria transmission, or intermittent preventive therapy, does provide an opportunity to diversify drug pressure. We thus propose an integrated approach to drug deployment, which minimises direct selective pressure on parasite populations from any one drug component. This approach is suitable for qualitatively and quantitatively different burdens of malaria, and should be supported by a programme of routine surveillance for emerging resistance.     

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

Trichomoniasis is the most common, sexually transmitted infection.It is caused by the flagellated protozoan parasite Trichomonas vagina/is. Symptoms include vaginitis and infections have been associatedwith preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDSand cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved,effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, withsome cases remaining unresolved. The mechanism of metronidazole resistance in T. Vagina/is from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasingmet ronidazole pressure. In the latter situation, hydrog enosomal function which is involved in activationof the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal ofdrug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintaintheir resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded asa laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. Vagina/is appears to be on the increase and improved surveillance of treatment failures is urged.     

The mode of action of chloroquine and related malarial schizontocides

Use of fast-acting blood schizontocidal drugs such as chloroqune, amodiaquine, mepacrine or quinine, is essential for the treatment of acute malaria infections. The spread of resistance in Plasmodium falciparum to chloroquine, the most useful of these drugs, has been a serious problem since the 1960s, and the resistant strains show various degrees of cross-resistance to other drugs. Design of replacement drugs requires knowledge of their modes of action and mechanisms of resistance. At present, there are two theories to explain the mode of action of chloroquine (Box 1). In this debate, Coy Fitch advances the hypothesis that chloroquine acts by delaying the sequestration of Ferriprotoporphyrin IX (FP) into malaria pigment, thereby allowing FP to exert its intrinsic cellular toxicity. In contrast, David Warhurst proposes a new 'Permease theory' suggesting that chloroquine is imported into the parasite cytoplasm on a membrane carrier (the permease) under the influence of a proton gradient; the drug would then interfere with lysosomal digestion of haemoglobin, thus starving the parasite of amino acids for protein synthesis.     

Identification of Selective Inhibitors of the Plasmodium falciparum Hexose Transporter PfHT by Screening Focused Libraries of Anti-Malarial Compounds

Development of resistance against current antimalarial drugs necessitates the search for novel drugs that interact with different targets and have distinct mechanisms of action. Malaria parasites depend upon high levels of glucose uptake followed by inefficient metabolic utilization via the glycolytic pathway, and the Plasmodium falciparum hexose transporter PfHT, which mediates uptake of glucose, has thus been recognized as a promising drug target. This transporter is highly divergent from mammalian hexose transporters, and it appears to be a permease that is essential for parasite viability in intra-erythrocytic, mosquito, and liver stages of the parasite life cycle. An assay was developed that is appropriate for high throughput screening against PfHT based upon heterologous expression of PfHT in Leishmania mexicana parasites that are null mutants for their endogenous hexose transporters. Screening of two focused libraries of antimalarial compounds identified two such compounds that are high potency selective inhibitors of PfHT compared to human GLUT1. Additionally, 7 other compounds were identified that are lower potency and lower specificity PfHT inhibitors but might nonetheless serve as starting points for identification of analogs with more selective properties. These results further support the potential of PfHT as a novel drug target.     

Leishmania: role of P glycoprotein in drug resistance and reversion strategies

Protozoan parasites are important causative agents of morbidity and mortality throughout the world--a problem further complicated by the emergence of drug resistance in these parasites. Mechanisms of drug resistance include the following: decreased uptake of the drug into the cell, loss of drug activation, alterations in the drug target, and over-expression of a well-known multiple drug transporter proteins. In this review, two critical components of resistance are stressed: (1) the role of ATP binding cassette proteins, such as P-glycoproteins, in mediating drug resistance in Leishmania and other protozoans, followed by development of cross-resistance to many structurally and functionally unrelated drugs, and (2) some concepts concerning the reversal mechanism of multidrug resistance by drugs and natural products. Several modulators or chemosensitizers alter the capacity of P-glycoproteins to maintain subtoxic intracellular drug concentrations. Calcium channel blockers such as verapamil act in this mode; however, high concentrations are required for an efficient and effective inhibition and, in addition, produce undesirable side effects. The discovery of new, natural product modulators of P-glycoproteins is stressed. This category of modulators offer potentially improved efficacy and lowered toxicity for the mammalian host.     

The pharmacology of anthelmintics in livestock

The recent development of ivermectin, netobimin, closantel, triclabendazole and clorsulon as well as new ways of using established anthelmintics has significantly improved the potential for parasite control in livestock. It is of interest to note that potential control of F. hepatica has been particularly improved with four of these new anthelmintic drugs being fasciolicidal. In addition to an appreciation of the spectrum of action of the new anthelmintics, a considerable amount of information on the pharmaco-kinetic behaviour, metabolism and mode of action of these drugs has been published. New information on pharmacology of established anthelmintics has provided new insights into the biology of parasites, drug resistance and the options for improved chemotherapy by manipulation of pharmacokinetics and metabolism. Much recent pharmacological research has been concentrated on the BZs and this has been considered here in an attempt to provide a better understanding of this significant group of anthelmintics. The development of new delivery systems, such as intraruminal slow release devices, is potentially useful with most anthelmintics. So far, this new approach to parasite control has been applied most successfully with morantel tartrate.     

Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin

Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field.     

An Epigenetic Antimalarial Resistance Mechanism Involving Parasite Genes Linked to Nutrient Uptake

Acquired antimalarial drug resistance produces treatment failures and has led to periods of global disease resurgence. In Plasmodium falciparum, resistance is known to arise through genome-level changes such as mutations and gene duplications. We now report an epigenetic resistance mechanism involving genes responsible for the plasmodial surface anion channel, a nutrient channel that also transports ions and antimalarial compounds at the host erythrocyte membrane. Two blasticidin S-resistant lines exhibited markedly reduced expression of clag genes linked to channel activity, but had no genome-level changes. Silencing aborted production of the channel protein and was directly responsible for reduced uptake. Silencing affected clag paralogs on two chromosomes and was mediated by specific histone modifications, allowing a rapidly reversible drug resistance phenotype advantageous to the parasite. These findings implicate a novel epigenetic resistance mechanism that involves reduced host cell uptake and is a worrisome liability for water-soluble antimalarial drugs.     

A BODIPY-embedding miltefosine analog linked to cell-penetrating Tat(48-60) peptide favors intracellular delivery and visualization of the antiparasitic drug

Therapeutic application of many drugs is often hampered by poor or denied access to intracellular targets. A case in point is miltefosine (MT), an orally active antiparasitic drug, which becomes ineffective when parasites develop dysfunctional uptake systems. We report here the synthesis of a fluorescent BODIPY-embedding MT analogue with appropriate thiol functionalization allowing linkage to the cell-penetrating Tat(48-60) peptide through disulfide or thioether linkages. The resulting constructs are efficiently internalized into the otherwise MT-invulnerable R40 Leishmania strain, resulting in fast parasite killing, and hence successful avoidance of the resistance. In the disulfide-linked conjugate, an additional fluoro tag on the Tat moiety allows to monitor its reductive cleavage within the cytoplasm. Terminally differentiated cells such as peritoneal macrophages, impervious to MT unless infected by Leishmania, can uptake the drug in its Tat-conjugated form. The results afford proof-of-principle for using CPP vectors to avert drug resistance in parasites, and/or for tackling leishmaniasis by modulating macrophage uptake.     

Malarial parasites vs. antimalarials: never-ending rumble in the jungle

Development of resistance is an increasing problem for antimalarial chemotherapy because resistance against most available drugs has developed in the majority of world-wide parasite populations. Therefore, several strategies to counteract resistance-development are in place. From the pharmaceutical side, identification of new targets and compounds, development of structural relatives of known antimalarials, and fixed combination therapy are pursued. On the other hand, clinical studies focus on novel regimens, distribution schemes and drug combinations. A third possibility to diminish progression of resistance is the application of evolutionary concepts to design new strategies for validation, monitoring and interference with the selection-process that leads to the spread of multidrug-resistance. Since the pharmacologic and clinical side of antimalarial chemotherapy is covered by recent reviews we refer to the newest developments only and lay our focus on determinants of selection for drug resistance in human malaria.     

Current perspectives on the mechanism of action of artemisinins

Artemisinin derivatives are the most recent single drugs approved and introduced for public antimalarial treatment. Although their recommended use is for treatment of Plasmodium falciparum infection, these drugs also act against other parasites, as well as against tumor cells. The mechanisms of action attributed to artemisinin include interference with parasite transport proteins, disruption of parasite mitochondrial function, modulation of host immune function and inhibition of angiogenesis. Artemisinin combination therapies are currently the preferred treatment for malaria. These combinations may prevent the induction of parasite drug resistance. However, in view of the multiple mechanisms involved, especially when additional drugs are used, the combined therapy should be carefully examined for antagonistic effects. It is now a general theory that the crucial mechanism is interference with plasmodial SERCA. Therefore, future development of resistance may be associated with overproduction or mutations of this transporter. However, a general mechanism, such as alterations in general drug transport pathways, is feasible. In this article, we review the evidence for each mechanism of action suggested.     

Sequence signatures involved in targeting the male-specific lethal complex to X-chromosomal genes in Drosophila melanogaster

Background

Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance. A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.

Results

Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.

Conclusions

Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.     

Reduced permeability in CHO cells as a mechanism of resistance to colchicine

Colchicine resistant (CH^R) lines of stable phenotype have been isolated from cultured Chinese hamster (CHO) cells. Successive single-step selections for increasing resistance were performed by isolating resistant colonies at each step. Two complementary assays involving [³H] colchicine uptake by whole cells and binding of [³H] colchicine by cytoplasmic extracts were developed to test for altered permeability and altered intracellular target protein, respectively. All clones isolated appeared to have decreased permeability to the drug while their colchicine-binding ability was not reduced. The amount of reduction in colchicine uptake correlated strongly with cellular resistance. The CH^R lines were also cross resistant to other drugs such as actinomycin D, vinblastine and Colcemid; furthermore, the degree of cross resistance was positively correlated with the degree of colchicine resistance. The non-ionic detergent Tween 80 potentiated the cytotoxic action of colchicine on mutant cells as well as its rate of uptake into whole cells.     

New perspectives on the use of nucleic acids in pharmacological applications: inhibitory action of extracellular self-DNA in biological systems

The research for new products against pathogens, parasites and infesting species, in both agriculture and medicine, implies huge and increasing scientific, industrial and economic efforts. Traditional approaches are based on random screening procedures searching for bioactive compounds. However, the success of such methodologies in most cases has been strongly limited by side-effects of the potential new drugs, especially toxicity and pharmacological resistance. The use of nucleic acids in drug development has been introduced searching for target-specific effect. In addition, a recent discovery revealed that randomly fragmented extracellular self-DNA may act as highly species-specific inhibitory product for different species, suggesting an unprecedented use of DNA for biological control. On this base, a new scenario of pharmacological applications is discussed.     

抗结核药物及其耐药相关突变位点的研究进展

结核病是结核分枝杆菌复合物引起的传染性疾病,致死率、致残率高,在全球传染病中居第2位。近年来耐药结核病所占比例逐年升高,成为消灭结核病面临的巨大挑战之一。传统的耐药诊断方法基于培养,费时费力,所需技术要求高;而现有分子检测方法仅能检测少量抗结核药物的少数耐药基因。因此,更好地理解抗结核药物的耐药机制有助于全面耐药诊断。本文对临床中使用频率较高的11类一线和二线抗结核药物及其相应耐药相关基因、突变位点的研究进展进行总结,尤其是对环丝氨酸、利奈唑胺、氯法齐明等二线药物的近期研究做了系统描述,为全面耐药诊断、精准治疗指导、新药研发及耐药机制深入研究提供了前期工作基础。     

Comparative folate metabolism in humans and malaria parasites (part I): pointers for malaria treatment from cancer chemotherapy

New inhibitors are urgently needed to overcome the burgeoning problem of drug resistance in the treatment of Plasmodium falciparum infection. Targeting the folate pathway has proved to be a powerful strategy for drug development against rapidly multiplying systems such as cancer cells and microorganisms. Antifolates have long been used for malaria treatment but, despite their success, much less is known about parasite folate metabolism than about that of the human host. In this article, we focus on folate enzymes used clinically as anticancer drug targets, in addition to those that have potential to be used as drug targets, for which there are inhibitors at various stages of development. We discuss how this information could lead to the identification of new targets in malaria parasites.