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Project Descriptions

1:	Understanding the cellular basis for quinoline/peroxide antimalarial interactions and their implications for drug efficacy and toxicity.
2:	Molecular and biochemical basis of Quinoline resistance in P.falciparum and P. vivax
3:	Role of iron in the antimalarial activity of trioxanes
4:	New antimalarial endoperoxide lead compounds from both natural and synthetic origin
5:	Antimalarial chemotherapy inhibiting phospholipidic metabolism effectors, valorisation of N-hydroxylated pro-drug structures.
6:	Drug-heme interactions in lipid and aqueous environments and their implications.
7:	Functional analysis of metabolite and drug carrier systems in P. falciparum
8:	Nucleoside Drug Targets in Plasmodium falciparum
9:	Pharmacokinetic studies on FR-900098, an improved inhibitor of the mevalonate-independent isoprenoid biosynthesis pathway of P. falciparum
10:	Modulation of monocyte CD36 expression, PPARγ activation and RBC phagocytosis by different families of anti-malarial drugs.
11:	Structure-based design of inhibitors against selected Plasmodium falciparum protein kinases

Project 1: Understanding the cellular basis for quinoline/peroxide antimalarial interactions and their implications for drug efficacy and toxicity.

Supervisors:

D Taramelli, University of Milan, Italy, donatella.taramelli@unimi.it
D Monti, University of Milan, Italy, diego.monti@istm.cnr.it
S A Ward, Liverpool School of Tropical Medicine, UK, saward@liv.ac.uk

There is general agreement in the malaria community that combination chemotherapy is the way forward in the treatment of malaria. Furthermore it is argued that one component of such combinations should be an artemisinin/peroxide. Consequently there are quinoline/artemisinin combinations which are currently under evaluation, and it is anticipated that new quinoline and peroxide based drugs would be used in combination in the future. There is good evidence that these two drug classes are antagonistic against P. falciparum in culture and that both rely to some degree on the generation of heme within the parasite in order to exert their full effects.

The aims of this project are to probe the chemical and molecular basis of this drug:drug interaction in a range of model systems.

Chemical interactions - Biomimetic chemistry has demonstrated the chemical reactivity of the artemisinins and peroxides in the presence reducing agents such as FeII. Using cell free systems and a range of reducing conditions the ability of quinolines to modify the rates and routes of biomimetic peroxide cleavage will be investigated.

Antimalarial drug interactions - The quinolines and the artemisinin peroxides act antagonistically in vitro against P.falciparum in culture. We propose to investigate this interaction at the level of potential drug: heme interactions, under conditions of varying oxygen tension and at the level of PfATPase6 a key target in artemisnin activity.

Toxicity and drug interactions – although generally considered safe the artemisnins do carry a potential reprotoxic and neurotoxic potential as demonstrated by experiments in a range of models systems including, toxicity against neuroblastoma cells in vitro, inhibition of angiogenesis in a human endothelial cell model and embryolethality and teratogenicity in rats and rabbits. There is increasing evidence that all of these effects are influenced by heme / iron in the experimental system but the potential modifying role of the quinolines is yet to be evaluated.

These studies will provide fundamental data on quinoline / artemisinin interactions as they relate to both drug activity and toxicity and will help guide the next generation of quinoline and peroxide based antimalarials.

Project 2: Molecular and biochemical basis of Quinoline resistance in P. falciparum and P. vivax

Supervisors:

CHM Kocken, Biomedical Primate Research Centre, The Netherlands, kocken@bprc.nl
SA Ward, Liverpool School of Tropical Medicine, UK, saward@liv.ac.uk
M Lanzer, University of Heidelberg, Germany, michael_lanzer@med.uni-heidelberg.de
4SC, Germany, daniel.vitt@4sc.com

The acquisition and evolution of resistance to antimalarial drugs is the principle reason for the current problems we face with this disease. The work of this PhD Project will focus on understanding the molecular and biochemical basis of resistance to new quinoline (derivatives) in malaria parasites to provide insight into the types of resistance mechanism the parasite can call on.

The main workhorse of this Project will be the versatile P. knowlesi in vitro transfection system.

We will exploit this to analyse the role of Pfcrt (and other genes involved in chloroquine resistance) in the sensitivity of parasites to novel quinoline antimalarials generated as part of the Antimal initiative and to analyse the molecular basis of resistance in P. vivax. Throughout the project malaria transfection technology will be further optimised and a range of state-of-the-art molecular and biochemical procedures will be used.

The aims of this project are to probe:

Quinoline resistance in P. falciparum. The role of Pfcrt in resistance to traditional quinolines such as chloroquine and amodiaquine and novel quinolines generated by Antimal will be investigated by allelic exchange of Pfcrt cDNA from chloroquine sensitive and resistant P. falciparum strains into P. knowlesi. In addition, chimeric molecules between Pf and Pkcrt, as well as mutagenised Pfcrt cDNA will be used for allelic exchange in P. knowlesi. Transfected parasites will be screened for sensitivity to quinolines. Following thorough biochemical analyses, such replacement parasites will be used to further study the role of Pfcrt mutations in conferring resistance to antimalarial drugs.

Additional genes implicated in CQR will also be investigated using this molecular strategy. Chloroquine/quinoline resistance in P. vivax. Several P. vivax isolates have displayed enhancedresistance to chloroquine without any mutations in Pvcrt). cDNA and gDNA expression libraries from such isolates will be generated in transfection plasmids and the libraries will be transfected into P. knowlesi using optimised transfection systems and selected with chloroquine to isolate cDNA’s involved in chloroquine resistance in P. vivax. Genotypic, phenotypic and biochemical analyses will shed light on the mechanisms of quinoline ressiatnce in P. vivax.

Project 3: Role of iron in the antimalarial activity of trioxanes

Supervisors:

B Meunier, Centre National de la Recherche Scientifique, France, bmeunier@lcc-toulouse.fr
A Robert, Centre National de la Recherche Scientifique, France, arobert@lcc-toulouse.fr
P O’Neill, University of Liverpool, UK, P.M.Oneill01@liv.ac.uk
D Caussanel, Palumed, France.

Upon reductive activation by iron (II)-heme, antimalarial trioxanes are efficient alkylating agents toward heme itself. Covalent heme-artemisinin adducts, resulting from the alkylation of the four meso position of heme by a drug-derived C-centered radical have been isolated and characterized.1-3 These adducts are also formed in malaria-infected mice treated with artemisinin at pharmacologically relevant dose, but are absent in healthy mice treated in the same conditions. This clearly indicates that the alkylating ability of artemisinin can occur in mammals, and that this effect is triggered by the parasite.4 New drugs named « trioxaquines »®, which combine within a single molecule an aminoquinoline and a trioxane residue, are efficient to cure infected mice by oral administration.5 Trioxaquines are also able to alkylate heme in vitro. 6 The possible role or iron (heme and also other iron-containing biological entities) for the antimalarial activity of artemisinin and trioxaquines will be further investigated. The alkylating properties of trioxaquines toward heme will be evaluted in infected mice and correlated with their antimalarial activity, in order to provide the mechanistic support for the pre-clinical development of trioxaquines, and the design of a new generation of trioxane-containing drug candidates.

The final year of the project will involve the preparation of fluorescent probe acridine variants for use in confocal microscopy studies. We will then examine the distribution of « tagged molecules »in living parasites using methods previously developed.7

1. Robert, A.; Cazelles, J.; Meunier, B. Angew. Chem. Int. Ed. 2001, 40, 1954-1957.
2. Robert, A.; Cazelles, J.; Dechy-Cabaret, O.; Meunier, B. Acc. Chem. Res. 2002, 35, 167-174.
3. Laurent, S. A.-L.; Robert, A.; Meunier, B. Angew. Chem. Int. Ed. 2005, 44, 2060-2063.
4. Robert, A.; Benoit-Vical, F.; Claparols, C.; Meunier, B., Proc. Natl. Acad. Sci. USA 2005, 102, 13676-13680.
5. Dechy-Cabaret, O.; Benoit-Vical, F.; Loup, C.; Robert, A.; Gornitzka, H.; Bonhoure, A.; Vial, H.; Magnaval, J.-F.; Séguéla, J.-P.; Meunier, B. Chem. Eur. J. 2004, 10, 1625-1636.
6. Laurent, S. A.-L.; Loup, C.; Mourgues, S.; Robert, A.; Meunier, B. ChemBioChem 2005, 6, 653- 658.
7. Eckstein-Ludwig, U.; Webb, R. J.; van Goethem, I. D. A.; East, J. M.; Lee, A. G.; Kimura, M.;O'Neill, P.M.; Bray, P. G.; Ward, S. A.; Krishna, S.. Artemisinins target the SERCA of Plasmodium falciparum Nature 2003, 424, 957-961

Project 4: New antimalarial endoperoxide lead compounds from both natural and synthetic origin

Supervisors:

G Campiani, Siena University, Italy, campiani@unisi.it
PM O’Neill, University of Liverpool, UK, P.M.Oneill01@liv.ac.uk
B Meunier, Centre National de la Recherche Scientifique, France, bmeunier@lcc-toulouse.fr
A Robert, Centre National de la Recherche Scientifique, France, arobert@lcc-toulouse.fr

Recently, the antimalarial activity of a novel class of endoperoxide, isolated from the marine sponge Plakortis simplex, and structurally characterized by researchers of the research partner NatSyn has been disclosed. (J. Antimicrob. Chemother., 2002 50,883). Among the secondary metabolites isolated and structurally characterized, Plakortin and Dihydroplakortin showed the most potent acivities against chloroquine-resistant P. f.strains (activity determined by Donatella Taramelli). Preliminary structure/activity relationships in the Plakortin series were drawn on the basis of the isolated minor metabolites from Plakortis species providing valuable information about the mechanism of action of this class of endoperoxide antimalarial.

At the University of Liverpool, we have described three different synthetic methods for the synthesis of the 1,2,4-trioxane pharmacophore. 1-3 The first of these methods 2 employs bis (2,2,6,6-tetramethyl-3, 5-heptanedionato), cobalt (II) (Co(thd) 2), a -diketonate catalyst prepared in a simple one-step procedure. In contrast to? the commercially available Co(acac) 2 the key finding that Co(thd) 2 can catalyse the regioselective hydroperoxysilylation of cyclic allylic alcohols has enabled us to broaden this approach to the synthesis of some potent spiro 1,2,4-trioxane antimalarials ; a total of 50 trioxanes have been prepared. In addition to this synthetic methodology, we have also prepared a small library of racemic and chiral analogues by a Thiol Olefin Co-oxidation (TOCO) strategy 3,4 and by singlet oxygenation of chiral allylic alcohols. By examination of SAR relationships for these novel series of analogues we now wish to optimise our lead compounds and develop QSAR analyses for these classes of endoperoxides.

Aims of the PhD program Supervisors NatSyn-PON-BM-AR Y2-Y3, 12 months at NatSyn Labs

Chemistry program at Natsyn (12 months)

The main goal in the first phase of this PhD studentship will be to develop a versatile synthetic strategy to plakortine, dihydroplakortine and to plakortine-related derivatives with simplified structure. Retrosynthetic analysis of structures of plakortin-like has shown that a versatile and innovative strategy of synthesis could be developed, enabling easy creation of natural products and structural analogues. Since the natural products contain four chiral centres, synthesis must be stereoselective, using specific chiral auxiliaries. The new strategy of synthesis may provide a sufficient quantity of natural products (e.g. plakortine and dihydroplakortine) for more detailed pharmacological analysis and for study of mechanisms of antimalarial activity.

Synthetic and mechanistic studies at Liverpool and Laboratoire de Chimie de Coordination du CNRS (18 months)

The clear strategy will be to produce a drug candidate that can be prepared in achiral (or chiral form) in only a few steps from readily available starting materials. Candidates will be critically assessed and ranked by examination of in vitro and in vivo antimalarial activity data. Mechanistic work (iron degradation and heme alkylation studies) will follow on one selected lead compound in the labs of Professor Meunier. If time allows, we will also prepare a BODIPY tagged analogue for confocal microscopy studies.

Computational chemistry at Natsyn (6 months)

Identification of the key chemical physical properties responsible for the antimalarial activity of two selected endoperoxide leads from the Natsyn and Liverpool Group.

sampling of the conformational space by means of molecular dynamics calculations followed by an energy minimization protocol
selection of the energetically accessible conformations
hypothesis of bioactive conformation
calculation of the electronic properties of the ligand and its activating target heme through quantum chemistry methods. The role of iron: investigation of the reactivity of FeII/FeIII in different chemical environment.
flexible docking studies on ligand/heme complex
investigation of the hypothetical radicalic mechanism of action through the application of ab initio calculations.

To these aims, molecular modelling techniques and specific new softwares (InsightII, Catalyst, MSI; Sybyl, Tripos; Gaussian) will be used.

References

1. O’ Neill, P.M.; Pugh, M.; Davies, J.; Ward, S.A.; Park, B.K.; Regioselective Mukaiyama hydroperoxysilylation of 2-alkyl or 2-aryl-prop-2-en-1-ols: application to a new synthesis of 1,2,4-trioxanes, Tetrahedron. Lett., 2001, 42, 4569-4571

2. O'Neill, P. M.; Hindley, S.; Pugh, M. D.; Davies, J.; Bray, P. G.; Park, B. K.; Kapu, D. S.; Ward, S. A.; Stocks, P. A., Co(thd)(2): a superior catalyst for aerobic epoxidation and hydroperoxysilylation of unactivated alkenes: application to the synthesis of spiro-1,2,4-trioxanes, Tetrahedron Lett., 2003, 44, 8135-8138

3. O’Neill, P.M.; Mukhtar, A.; Ward, S.A.; Bickley, J.F.; Davies, J.; Bachi, M.D.; O’Neill, P.M., Application of Thiol Olefin Co-Oxidation Methodology to a New Synthesis of the 1,2,4-Trioxane Phramacophore, Org. Lett., 2004, 6, 3035-3038

4. O’Neill, P.M., Stocks, P.A.; Pugh, M.D.; Araujo, N.C; Korshin, E.E.; Bickley, J.F.; Ward, S.A.; Bray, P.G.; Pasini, E.; Davies, J.; Verissimo, E.; Bachi, M.D. Design and Synthesis of Endoperoxide Antimalarial Pro-Drug Models: Prototypes for Selective Intraparasitic Generation of Cysteine Protease Inhibitors and Other Parasitocidal Species, Angew. Chem. Int. Ed., 2004, 116, 4289-4293

5. Eckstein-Ludwig, U.; Webb, R. J.; van Goethem, I. D. A.; East, J. M.; Lee, A. G.; Kimura, M.; O'Neill, P.M.; Bray, P. G.; Ward, S. A.; Krishna, S.. Artemisinins target the SERCA of Plasmodium falciparum Nature 2003, 424, 957-961

Project 5: Antimalarial chemotherapy inhibiting phospholipidic metabolism effectors, valorisation of N-hydroxylated pro-drug structures.

Supervisors:

T Durand, Centre National de la Recherche Scientifique, France, thierry.durand@univ-montp1.fr
PM O’Neill,P.O. University of Liverpool, UK, P.M.Oneill01@liv.ac.uk
F Bressolle, Centre National de la Recherche Scientifique, France, Fbressolle@aol.com

To fight multiresistant Plasmodium falciparum malaria, a new chemotherapy has been developed with P. falciparum et P. vivax models. We have designed bis-cations which specifically target and accumulate in infected cells. These compounds exert their antimalarial activity by two ways: they inhibit plasmodial phospholipidic metabolism which is crucial for the membrane neogenesis of the intra-erythrocytic parasite, and they also interact with the parasitic pigment (hemozoin).

This antimalarial approach was first designed through quaternary ammonium salts (first generation compounds). To improve tolerance and oral absorption, bis-amidines and bis-guanidines (second generation compounds) were synthesized. Currently, we are developing a pro-drug strategy to generate a new class of orally available antimalarials.

During this PhD work, uncharged derivatives (e.g. carbamate, N-hydroxylated, N-nitrilate) of active charged drugs will be prepared in various series: bis-C-alkylamidines (eventually N-substituted) reverse bis-N-alkylamidines and bis-guanidines... The aim of our pro-drug design strategy will be to define pro-drug motifs that are readily metabolised to produce the active biscationic form of the drug.

Pro-drugs will be selected upon the criteria of their transformation into drugs and of antimalarial activity after oral administration in animal models. Then, structures will be ranked based on their antimalarial activity and pharmacokinetic parameters.

The purpose of this research task is to rationalise research of orally active antimalarials and to select optimized structures for a thorough antimalarial activity including studies in the monkey. This approach should lead to compounds with substantially-improved oral absorption and thus to realistic antimalarial drug candidates.

Project 6: Drug-heme interactions in lipid and aqueous environments and their implications.

Supervisors:

P Bray, Liverpool School of Tropical Medicine, UK, p.g.bray@liv.ac.uk
H Vial, Centre National de la Recherche Scientifique, France, vial@univ-montp2.fr

During its intra-erythrocytic cycle, the malarial parasite, Plasmodium falciparum, ingests anddegrades most of the host cell haemoglobin, releasing large quantities of haeme or ferriprotoporphyrin IX (FP) which is then detoxified by crystallization into regular crystallites of haemozoin (HZ). Chloroquine (CQ) and other quinolines are known to inhibit the formation of HZcrystals, causing FP to accumulate and kill the parasite. FP is highly lipid-soluble and lipids and membranes are known to promote HZ formation in vitro. A number of other antimalarial drugs are also known to interact with FP, including a range of novel and potent biscationic phospholipids (PL) analogues. Unlike quinolines, these compounds clearly inhibit parasite PL metabolism as well as interacting with FP and the relative contribution of these activities to the antimalarial mode of action is not clear. This project will focus on the characterization of the FP-binding properties of the biscationic choline analogues.

The FP-antimalarial interaction in the potentially crucial lipid membrane environment will be studied and we will compare the interaction of antimalarial biscationic drugs with FP in both aqueous and lipid environments.

We will use established methods to study the interaction of the novel biscations with FP in an aqueous environment. In parallel, we will study interactions within the membrane environment. As an example, FP binds strongly to lipids, with the polar side chains at the interface and the vinyl groups towards the less polar interior. Initial experiments will use small unilamellar vesicles of phosphatidyl choline (PC), which are readily prepared and optically transparent.Having established the basic features of recognition in neutral PC vesicles, other lipids and cholesterol will then be added to reproduce more closely the lipid composition of infected erythrocytes. Anionic lipids (phosphatidic acid, phosphatidyl serine/inositol/glycerine etc.) and neutral lipids are of particular interest, as these would be expected to increase drug partitioning.

Eventually, membrane preparations from erythrocytes will also be employed. It is essential to understand the chemistry of the drug -FP interaction within this physiologically relevant environment if we are to fully understand the mode of action of these drugs and establish a meaningful basis for rational drug design.

Project 7: Functional analysis of metabolite and drug carrier systems in P. falciparum

Supervisors:

S Krishna, St George’s Hospital Medical School, UK, sgjf100@sgul.ac.uk
M Lanzer, University of Heidelberg, Germany, michael_lanzer@med.uni-heidelberg.de

Carrier-mediated transport processes play an important role in the pathophysiology of the human malarial parasite Plasmodium falciparum and are involved, for example, in nutrient uptake and drug resistance mechanisms. The objective of this project is to investigate two model carrier systems of the parasite - the principle hexose transporter PfHT and the P. falciparum mdr1 homolog. PfHT has been extensively characterised in Xenopus oocytes and is a validated drug target in vitro and in vivo.

Polymorphisms within PfMDR1 have been implicated in altered responses to a wide range of different antimalarial drugs including quinine, chloroquine, mefloquine, arthemisinin and halofantrine. To better define the biological functions of these two carriers during the parasite’s life cycle, as well as their roles as drug targets and mediators of resistance mechanisms, we propose to generate constitutional knock-out mutants in which expression of these respective genes is under the control of an inducible promoter. The project combines transfection technology with state-of the-art life cell imaging and biochemical approaches. The project will be jointly executed by the Universities of London and Heidelberg.

Project 8: Nucleoside Drug Targets in Plasmodium falciparum.

Supervisors:

I. Gilbert, University of Dundee, UK, i.h.gilbert@dundee.ac.uk
D. González-Pacanowska, CSIC Madrid, E dgonzalez@ipb.csic.es
F. Mulaa, University of Nairobi, EAK, mulaafj@uonbi.ac.ke

Pyrimidines are essential for all cells. They are required not only for DNA and RNA biosynthesis, but also for the synthesis of phospholipids and glycoproteins. In the case of Plasmodium, de novo pyrimidine nucleotide biosynthesis is a major route for therapeutic intervention since, unlike mammalian cells, the parasite lacks the enzymes required for pyrimidine base and nucleoside salvage and hence completely depends on de novo synthesis to meet its metabolic requirements. The essential character of this process has been shown for certain enzymes of the pathway and several compounds used routinely as antimalarials act at this level. Thus, dihydrofolate reductase is a validated target for the treatment of the disease. Furthermore a number of inhibitors of thymidylate synthesis, such as 5-fluorotate are effective against the parasite, and these deleterious effects cannot be reversed by the presence of thymidine.

The aim of this project is to explore nucleoside metabolism as a source of drug targets against Plasmodium and optimise nucleoside analogues that are existing leads. The objectives to achieve this goal will be: (1) Identification and of novel targets connected with nucleoside metabolism using a proteomics based approach and/or a yeast three-hybrid based compound/protein display system. (2) Characterization of nucleoside/nucleotide kinases which are important in intracellular activation of nucleosides. (3) Perform screening activities for identification of nucleoside analogues with anti-plasmodial activity. (4) Validation of new targets.

This is an interdisciplinary project which will involve the student working in a number of different disciplines and labs.

(1)Target Identification : Specifically, a series of novel nucleoside analogues with anti-plasmodial activity have been identified for which the drug target is not known. A two-fold strategy will be used for target identification:

(a) These compounds will be used in chemical proteomics studies in order to establish new pyrimidine binding proteins. Nucleoside analogues and a series of pyrimidine nucleosides will be coupled to solid supports to allow isolation of proteins. Proteins which bind to these “probes” will be identified by a combination of gel-electrophoresis and mass spectrometry.

(b) The three yeast three-hybrid (Y3H)-based compound/protein display system will be used to screen the P. falciparum proteome for targets of nucleoside inhibitors. Various known pyrimidine nucleoside analogs, will be displayed in the form of methotrexate-based hybrid ligands and deployed in P. falciparum cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell pyrimidine binding proteins as well as novel candidate unrelated targets could be identified, many of which will be independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system may prove generally useful in the discovery of candidate drug targets.

Activities :
Probe synthesis and coupling to solid supports: University of Dundee
Affinity chromatography, 2D Gel electrophoresis and protein identification: CSIC.
Hybrid ligand synthesis: University of Dundee

Yeast three hybrid analysis:

(2)Target Characterisation : Proteins identified by the proteomics approach will be cloned, over-expressed and kinetically characterised. Simultaneously, several enzymes involved in nucleoside metabolism, which can be identified from the genome, will be cloned and over-expressed. Specifically we shall characterize substrate specificity and inhibition profiles of Plasmodium falciparum thymidylate and uridylate/cytidylate kinases, enzymes which have been exploited as drug targets in other organisms.

Activities :
Cloning and expression system development of target proteins:
Substrate specificity studies and inhibition profile:

(3)Hit/ Lead Identification : Where appropriate, preliminary screening programmes will be undertaken to develop drug leads suitable for further development and optimisation. In vitro activity and efficacy in animal models of the disease will be evaluated.

Activities :
Design and chemical synthesis:
Optimization:
In vitro and in vivo activity:

(4)Target validation : Inhibitor interaction studies will be performed in detail with identified targets in order to understand structural requirements of inhibitor potency and selectivity. Mode of action of selected compounds will be analysed in the parasite by studying perturbations in nucleotide pools using the DNA polymerase based assay or high-performance liquid chromatography. DNA precursor asymmetries that result from drug action will be established.

Activities:
Inhibition profiles:
Intracellular nucleotide pools measurements:

Project 9: Pharmacokinetic studies on FR-900098, an improved inhibitor of the mevalonate-independent isoprenoid biosynthesis pathway of P. falciparum

Supervisors:

H Jomaa, University of Giessen, Germany, hassan.jomaa@uniklinikum-giessen.de
F Jehl, University of Strasbourg, France, francois.jehl@medecine.u-strasbg.fr
S Van Calenbergh, Ghent University, Belgium, serge.vancalenbergh@ugent.be

The isoprenoid biosynthesis of P. falciparum depends on the mevalonate-independent 1-deoxy-Dxylulose 5-phosphate (DOXP) pathway. This pathway was discovered only recently and is typically used by the plastids of plants and the majority of eubacteria. In P. falciparum the enzymes of the DOXP pathway is located inside the plastid-like organelle, the so-called apicoplast. The natural antibiotic fosmidomycin was identified as an inhibitor of DOXP reductoisomerase, the second enzyme of the DOXP pathway. In recent clinical phase II studies conducted in Gabon and Thailand fosmidomycin was demonstrated to represent a potent antimalarial drug.

The overall goal of the project is the preclinical and early clinical development of FR-900098, which was identified as a fosmidomycin derivative with increased antimalarial activity. The specific aim of this PhD project will be the development of analytical methods for the conduct of pharmacokinetic studies on FR-900098.

Preferentially, a method based on capillary electrophoresis will be applied. However, also other techniques such as anion exchange HPLC will be evaluated. As an additional task, pharmacokinetic methods for established antimalarial drugs potentially co-administered with FR-900098 will be established, in particular for artesunate and clindamycin, including the determination of different degradation and biotransformation products.

This work will be part of the preclinical studies providing the data packages required for first human trials.

Project 10: Modulation of monocyte CD36 expression, PPARγ activation and RBC phagocytosis by different families of anti-malarial drugs.

Supervisors:

L Vivas, London School of Hygiene and Tropical Medicine, UK, livia.vivas@lshtm.ac.uk
D Taramelli, University of Milan, Italy, donatella.taramelli@unimi.it
F Omodeo Salè, University of Milan, Italy
D Monti, University of Milan, Italy, diego.monti@istm.cnr.it

Malaria pathogenesis is influenced by the balance between parasite burden and host immune response. In addition, bone marrow and red blood cell (RBC) dysfunction induced by parasite products contribute to severe malaria anaemia. Well known anti-malarial drugs have been shown to affect monocyte functions and the release of pro-inflammatory cytokines, whereas the effect on erythropoiesis and RBC activities is less clear.

The monocyte CD36 scavenger receptor is involved in the removal of pathological RBC in different haemoglobinopathies as well as parasitized red cells, via non-opsonic mediated phagocytic uptake of infected RBC. This can significantly affect the resolution of malarial infection in non-immune individuals who lack the opsonizing effects of specific anti-malarial antibodies or may play a role in severe malaria anemia. It has also been shown that mutations in CD36 are associated with susceptibility to severe malaria. Up-regulation of the CD36 scavenger receptor by peroxisome proliferator activated receptor ?-retinoic-X-receptor (PPAR-?-RXR) agonists, enhances phagocytosis of P. falciparum parasitised erythrocytes in vitro and also results in the inhibition of NF?B, and therefore of pro-inflamatory cytokine production.

The aims of this project are to determine the modulatory effects of antimalarial drugs on PPAR-? activation, CD36 expression, oxidative burst, and release pro-inflammatory cytokines by monocytes/macrophages and phagocytosis of infected and uninfected red cells. In addition, the structural and metabolic modifications of normal RBC induced by parasite products and/or antimalarial drugs and their susceptibility to CD36-mediated phagocytosis will be studied.

This study will provide at least two sets of information: 1) in non-immune individuals, it may lead to the development of a prophylactic therapy by increasing non-opsonic phagocytosis of infected red cells through the activation of PPAR-? and CD36 expression, and regulation of the pro-inflammatory response induced by the malaria infection; 2) in the population at risk of severe malaria (children and pregnant woman) may help finding adjunct therapies to ameliorate anaemia.

These effects will be also investigated using in vivo rodent malaria models. To differentiate the effect of malaria immunity from CD36 mediated parasite clearance, drugs will be administered to naïve and semi-immune mice for different periods prior to infection and their peripheral or splenic monocytes used to measure phagocytosis of infected and uninfected red cells, CD36 and PPAR-? expression and levels of pro-inflamatory cytokines. The effects of these antimalarials on anaemia in treated and untreated mice will also be investigated.

Project 11: Structure-based design of inhibitors against selected Plasmodium falciparum protein kinases.

Supervisors:

C Doerig, INSERM U609, Wellcome Centre for Molecular Parasitology, University of Glasgow, UK. cdoer001@udcf.gla.ac.uk
K Wilson, University of York, UK, keith@usbl.york.ac.uk
Laurent Meijer, CNRS Roscoff, France, meijer@sb-roscoff.fr

The recent surge of interest in protein kinases as targets for chemotherapeutic intervention in diseases such as cancer and neurodegenerative disorders has stimulated research aimed at determining whether enzymes of this class might also be considered as targets in the context of malaria. The 85 protein kinases encoded in the genome of Plasmodium falciparum include orthologues of enzymes known to play essential roles in fundamental cellular processes in higher eukaryotes, and therefore represent attractive potential targets for novel antimalarials. Several of these have been expressed as active recombinant enzymes, allowing medium-throughput screening of chemical libraries, and in a few instances compounds have been identified that display selectivity against the plasmodial (versus human) orthologue.

The general objective of this project is to obtain structural data (i) to explain the differential susceptibility of plasmodial versus human enzymes to previously identified inhibitory compounds, and (ii) to provide a structural basis for the design of new inhibitors against novel protein kinases.

Specific aims will be as follows:

-clone, express, characterise (assay development) and crystallize the catalytic domains of two novel plasmodial enzyme of the tyrosine-kinase-like (TKL) family (in Glasgow). If time permits, the recombinant enzymes will be used in medium-throughput screening operations in Roscoff.

-obtain crystals of PfGSK3, a P. falciparum orthologue of GSK3, an enzyme involved in the control of cell proliferation and development in higher eukaryotes (an expression protocol is already available for this protein).

-obtain co-crystals of PfGSK3 with species-specific inhibitors that were identified previously in the Roscoff laboratory

-use the crystals for structure solution using X-ray diffraction. The structure of the human GSK3 homologue is already in the PDB so it should be possible to solve the structure using molecular replacement (structural biology will be done in York).

This project will be run in parallel with, and is complementary to, work that is ongoing in the Glasgow laboratory, aiming at validating these potential targets by reverse genetics approaches. York is also a participant in two other malaria projects (1) on the dUTPase from P. falciparum funded by the EC and (2) a functional genomics project funded by the Wellcome Trust. Roscoff and Glasgow are involved in the SIGMAL (EC STREP) project.

Training:The project will provide training in a range of areas in modern structural biology, from cloning to 3D structure determination. Together, the three laboratories have excellent facilities to host a programme comprising structural biology, as well as kinase biochemistry and screening.