Rozprawy doktorskie na temat „Antimalaria- Drug”

Of the plethora of parasitic diseases that afflict mankind, malaria remains the most significant with 100-300 million cases reported annually and 600,000 fatalities. Treatment and control measures have been hampered by the emergence of drug resistance to most antimalarial therapies. Early signs of drug resistance to the current frontline option, the artemisinins, make it imperative that novel drug candidates are discovered. One possible short-term solution is drug repositioning, via screening existing FDA-approved (Food and Drug Administration agency) drug libraries for antimalarial activity. Towards this goal, two, fast, simple, and reliable in vitro SYBR Green-based drug susceptibility assays were optimised. The first, the SYBR Green microplate method offered a medium throughput option that was used to screen two FDA-approved libraries (Z score = 0.68 +0.06), LOPAC and ENZO (~700 compounds). Approximately 60 hits, defined as > 50 % inhibition at 2.5 µM, were identified by the preliminary screen. The SYBR Green flow cytometer method, capable of providing direct parasitaemia estimates and stage-specific information, was used for second-phase characterisation of the hits. From these, antiamoebic compound emetine dihydrochloride hydrate was identified as a potent inhibitor of the multidrug resistant Plasmodium falciparum, strain K1, with an IC50 of 47 nM (95 % confidence interval 44.92-49.17). Further characterisation of the compound involved analysis of the parasite killing profile, to determine the parasite reduction ratio (PRR) and parasite clearance time (PCT) as well drug interaction analysis with existing antimalarials. Emetine was shown to have a similar killing profile to atovaquone inferring a similar mitochondrial mode of action, corroborated by fluorescence staining with the JC-1 mitochondrial probe. Taken together, emetine’s pharmacokinetic matching and synergy with atovaquone provide an exciting drug combination for further investigation. The relatively high hit rate presented in the study, and in vitro workflow outlined for emetine, also showed drug repositioning to be a promising option for antimalarial drug discovery.

Malaria remains a major global health problem causing >600,000 deaths annually [1]. Efforts to control malaria are hampered by parasite drug resistance, insecticide resistance in mosquitoes, and the lack of an effective vaccine. In the last decade only one new chemotype, the spiroindolones, has progressed to clinical trials for malaria treatment [2, 3]. To address this significant problem the identification and development of new antimalarial agents is a priority. Part of this process includes ensuring that sufficient drug leads are available to prime the drug discovery pipeline, particularly those with novel modes of action in order to limit issues of cross-resistance with existing drugs [4]. While high throughput screening campaigns have identified thousands of potential antimalarial compounds from big Pharma libraries [5, 6], antimalarial target information on most compounds are lacking. Screening different libraries and pharmacophores is also recognised as being crucial to ensure chemical diversity [7]. There is also an increasing interest in repurposing existing drugs or drug classes or using them as starting point for discovery of new antimalarial agents. One of the aims of this thesis was to address the need for new antimalarial drug leads by investigating three compound classes with demonstrated clinical efficacy against cancer, HIV and other human diseases in a so called “piggyback” approach.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text

Plasmodiumfalciparum malaria poses one of the most important disease problems in the world. Despite decades of effort to improve disease outcome, the emergence and rapid dissemination of multi-drug resistant parasites has led to a disturbing increase in malaria mortality and morbidity. A critical limitation in managing multi-drug resistant falciparum malaria has been the incomplete understanding of both the underlying molecular mechanisms of drug resistance and the mode of action of widely used drugs. This study aimed to characterise the molecular mechanisms underlying multi- drug resistant malaria by studying the role of gene amplification in the P. falciparum multi-drug resistance gene 1 (pfmdrl) in determining parasite response to a variety of antimalarials in vitro and in vivo. In addition, P. falciparum ATPase 6 (PfATP6), a putative drug target of the widely used artemisinins, was also examined for possible drug-modulating mutations. First a real-time peR technique to measure amplification of pfmdri was developed and validated. This technique was used to determine pfmdri copy number in a unique set of field sample set (n = 600) collected in Northern Thailand, an area harbouring the world's most drug-resistant parasites. This allowed a comprehensive analysis of the importance of pfmdri amplification in (1) in vitro resistance to drugs, (2) in vivo response to mefloquine or mefloquine- artesunate therapy, (3) evolution of amplification in pre- and post-treatment samples. Subsequent studies also investigated the prevalence of pfmdrt amplification in Gabon, a Sub-Saharan country with very little mefloquine resistance. In addition, P. falciparum field isolates were studied for possible polymorphisms in PfATP6 and plasmid constructs generated to study the role of single nucleotide point mutations in the putative active site of the enzyme.

Includes bibliographical references (leaves 132-137).
Malaria ranks among the world's most important tropical parasitic diseases with world prevalence figures between 350 and 550 million clinical cases per annum. [WHO, 2008a] 'Treatment and prevention of malaria places a considerable burden on struggling economies where the disease is rampant. Research in malaria does not stop as the change in response to antimalarial drug treatment requires the development of new drugs and innovation in the use of old drugs. This thesis focused on building a model of the spread of resistance to Sulfadoxine/Pyrimethamine (SP) in a setting where both SP and SP in artemisinin-based combination therapy (ACT) are the first line therapies for malaria. The model itself is suitable to any low transmission setting where antimalarial drug resistance exists but the country of choice in this modeling exercise was Mozambique. The model was calibrated using parameters specific to the malaria situation in Mozambique. This model was intended to be used to aid decision making in countries where antimalarial drug resistance exists to help prevent resistance spreading to such an extent that drugs lose their usefulness in curing malaria. The modeling technique of choice was differential equation modeling; a simulation technique that falls under the System Dynamics banner in the Operations Research armamentarium. It is a technique that allowed the modeling of stocks and flows that represent different stages or groupings in the disease process and the rate of movement between these stages respectively. The base model that was built allowed infected individuals to become infectious, to be treated with SP or ACT and to be sensitive to or fail treatment. Individuals were allowed a period of temporary immunity where they would not be reinfected until the residual SP had been eliminated from their bloodstream. The base model was then further developed to include the pharmacokinetic properties of SP where individuals were allowed to be reinfected with certain strains of infection given the level of residual drug in their bloodstream after their current infection had been cleared. The models used in this thesis were built with idea of expanding on previous models and using available data to improve parameter estimates. The model at its core is similar to the resistance model used in Koella and Antia [2003] where differential equation modeling was used to monitor a population as it became infected with a sensitive or resistant infection and then University of Cape Town recovered. The inclusion in the model of the PK component was derived from Prudhomme-O'Meara et al. [2006] where individuals could be reinfected depending on the residual drug in their bloodstream. Rather than modeling simply sensitive and resistant infections, mutations categories were used as was the case in Watkins et al. [2005] population genetics model. The use of mutation categories allowed one to use parameters specific to these categories rather than the sensitive/resistant stratification and this is particularly relevant in Mozambique where all mutation categories still exhibit some degree of sensitivity to treatment i.e. total resistance has not yet developed for any particular mutation category. The last adaptation of the model was to use gametocyte information directly to determine human infectiousness rather than through using a gametocyte switching rate (constant multiplier used to convert parasite density to gametocyte density) as was done in Pongtavompinyo [2006]. The models developed in this thesis found that the existing vector control and drug policy in Mozambique had the major effect of decreasing total prevalence of malaria by approximately 70% in the 11 year period. The distribution of Res3 (presence of DHFR triple) and Res5 (presence of DHFR triple and DHPS double) infections changed over the 11 year period with Res3 infections initially increasing and then decreasing while Res5 infections started low and increased to overtake Res3 infections. The timing of the change in this composition of infection corresponds with the introduction of ACT and thus it appears that the use of ACT prompted the increased prevalence of quintuple parasites over DHFR triple and sensitive parasites. The total number of failures decreased substantially after the introduction of ACT to 17% of its previous level. The results of the base model corresponded with the observed data from the SEACAT study in terms of the magnitude and the trends of the impact of the change to ACT policy, but underestimated the impact of the vector control strategies compared to rapid effect noted in Sharp et al. [2007]. The Scenario testing of the base model showed that vector control is an effective strategy to reduce prevalence and that it is sensitive to the time at which the control is started as it decreased prevalence very gradually. The Scenario testing of the base model also showed that the introduction of ACT in Mozambique had a greater impact on reducing prevalence and that the start time of the ACT strategy did not decrease the effect on prevalence though earlier start times decreased the total number of resistance cases. The ratio of Res5 to Res3 infections increased faster when ACT was the treatment policy than when SP was the policy. Thus higher values of this ratio are associated with ACT being the treatment strategy in place. Thus differential equation modeling is an effective modeling tool to capture the spread of disease and to test the effects of policy interventions as it allows one to assess these effects on populations and averages out individual-level intricacies to better inform policy decisions.

Malaria remains a major public health concern for billions of people worldwide. Achieving the ambitious goal of malaria eradication requires co-ordination of control strategies dealing with a range of parasite, vector, human, social and environmental factors. Availability of effective antimalarial treatment is a key component in malaria control. However the number of drugs available is limited and drug resistance, particularly in Plasmodium falciparum, has now been reported for all currently available antimalarials. Mathematical models provide the opportunity to explore key features underlying antimalarial drug action, effectiveness and resistance. They further allow investigation into questions that cannot otherwise be easily addressed, either because they are too expensive, unethical or logistically too complex. This thesis aims to develop pharmacological models to investigate antimalarial drug treatment. In Chapter 2 we develop a pharmacokinetic-pharmacodynamic (PK/PD) model of antimalarial drug treatment (calibrated using published data) and use it to investigate the efficacy of artemisinin combination therapies (ACTs). Chapter 3 addresses two assumptions built into the methodology that limit the models future application. The model now allows for (i) time lags and drug concentration profiles for drugs absorbed across the gut wall and, if necessary, converted to another active form (ii) multiple drugs within a treatment regimen (iii) differing modes of drug action in combinations (iv) modelling drugs converted to an active metabolite with similar modes of action. In Chapter 4 we extend the methodology to allow for i) the presence of more than one clone when treatment begins (ii) the acquisition of new clones during treatment follow-up (iii) the tracking of individual clones using molecular markers. We then use these extensions to simulate clinical trial data to determine the best methods of analysis. Chapter 5 details how the drug action components of the extended PK/PD model were incorporated into OpenMalaria; a mathematical model of malaria epidemiology allowing investigation of the effects of various intervention strategies including malaria vaccines, vector control strategies and antimalarial drug treatment. In Chapter 6 we investigate the ability of clinical trials to accurately estimate (WoS) using the extended PK/PD model. Windows of selection (WoS) are often used to quantify the genetic process whereby parasites evolve increasing tolerance to antimalarial drugs. We noted a conspicuous lack of comprehensive, good-quality PK datasets currently available in the literature. Despite this, the models produced results highly consistent with field data. They were applied to investigate the potential implications of drug resistance and to make predications about the future effectiveness of antimalarials. We emphasise the value of mathematical models by simulating ‘field data’ to assess the best methods of analysing clinical trials and to investigate the predictive ability of WoS. While we do not suggest models can replace the information gained in clinical trials, this work does demonstrate the importance of mathematical models capable of generating results consistent with field data.

Malaria is a devastating global health issue that affects approximately 200 million people yearly and over half a million deaths are caused by this parasitic protozoan disease. Most commercially available drugs only target the blood stage form of the parasite, but the only way to ensure proper elimination is to treat the exoerythrocytic stages of the parasite development cycle. There is a demand for the discovery of new liver stage antimalarial compounds as there are only two current FDA approved drugs for the treatment of liver stage parasites, one of which fails to eliminate dormant forms and the other inducing hemolytic anemia in patients with G6PD deficiency. In efforts to address the dire need for liver stage drugs, we developed a high-throughput liver stage drug-screening assay to identify liver stage active compounds from a wide variety of chemical libraries with known blood stage activity. The liver stage screen led us to further investigate an old, abandoned compound known as menoctone. Menoctone was developed as a liver stage active antimalarial, however, the development of more potent compounds led to the abandonment of further menoctone research. Our research demonstrated that resistant parasites can transmit mutations through mosquitoes, which was previously believed to not be possible. Furthermore, we studied a novel genetic marker that may indicate potential resistance against malaria parasite infection and the cytotoxic effects associated with the disease. Future experiments aim to identify and advance our methods for the elimination of Plasmodium exoerythrocytic parasites.

A survey was undertaken to assess the knowledge, attitudes and practices, with respect to malaria, of 300 women from six randomly selected rural communities in Central Ethiopia from December 1991 to February 1992. Eighty-five per cent were able to recognize one or more of the common symptoms of malaria. Transmission was generally misunderstood and only 23% believed it could be prevented. More women preferred to obtain antimalarials from government clinics than from private drug shops, mission clinics, unofficial injectors, open markets or from leftover sources. Children under five were identified as the most malaria-vulnerable group and given priority for treatment. Severity of illness was the principal determinant in seeking treatment. Decisions were generally made jointly by both parents.
As distance from a health unit increased, knowledge about transmissibility of malaria decreased (OR =.48; 95% CI.27,.86). Logistic regression analysis showed literacy and village to be the most important variables associated with knowledge of prevention.

The increasing emergence of resistance to commonly used therapies has placed a huge strain on the prevention and control of malaria; therefore, there is an urgent need to develop novel antimalarial agents. The aim of this research was to design and synthesise a library of potent antimalarial compounds, with desirable pharmacokinetic profiles, in order to identify a drug candidate suitable for preclinical development. This research was divided into two main sections: x The synthesis of compounds deigned to inhibit IspD, a novel target in antimalarial drug discovery x The late stage development of a series of endoperoxide-based antimalarials, which are derived from the structure of artemisinin A library of benzisothiazolinone compounds was generated to target the IspD enzyme. Many of these compounds displayed low micromolar inhibitory activity against both enzymatic and phenotypic assays in vitro and an investigation into structure-activity relationships around the core of these benzisothiazolinones was also conducted. The most potent compound to emerge, a CH2 linked benzisoselenazolone, had an IC50 of 0.17 μM against PfIspD and 5.54 μM against Pf3D7. These compounds represent a novel class of IspD inhibitor, which have the potential for further development as antimalarial agents. A number of 1,2,4,5-tetraoxane analogues were also prepared in order to develop an antimalarial agent suitable for a single-dose cure. The most potent analogue, N205, had an IC50 of 1.3 nM and an average mouse survival of 26.3 days (66% cure rate) following a single dose. A less than optimal stability profile for N205 led to the further development of another potent tetraoxane analogue, E209. Optimisation of the synthetic pathway led to the generation of E209 in a series of five high-yielding steps that are suitable for large-scale production. E209 represents the first 1,2,4,5-tetraoxane that is comparable, in terms of both efficacy and PK/PD profiles, to OZ439, and is a candidate for pre-clinical development.

Malaria remains one of the deadliest human infectious diseases. In 2020 alone, there were an estimated 241 million clinical cases of malaria which resulted in approximately 627,000 deaths. With 3.2 billion people at risk of malaria and a global agenda focused on eradication there is a desperate need to improve malaria prevention and control strategies. As there is no highly effective vaccine, the treatment and prophylaxis of malaria are dependent on chemotherapy which is associated with increasing levels of parasite resistance. Given the lengthy development times for new drugs, strategies to safeguard current drugs are essential. This includes the development of resistance monitoring strategies and the rational selection of combination partners which should ideally be guided by mode of action information. The antimalarial drug proguanil is used in combination with the cytochrome bc1 inhibitor atovaquone (as Malarone®) for malaria prevention and treatment. While proguanil was developed as a pro-drug and is metabolized in vivo to the potent dihydrofolate reductase (DHFR) inhibitor cycloguanil, recent studies in our laboratory have demonstrated that it, and a cyclization-blocked analogue (tBuPG) that cannot be metabolized to cycloguanil, have potent slow action antiplasmodial activity independent of cycloguanil. Data has demonstrated that this activity is different to other slow action antimalarial agents and preliminary unpublished metabolomics work has shown that deoxythymidine monophosphate (dTMP) accumulates in proguanil and tBuPG treated parasites raising the hypothesis that P. falciparum thymidylate kinase (PfTMPK), the enzyme responsible for conversion of dTMP to deoxythymidine diphosphate (dTDP) may be a target of proguanil. However, the mechanism of proguanil’s slow action activity remains unknown. As proguanil’s intrinsic slow action activity may have clinical implications and there have been recent reports of resistant P. falciparum parasites in Uganda, the current work aimed to further investigate the slow action activity of proguanil. Investigations included examining the role of P. falciparum thymidylate kinase (PfTMPK) in the mode of action of proguanil, investigating the genome of P. falciparum parasites selected for in vitro resistance to tBuPG and examining the thermostability of proteins in the presence and/or absence of proguanil. P. falciparum thymidylate kinase (PfTMPK) was investigated as a target of proguanil as preliminary metabolomics work performed prior to this project (unpublished; collaboration with Professor Malcolm McConville, Melbourne University) raised the hypothesis that this enzyme is a target of proguanil. However, when tested during the current study, supportive data for this hypothesis was not generated. The overexpression of PfTMPK in P. falciparum 3D7 parasites did not impact proguanil or tBuPG activity (proguanil IC50 values of 0.094 μM versus 0.145 μM; P>0.05; tBuPG IC50 0.058 μM versus 0.046 μM, P>0.05). Proguanil and tBuPG failed to stabilize PfTMPK using two separate CETSA approaches and proguanil was shown to have no direct inhibitory activity against PfTMPK in in vitro activity studies. While these studies were not without limitations, together they suggest that proguanil does not directly target PfTMPK. However, there is still the possibility that PfTMPK could be indirectly associated with proguanil action including impacting PfTMPK localization which could lead to reduced conversion of dTMP to dTDP. Alternatively, adenosine triphosphate (ATP), which is required for the conversion of dTMP to dTDP by PfTMPK, could be affected by proguanil. This would align with a previously proposed hypothesis that proguanil may target ATP synthase. However, an impact on ATP would be expected to result in pleiotropic metabolic effects, which were not seen. In addition, while metabolomics data provided information on total ATP pools and not specific sub-cellular changes (e.g., mitochondria), they remained unchanged in proguanil or tBuPG treated versus wildtype parasites.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

Background and Rationale: Although the majority of patients with endometrial cancer (EC) are diagnosed early when disease is confined in the uterus and prognosis is excellent, there is a subset of patients with dismal prognosis. Carboplatin and paclitaxel is the standard chemotherapeutic regimen for EC. Given that response to chemotherapy impacts disease prognosis, especially in advanced, recurrent and metastatic disease, novel chemotherapeutic agents with improved safety profile are necessary to improve response rates and outcomes in these patients. Quinacrine (QC) is an inexpensive antimalarial drug with a predictable safety profile which recently surfaced as a promising anticancer agent thought to be associated with decreased risk of developing chemo-resistance through targeting multiple pathways simultaneously.

Objective: To generate preclinical data on the effect of QC in inhibiting tumorigenesis in EC both in vitro and in vivo as well as explore its role as an adjunct to standard chemotherapy in vivo in an EC mouse xenograft model.

Methods: Five different EC cell lines (Ishikawa, Hec-1B, KLE, ARK-2, and SPEC-2) representing different histologies, grades of EC, sensitivity to cisplatin and p53 status were used for the in vitro studies. MTT and colony formation assays were used to examine QC’s ability to inhibit cell viability in vitro. Drug combination studies were performed and the Chou-Talalay methodology was employed in order to examine synergism between QC and cisplatin, carboplatin or paclitaxel. A cisplatin-resistant EC subcutaneous mouse xenograft model was used in order to explore QC’s anticancer activity in vivo and assess its role as maintenance therapy.

Results: QC exhibited strong synergism in vitro when combined with cisplatin, carboplatin or paclitaxel with the highest level of the synergism being observed in the most chemo-resistant EC cell line. Neither QC monotherapy nor standard chemotherapy significantly delayed tumor growth in the mouse xenografts. Co-administration of QC with standard chemotherapy significantly augmented the antiproliferative ability of these chemotherapeutic agents as evidenced by the significant decrease in tumor burden. Combination treatment was associated with a 14-week prolongation of median survival compared to standard chemotherapy alone. Maintenance therapy with QC following standard chemotherapy was proven superior to standard chemotherapy as it resulted in long-term stabilization of disease evidenced by lack of significant tumor progression and further prolongation of overall survival. QC treatment alone, in combination with standard chemotherapy or as maintenance therapy was well-tolerated and was not associated with weight loss compared to control mice. A yellow skin discoloration was noted during active treatment with QC which was entirely reversible within a few days upon discontinuation of treatment.

Conclusions: QC exhibited significant antitumor activity against EC cell lines in vitro and was successful as maintenance therapy in chemo-resistant EC mouse xenografts. This preclinical data suggest that QC may be an important adjunct to standard platinum-based chemotherapeutic regimens for patients with recurrent EC.

Phosphodiesterases are key enzymes in cyclic-nucleotide signalling pathways, regulating the levels of cAMP and cGMP in the cell. Cyclic nucleotides play an important role in regulating progression of the complex parasite life cycle. There are four Plasmodium PDEs, PDEα-δ. PDEβ has proved refractory to deletion and is predicted to be essential in asexual blood stages. PDEs α, γ and δ have been successfully disrupted in previous studies, which revealed stage-specific roles for PDEγ and PDEδ in the mosquito. PDEβ is a promising drug target due to the proven ability to create specific inhibitors for individual human PDEs (e.g. Viagra inhibits human PDE5), the established safety record of current inhibitors and the predicted essential nature of PDEβ in P. falciparum blood stages. The lack of useful reagents; knockout strains, tagged lines and recombinant proteins, has severely limited progress in this field. Conditional genetic disruption (required to study essential genes) is notoriously difficult in P. falciparum. This project has attempted to generate a conditional knock-out using the destabilisation domain system and the novel DiCre system, which involves recombinase-mediated genomic excision of the target DNA upon introduction of a drug. A PDEβ haemagglutanin (HA) tagged line has been successfully generated and used to investigate the cellular biology of PDEβ, including, the subcellular localisation and cyclic nucleotide specificity of PDEβ, which until now has remained speculative. A small library of PDE inhibitors generated by Pfizer has been evaluated using a parasite growth inhibition assay and a PDE assay, with compounds active at sub-micromolar concentrations against the parasite and the protein. These assays have used wild type parasites and also a PDEα KO line which has no obvious phenotype in blood stage parasites.

Histone deacetylases (HDACs) are recognised as potential drug targets for many diseases including cancer, inflammatory diseases and some parasitic diseases including malaria. In eukaryotic cells, these enzymes play an important role in transcriptional regulation through modification of chromatin structure. Inhibitors of mammalian HDAC enzymes including trichostain A and apicidin are active against P. falciparum parasites, however these compounds are not selective for malaria parasites versus normal cell lines. The aims of this study were to examine the antimalarial potential of new hydroxamate-based HDAC inhibitors and to investigate a P. falciparum HDAC, PfHDAC1, as a potential new antimalarial drug target.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Science
Griffith Health
Full Text

Malaria is considered as one of the most prevalent and debilitating diseases affecting humans. Plasmodium falciparum is the most virulent form of the parasite which developed resistance to several antimalarial drugs. Chloroquine is one of the most successful antimalarials developed that is safe, effective, and cheap. However, its use has been limited due to the emergence of drug resistance. Click chemistry, particularly, the copper(I)-catalyzed reaction between azides and alkynes has shown to have a cutting-edge advantage in medicinal chemistry by its reliability, selectivity and biocompatibility. Triazole-based antimalarials were synthesized via copper(I)-catalyzed alkyne-azide cycloaddition reaction by modifying the aliphatic chains terminal of chloroquine. The compounds synthesized contain triazole ring directly connected to an aromatic ring or via a piperazine linker. When tested for their in vitro antimalarial activity against D6, Dd2 and 7G8 strains of P. falciparum, 12 out of 28 compounds showed better activity against chloroquine resistant strains. Particularly, PL403 and PL448 exhibited potent activity than chloroquine against CQ-resistant strains Dd2 and 7G8, with IC50 values of 12.8 & 14.5 nM, and 15.2 & 11 nM respectively. The efficiency of synthesizing several triazole-based antimalarials have proven click chemistry to be fast and efficient reaction. Generally, para-substitutions and di-substitutions with electron-withdrawing groups were found to be beneficial for having better antimalarial activity for these group of click compounds. Moreover, the incorporation of piperazine linker has brought an enhanced antimalarial activity.

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New antimalarial drugs are urgently needed to combat emerging multidrug resistant strains of malaria parasites. This Highlight focuses on plant-derived natural products that are of interest as potential leads towards new antimalarial drugs including synthetic analogues of natural compounds, with the exception of artemisinin derivatives, which are not included due to limited space. Since effective antimalarial treatment is often unavailable or unaffordable to many of those who need it, there is increasing interest in the development of locally produced herbal medicines; recent progress in this area will also be reviewed in this Highlight.

Allometric scaling was found as a plausible technique for dose determination in children. Permeability and P-glycoprotein efflux transport of antimalarials were determined using in-vitro Caco-2 cells. Mefloquine showed P-glycoprotein inhibition. Amodiaquine, artesunate and artemisone were not P-glycoprotein substrates or inhibitors. Methylene-blue showed some P-glycoprotein mediated efflux. Permeability was high for amodiaquine and artemisone, medium for mefloquine and artesunate and low for methylene-blue. P-glycoprotein was up-regulated when exposed to dihydroartemisinin/artemisone in combinations with amodiaquine/mefloquine.

A collaborative study between the University of Salford and the National Institute of Pharmaceutical Research and Development (NIPRD), Nigeria, involving in vitro tests at the University of Salford and the use of facilities at NIPRD for invivo tests has led to the evaluation of a range of traditional “fever cures” for antimalarial efficacy. The main objective of this study is to identify a suitable antimalarial plant candidate towards the development of a phytopharmaceutical drug (a plant-based medicinal mixture/compound used in preventive or therapeutic medicine) for the treatment of malaria as a cheaper and more accessible alternative, inline with the WHO resolution (WHA 3049) urging nations to use their traditional systems of medicine as part of their healthcare systems. Traditionally, the mainstay of the antimalarial drug discovery process has been natural products. Their use however diminished over the past few decades due to several advances in molecular targets and technical difficulties encountered in high-thoughput screens of natural product leads. Natural products have played a very important role as a source of antimalarials (e.g. quinine and artemisinin derivatives). In this present study, fluorescent-based in vitro antimalarial assays including flow cytometry (FCM) and SYBR green microtitre assay (SG) were optimized to screen some aqueous plant extracts which were selected based on their ethnopharmacological usage. Giemsa light microscopy was used to validate the assays. Plasmodium berghei malaria animal model was also used to evaluate the anti-plasmodial activity of extracts in vivo. The results showed a strong antimalarial activity in all the six extracts. Bryocarpus coccineus and Bridelia ferruginea were chosen for further investigation due to their efficacy and the collaborative nature of the study. The IC50 values obtained in the in vitro antimalarial studies in the region of 70 µg/ml and 15 µg/ml. Bridelia ferruginea aqueous and methanolic extract was compared to determine any differences in IC50. In vitro comparison of the aqueous and methanolic extracts of the extract revealed an IC50 value in the region of 25.69 µg/ml for the aqueous extract and 15-16 µg/ml for the methanolic extract. Qualitative phytochemical screening of both extracts revealed the presence of various bioactive compounds including tannins, flavonoids, saponins and cardiac glycosides amongst others in both the aqueous and methanolic extract of B. ferruginea. Anthocyanins were found present in the methanolic extract only. Further investigation of the mechanism of action of the methanolic B. ferruginea extract showed that the extract inhibited β-haematin formation, indicating the inhibition of haemazoin formation in the parasite. Lastly, the methanolic extract was fractionated using HPLC analysis. Various resolved peaks were obtained and subsequent bioassays of the collected fractions revealed that antimalarial activity was distributed across the fractions. This may suggests that isolating a single active compound might not be advantageous, making a case for a phytopharmaceutical drugs.

Introduction: Antimalarial resistance, particularly artemisinin resistance, is a major threat to P. falciparum malaria elimination efforts worldwide. Urgent intervention is required to tackle artemisinin resistance but field data on which to base planning of strategies are limited. The aims were to collect available field data and develop population level mathematical models of P. falciparum malaria treatment and artemisinin resistance in order to determine the optimal strategies for elimination of artemisinin resistant malaria in Cambodia and treatment of pre-hospital and severe malaria in Cambodia and Bangladesh. Methods: Malaria incidence and parasite clearance data from Cambodia and Bangladesh were collected and analysed and modelling parameters derived. Population dynamic mathematical models of P. falciparum malaria were produced. Results: The modelling demonstrated that elimination of artemisinin resistant P. falciparum malaria would be achievable in Cambodia in the context of artemisinin resistance using high coverages with ACT treatment, ideally combined with LLITNs and adjunctive single dose primaquine. Sustained efforts would be necessary to achieve elimination and effective surveillance is essential, both to identify the baseline malaria burden and to monitor parasite prevalence as interventions are implemented. A modelled policy change to rectal and intravenous artesunate in the context of pre-existing artemisinin resistance would not compromise the efficacy of ACT for malaria elimination. Conclusions: By being developed rapidly in response to specific questions the models presented here are helping to inform planning efforts to combat artemisinin resistance. As further field data become available, their planned on-going development will produce increasingly realistic and informative models which can be expected to play a central role in planning efforts for years to come.

Preclinical pharmacokinetics relies on efficient and accurate screening to select clinical candidates from early leads. Poor pharmacokinetic interpretation can disadvantage drug discovery by promoting inadequate compounds and expelling potential drug candidates. Objectives of this project included pharmacokinetic evaluation of antimalarial and anti-tuberculosis lead compounds with techniques aimed at improving preclinical pharmacokinetic outcomes. This included mechanistic pharmacokinetic approaches such as non-linear mixed effects (NLME) modelling in comparison with traditional non-compartmental analysis. Where appropriate, pharmacokinetic methods were expanded to include organ distribution and capsule dosing in mice to bridge our techniques from discovery to early development. Three benzoxazole amodiaquine analogues possessing equipotent in vitro antiplasmodial activity and showed diverse in vivo efficacy in a malaria mouse model. Evaluation of their respective pharmacokinetics in mice showed their in vivo exposures could translate to in vivo efficacy. Retrospective PK/PD simulations point to a time above IC50 drive in efficacy. Pharmacokinetic evaluation of an aminopyridine antimalarial compound in its cyclodextrin inclusion complex revealed a pH dependent increase in solubility that reduced variance, likely due to favoured intestinal absorption. Investigation of two novel fusidic acid C-3 ester prodrugs aimed at repositioning fusidic acid for tuberculosis, showed high concentrations of the rodent specific 3-epifusidic acid metabolite that greatly reduced exposure of fusidic acid in mice. Further organ distribution studies showed a prodrug strategy is still viable for repositioning fusidic acid for tuberculosis, but that rodent models are inappropriate for further evaluation. NLME modelling successfully provided unique mechanistic and mathematical insight of pharmacokinetic profiles of new leads. The level of interpretation on pharmacology parameters improved and aided in understanding why drug leads are likely to fail or succeed, assisting future compound optimisation.

The UCT Drug Discovery and Design Centre, H3D, provided anti-tubercular and antimalarial drug leads that display potent in vitro and in vivo activity, but with unfavourable physicochemical properties. The primary objective of this study was to prepare supramolecular derivatives of the drug leads in an attempt to improve their physicochemical properties. Any new solid forms were to be characterized by a variety of analytical techniques, including X-ray analysis, spectroscopic and thermal techniques. Where possible, such derivatives would be tested for any enhancement in the aqueous solubility or biological activity of the drug lead. The second objective of this study was to employ supramolecular intervention in the early stages of the drug discovery and development process to help streamline the process by distinguishing between compounds that might be amenable to beneficiation via supramolecular modification and those that might not. The crystal structure of a novel anti-tubercular drug lead, DL2, was solved and the compound was fully characterized using thermal and X-ray techniques. This compound displayed very poor solubility in both aqueous and organic media. Phase solubility studies were performed with anti-tubercular drug lead DL3 and selected cyclodextrins (CDs). The apparent solubility of DL3 increased by a factor of more than 300 at the highest concentration of hydroxypropyl-β-CD (HPβCD) and β-CD investigated. Three salts of antimalarial drug lead DL4 and carboxylic acids were prepared. The salts were characterized by X-ray and thermal techniques. A salt of citric acid and DL4 could be prepared by the liquid-assisted grinding method. The equilibrium solubility of this salt was 48 times greater than that of DL4.

Malaria remains one of the world’s most important infectious diseases causing over 600,000 deaths annually, mainly in African children under the age of five [1]. In the absence of a licenced vaccine, malaria prevention and treatment relies on drugs and vector control [1]. Unfortunately malaria parasite resistance has emerged to currently used antimalarial drugs, including the current gold standard artemisinin combination therapies (ACTs) [1-3]. Added to this, the majority of agents in the current antimalarial drug discovery and development portfolio are based on known antimalarial pharmacophores [4], which may compromise their widespread use due to potential issues of cross resistance. With only one new antimalarial chemical class (chemotype) presently under development, the spiroindolones [5], there is an urgent need to ensure that the antimalarial drug discovery pipeline is primed with new chemotypes, ideally with novel modes of action in order to combat resistance. In this thesis project this problem was addressed by investigating the antimalarial potential of primary sulfonamides (PS), a chemotype not currently used for malaria, but with a proven track record for treatment of other diseases, including glaucoma, renal disorders and epilepsy [6].
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
Full Text

In spite of great effort aimed at eradication, the malaria epidemic still claims over 600,000 lives each year, and 50% of the world is at risk of contracting the disease. The most deadly form of malaria is caused by Plasmodium falciparum, which is spread from human to human via the female Anopheles mosquito. P. falciparum's lifecycle, which includes both sexual and asexual reproduction, facilitates rapid evolution in response to drug pressure, resulting in the emergence of resistant strains against every antimalarial medication that has been deployed. There is a great need for new antimalarial drugs. Chloroquine (CQ), an aminoquinoline drug deployed in the 1940s, was an inexpensive, effective and safe drug but now has been rendered ineffective throughout much of the tropical regions due to the emergence of CQ-resistant strains of P. falciparum. A new class of hybrid drugs, called Reversed-CQs, has been developed by linking a molecule with a CQ-like moiety to a molecule with a reversal agent (RA) moiety; an RA is a chemosensitizer that can reverse CQ-resistance. The prototype Reversed-CQ, PL01, was shown to be effective in vitro against sensitive and resistant P. falciparum cell cultures, with IC50 values of 2.9 and 5.3 nM, respectively, in comparison to IC50 values for CQ which were 6.9 and 102 nM, respectively. In the course of the Reversed-CQ research, PL74 was synthesized with a pyridine ring replacing the quinoline ring. It was expected that PL74 would display reversal agent activity but would not display antimalarial activity. However PL74 showed antimalarialactivity with IC50 values of 185 and 169 nM in vitro against CQ-sensitive and CQ-resistant strains, respectively. In the investigation of PL74 it has been found that this molecule has a pyridinium salt structure, novel to the Reversed-CQ compounds, and through a structure-activity relationship (SAR) study, it was shown to have activity that may indicate a mode of action different from the Reversed-CQ compounds. A study of the literature revealed that pyridinium salt compounds, with some similarity to PL74, were found to operate as choline analogs inhibiting the biosynthesis of phosphatidylcholine as their main antimalarial mode of action.

Malaria is one of the most life-threatening diseases affecting mankind, with over 3 billion people being at risk of infection, with most of these people living in Africa, South America and Asia. As the malaria parasite is rapidly becoming resistant to many of the possible treatments on the market, it is of upmost importance to identify new possible drug targets and describe drugs against these that are inexpensive, easy to manufacture and have a long shelf-life in order to combat malaria. One such target is the polyamine pathway. The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation. Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections. Usually, both enzymes are individually transcribed and highly regulated as monofunctional proteins. However, ODC and AdoMetDC from P. falciparum (PfODC and PfAdoMetDC, respectively) are found as a unique bifunctional protein (PfAdoMetDC/ODC) in the malaria parasite, making it an enticing target for new, selective antimalarial chemotherapies. In order to apply structure-based drug discovery strategies to design inhibitors for PfAdoMetDC/ODC, the atomic resolution structures of these proteins are needed. Each individual domain has had its structure proposed through homology modelling; however atomic resolution structures of these domains are not yet available. The homology model of PfAdoMetDC/ODC has not yet been elucidated due to the interactions between the domains of the bifunctional protein not being fully understood. High levels of recombinant expression of the bifunctional protein have been either unsuccessful or resulted in the formation of insoluble proteins being produced. The purpose of this project is to optimise the recombinant expression of PfAdoMetDC/ODC, and the PfODC domain, to produce high yields of pure, soluble protein for subsequent atomic resolution structure determination. Ultimately, this will enable the utilisation of PfAdoMetDC/ODC in structure-based drug discovery strategies. Overexpression of P. falciparum proteins in E. coli is notoriously difficult, mainly due to the codon bias between the two species. Comparative studies were performed on four constructs of the PfAdoMetDC/ODC gene, containing either the wild-type, fully codon harmonised, or partially codon harmonised gene sequences to analyse the effect codon harmonisation had on protein expression and activity of both domains of PfAdoMetDC/ODC as well as on the monofunctional PfODC domain. Codon harmonisation did not improve the expression levels or the purity of recombinantly expressed PfAdoMetDC/ODC or the monofunctional PfODC domain. Truncated versions of both proteins, and contamination by the E. coli chaperone proteins DnaK and GroEL, were present in the protein samples even after purification by affinity chromatography. However, codon harmonisation improved the activity levels of the PfAdoMetDC domain, while decreasing the activity of the PfODC domain of PfAdoMetDC/ODC. Harmonisation of the monofunctional PfODC domain resulted in a decrease in the activity of the protein. In order to identify possible inhibitors of the PfODC domain of the bifunctional protein, a structure-based drug discovery study was initiated based on a homology model for PfODC. Four hundred compounds with known antimalarial activity were virtually screened against the PfODC homology model and the top two scoring compounds were selected for enzyme inhibition assays based on their predictive binding affinity against the enzyme, and two medium scoring compounds were selected as controls. Enzyme inhibition studies were performed on the bifunctional PfAdoMetDC/ODC to determine the effect the compounds had on both domains of the protein. Of the compounds assayed one of the compounds significantly reduced the activity levels of both domains of PfAdoMetDC/ODC. Additionally, one compound significantly reduced the activity level of the PfAdoMetDC domain of PfAdoMetDC/ODC. This work therefore contributes towards characterisation of the unique PfAdoMetDC/ODC in malaria parasites as a novel drug target.
Dissertation (MSc)--University of Pretoria, 2012.
Biochemistry
unrestricted

Although global cases and death from malaria have reduced over the last ten years, malaria is still a significant infectious disease. This disease kills about 2000 people per day. There is currently no licenced vaccine and current drugs are failing due to parasite drug resistance. Thus, there is an urgent need to develop new drugs to prevent and treat this disease. Natural products and their derivatives have played a significant role in drug discovery, since they have been an important source or inspiration for numerous current drugs. Australian macrofungi have rarely been studied for their potential as sources of new bioactive natural products, and in the antimalarial drug discovery realm, this is a pioneering study. As part of a research program aimed at identifying new antimalarial lead compounds or drugs from nature, a pre-fractionated fungal library was screened for antimalarial activity. All macrofungi used during these studies were collected from a variety of ecosystems found within the state of Queensland, Australia. A taxonomically diverse set of fungi were used with 37 families and 62 genera represented. The library consisted of 2,035 fractions obtained from C18 HPLC fractionation of 407 DCM/MeOH fungal extracts, with five fractions collected for each extract. A radiometric growth inhibition assay was used to screen the fractions against the chloroquine sensitive Plasmodium falciparum 3D7 malaria parasite line. Of the 2,035 fractions screened, 20 displayed inhibition of >80% towards P. falciparum, the cut off selected for pursuing lead fractions. Bioassay- or UV-guided fractionations were performed on three fungal samples, and several antimalarial natural product compounds were purified and their chemical structures determined using a combination of 1D/2D NMR, MS, and UV data.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text

Malaria is still an epidemic in many parts of the world-about 220 million people are still infected with malaria worldwide and about 700 thousand people die from this disease per year. Most of the drugs used to treat malaria work well if they are used as required and they contain the right amounts of the active ingredient; however, it is estimated that more than 10% of drugs traded worldwide are counterfeits including 38% to 53% of antimalarial tablets produced in China and India. Due to the lack of data covering the extent of counterfeit antimalarial drugs in Ghana, the purpose of this quantitative study was to determine the percentage of counterfeit antimalarial drugs sold in Ghana by assessing the amounts of the 2 most common antimalarial drugs, artemether (ATMT) and lumefantrine (LMFT) in drugs sold in Ghana retail outlets. These drugs were purchased from retail outlets in Ghana and analyses at the Mayo Clinic Pharmacology core lab (Rochester, MN). The quality of the drugs were characterized by comparing the actual amount of ATMT & LMFT in each tablet to the expected amount. Using explanatory theory along with dose response-response occupancy theory, the researcher addressed quantitative solutions to questions related to the percentage and distribution of counterfeit ATMT and LMFT tablets. The results revealed that overall 20% of the drugs are counterfeit; this is not dependent on the location or kind of outlet but rather depends on whether the tablets were imported or locally manufactured and whether the tablets had a pedigree scratch panel. This study provides a better understanding of how much antimalarial medication is counterfeit in Ghana, which will aid interventions to minimize the adverse effects of counterfeit antimalarial medication in Ghana

Malaria is arguably one of the main medical concerns worldwide because of the numbers of people affected, the severity of the disease, and the complexity of the life cycle of its causative agent, the protozoan Plasmodium sp. The clinical, social and economic burden of malaria has led for the last 100 years to several waves of serious efforts to reach its control and eventual eradication, without success to this day. At present, administration methods of antimalarial drugs release the free compound in the blood stream, from where it can be significantly removed by many tissues and organs, thus reducing its availability for Plasmodium-infected erythrocytes. Due to this lack of specificity regarding the target cells, current oral or intravenous delivery approaches for most antimalarial drugs require high doses. However, unspecificity of toxic drugs demands low concentrations to minimize undesirable side-effects, thus incurring the risk of sublethal doses favouring the appearance of resistant pathogen strains. Targeted nanovector systems can fulfill the objective of achieving the intake of total doses sufficiently low to be innocuous for the patient but that locally are high enough to be lethal for the malaria parasite. With the advent of nanoscience, renewed hopes have appeared of finally obtaining the long sought-after magic bullet against malaria in the form of a nanovector for the targeted delivery of antimalarial drugs exclusively to Plasmodium-infected cells. We work on the development of antimalarial drug-carrying nanovectors specifically targeted to Plasmodium-infected red blood cells (pRBCs). Our first immunoliposomal prototype delivers its contents exclusively to pRBCs containing the P. falciparum late forms trophozoites and schizonts, and improves on average tenfold the efficacy of the antimalarial drugs chloroquine and fosmidomycin. Using chloroquine concentrations well below its IC50, and by modifying parameters such as liposome size, density of targeting antibodies on the liposome surface, targeted antigen, and intraliposomal drug concentration, we approach 100% of parasitemia reduction both in vitro and in vivo using a murine model for P. falciparum malaria. We are working in the improvement of the nanovector through modification of (i) the targeting element: better antibodies, non-protein molecules such as DNA aptamers and polysaccharides, (ii) the encapsulated drug(s), and (iii) the type of nanocapsule, making special emphasis on polymeric structures. Our objective in the short term is the design of a nanostructure adequate to enter the preclinical pipeline as an economically affordable new antimalarial therapy.
Desarrollo de nanovectores para la liberación dirigida de antimaláricos Los métodos actuales de administración oral o intravenosa requieren dosis elevadas que a menudo desencadenan efectos secundarios perniciosos. Por el contrario, el riesgo de suministrar dosis subletales a causa de dichas concentraciones terapéuticas críticas o por razones de inestabilidad del compuesto, favorece la aparición de cepas resistentes de Plasmodium. La liberación dirigida de antimaláricos es una aproximación prometedora para evitar ese riesgo. El trabajo presentado en esta tesis doctoral tiene como objetivo principal el desarrollo de un nanovector para la mejora de la eficacia de los antimaláricos existentes y la comprensión de los parámetros fundamentales de su diseño que determinan la eficacia de dicho nanovector. Liposomas con quantum dots en su interior y que han sido funcionalizados con hemi-anticuerpos contra formas tardías del parásito se unen en menos de 90 minutos a eritrocitos infectados por Plasmodium y liberan su contenido en el interior de las células diana. Cuando se encapsulan fármacos antimaláricos en el modelo inmunoliposomal, se incrementa hasta diez veces la eficacia de los fármacos. La formulación para administración oral de anticuerpos y liposomas es complicada, nanovectores adecuados para esta vía de administración serían una contribución valiosa para el tratamiento de la malaria en zonas endémicas, alejadas de centros de salud. Durante la última parte de esta tesis, nos hemos centrado en el desarrollo de nuevos nanovectores poliméricos que liberen de forma específica los fármacos a pRBCs, ya que las nanopartículas poliméricas pueden ser formuladas para administración oral más fácilmente que los liposomas. Las diferentes partes de futuros nanovectores (moléculas direccionalizadoras, formulación liposomal, recubrimiento exterior, fármaco encapsulado) están diseñadas de tal manera que puedan ser sustituidas por nuevos elementos para su utilización contra diferentes especies del parásito o para reconocer diferentes dianas intracelulares.

Malaria is a leading cause of morbidity and mortality, causing more than 400,000 deaths per year. Malaria is caused by parasites of the Plasmodium genus with most deaths due to P. falciparum infection. The control of malaria is complicated by the lack of a widely effective vaccine, the spread of mosquito resistance to insecticides and Plasmodium parasite resistance to available drugs, including the gold standard artemisinin-combination therapies. Thus, there is an urgent requirement for the development of new antimalarials, in particular those with different modes of action to existing drugs to limit potential problems of cross-resistance. Plasmodium species have a complex lifecycle that includes transmission from the female Anopheles mosquito vector to a human host requiring significant morphological changes. These morphological changes are associated with stage-specific changes in transcription regulated by epigenetic mechanisms. The proteins involved in these processes are potential new therapeutic targets for malaria. This includes histone deacetylases (HDACs), which together with histone acetyltransferases (HATs), are involved in reversible posttranslational acetylation of histone and non-histone proteins, regulating transcription and other cellular processes. To date, over 650 HDAC inhibitors have been investigated for in vitro activity against malaria parasites. Some inhibitors, particularly those with a hydroxamic acid zinc-binding group that targets inhibitors to the HDAC active site, have demonstrated low nM in vitro potency against P. falciparum and selectivity for the parasite over human cells. However, antiplasmodial HDAC inhibitor drug development has been hindered by factors including the lack of recombinant P. falciparum HDACs (only one available and purity is low), the lack of HDAC crystal structures (none available) and low throughput activity assays that are largely indirect measures of HDAC inhibition. Without these tools, mode of action studies, the rational design of new and improved inhibitors and the prioritisation of compounds for preclinical testing remains difficult. To address some of these challenges and further progress the development of antimalarial HDAC inhibitors, the current study employed a multi-pronged approach, including: (i) investigating the in vitro and in vivo activity of new HDAC inhibitors; (ii) establishing a higher throughput ELISA method to analyse P. falciparum lysine acetylation alterations and; (iii) developing a quantitative structure-activity relationship (QSAR) model based on classification algorithms. HDAC inhibitors typically have a pharmacophore comprising a zinc-binding group that interacts with the zinc ion in the active site of the enzyme, a linker unit and a cap group promoting hydrophobic interaction with amino acid residues at the entry of the active site. Here, a set of 26 new HDAC inhibitors with a peptoid-based scaffold was tested in vitro against drug sensitive asexual intraerythrocytic-stage P. falciparum 3D7 parasites. The set are analogues of compounds that have previously shown in vitro dual-stage antiplasmodial activity against asexual intraerythrocytic and exoerythrocytic stages and includes 16 compounds with a hydroxamic acid zinc-binding group and 10 prodrugs of this compound class. The unprotected hydroxamate-based inhibitors demonstrated growth inhibition of P. falciparum 3D7 asexual intraerythrocytic-stage parasites in the nanomolar to micromolar range (50% growth inhibition values (PfIC50) 0.008-1.04 μM) and up to 1,250-fold selectivity (selectivity indices (SI; PfIC50/human cell IC50): 10-1,250) for the parasite compared to human cells. Structure-activity relationship (SAR) analysis of cap region residues (carbonyl region, carboxylic region and isocyanide region) indicated that benzyl groups in the isocyanide region and alkyl groups in the para position of the carboxylic region are associated with increased antiplasmodial activity. In addition, methyl groups in the carbonyl region of the cap group demonstrated reduced cytotoxicity against neonatal foreskin fibroblasts (NFF), however, also somewhat reduced activity against asexual blood-stage parasites. Work by collaborators demonstrated micromolar in vitro activity of several compounds of this set against exoerythrocytic P. berghei parasite forms indicating dual-stage activity. The compound with the greatest dual-stage activity displayed an IC50 of 8 nM against asexual blood-stage P. falciparum and an IC50 of 60 nM against exoerythrocytic P. berghei in vitro. Compounds with PfIC50 of 100 nM or lower were tested against the multi-drug resistant P. falciparum Dd2 line (resistant to chloroquine, pyrimethamine, mefloquine, and other antimalarial drugs), and demonstrated a resistance index (RI) <1 indicating a lack of cross-resistance by this parasite line. The same subset of compounds was investigated for their ability to hyperacetylate P. falciparum histone H4; differential effects were observed with some compounds causing up to ~2.5-fold hyperacetylation compared to untreated controls. 10 prodrug peptoid-based HDAC inhibitors were also investigated. The prodrug strategy seeks to make the hydroxamic acid-based inhibitors more stable and bioavailable for in vivo applications as they are prone to degradation processes such as hydrolysis or reduction. These compounds were synthesised with masked hydroxamate functionalities that may undergo activation in vitro. Preliminary data demonstrated in vitro PfIC50 of 0.014-1.75 μM and 6-642-fold selectivity for the parasite over human fibroblasts. Three of these compounds displayed PfIC50 <0.1 μM and SI >100 and may therefore be of interest in further studies. Based on the in vitro antiplasmodial activity, selectivity and chemical diversity in the cap region, five peptoid-based compounds (3a, 3c, 3f, 3m, 3n, Pf3D7 IC50 0.008-0.034 μM, SI 97-625) were further investigated for in vivo efficacy against Plasmodium parasites. In addition, four analogues of the tethered phenylbutyrate-based HDAC inhibitor AR42 (Pf3D7 IC50 0.02 μM, SI 39) were also investigated in vivo (JT21b, JT83, JT92a, JT94; Pf3D7 IC50 0.005-0.21 μM, SI 55-118, (data generated by Dr MJ Chua, personal communication)). AR42 is currently in phase 1 clinical trials against various types of cancer and demonstrates an improved pharmacokinetic profile compared to a number of clinically approved HDAC inhibitors (e.g. AR42 Cmax 14.7 μM compared to vorinostat Cmax 1.9 μM, AR42 t1/2 11.1 h compared to vorinostat t1/2 0.75 h; tested in mice). AR42 analogues were of interest as AR42 has previously been shown to cure Plasmodium infections in mice (Dr MJ Chua, Griffith Institute for Drug Discovery; unpublished). While the two analogue sets differ significantly in linker and cap group, both bear a hydroxamic acid zinc-binding group. Compounds were tested in groups of two female BALB/c mice infected with P. berghei ANKA infected erythrocytes. Dosing was via oral gavage at 25 mg/kg twice daily with four hours between dosing (beginning 2 h post infection) for four consecutive days. Peripheral blood parasitemia was monitored by microscopic evaluation of stained thin blood films from day four post infection. None of the peptoid-based HDAC inhibitors attenuated P. berghei growth in BALB/c mice by more than 33% (3f (31%) and 3n (33%) on day 6 post infection). Data from collaborators demonstrated 3n to have the best metabolic stability (t1/2 271 min, Clint 6 μL/min/mg in mice; Prof Finn Hansen, University of Bonn, Germany) which may have contributed to this compound’s improved activity compared to some other analogues. In comparison, AR42 and two if its analogues cured mice of infection (AR42, 1 of 2 mice; JT21b, 2 of 2 mice; JT83 2 of 2 mice), up until day 24 post infection, at which point the mice were euthanised. AR42 and analogues are the first demonstration of oral cures in mice with a HDAC inhibitor (manuscript in preparation) and these data will be pursued in future work to further develop this HDAC inhibitor chemotype for malaria. One of the current limitations in the field is the lack of recombinant P. falciparum HDACs and the need to rely on low throughput assays to demonstrate HDAC inhibitor action via reduced total deacetylase activity or in situ lysine acetylation alterations. While deacetylase assays do not allow the differentiation of compound effects, Western blot using different acetyl-lysine antibodies can reveal compound specific acetylation profiles. Here, two higher throughput methods, dot blot and ELISA, were investigated to assess the effects of HDAC inhibitors on lysine acetylation. Using the control hydroxamate HDAC inhibitor vorinostat (first HDAC inhibitor clinically approved for cancer), the ELISA method was demonstrated to be more reliable than dot blot in detecting acetylation changes in protein lysates from P. falciparum trophozoites exposed to compound for 3 h. ELISA was therefore used to investigate histone H3 and H4 lysine acetylation alterations following exposure of P. falciparum to six commercially available anti-cancer HDAC inhibitors (vorinostat, panobinostat, trichostatin A, romidepsin, entinostat and tubastatin A). All compounds have in vitro activity against asexual intraerythrocytic P. falciparum parasites (Pf3D7), with tubastatin A activity reported for the first time here (PfIC50 0.15 ± 0.03 μM). All compounds were also shown to inhibit >84% deacetylase activity using P. falciparum protein lysates in an in vitro assay at 1 μM, with the exception of entinostat (~50% inhibition at 1 μM); this compound was also the least active against the parasite (PfIC50 11.5 μM). Using ELISA, vorinostat, panobinostat, trichostatin A, romidepsin and entinostat were all found to cause a ~3-fold increase in the signal detected using an anti-tetra-acetyl-lysine antibody. In comparison, the only human HDAC6-specific inhibitor tested, tubastatin A, caused 1.8-fold histone H4 hyperacetylation compared to the control. Further investigations of the individual N-terminal H4 lysine residues using antibodies specific to acetylated lysine 5, 8, 12 or 16 revealed that all compounds, except tubastatin A, caused hyperacetylation using each antibody. No differential effect was observed for histone H3 acetylation, with all compounds causing an ~1.8-fold increased signal using an acetyl-H3 antibody. The new ELISA method developed here provides a higher throughput way to assess differential compound induced lysine acetylation alterations in P. falciparum and therefore represents a valuable new tool to aid the investigation of HDAC inhibitors for malaria. As discussed above, the lack of tools, such as recombinant P. falciparum HDAC proteins, crystal structures and homology models, has meant that the identification of antiplasmodial HDAC inhibitors has been limited to whole-cell screening approaches which can be time-consuming and costly. To begin to address this problem, quantitative structure-activity relationship (QSAR) models were developed based on logistic algorithms with the aim of providing a new tool to triage compounds for in vitro testing. A database of 457 antiplasmodial HDAC inhibitors was assembled with published data on PfIC50 and, for 292 of those compounds with data on plasmodial selectivity. Two independent prediction algorithms based on logistic regression were developed to classify (1) antiplasmodial activity or (2) selectivity of hydroxamate-based HDAC inhibitors. Seven different activity and five different selectivity models were built, each with individual decision cut-offs defining active/selective and non-active/unselective compounds (e.g. PfIC50: active compound <0.1 μM> non-active compound; SI: selective compound >100< unselective compound). Activity model A7 revealed the highest prediction performance by predicting 93% of the training compound set and 87% of the external test compound set correctly. Cross validation revealed a prediction accuracy of 91%. The most accurate selectivity model S4 demonstrated a slightly poorer prediction performance due to a much smaller initial data set as not all the HDAC inhibitors had reported selectivity information (64%). Despite this, the selectivity model demonstrated an internal prediction accuracy of 91%, a cross-validated (internal) prediction accuracy of 82% and an external prediction accuracy of moderate 72%. To validate the prediction performance of the activity model further, they were applied to a set of 22 experimentally untested compounds (validation set) and the prediction performance compared to their experimental antiplasmodial activity. Applying prediction model A7 to this compound set predicted three hit compounds (two of which were confirmed by experimental assay data) and 12 non-actives (confirmed for 11 based on experimental assay data). The experimental PfIC50 assessment revealed asexual blood-stage PfIC50s for the whole set in the nanomolar to micromolar range (PfIC50 0.006-8.45 μM; data from Dr MJ Chua), with the correctly predicted hits (S2_E10 and LD016) having PfIC50 <0.008 μM. Overall, virtual screen using QSAR model A7 identified 87% of the validation compounds correctly and revealed high prediction specificity, identifying 92% of the non-active compounds correctly. Due to a lack of available data sets with selectivity index information (and time constraints for this project), the selectivity models were not able to be tested with an external set. These activity and selectivity QSAR models are the first generated for antiplasmodial HDAC inhibitors. These models will aid the in silico assessment of antiplasmodial activity and selectivity of hydroxamate-based HDAC inhibitors and therefore represent useful new tools in the investigation of HDAC inhibitors for malaria. In summary, data presented in this thesis include the identification of novel antiplasmodial HDAC inhibitors with activity against asexual intraerythrocytic-stage P. falciparum parasites, in vivo data demonstrating oral cures in mice for two analogues of the anti-cancer HDAC inhibitor AR42, a new ELISA method to allow higher throughput assessment of HDAC inhibitor induced changes to histone lysine residues and the first antiplasmodial HDAC inhibitor QSAR models. HDAC inhibitors identified in this study with promising in vitro and in vivo antiplasmodial activity profiles are new starting points for further development of HDAC inhibitors for malaria. In addition, the in vitro and in silico approaches developed in this study are useful new tools to facilitate the discovery of HDAC inhibitors and the understanding of their biological effects on the parasite.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Malaria is responsible of almost half a million deaths every year. Currently, campaigns for the control and elimination of malaria are implemented in malaria endemic areas. However, drug resistance is one of the major impediments to achieve malaria elimination. In this thesis we have investigated how P. falciparum parasites develop resistance to some toxic compounds by functional variation linked to epigenetic regulation of clag3 genes. These genes present clonally variant expression and determine the formation of the main channel for the transport of solutes at the membrane of the infected RBC: Plasmodium Surface Anion Channel (PSAC). Hence, we hypothesized that P. falciparum parasites can modify the permeability of the membrane to specific solutes by epigenetic regulation of clag3 genes expression; this way, parasites could develop resistance to antimalarial drugs. To test this hypothesis, we have investigated the role of switches in clag3 expression in the acquisition of resistance to the antibiotic BS, the dynamics of clag3 genes expression in human infections and we have tested drugs susceptible to failure by this drug resistance mechanism. First, we show that BS pressure at low concentrations selected for parasites expressing clag3.1, whereas parasites exposed to higher concentrations of BS had repressed the expression of both clag3 genes. We did not find any mutation in the genome of these parasites that could explain the change in their phenotype. Thus, we concluded that parasites can develop resistance to toxic compounds through epigenetic regulation of clag3 genes. Then, we found that parasites collected from patients with uncomplicated malaria predominantly express one of the two paralogues, consistent with the property of mutually exclusive expression, previously described in lab-adapted parasite lines. Adaptation to culture conditions or selection with toxic compound results in isolate-dependent changes in clag3 expression, implying functional differences between the proteins encoded. We also observed that samples collected at day 9 post-infection in human experimental infections (when parasites had been in the peripheral blood for approximately one erythrocytic cycle) showed a mix of parasites expressing either clag3.1 or clag3.2, suggesting that the epigenetic memory of clag3 genes is reset during transmission stages. Finally, we tested whether other drugs, that are suspected to require facilitated transport to reach the cell, could be susceptible of failure by this drug resistance mechanism. We found that the antimalarial compounds T3 and T16 (bis-thiazolium salts) require the product of clag3 genes to enter the infected erythrocyte and that P. falciparum populations can develop resistance to these compounds by selection of parasites with dramatically reduced expression of both genes. The rest of the drugs that we tested might use alternative routes in which clag3 genes are not involved. We have described for the first time an antimalarial drug resistance mechanism regulated at the epigenetic level in P. falciparum parasites. This phenomenon may be of relevance for parasite adaptation to the presence of toxic compounds in human blood, selecting rapidly those parasites that present the less permeable phenotype and developing drug resistance in a single infection.
Actualmente, la resistencia a los medicamentos antimaláricos es uno de los principales impedimentos para lograr la eliminación de la malaria. En esta tesis hemos investigado cómo los parásitos de P. falciparum desarrollan resistencia a algunos compuestos tóxicos por variación funcional relacionada con la regulación epigenética de los genes clag3 (clag3.1 y clag3.2), los cuales presentan expresión clonal variante y mutuamente exclusiva (en condiciones normales sólo uno de los dos genes está en estado activo). clag3 determinan la formación del canal principal para el transporte de solutos a través de la membrana del eritrocito infectado: PSAC. En este trabajo, primero observamos que la aplicación de bajas concentraciones del antibiótico blasticidina en cultivos de P. falciparum resultó en la selección de parásitos que expresan clag3.1, mostrando una IC50 a este compuesto más elevada que aquellas líneas que expresan clag3.2. Por otro lado, parásitos expuestos a concentraciones más altas de blasticidina reprimieron la expresión de ambos clag3 y mostraron altos niveles de resistencia al fármaco. No encontramos ninguna mutación en el genoma de estos parásitos que explicase el cambio de fenotipo, sugiriendo que se trata de un mecanismo regulado a nivel epigenético. El estudio de clag3 en parásitos recolectados de pacientes con malaria no complicada mostró que P. falciparum en infecciones naturales expresa predominantemente uno de los dos parálogos: clag3.2, indicando que este patrón de expresión confiere una ventaja fenotípica en sangre humana. Por otro lado, el análisis de muestras recogidas de infecciones humanas experimentales determinó que la memoria epigenética de los genes clag3 se restablece durante las etapas de transmisión, seleccionándose en pocos ciclos aquellos parásitos que presentan el patrón de expresión más favorable en sangre humana: clag3.2. Finalmente, probamos si otros fármacos que requieren transporte facilitado para llegar a la célula podrían ser susceptibles de fracaso terapéutico a través de este mecanismo de resistencia. Hayamos que los compuestos antipalúdicos T3 y T16 (sales de bis-tiazolio) requieren el producto de los genes clag3 para ingresar en el eritrocito infectado y que poblaciones de P. falciparum puedan desarrollar resistencia a estos compuestos mediante la selección de parásitos con expresión reducida de ambos genes.

Malaria, an illness caused by protozoan parasites of the genus Plasmodium, continues to be a key global health issue; around 40% of the world’s population are at risk and more than one million people are killed each year according to the World Health Organisation (WHO). It is transmitted via bites of infected female mosquitoes (Anopheles) and its severest form, falciparum malaria, can lead to death if left untreated. Effective malarial treatment is complex due to drug resistance and socioeconomic issues in many of the most affected areas. An enzyme from the parasite, myristoyl CoA:protein N-myristoyl transferase (NMT), has been identified as a potential target for antimalarial drugs. N-Myristoyl transferase, which catalyses the co-translational transfer of myristic acid to an N-terminal glycine of certain substrate proteins, has been shown to be essential for various pathogens. This thesis demonstrates the design, synthesis and analysis of potential inhibitors of Plasmodium falciparum NMT. Approximately 50 inhibitors with systematic variations based on a benzothiazole scaffold have been synthesised. It is known that these benzothiazoles compete with binding of peptide substrate within the NMT enzyme binding cleft. Differences between the peptide binding pockets of P. falciparum and human NMTs were exploited to design effective and selective new antimalarial treatments. The level of inhibition was measured using SPA that monitors the transfer of 3H-labelled myristoyl CoA to the N-terminus of a polypeptide substrate. A plot of enzyme activity as a function of inhibitor concentration gave inhibition curves from which IC50-values were derived. In vitro tests resulted in four hits with improved activity in the low micromolar region against P. falciparum NMT compared to the lead compound. Nevertheless, the inhibitors were not exceptionally selective over Homo sapiens NMT with an IC50 in the low micromolar region also. Selections of the most promising inhibitors have been tested in vivo and considerable reductions in parasitemia were noted.

Malaria remains one of the most common causes of illness and death in developing countries.1 The development of new drugs to combat the disease is becoming one of the fastest growing research areas. Hydroxypiperaquine (HPQ) is an antimalarial bisquinoline compound related to the emerging antimalarial drug Piperaquine (PQ).2 Various research programs are being conducted internationally in efforts to prepare PQ for possible clinical use in combination with artemisinin derivatives. The hydroxy compound (HPQ) has been described in the Chinese literature but no data exists for this compound within the Western literature.3The primary aim of this research project was to synthesise Hydroxypiperaquine via alternative synthetic pathways to that briefly described by Xu et al3 by exploring various synthetic strategies based on literature synthetic procedures involving similar compounds. HPQ was synthesised through a three step synthetic process. In the first step, tertiary butoxy carbonyl (tBOC) piperazine was coupled with 4,7-dichloroquine (4,7-DCQ) to produce the intermediate 7-chloro-4-(tBOC piperazin-1-yl)quinoline. The second synthetic step involved the deprotection of 7-chloro-4-(tBOCpiperazinyl)quinoline to remove the tertiary butoxy carbonyl (tBOC) protecting group.The deprotected intermediate, 7-chloro-4-(piperazin-1-yl)quinoline, was subsequently reacted with 1,3-dichloropropanol in 1-pentanol to yield HPQ in the third step. This three step synthetic approach provides an alternative and efficient process to synthesise HPQ. The research provides important and specific details for the synthetic methodology involved in the synthesis of HPQ for future synthetic and biological research.

Malaria has long affected the world both socially and economically. Annually, there are 1.5-2.7 million deaths and 300-500 million clinical infections (WHO, 1998). Several antimalarial agents (such as chloroquine, quinine, pyrimethamine, cycloguanil, sulphadoxine and others) have lost their effectiveness against this disease through drug resistance being developed by the malarial parasites (The- Wellcome-Trust, 1999). Although there is no hard-core evidence of drug resistance shown on the new antimalarial compounds (artemisinin and artesunate), induced resistant studies in animal models have demonstrated that the malarial parasites have capabilities to develop resistance to these compounds (Ittarat et al., 2003; Meshnick, 1998; Meshnick, 2002; Walker et al., 2000). Furthermore, a useful vaccine has yet to be developed due to the complicated life cycle of the malarial parasites (The- Wellcome-Trust, 1999). As such, the re-emergence of this deadly infectious disease has caused an urgent awareness to constantly look for novel targets and compounds. In this present study, Plasmodium falciparum (clone 3D7) was cultured in vitro in human red blood cells for extraction of total RNA which was later reverse transcribed into cDNA. The áI-, áII- and â-tubulin genes of the parasite were then successfully amplified and cloned into a bacterial protein expression vector, pGEX- 6P-1. The tubulin genes were then sequenced and analysed by comparison with previously published homologues. It was found that the sequenced gene of áItubulin was different at twelve bases, of which only six of these had resulted in changes in amino acid residues. áII- and â-tubulin genes demonstrated 100% sequence similarity with the published sequences of clone 3D7, but differences were observed between this clone and other strains (strains NF54 & 7G8) of â-tubulin. Nevertheless, the differences were minor in áI- and â-tubulins and there was greater than 99% homology. Subsequently, all three Plasmodium recombinant tubulin proteins were separately expressed and purified. Insoluble aggregates (inclusion bodies) of these recombinant tubulins were also refolded and have been tested positive for their structural characteristics in Western blot analysis. Both soluble and refolded recombinant tubulins of malaria were examined in a drugtubulin interaction study using sulfhydryl reactivity and fluorescence quenching techniques. Known tubulin inhibitors (colchicine, tubulozole-c and vinblastine) and novel synthetic compounds (CCWA-110, 239 and 443) were used as the drug compounds to determine the dynamics and kinetics of the interactions. In addition, mammalian tubulin was also used to determine the potential toxicity effects of these compounds. Similarities were observed with other published reports in the binding of colchicine with the recombinant tubulins, hence confirming proposed binding sites of this compound on the Plasmodium recombinant tubulins. Two synthetic compounds (CCWA-239 and 443) that have previously tested positive against P. falciparum in vitro were found to bind effectively with all three tubulin monomers, while displaying low binding interactions with the mammalian tubulin, thus indicating that these compounds have potential antimalarial activity. Therefore, this study has satisfied and fulfilled all the aims and hypotheses that have previously been stated.

Malaria is the most important parasitic disease in man and it kills approximately 2,000 people each day. Pregnant women are especially vulnerable to malaria with increased incidence and mortality rates. There are indications that pregnancy alters the pharmacokinetic properties of many antimalarial drugs. This is worrisome as lower drug exposures might result in lowered efficacy and lower drug exposures can also accelerate the development and spread of resistant parasites. The aim of this research was to study the pharmacokinetics and pharmacodynamics of the most commonly used drugs for the treatment of uncomplicated Plasmodium falciparum malaria during the second and third trimester of pregnancy using a pharmacometric approach. This thesis presents a number of important findings that increase the current knowledge of antimalarial drug pharmacology and that may have an impact in terms of drug efficacy and resistance. (1) Lower lumefantrine plasma concentrations at day 7 were evident in pregnant women compared to that in non-pregnant patients. Subsequent in-silico simulations with the final pharmacokinetic-pharmacodynamic lumefantrine/desbutyl-lumefantrine model showed a decreased treatment failure rate after a proposed extended artemether-lumefantrine treatment. (2) Dihydroartemisinin exposure (after intravenous and oral administration of artesunate) was lower during pregnancy compared to that in women 3 months post-partum (same women without malaria). Consecutive in-silico simulations with the final model showed that the underexposure of dihydroartemisinin during pregnancy could be compensated by a 25% dose increase. (3) Artemether/dihydroartemisinin exposure in pregnant women was also lower compared to literature values in non-pregnant patients. This further supports the urgent need for a study in pregnant women with a non-pregnant control group. (4) Quinine pharmacokinetics was not affected by pregnancy trimester within the study population and a study with a non-pregnant control group is needed to evaluate the absolute effects of pregnancy. (5) Finally, a data-dependent power calculation methodology using the log likelihood ratio test was successfully used for sample size calculations of mixed pharmacokinetic study designs (i.e. sparsely and densely sampled patients). Such sample size calculations can contribute to a better design of future pharmacokinetic studies. In conclusion, this thesis showed lower exposures for drugs used to treat uncomplicated Plasmodium falciparum malaria during the second and third trimester of pregnancy. More pharmacokinetic studies in pregnant women with a non-pregnant control group are urgently needed to confirm the current findings and to enable an evidence-based dose optimisation. The data-dependent power calculation methodology using the log likelihood ratio test can contribute to an effective design of these future pharmacokinetic studies.

With the increase in recent years in the prevalence of malaria, and in drug resistance of Plasmodium falciparum, there has been much interest in natural plant products for new antimalarials with novel modes of action against Plasmodium. Artemisinin or Qinghaosu is one such antimalarial isolated from a Chinese herb, Anemisia annua (Asteraceae) and it is currently undergoing phase I and II clinical trials. The Southern African species, Artemisia afra (African wormwood, wildeals, lengana) is commonly used by local traditional healers for symptoms of malaria, in particular fever. Thus it seemed appropriate to investigate this species for antimalarial activity. Crude petroleum ether soxhlet extracts of Anemisia afra had demonstrated antimalarial activity against Plasmodium falciparum, FCR-3, cultured in vitro. The IC₅₀ values ranged from 5-13μg/ml. The extract from leaves and flowers was then screened against D10 (chloroquine-sensitive) and FAC8 (chloroquineresistant) P. falciparum, in vitro, with IC₅₀ values of 1.03μg/ml and l.5μg/ml respectively. This extract was fractionated by column chromatography using silica gel-60 and the fractions obtained were screened for antimalarial activity. The most active fraction had an IC₅₀ of 0.5μg/ml against D10 and FAC8. Using TLC and HPLC-UV analysis with pure artemisinin as a standard, no artemisinin could be detected in this fraction. This result was confirmed by thermospray LC-MS analyses. Purification of this fraction yielded ultimately a single pure compound; a clear colourless oil identified by MS and NMR analyses as hydroxydavanone. The compound was screened against a variety of P. falciparum strains with varying degrees of sensitivity and resistance to both chloroquine and mefloquine. Their sensitivity against artemisinin was also established. IC₅₀ values obtained for the isolated pure compound against P. falciparum ranged from 0.87 to 2.54μg/ml. The IC₅₀ values obtained for general cytotoxicity of the crude extract and isolated pure compound against RAT-I fibroblast cells were 34.78 ± 8.23 and 6.29 ± 0.95 μg/ml (n=4) respectively. Thus the crude extract and isolated pure compound exhibited a greater antimalarial than cytotoxic effect. Hence, there are implications for A. afra to be used as a phytomedicine for the treatment of malaria. In vivo studies are recommended for hydroxydavanone in order to fully assess its potential for clinical use.

Le paludisme est une maladie parasitaire tropicale menaçant les populations dans les zones tropicales et sub-tropicales, en particulier les jeunes enfants en Afrique. En raison des résistances aux médicaments antipaludiques qui se sont propagées dans le monde entier au cours des 50 dernières années, de nouveaux médicaments sont vraiment nécessaires. La plasmodione (série benzylmenadione) a été identifiée comme un médicament-candidat antipaludique puissant, agissant selon une bioactivation rédox sur les stades sanguins asexués et sexués jeunes, mais son métabolisme est inconnu. Par conséquent, afin d'identifier les structures des métabolites actifs générés par la plasmodione antipaludique, la synthèse complète de la plasmodione 13C18-enrichie a été conçue et réalisée en 10 étapes. En outre, le procédé d'extraction pour l'étude du métabolisme de la molécule a été établi à partir de globules rouges parasités traités par la plasmodione 13C18-enrichie. D'autre part, la préparation de dérivés oxétane et N-alkylaryl de plasmodione avec une solubilité potentielle améliorée a également été réalisée par substitution nucléophilie aromatique (SNAr) et réaction de couplage Buchwald-Hartwig catalysée par le palladium, respectivement. Enfin, un complexe d'or (I) phosphole, connu comme un inhibiteur irréversible et puissant de la thiorédoxine réductase séléno-dépendante humaine, a été synthétisé et son profil antiparasitaire a été étudié sur de nombreux parasites pathogènes pour l’homme, protozoaires et helminthes en cultures
Malaria is a tropical parasitic disease threatening populations in tropical and sub-tropical areas, especially young children in Africa. Due to the drug resistance spread all over the world in the past 50 years, new drugs are urgently needed. Plasmodione (benzylmenadione series) had been identified as a potent anti-malarial early lead drug, acting through a redox bioactivation on asexual and young sexual blood stages, but its drug metabolism is unknown. Therefore, in order to identify the structures of the active drug metabolites generated from the antimalarial plasmodione, fully 13C18-enriched-plasmodione synthesis was designed and performed in 10 steps. Furthermore, the extraction method for the drug metabolism study was established from 13C18-enriched plasmodione-treated parasitized red blood cells. On the other hand, the preparation of oxetane and N-alkylaryl derivatives of plasmodione with potential improved solubility was also investigated through aromatic nucleophilic substitution (SNAr) and palladium-catalyzed Buchwald-Hartwig coupling reaction, respectively. Finally, a gold(I) phosphole complex, known as an irreversible and potent inhibitor of the human seleno-dependent thioredoxin reductase, was synthetized and its antiparasitic profile investigated against a panel of parasites, protozoans and helminthes in cultures

The malaria parasite, Plasmodium falciparum, has a demonstrated history of adaptation to antimalarials and host immune pressure. This ability unraveled global eradication programs fifty years ago and seriously threatens renewed efforts today. Despite the magnitude of the global health problem, little is known about the genetic mechanisms by which the parasite evades control efforts. Population genomic methods provide a new way to identify the mutations and genes responsible for drug resistance and other clinically important traits.