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ABSTRACT: A series of phenoxyoxazaphospholidine, phenoxyoxazaphosphinane and benzodioxaphosphininamine sulfides and related cyclic organophosphorus compounds based on the lead anti-tubulin herbicides amiprophos methyl and butamifos were synthesised and evaluated for anti-malarial activity. Of these compounds, while none of the phenoxyoxazaphospholidines, phenoxyoxazaphosphinanes or benzodioxaphosphininamine sulphides were more potent than APM, phosphorothioamidate 30, a dual compound also bearing an aminoquinoline motif, showed promising activity against Plasmodium falciparum (IC50 0.038μM) and warrants further study.
Bioorganic & medicinal chemistry letters 04/2013; · 2.65 Impact Factor
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ABSTRACT: Malarial parasites are exquisitely susceptible to a number of microtubule inhibitors but most of these compounds also affect human microtubules. Herbicides of the dinitroaniline and phosphorothioamidate classes however affect some plant and protozoal cells but not mammalian ones. We have previously shown that these herbicides block schizogony in erythrocytic parasites of the most lethal human malaria, Plasmodium falciparum, disrupt their mitotic spindles, and bind selectively to parasite tubulin. Here we show for the first time that the antimitotic herbicides also block the development of malarial parasites in the liver stage. Structure-based design of novel antimalarial agents binding to tubulin at the herbicide site, which presumably exists on (some) parasite and plant tubulins but not mammalian ones, can therefore constitute an important transmission blocking approach. The nature of this binding site is controversial, with three overlapping but non-identical locations on α-tubulin proposed in the literature. We tested the validity of the three sites by (i) using site-directed mutagenesis to introduce six amino acid changes designed to occlude them, (ii) producing the resulting tubulins recombinantly in Escherichia coli and (iii) measuring the affinity of the herbicides amiprophosmethyl and oryzalin for these proteins in comparison with wild-type tubulins by fluorescence quenching. The changes had little or no effect, with dissociation constants (Kd) no more than 1.3-fold (amiprophosmethyl) or 1.6-fold (oryzalin) higher than wild-type. We conclude that the herbicides impair Plasmodium liver stage as well as blood stage development but that the location of their binding site on malarial parasite tubulin remains to be proven.
Molecular and Biochemical Parasitology 03/2013; · 2.55 Impact Factor
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Angus Bell
Current pharmaceutical design 09/2012; · 4.41 Impact Factor
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ABSTRACT: Antimalarial drugs have in the past fallen prey to resistance and this problem is likely to continue in the future. One approach to developing drugs that might be less prone to resistance might be to target the disease rather than the parasite itself. The rationale for this idea, which has been somewhat developed in antibacterial chemotherapy, is that drugs that can alleviate disease pathogenesis while not compromising the survival, growth or transmission of the pathogen should not exert selective pressure that would encourage the emergence and spread of resistance. This review considers (concentrating on possible interventions at the parasite level) whether such 'anti-disease' therapy could be developed for severe Plasmodium falciparum malaria, and if so whether it might be less prone to resistance. Several anti-adhesive treatments, aiming to reduce the tissue sequestration of P. falciparum-parasitised erythrocytes that is associated with cerebral malaria and other complications, have been investigated as 'adjunctive' therapies. These therapies are however unlikely to be 'resistance proof' because sequestration appears to enhance parasite survival in the host. Severe malarial anaemia is another potentially fatal complication of malaria that results not only from lysis of host erythrocytes by intracellular parasites but to a greater extent from lysis of unparasitised erythrocytes and impaired erythropoiesis. The possibility of therapy interfering with the last of these processes, which may be more 'resistance proof', is discussed in detail.
Current pharmaceutical design 09/2012; · 4.41 Impact Factor
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ABSTRACT: The cyclophilins are a large family of proteins implicated in folding, transport and regulation of other proteins and are potential drug targets in cancer and in some viral and parasitic infections. The functionality of cyclophilins appears to depend on peptidyl-prolyl cis-trans isomerase (foldase) and/or molecular chaperone activities. In this study we assessed the peptidyl-prolyl isomerase and chaperone activities of 8 members of the Plasmodium falciparum cyclophilin family, all produced recombinantly using a common host/vector system. While only two of these proteins had isomerase activity, all of them displayed chaperone function as judged by the ability to prevent the thermal aggregation of model substrates. We suggest that the cyclophilins constitute a family of molecular chaperones in malarial parasites that complement the functions of other chaperones such as the heat-shock proteins.
Molecular and Biochemical Parasitology 04/2012; 184(1):44-7. · 2.55 Impact Factor
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ABSTRACT: Malaria represents a global health, economic and social burden of enormous magnitude. Chemotherapy is at the moment a largely effective weapon against the disease, but the appearance of drug-resistant parasites is reducing the effectiveness of most drugs. Finding new drug-target candidates is one approach to the development of new drugs. The family of cyclophilins may represent a group of potential targets. They are involved in protein folding and regulation due to their peptidyl-prolyl cis-trans isomerase and/or chaperone activities. They also mediate the action of the immunosuppressive drug cyclosporin A, which additionally has strong antimalarial activity. In the genome database of the most lethal human malarial parasite Plasmodium falciparum, 11 genes apparently encoding cyclophilin or cyclophilin-like proteins were found, but most of these have not yet been characterized. Previously a pET vector conferring a C-terminal His₆ tag was used for recombinant expression and purification of one member of the P. falciparum cyclophilin family in Escherichia coli. The approach here was to use an identical method to produce all of the other members of this family and thereby allow the most consistent functional comparisons. We were successful in generating all but three of the family, plus a single amino-acid mutant, in the same recombinant form as either full-length proteins or isolated cyclophilin-like domains. The recombinant proteins were assessed by thermal melt assay for correct folding and cyclosporin A binding.
Protein Expression and Purification 08/2011; 78(2):225-34. · 1.59 Impact Factor
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ABSTRACT: A series of phosphoramidate and phosphorothioamidate compounds based on the lead antitubulin herbicidal agents amiprophos methyl (APM) and butamifos were synthesised and evaluated for antimalarial activity. Of these compounds, phosphorothioamidates were more active than their oxo congeners and the nature of both aryl and amido substituents influenced the desired activity. The most active compound was 46, O-ethyl-O-(2-methyl-4-nitrophenyl)-N-cyclopentyl phosphorothioamidate, which was more effective than the lead compound.
Bioorganic & medicinal chemistry letters 07/2011; 21(20):6180-3. · 2.65 Impact Factor
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ABSTRACT: We describe the application of ligand based virtual screening technologies towards the discovery of novel plasmepsin (PM) inhibitors, a family of malarial parasitic aspartyl proteases. Pharmacophore queries were used to screen vendor libraries in search of active and selective compounds. The virtual hits were biologically assessed for activity and selectivity using whole cell Plasmodium falciparum parasites and on target in PM II, PM IV and the closely related human homologue, Cathepsin D assays. Here we report the virtual screening highlights, structures of the hits and their demonstrated biological activity.
Bioorganic & medicinal chemistry letters 06/2011; 21(11):3335-41. · 2.65 Impact Factor
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ABSTRACT: Blood-stage malarial parasites (Plasmodium falciparum) digest large quantities of host haemoglobin during their asexual development in erythrocytes. The haemoglobin digestion pathway, involving a succession of cleavages by various peptidases, appears to be essential for parasite development and has received much attention as an antimalarial drug target. A variety of peptidase inhibitors that have potent antimalarial activity are believed to inhibit and/or kill parasites by blocking haemoglobin digestion. It has not however been established how such a blockage might lead to parasite death. The answer to this question should lie in identifying the affected physiological function, but the purpose of excess haemoglobin digestion by P. falciparum has for many years been the subject of debate. The process was traditionally believed to be nutritional until Lew et al. [Blood 2003;101:4189-94] suggested that it is linked to volume control of the infected erythrocyte and is necessary to prevent premature osmotic lysis of the host cell. Their model predicts that sufficient inhibition of haemoglobin degradation should result in premature haemolysis. In this study we examined the downstream effects of reduced haemoglobin digestion on osmoprotection and nutrition. We found that inhibitors of haemoglobinases (plasmepsins, falcipains and aminopeptidases) did not cause premature haemolysis. The inhibitors did however block parasite development and this effect corresponded to a strong inhibition of protein synthesis. The effect on protein synthesis (i) occurred at inhibitor concentrations and times of exposure that were relevant to parasite growth inhibition, (ii) was observed with different chemical classes of inhibitor, and (iii) was synergistic when a plasmepsin and a falcipain inhibitor were combined, reflecting the well-established antimalarial synergism of the combination. Taken together, the results suggest that the likely primary downstream effect of inhibition of haemoglobin degradation is amino acid depletion, leading to blockade of protein synthesis, and that the parasite probably degrades globin for nutritional purposes.
Molecular and Biochemical Parasitology 10/2010; 173(2):81-7. · 2.55 Impact Factor
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ABSTRACT: With the rapid spread of drug-resistant strains of Plasmodium falciparum, the development of new antimalarials is an urgent need. As malaria parasites live in a highly pro-oxidant environment, their anti-oxidant defences have frequently been suggested as candidate drug targets. A key point in such defences is the production of NADPH e.g. for maintaining anti-oxidant glutathione in the reduced state. Some authors have attributed this function in P. falciparum to a glutamate dehydrogenase, therefore proposed as a potential drug target. Here we show that isophthalic acid inhibits both Plasmodium GDH and bovine GDH but showing marked discrimination (70-fold lower K(i) for the parasite GDH). Isophthalic acid impairs intra-erythrocytic growth of P. falciparumin vitro whilst o-phthalic acid, not a GDH inhibitor, shows no effect. This offers hope that with careful design or thorough screening it should be possible to find inhibitors with the necessary selectivity between parasite and human GDHs.
Molecular and Biochemical Parasitology 08/2010; 172(2):152-5. · 2.55 Impact Factor
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Malaria Journal. 01/2010;
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Angus Bell
Biochemical and Biophysical Research Communications 12/2008; 378(3):678-9; author reply 680-1. · 2.48 Impact Factor
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ABSTRACT: During its intraerythrocytic phase, the most lethal human malarial parasite, Plasmodium falciparum, digests host cell hemoglobin as a source of some of the amino acids required for its own protein synthesis. A number of parasite endopeptidases (including plasmepsins and falcipains) process the globin into small peptides. These peptides appear to be further digested to free amino acids by aminopeptidases, enzymes that catalyze the sequential cleavage of N-terminal amino acids from peptides. Aminopeptidases are classified into different evolutionary families according to their sequence motifs and preferred substrates. The aminopeptidase inhibitor bestatin can disrupt parasite development, suggesting that this group of enzymes might be a chemotherapeutic target. Two bestatin-susceptible aminopeptidase activities, associated with gene products belonging to the M1 and M17 families, have been described in blood-stage P. falciparum parasites, but it is not known whether one or both are required for parasite development. To establish whether inhibition of the M17 aminopeptidase is sufficient to confer antimalarial activity, we evaluated 35 aminoalkylphosphonate and phosphonopeptide compounds designed to be specific inhibitors of M17 aminopeptidases. The compounds had a range of activities against cultured P. falciparum parasites with 50% inhibitory concentrations down to 14 muM. Some of the compounds were also potent inhibitors of parasite aminopeptidase activity, though it appeared that many were capable of inhibiting the M1 as well as the M17 enzyme. There was a strong correlation between the potencies of the compounds against whole parasites and against the enzyme, suggesting that M17 and/or M1 aminopeptidases may be valid antimalarial drug targets.
Antimicrobial Agents and Chemotherapy 06/2008; 52(9):3221-8. · 4.84 Impact Factor
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ABSTRACT: Malaria is a disease in desperate need of new chemotherapeutic approaches. Certain microtubule inhibitors, including vinblastine and taxol, have highly potent activity against malarial parasites and disrupt the normal microtubular structures of intra-erythrocytic parasites at relevant concentrations. While these inhibitors are useful tools, their potential as anti-malarial drugs is limited by their high toxicity to mammalian cells. In contrast, two classes of antimitotic herbicide, namely dinitroanilines (e.g. trifluralin and oryzalin) and phosphorothioamidates (e.g. amiprophosmethyl), exhibit moderate activity against the major human malarial parasite Plasmodium falciparum in culture but very low mammalian cytotoxicity. We examined the dynamics and kinetics of uptake and subcellular compartmentation of [14C]trifluralin in comparison with [3H]vinblastine. We wished to determine whether the relatively modest activity of trifluralin was the consequence of poor uptake into parasite cells. Trifluralin accumulated in parasite-infected erythrocytes to approximately 300 times the external concentration and vinblastine at up to approximately 110 times. Accumulation into uninfected erythrocytes was much lower. Uptake of trifluralin was rapid, non-saturable and readily reversed. It appears that the hydrophobic nature of trifluralin leads to accumulation largely in the membranes of the parasite, reducing the levels in the soluble fraction and limiting access to its microtubular target. By contrast, vinblastine accumulated predominantly in the soluble fraction and uptake was saturable and mostly irreversible, consistent with binding predominantly to tubulin. The results indicate that synthesis of more polar trifluralin derivatives may be a promising approach to designing microtubule inhibitors with more potent antimalarial activity.
Biochemical pharmacology 05/2008; 75(8):1580-7. · 4.25 Impact Factor
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ABSTRACT: Artemisinin was reduced to dihydroartemisinin and coupled to a carboxylic acid derivative of quinine via an ester linkage. This novel hybrid molecule had potent activity against the 3D7 and (drug-resistant) FcB1 strains of Plasmodium falciparum in culture. The activity was superior to that of artemisinin alone, quinine alone, or a 1:1 mixture of artemisinin and quinine.
Bioorganic & Medicinal Chemistry Letters 08/2007; 17(13):3599-602. · 2.55 Impact Factor
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Colin M Stack,
Jonathan Lowther,
Eithne Cunningham,
Sheila Donnelly,
Donald L Gardiner,
Katharine R Trenholme,
Tina S Skinner-Adams,
Franka Teuscher,
Jolanta Grembecka,
Artur Mucha,
Pawel Kafarski,
Linda Lua, Angus Bell,
John P Dalton
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ABSTRACT: Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs.
Journal of Biological Chemistry 02/2007; 282(3):2069-80. · 4.77 Impact Factor
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Colin M. Stack,
Jonathan Lowther,
Eithne Cunningham,
Sheila Donnelly,
Donald L. Gardiner,
Katharine R. Trenholme,
Tina S. Skinner-Adams,
Franka Teuscher,
Jolanta Grembecka,
Artur Mucha,
Pawel Kafarski,
Linda Lua, Angus Bell,
John P. Dalton
[show abstract]
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ABSTRACT: Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for
protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino
acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial
drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral
or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic
amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites,
a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical
methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated
merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and
also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors.
Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future
design/discovery of new antimalarial drugs.
Journal of Biological Chemistry 01/2007; 282(3):2069-2080. · 4.77 Impact Factor
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ABSTRACT: Immunophilin is the collective name given to the cyclophilin and FK506-binding protein families. As the name suggests, these include the major binding proteins of certain immunosuppressive drugs: cyclophilins for the cyclic peptide cyclosporin A and FK506-binding proteins for the macrolactones FK506 and rapamycin. Both families, although dissimilar in sequence, possess peptidyl-prolyl cis-trans isomerase activity in vitro and can play roles in protein folding and transport, RNA splicing and the regulation of multi-protein complexes in cells. In addition to enzymic activity, many immunophilins act as molecular chaperones. This property may be conferred by the isomerase domain and/or by additional domains. Recent years have seen a great increase in the number of known immunophilin genes in parasitic protozoa and helminths and in many cases their products have been characterised biochemically and their temporal and spatial expression patterns have been examined. Some of these genes represent novel types: one example is a Toxoplasma gondii gene encoding a protein with both cyclophilin and FK506-binding protein domains. Likely roles in protein folding and oligomerisation, RNA splicing and sexual differentiation have been suggested for parasite immunophilins. In addition, unexpected roles in parasite virulence (Mip FK506-binding protein of Trypanosoma cruzi) and host immuno-modulation (e.g. 18-kDa cyclophilin of T. gondii) have been established. Furthermore, in view of the potent antiparasitic activities of cyclosporins, macrolactones and non-immunosuppressive derivatives of these compounds, immunophilins may mediate drug action and/or may themselves represent potential drug targets. Investigation of the mechanisms of action of these agents may lead to the design of potent and selective antimalarial and other antiparasitic drugs. This review discusses the properties of immunophilins in parasites and the 'animal model'Caenorhabditis elegans and relates these to our understanding of the roles of these proteins in cellular biochemistry, host-parasite interaction and the antiparasitic mechanisms of the drugs that bind to them.
International Journal for Parasitology 04/2006; 36(3):261-76. · 3.39 Impact Factor
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ABSTRACT: Microtubules play important roles in cell division, motility and structural integrity of malarial parasites. Some microtubule inhibitors disrupt parasite development at very low concentrations, but most of them also kill mammalian cells. However, the dinitroaniline family of herbicides, which bind specifically to plant tubulin, have inhibitory activity on plant cells but are relatively non-toxic to human cells. Certain dinitroanilines are also inhibitory to various protozoal parasites including Plasmodium. Here we demonstrate that the dinitroanilines trifluralin and oryzalin inhibited progression of erythrocytic Plasmodium falciparum through schizogony, blocked mitotic division, and caused accumulation of abnormal microtubular structures. Moreover, radiolabelled trifluralin interacted with purified, recombinant parasite tubulins but to a much lesser extent with bovine tubulins. The phosphorothioamidate herbicide amiprophos-methyl, which has the same herbicidal mechanism as dinitroanilines, also had antimalarial activity and a similar action on schizogony. These data suggest that P. falciparum tubulin contains a dinitroaniline/phosphorothioamidate-binding site that is not conserved in humans and might be a target for new antimalarial drugs.
Molecular and Biochemical Parasitology 03/2006; 145(2):226-38. · 2.55 Impact Factor
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Angus Bell
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ABSTRACT: Interactions between antimicrobial agents provide clues as to their mechanisms of action and influence the combinations chosen for therapy of infectious diseases. In the treatment of malaria, combinations of drugs, in many cases acting synergistically, are increasingly important in view of the frequency of resistance to single agents. The study of antimalarial drug interactions is therefore of great significance to both treatment and research. It is therefore worrying that the analysis of drug-interaction data is often inadequate, leading in some cases to dubious conclusions about synergism or antagonism. Furthermore, making mechanistic deductions from drug-interaction data is not straightforward and of the many reported instances of antimalarial synergism or antagonism, few have been fully explained biochemically. This review discusses recent findings on antimalarial drug interactions and some pitfalls in their analysis and interpretation. The conclusions are likely to have relevance to other antimicrobial agents.
FEMS Microbiology Letters 01/2006; 253(2):171-84. · 2.04 Impact Factor
