Drug-resistant Parasites Research Articles

Effect of anti-hyperlipidemia drugs on the alpha-tocopherol concentration and their potential for murine malaria infection.

The current preventions of malaria are protection against mosquito bites and taking chemoprophylactic anti-malarial drugs. However, drug therapies are usually associated with adverse events and emergency of drug-resistant malaria parasites. Previous study showed that host plasma alpha-tocopherol deficiency enhanced resistance against malaria infection in mice. Here, we report a new prevention strategy against malaria by using anti-hyperlipidemia drugs, ezetimibe, berberine, cholestyramine, and probucol to modify the host plasma alpha-tocopherol concentration. The drugs were mixed with diet and fed to C57BL/6J mice for 2 weeks. Although all drugs reduced plasma alpha-tocopherol concentration after 2 weeks of feeding, probucol-treated mice showed 90 % reduction and it was the lowest alpha-tocopherol concentration among the four drugs. Ezetimibe, berberine, and combination of ezetimibe and berberine pretreatment for 2 weeks were not effective against infection of Plasmodium yoelii XL17, a lethal strain, for survival and parasitemia in mice. Two-week pretreatment and 1-week treatment after infection of cholestyramine had also no effect on malaria infection. Survival rates of cholestyramine, ezetimibe, and/or berberine treated mice were 0-22 %. However, probucol caused significant decrease in parasitemia and increased in mice survival following 2-week pretreatment and 1-week treatment after infection. All control mice died while all probucol treated mice survived during the course of infection. Thus, probucol which reduced plasma alpha-tocopherol concentration was effective in enhancing the host to resist malaria infection in mice. Our finding indicates that plasma alpha-tocopherol reducing drugs like probucol might be a candidate for beneficial prevention strategy for travelers from malaria-free area.

Systems analysis of host-parasite interactions.

Parasitic diseases caused by protozoan pathogens lead to hundreds of thousands of deaths per year in addition to substantial suffering and socioeconomic decline for millions of people worldwide. The lack of effective vaccines coupled with the widespread emergence of drug‐resistant parasites necessitates that the research community take an active role in understanding host–parasite infection biology in order to develop improved therapeutics. Recent advances in next‐generation sequencing and the rapid development of publicly accessible genomic databases for many human pathogens have facilitated the application of systems biology to the study of host–parasite interactions. Over the past decade, these technologies have led to the discovery of many important biological processes governing parasitic disease. The integration and interpretation of high‐throughput ‐omic data will undoubtedly generate extraordinary insight into host–parasite interaction networks essential to navigate the intricacies of these complex systems. As systems analysis continues to build the foundation for our understanding of host–parasite biology, this will provide the framework necessary to drive drug discovery research forward and accelerate the development of new antiparasitic therapies. WIREs Syst Biol Med 2015, 7:381–400. doi: 10.1002/wsbm.1311For further resources related to this article, please visit the WIREs website.

Unlocking artemisinin's biosynthetic secrets

Plant Science![Figure][1] The malaria drug artemisinin is isolated from Artemisia annua plants PHOTO: © MANFRED RUCKSZIO/ALAMY The emergence of drug-resistant parasites presents a major barrier to controlling human malaria. Artemisinin is the recommended first-line treatment for uncomplicated mal aria, but demand outstrips drugmakers' ability to cultivate the source, the Chinese herb Artemisia annua . A better understanding of the biosynthetic pathway could enhance scientists' ability to produce artemisinin from yeast, increasing the drug's availability. Bryant et al. explored the proteome of artemisinin-producing hairs stripped off the plant, cross-matching with genomic information to identify proteins. Key to the final steps of artemisinin biosynthesis may be enzymes like cyclophilins and peroxidases, hinting that steps that can proceed nonenzymatically may nonetheless get an enzymatic turbo-boost. BMC Plant Biol. 15 , 175 (2015). [1]: pending:yes

Editorial: The Schistosomiasis Vaccine - It is Time to Stand up.

Schistosomiasis is a severe parasitic disease, endemic in 74 developing countries with up to 600 million people infected and 800 million, mostly children, at risk of contracting the disease following infection predominantly with Schistosoma mansoni, Schistosoma haematobium, or Schistosoma japonicum. The disease burden is estimated to exceed 70 million disability-adjusted life-years, and leads to remarkably high YLD (years lived with disability) rates. Even more importantly, people with schistosomiasis are highly susceptible to malaria, tuberculosis, and hepatic and acquired immunodeficiency viruses. There is only one drug, praziquantel, currently available for treatment and it has high efficacy, low cost, and limited side effects. However, only 13% of the target population has received the drug, and those treated are at continuous risk of reinfection necessitating repeated drug administration and the emergence of drug-resistant parasites is a constant threat (1). Currently there is no vaccine. The a priori requirements for discovery of a vaccine formulation include the following: identification of protective key immune players in humans; characterization and isolation of target antigens; establishment of efficacy in terms of reduction of parasite burden as well as amelioration of immunopathology; establishment of safety; and finally, provision of considerable funds along with physical infrastructure and qualified personnel to carry out clinical trials.

Basigin is a druggable target for host-oriented antimalarial interventions.

Plasmodium falciparum is the parasite responsible for the most lethal form of malaria, an infectious disease that causes a large proportion of childhood deaths and poses a significant barrier to socioeconomic development in many countries. Although antimalarial drugs exist, the repeated emergence and spread of drug-resistant parasites limit their useful lifespan. An alternative strategy that could limit the evolution of drug-resistant parasites is to target host factors that are essential and universally required for parasite growth. Host-targeted therapeutics have been successfully applied in other infectious diseases but have never been attempted for malaria. Here, we report the development of a recombinant chimeric antibody (Ab-1) against basigin, an erythrocyte receptor necessary for parasite invasion as a putative antimalarial therapeutic. Ab-1 inhibited the PfRH5-basigin interaction and potently blocked erythrocyte invasion by all parasite strains tested. Importantly, Ab-1 rapidly cleared an established P. falciparum blood-stage infection with no overt toxicity in an in vivo infection model. Collectively, our data demonstrate that antibodies or other therapeutics targeting host basigin could be an effective treatment for patients infected with multi-drug resistant P. falciparum.

Systems of Populations with Multiple Structures: Modeling and Analysis

In this paper a class of functional–differential equations is considered, arising in modeling the dynamics of structured populations. This class includes model equations for diseases spreading by interactions of hosts, vectors and parasites considered in An (Drug Resistance in Infectious Diseases: Modeling, Analysis and Simulation, Doctoral thesis, University of Heidelberg, 2012) and An et al. (A structured population model for malaria including drug resistance. In preparation), where drug sensitive and drug resistant parasites and their effects on the infected host and vector populations are modeled via structural variables. In this paper, the evolution of n structured and m unstructured interacting populations is described by a coupled system of n functional–partial differential equations and m functional–ordinary differential equations. In general, besides initial conditions additional “boundary” conditions are required in order to determine missing data for the structure dependent variables. As the main result of this paper, we are going to present the analysis for the existence of unique, positive solutions.

Long-acting drug to treat resistant malaria

Drug Discovery Malaria kills 0.6 million people annually. Currently available drugs are no longer fully effective, because the malarial parasite has developed resistance. Now, Phillips et al. have identified a drug, DSM265, that kills both drug-sensitive and drug-resistant parasites by targeting their ability to synthesize precursors required for synthesis of DNA and RNA. DSM265 kills parasites in the blood and the liver and is sufficiently long-acting that it could potentially cure malaria after a single dose or provide effective chemo-prevention if given weekly. Sci. Transl. Med. 7 , 296ra111 (2015).

Malaria and the mobile and migrant population in Cambodia: a population movement framework to inform strategies for malaria control and elimination.

BackgroundThe relationships between human population movement (HPM) and health are a concern at global level. In the case of malaria, those links are crucial in relation to the spread of drug resistant parasites and to the elimination of malaria in the Greater Mekong sub-Region (GMS) and beyond. The mobile and migrant populations (MMP) who are involved in forest related activities are both at high risk of being infected with malaria and at risk of receiving late and sub-standard treatment due to poor access to health services. In Cambodia, in 2012, the National Malaria Control Programme (NMCP) identified, as a key objective, the development of a specific strategy for MMPs in order to address these challenges. A population movement framework (PMF) for malaria was developed and operationalized in order to contribute to this strategy.MethodsA review of the published and unpublished literature was conducted. Based on a synthesis of the results, information was presented and discussed with experienced researchers and programme managers in the Cambodian NMCP and led to the development and refinement of a PMF for malaria. The framework was “tested” for face and content validity with national experts through a workshop approach.ResultsIn the literature, HPM has been described using various spatial and temporal dimensions both in the context of the spread of anti-malarial drug resistance, and in the context of malaria elimination and previous classifications have categorized MMPs in Cambodia and the GMS through using a number of different criteria. Building on these previous models, the PMF was developed and then refined and populated with in-depth information relevant to Cambodia collected from social science research and field experiences in Cambodia. The framework comprises of the PMF itself, MMP activity profiles and a Malaria Risk Index which is a summation of three related indices: a vulnerability index, an exposure index and an access index which allow a qualitative ranking of malaria risk in the MMP population. Application of currently available data to the framework illustrates that the highest risk population are those highly mobile populations engaged in forest work.ConclusionThis paper describes the process of defining MMPs in Cambodia, identifying the different activities and related risks to appropriately target and tailor interventions to the highest risk groups. The framework has been used to develop more targeted behaviour change and outreach interventions for MMPs in Cambodia and its utility and effectiveness will be evaluated as part of those interventions.

In vitro and in vivo evaluation of antitrypanosomal activity of Annona muricata stem bark extracts.

Summary The control of trypanosomosis in animals and humans based on chemotherapy is limited and not ideal, since the agents used are associated with severe side effects, and emergence of relapse and drug resistant parasites. The need for the development of new, cheap and safe compounds stimulated this study. Three concentrations (211, 21.1 and 2.11 mg per ml) of chloroform stem bark extract of Annona muricata were screened for trypanocidal activity against Trypanosoma brucei brucei in vitro. Also, two doses (200 mg per kg and 100 mg per kg) of the extract were evaluated for trypanocidal activity in rats infected with the parasite. Haematological parameters were determined on day 1 post infection and on days 1, 6 and 30-post extract treatment. The extracts inhibited parasite motility and totally eliminated the organisms at the concentrations used in vitro. The extract also showed promising in vivo trypanocidal activity. The observed in vitro and in vivo trypanocidal activities may be due to the presence of bioactive compounds present in the extracts as seen in this study. The extract also improved the observed decreases in haematological parameters of the treated rats, which may be due to their ability to decrease parasite load. The observed oral LD50 of 1,725.05 mg per kg of the chloroform A. muricata extract using up and down method is an indication of very low toxicity, implying that the extract could be administered with some degree of safety.

The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism.

Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 μM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.

Insights into the Interactions of Fasciola hepatica Cathepsin L3 with a Substrate and Potential Novel Inhibitors through In Silico Approaches.

Background Fasciola hepatica is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. F. hepatica cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of F. hepatica.Methodology/Principal FindingsStarting with a three dimensional model of FhCL3 we performed a structure-based design of novel inhibitors through a computational study that combined virtual screening, molecular dynamics simulations, and binding free energy (ΔGbind) calculations. Virtual screening was carried out by docking inhibitors obtained from the MYBRIDGE-HitFinder database inside FhCL3 and human cathepsin L substrate-binding sites. On the basis of dock-scores, five compounds were predicted as selective inhibitors of FhCL3. Molecular dynamic simulations were performed and, subsequently, an end-point method was employed to predict ΔGbind values. Two compounds with the best ΔGbind values (-10.68 kcal/mol and -7.16 kcal/mol), comparable to that of the positive control (-10.55 kcal/mol), were identified. A similar approach was followed to structurally and energetically characterize the interface of FhCL3 in complex with a peptidic substrate. Finally, through pair-wise and per-residue free energy decomposition we identified residues that are critical for the substrate/ligand binding and for the enzyme specificity.Conclusions/SignificanceThe present study is the first computer-aided drug design approach against F. hepatica cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds as FhCL3 inhibitors. Overall, these results will foster the future rational design of new inhibitors against FhCL3, as well as other F. hepatica cathepsins.

Anti-Toxoplasma activity of natural products: a review.

Toxoplasma gondii is an intracellular parasite widely distributed in nature. Infection is asymptomatic in immunocompetent individuals; however, various clinical manifestations may occur in immunocompromised individuals. Although there are medications for the treatment of toxoplasmosis, such as pyrimethamine and sulfonamide, they cannot always be used due to adverse reactions or to therapeutic failures related to intolerance or malabsorption of drugs and to parasite drug resistance. In recent years, the search for new antimicrobial agents derived from plants has intensified because a quarter of synthetic drugs that are currently prescribed have been isolated from a plant source, demonstrating that natural products are important in the development of new drugs. A systematic literature search was conducted to evaluate the use of natural products as an alternative for the treatment of T. gondii infection. The search was conducted for the 2000-2014 period in Science Direct, Scopus, PubMed, EMBASE, and SciELO databases, using the following MeSH terms: anti-Toxoplasma activity, toxoplasma AND natural products, toxoplasma AND plant extracts. Ethnobotanical and experimental evidence (in vitro/in vivo) was found supporting the use of natural products as a source for the discovery of new therapies against T. gondii.

A non-cytotoxic N-dehydroabietylamine derivative with potent antimalarial activity

Malaria caused by the Plasmodium parasites continues to be an enormous global health problem owing to wide spread drug resistance of parasites to many of the available antimalarial drugs. Therefore, development of new classes of antimalarial agents is essential to effectively treat malaria. In this study, the efficacy of naturally occurring diterpenoids, dehydroabietylamine and abietic acid, and their synthetic derivatives was assessed for antimalarial activity. Dehydroabietylamine and its N-trifluoroacetyl, N-tribromoacetyl, N-benzoyl, and N-benzyl derivatives showed excellent activity against P. falciparum parasites with IC50 values of 0.36 to 2.6 µM. Interestingly, N-dehydroabietylbenzamide showed potent antimalarial activity (IC50 0.36), and negligible cytotoxicity (IC50 >100 µM) to mammalian cells; thus, this compound can be an important antimalarial drug. In contrast, abietic acid was only marginally effective, exhibiting an IC50 value of ~82 µM. Several carboxylic group-derivatives of abietic acid were moderately active with IC50 values of ~8.2 to ~13.3 µM. These results suggest that a detailed understanding of the structure–activity relationship of abietane diterpenoids might provide strategies to exploit this class of compounds for malaria treatment.

Relevance of undetectably rare resistant malaria parasites in treatment failure: experimental evidence from Plasmodium chabaudi.

Resistant malaria parasites are frequently found in mixed infections with drug-sensitive parasites. Particularly early in the evolutionary process, the frequency of these resistant mutants can be extremely low and below the level of molecular detection. We tested whether the rarity of resistance in infections impacted the health outcomes of treatment failure and the potential for onward transmission of resistance. Mixed infections of different ratios of resistant and susceptible Plasmodium chabaudi parasites were inoculated in laboratory mice and dynamics tracked during the course of infection using highly sensitive genotype-specific quantitative polymerase chain reaction (qPCR). Frequencies of resistant parasites ranged from 10% to 0.003% at the onset of treatment. We found that the rarer the resistant parasites were, the lower the likelihood of their onward transmission, but the worse the treatment failure was in terms of parasite numbers and disease severity. Strikingly, drug resistant parasites had the biggest impact on health outcomes when they were too rare to be detected by any molecular methods currently available for field samples. Indeed, in the field, these treatment failures would not even have been attributed to resistance.

Tracking of Coetaneous Leishmaniasis by Parasitological, Molecular and Biochemical Analysis

Background: In areas of endemicity without sufficient laboratory infrastructure, identification of Leishmaniaparasites is useful for control and preventive plan and very important since species differentiation is instrumental in selection of optimal therapy and treatment regimens. objectives: The present study was performed to identify the species and strain of Leishmaniaparasites isolated of different endemic areas. Methodology: In a consecutive series of 126 patients referred for a suspected CL lesion during October 2010 to December 2012 in five Iraqi provinces, direct smear and culture followed by molecular and biochemical analysis were done using nested-PCR and cellulose acetate electrophoresis. Results: Direct smears revealed that 90 (70%) patients gave positive results under light microscope. Out of 126 cases, 83 (467) gave positive growth in modified NNN medium and RPMI-1640 with fetal bovine serum followed by sub-culture in drosophila Schneider’s media. Out of 83 positive growth culture, only 52 specimens were studied by nested - PCR .It was found that 45 (89.5%) cases in the generation of a 560 bp DNA and 7(13.4%) patients displayed a fragment of 750 bp, corresponding to L. major and L. tropica, respectively. Cellulose acetate electrophoresis (CAE) was performed by 4 enzyme systems (glucose phosphate isomerize, leucilphosphate, mannose phosphate isomerize and 6 phosphogluconate dehydrogenase). The results showed that L. Major(LV39) isolate were in 20 mass cultivated culture Conclusion: Nested-PCR and cellulose acetate electrophoresis was an ideal method for discrimination of Leis mania. spp. and variants. Both L. major and L. tropica were the causative agents of cutaneous Leishmaniasis but L. major was the main species in study area. Recommendations: As a result of the observations made during the whole study period, nested- PCR is very suitable method, which can be used as a basis for future applications. Opening number of research centers situated in the country offer DNA applications for so-called “parasite tracking.” Such an application is probably more relevant for epidemiological purposes than for diagnosis. (e.g., in outbreak investigations or tracking drug-resistant parasite strains). After collection in the field, samples can be simply transferred to such a center for further analysis. More experimental studies for effective vaccine are developed for Leishmaniasis.

What is the mechanism of action of praziquantel and how might resistance strike?

Schistosomiasis, also known as bilharzia, is a neglected tropical disease affecting over 249 million people of which approximately 45% are school-aged children. Although there are significant numbers of people with the disease in Asia and South America, over 90% of those infected reside in Africa. The Global Burden of Disease Study 2010 estimated 11,700 deaths and 2,986,000 years lost to disability due to schistosomiasis in that year [1,2]. In 2012, the World Health Organization produced a ‘roadmap’ whose ultimate goal was the “...elimination of NTDs or reductions in their impacts to levels at which they are no longer considered public-health problems” [3]. Schistosomiasis was listed as a disease for potential elimination in the Eastern Mediterranean, Caribbean, Indonesia and Mekong River basin by 2015 and Brazil by 2020. Understandably, a more conservative note was sounded for Sub-Saharan Africa where worry over availability of medication was cited as the reason for failing to schedule elimination by 2020. The best-case scenario that could be made was with widespread availability of medication and strong political support schistosomiasis could be “...eliminated as a ‘public health problem’ in multiple countries in Africa by 2020.” The causative agents of schistosomiasis are digenetic trematode worms belonging the genus Schistosoma with Schistosoma mansoni, S. japonicum and S. haematobium being responsible for the majority of cases. Unfortunately, there is no vaccine available for the treatment of human schistosomiasis nor is the development of one on the horizon. Praziquantel (PZQ) is the only widely available drug for treatment of the disease and is thus at the heart of all control programs. It has been available for over 30 years yet the mechanism by which it kills schistosomes remains unknown and worries persist over the potential for mass administration campaigns to eventually diminish its usefulness. Here, we provide an introduction to schistosomiasis and discuss our current understanding of the mechanism of action of PZQ and the potential for widespread drug resistance.

Antimalarial drug resistance in Africa: key lessons for the future

Drug-resistant parasites repeatedly arise as a result of widespread use of antimalarial drugs and have contributed significantly to the failure to control and eradicate malaria throughout the world. In this review, we describe the spread of resistance to chloroquine and sulfadoxine-pyrimethamine, two old drugs that are no longer used owing to high rates of resistance, and examine the effect of the removal of drug pressure on the survival of resistant parasites. Artemisinin-resistant malaria is now emerging in Southeast Asia in a unique and unexpected pattern. We will review the most recent genomic and clinical data to help predict the behavior of resistance to new antimalarial medications and inform strategies to prevent the spread of drug-resistant malaria in Africa in the future.

Evaluation of the in vitro trypanocidal activity of methylated flavonoid constituents of Vitex simplicifolia leaves.

BackgroundTrypanosomiasis is a neglected tropical disease with complex clinical manifestations, tedious diagnosis, and difficult treatments. The drugs available for the treatment of this endemic disease are old, expensive, and associated with other problems including safety and drug resistant parasites. Therefore, there is an urgent need for the development of new, effective, cheap, and safe drugs for its treatment. Plants are potentially rich sources of leads for new drugs against trypanosomiasis.Vitex simplicifolia (Verbenaceae) is used traditionally for the treatment of tooth ache, edema, skin diseases, gout and trypanosomiasis in Nigeria. In a preliminary study, the methanol extract of Vitex simplicifolia was shown to exhibit a pronounced trypanocidal activity against T. b. rhodesiense.The present study was undertaken to investigate the active component responsible for the acclaimed activity of the leaves of Vitex simplicifolia in the traditional treatment of trypanosomiasis in Nigeria and other African countries. Our investigations aim at assessing the plant as a new source of potential trypanocidal compounds.MethodsThe crude extracts were prepared from the dried leaves using methanol, successive extraction with hexane, dichloromethane, ethylacetate and butanol was also done. The ethylacetate fraction was further fractionated and compounds isolated using preparative chromatographic technique and their structures were elucidated by NMR, mass spectrometry and comparison with literature data. Trypanocidal activities and cytotoxicity, using rat skeletal myoblast (L6) cells were investigated and their selectivity indices were determined.ResultsThe chromatographic separations of the methanol extracts gave rise to seven compounds. The isolated compounds 2, 3, 6 and 7 exhibited promising trypanocidal activity with IC50 values ranging from 4.7-12.3 μg/ml and cytotoxicity in the range of 1.58- 46.20 μg/ml. Compound 6, however, showed the most selective trypanocidal activity with a selectivity index of 9.8. This is the first report of trypanocidal activity of flavonoids from this plant genus.ConclusionsThe isolated compounds from Vitex simplicifolia exhibited noteworthy trypanocidal activities and hence may provide a source of new antitrypanosomal agents. These results also support the traditional use of Vitex simplicifolia in the treatment of trypanosomiasis. This is the first report of trypanocidal effect of flavonoids from this plant genus.

Identification of novel class of falcipain-2 inhibitors as potential antimalarial agents

Falcipain-2 is a papain family cysteine protease and an emerging antimalarial drug target. A pseudo-tripeptide scaffold I was designed using in silico screening tools and the three dimensional structures of falcipain-2, falcipain-3, and papain. This scaffold was investigated at four positions, T1, T2, T3, and T3′, with various targeted substitutions to understand the structure–activity relationships. Inhibitor synthesis was accomplished by first obtaining the appropriate dipeptide precursors with common structural components. The pyrrolidine moiety introduced interesting rotamers in a number of synthesized molecules, which was confirmed using high-temperature 1H NMR spectroscopy. Among the synthesized compounds, 61, 62, and 66 inhibited falcipain-2 activity with inhibition constants (Ki) of 1.8±1.1, 0.2±0.1 and 7.0±2.3μM, respectively. A group of molecules with a pyrrolidine moiety at the T2 position (68, 70, 71, 72, and 73) also potently inhibited falcipain-2 activity (Ki=0.4±0.1, 2.5±0.5, 3.3±1.1, 7.5±1.9, and 4.6±0.7μM, respectively). Overall, compound 74 exhibited potent anti-parasitic activity (IC50=0.9±0.1μM), corresponding with its inhibitory activity against falcipain-2, with a Ki of 1.1±0.1μM. Compounds 62 and 67 inhibited the growth of the drug resistant parasite Dd2 with better efficacy, and compound 74 exhibited a 7- to 12-fold higher potency against Dd2 and MCamp isolates, than the laboratory strain (3D7). These data suggest that this novel series of compounds should be further investigated as potential antimalarial agents.

Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei.

Drug resistance in pathogenic protozoa is very often caused by changes to the ‘transportome’ of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (DA; Berenil®), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (melarsoprol/pentamidine cross resistance, MPXR) is the result of loss of a separate high affinity pentamidine transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the “selectivity region” of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a possible structural rationale for this remarkable ability.