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Biomedical subjects

Michael P Barrett

Publications and source records attributed to Michael P Barrett.

At least 19 recordsLinked to original sources
PubMed2007

Crystal structures of a bacterial 6-phosphogluconate dehydrogenase reveal aspects of specificity, mechanism and mode of inhibition by analogues of high-energy reaction intermediates.

Crystal structures of recombinant Lactococcus lactis 6-phosphogluconate dehydrogenase (LlPDH) in complex with substrate, cofactor, product and inhibitors have been determined. LlPDH shares significant sequence identity with the enzymes from sheep liver and the protozoan parasite Trypanosoma brucei for which structures have been reported. Comparisons indicate that the key residues in the active site are highly conserved, as are the interactions with the cofactor and the product ribulose 5-phosphate. However, there are differences in the conformation of the substrate 6-phosphogluconate which may reflect distinct states relevant to catalysis. Analysis of the complex formed with the potent inhibitor 4-phospho-d-erythronohydroxamic acid, suggests that this molecule does indeed mimic the high-energy intermediate state that it was designed to. The analysis also identified, as a contaminant by-product of the inhibitor synthesis, 4-phospho-d-erythronamide, which binds in similar fashion. LlPDH can now serve as a model system for structure-based inhibitor design targeting the enzyme from Trypanosoma species.

Amino Acid Sequence↗
PubMed2006

Precision mapping of the metabolome.

The global study of the structure and dynamics of metabolic networks has been hindered by a lack of techniques that identify metabolites and their biochemical relationship in complex mixtures. The recent application of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to metabolomic analysis suggests a way to tackle the problem. A lower-cost alternative to high-field FTICR-MS, the Orbitrap mass analyzer, promises accelerated activity in this area. Here, we show how the ultra-high mass accuracy and resolution provided by this new generation of mass spectrometers can help to identify metabolites and connect them into metabolic networks. Data from perturbation studies and isotope-tracking experiments can complement this information to create metabolic maps de novo and chart unexplored areas of metabolism.

Algorithms↗
PubMed2006

Roles for the Trypanosoma brucei P2 transporter in DB75 uptake and resistance.

A novel trypanocide, 2,5-bis(4-amidinophenyl)furan (DB75), in its prodrug amidoxime-derivative form, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), is in trials as the first orally administered drug for human African trypanosomiasis. DB75 is a diamidine. Resistance to some diamidines correlates to loss of uptake via the P2 aminopurine transporter. We show here that uptake of DB75 into Trypanosoma brucei also occurs principally via the P2 transporter. Uptake of tritiated DB75 occurred via a high-affinity (K(m app), 3.2 microM) carriermediated route that was inhibited by adenosine, adenine, and pentamidine, all known substrates of the P2 transporter. Trypanosomes lacking the TbAT1 gene that encodes the P2 transporter demonstrated an 11-fold reduction in sensitivity to DB75 when measured under controlled in vitro conditions. These knockout cells were also less sensitive to DB75 than wild-type cells in mice. Initial uptake rates of DB75 into the Deltatbat1 knockout cell line were greatly reduced compared with rates in wild-type cells. A trypanosome cell line selected in vitro for DB75 resistance was shown to have lost P2-mediated DB75 uptake. The TbAT1 gene was mapped to chromosome V of the T. brucei genome and the DB75-resistant parasites were shown to have deleted both alleles of this gene. Fluorescence microscopy of DB75-treated trypanosomes revealed that DB75 fluorescence localizes rapidly within the DNA-containing organelles of wild-type trypanosomes, whereas no fluorescence was observed in Deltatbat1-null parasites or in the parasites selected for resistance to DB75.

Adenosine↗
PubMed2006

DNA binding affinity of bisguanidine and bis(2-aminoimidazoline) derivatives with in vivo antitrypanosomal activity.

A new antitrypanosomal hit compound that cures an acute (STIB 900) mouse model of Trypanosoma brucei rhodesiense trypanosomiasis is described. This bis(2-aminoimidazolinium) dicationic compound proved to be an excellent DNA minor groove binder, suggesting a possible mechanism for its trypanocidal activity. From these studies, the 4,4'-diaminodiphenylamine skeleton emerged as a good scaffold for antitrypanosomal drugs.

Animals↗
PubMed2006

Targeting of toxic compounds to the trypanosome's interior.

Drugs can be targeted into African trypanosomes by exploiting carrier proteins at the surface of these parasites. This has been clearly demonstrated in the case of the melamine-based arsenical and the diamidine classes of drug that are already in use in the treatment of human African trypanosomiasis. These drugs can enter via an aminopurine transporter, termed P2, encoded by the TbAT1 gene. Other toxic compounds have also been designed to enter via this transporter. Some of these compounds enter almost exclusively through the P2 transporter, and hence loss of the P2 transporter leads to significant resistance to these particular compounds. It now appears, however, that some diamidines and melaminophenylarsenicals may also be taken up by other routes (of yet unknown function). These too may be exploited to target new drugs into trypanosomes. Additional purine nucleoside and nucleobase transporters have also been subverted to deliver toxic agents to trypanosomes. Glucose and amino acid transporters too have been investigated with a view to manipulating them to carry toxins into Trypanosoma brucei, and recent work has demonstrated that aquaglyceroporins may also have considerable potential for drug-targeting. Transporters, including those that carry lipids and vitamins such as folate and other pterins also deserve more attention in this regard. Some drugs, for example suramin, appear to enter via routes other than plasma-membrane-mediated transport. Receptor-mediated endocytosis has been proposed as a possible way in for suramin. Endocytosis also appears to be crucial in targeting natural trypanocides, such as trypanosome lytic factor (TLF) (apolipoprotein L1), into trypanosomes and this offers an alternative means of selectively targeting toxins to the trypanosome's interior. Other compounds may be induced to enter by increasing their capacity to diffuse over cell membranes; in this case depending exclusively on selective activity within the cell rather than selective uptake to impart selective toxicity. This review outlines studies that have aimed to exploit trypanosome nutrient uptake routes to selectively carry toxins into these parasites.

Animals↗
PubMed2005

Chemotherapy of trypanosomiases and leishmaniasis.

New formulations, therapeutic switching of the established drugs amphotericin B and paromomycin, and the serendipitous discovery of miltefosine have markedly improved leishmaniasis chemotherapy in the past 21 years. The situation for the two trypanosomiases has been less encouraging. Apart from the introduction of eflornithine for the treatment of late-stage human African trypanosomiasis, with its serious limitations in terms of cost and difficulty of administration, no new drugs have been incorporated into the chemotherapeutic arsenal in the past 25 years, despite important advances in knowledge of the biology of the etiological agents and the pathophysiology of these diseases. In the case of Chagas disease, several classes of compound that target the validated biochemical pathways of the parasite (e.g. inhibitors of sterol biosynthesis and cysteine proteases) are in the pipeline. With the availability of complete genome sequences for all three pathogens, and methods for rapid validation of targets, it is hoped that much-needed amelioration will occur soon. Financial constraints continue to represent a major hurdle to drug development. However, the appearance of not-for-profit product-development partnerships offers a new paradigm for bringing new drugs to patients.

Animals↗
PubMed2005

Design and synthesis of a series of melamine-based nitroheterocycles with activity against Trypanosomatid parasites.

The parasites that give rise to human African trypanosomiasis (HAT) are auxotrophs for various nutrients from the human host, including purines. They have specialist nucleoside transporters to import these metabolites. In addition to uptake of purine nucleobases and purine nucleosides, one of these transporters, the P2 transporter, can carry melamine derivatives; these derivatives are not substrates for the corresponding mammalian transporters. In this paper, we report the coupling of the melamine moiety to selected nitro heterocycles with the aim of selectively delivering these compounds to the parasites. Some compounds prepared have similar in vitro trypanocidal activities as melarsoprol, the principal drug used against late-stage HAT, with 50% growth inhibitory concentrations in the submicromolar range. Selected compounds were also evaluated in vivo in rodent models infected with Trypanosoma brucei brucei and T. brucei rhodesiense and showed pronounced activity and in two cases were curative without overt signs of toxicity. Compounds were also tested against other trypanosomatid pathogens, Leishmania donovani and Trypanosoma cruzi, and significant activity in vitro was noted for T. cruzi against which various nitro heterocycles are already registered for use.

Animals↗
PubMed2005

Proline metabolism in procyclic Trypanosoma brucei is down-regulated in the presence of glucose.

Proline metabolism has been studied in procyclic form Trypanosoma brucei. These parasites consume six times more proline from the medium when glucose is in limiting supply than when this carbohydrate is present as an abundant energy source. The sensitivity of procyclic T. brucei to oligomycin increases by three orders of magnitude when the parasites are obliged to catabolize proline in medium depleted in glucose. This indicates that oxidative phosphorylation is far more important to energy metabolism in this latter case than when glucose is available and the energy needs of the parasite can be fulfilled by substrate level phosphorylation alone. A gene encoding proline dehydrogenase, the first enzyme of the proline catabolic pathway, was cloned. RNA interference studies revealed the loss of this activity to be conditionally lethal. Proline dehydrogenase defective parasites grew as wild-type when glucose was available, but, unlike wild-type cells, they failed to proliferate using proline. In parasites grown in the presence of glucose, proline dehydrogenase activity was markedly lower than when glucose was absent from the medium. Proline uptake too was shown to be diminished when glucose was abundant in the growth medium. Wild-type cells were sensitive to 2-deoxy-D-glucose if grown using proline as the principal carbon source, but not in glucose-rich medium, indicating that this non-catabolizable glucose analogue might also stimulate repression of proline utilization. These results indicate that the ability of trypanosomes to use proline as an energy source can be regulated depending upon the availability of glucose.

Adenosine Triphosphate↗
PubMed2005

The plastidic DNA replication enzyme complex of Plasmodium falciparum.

The replication and repair of organellar genomes in the malaria parasite Plasmodium falciparum is poorly understood. We have assessed the properties of an open reading frame Pfprex (formerly known as pom1) and confirm that it specifies a multi-domain polypeptide with DNA primase, DNA helicase, DNA polymerase and 3'-5' exonuclease activities. The sequence of the primase/helicase domain is phylogenetically related to the T7-bacteriophage gene 4 product and mammalian mitochondrial helicase, Twinkle. Despite that, the N-terminal sequence of this multi-domain polypeptide directs a green fluorescent protein reporter specifically to the P. falciparum apicoplast and not to the mitochondrion. Phylogenetic analysis placed the DNA polymerase sequence with the family A bacterial polymerases, most closely to those of the thermophilic Aquifex species. Notably, the malarial enzyme was optimally active at 75 degrees C. Pfprex is the first example of a gene encoding contiguous DNA polymerase, DNA primase and DNA helicase components. We propose it has a key role in replication of the malarial plastid genome, a validated drug target.

Animals↗
PubMed2005

Energy generation in insect stages of Trypanosoma brucei: metabolism in flux.

The generation of energy in African trypanosomes is a subject of undoubted importance. In bloodstream-form organisms, substrate-level phosphorylation of glucose is sufficient to provide the energy needs of the parasite. The situation in procyclic-form trypanosomes is more complex. For many years, it was accepted that glucose metabolism followed a conventional scheme involving glycolysis, the tricarboxylic acid cycle and ATP-producing oxidative phosphorylation linked to the electron-transport chain. However, progress in sequencing the Trypanosoma brucei genome and the development of gene-knockout and RNA interference technology has provided novel insight. Coupling these new technologies with classical approaches, including NMR and mass spectrometry to analyse glycolytic intermediates and end products, has yielded several surprises. In this article, we summarize how these recent data have helped to change the view of metabolism in procyclic-form T. brucei.

Animals↗
PubMed2005

Interaction of monobenzamidine-linked trypanocides with the Trypanosoma brucei P2 aminopurine transporter.

Single benzamidine group-carrying compounds were shown to interact with the Trypanosoma brucei P2 aminopurine transporter. Replacement of the amidine with a guanidine group decreased affinity. Trypanocidal activity was evident, but compounds were equally toxic against trypanosomes lacking the P2 transporter, which indicates additional uptake routes for monobenzamidine-derived compounds.

Animals↗
PubMed2004

Plasmodium interspersed repeats: the major multigene superfamily of malaria parasites.

Functionally related homologues of known genes can be difficult to identify in divergent species. In this paper, we show how multi-character analysis can be used to elucidate the relationships among divergent members of gene superfamilies. We used probabilistic modelling in conjunction with protein structural predictions and gene-structure analyses on a whole-genome scale to find gene homologies that are missed by conventional similarity-search strategies and identified a variant gene superfamily in six species of malaria (Plasmodium interspersed repeats, pir). The superfamily includes rif in P.falciparum, vir in P.vivax, a novel family kir in P.knowlesi and the cir/bir/yir family in three rodent malarias. Our data indicate that this is the major multi-gene family in malaria parasites. Protein localization of products from pir members to the infected erythrocyte membrane in the rodent malaria parasite P.chabaudi, demonstrates phenotypic similarity to the products of pir in other malaria species. The results give critical insight into the evolutionary adaptation of malaria parasites to their host and provide important data for comparative immunology between malaria parasites obtained from laboratory models and their human counterparts.

Amino Acid Motifs↗
PubMed2004

Transketolase from Leishmania mexicana has a dual subcellular localization.

Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent K(m) of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 A (1 A identical with 0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.

Amino Acid Sequence↗
PubMed2004

Selective inhibition of Trypanosoma brucei 6-phosphogluconate dehydrogenase by high-energy intermediate and transition-state analogues.

Two series of compounds were designed to mimic the transition state and high-energy intermediates (HEI) of the enzymatic reaction of 6-phosphogluconate dehydrogenase (6PGDH). Sulfoxide analogues (7-11) were designed to mimic the transition state during the oxidation of the substrate to 3-keto-6-phosphogluconate, an enzyme-bound intermediate of the enzyme. Hydroxamate and amide derivatives of d-erythronic acid were designed to mimic the 1,2-cis-enediol HEI of the 6PGDH reaction. These two series of compounds were assayed as competitive inhibitors of the Trypanosoma brucei and sheep liver enzymes, and their selectivity value (ratio sheep/parasite) was calculated. The sulfoxide transition-state analogues showed weak and selective inhibition of the T. brucei enzyme. The hydroxamic derivatives showed potent and selective inhibition of the T. brucei 6PGDH with a Ki in the nanomolar range.

Amides↗
PubMed2004

Interaction of substituted hexose analogues with the Trypanosoma brucei hexose transporter.

Glucose metabolism is essential for survival of bloodstream form Trypanosoma brucei subspecies which cause human African trypanosomiasis (sleeping sickness). Hexose analogues may represent good compounds to inhibit glucose metabolism in these cells. Delivery of such compounds to the parasite is a major consideration in drug development. A series of D-glucose and D-fructose analogues were developed to explore the limits of the structure-activity relationship of the THT1 hexose transporter of bloodstream form African trypanosomes, a portal that might be exploited for drug uptake. D-glucose analogues with substituents at the C2 and C6 position continued to interact with the exofacial hexose binding site of the transporter. There was a limit to the size at C6 which still permitted recognition, although compounds carrying large groups at position C2 were still recognised. However, radiolabelled N-acetyl-D-[1-14C] glucosamine was not internalised by trypanosomes, in spite of the ability of this compound to inhibit glucose uptake, indicating that there is a limit to the size of C2 substituent that allows translocation. Addition of an alkylating group (bromoacetyl) at position C2 in the D-glucose series and at position 6 in the D-fructose set, created two analogues which interact with the transporter and kill trypanosomes in vitro. This indicates that inhibition of the transporter may be a good means of killing trypanosomes.

Acetylglucosamine↗
PubMed2004

The trypanocide diminazene aceturate is accumulated predominantly through the TbAT1 purine transporter: additional insights on diamidine resistance in african trypanosomes.

Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals. It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite. We describe here the mechanisms by which [(3)H]diminazene is transported by Trypanosoma brucei brucei bloodstream forms. Diminazene was rapidly accumulated through a single transporter, with a K(m) of 0.45 +/- 0.11 micro M, which was dose dependently inhibited by pentamidine and adenosine. The K(i) values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter. Yeast expressing TbAT1 acquired the ability to take up [(3)H]diminazene and [(3)H]pentamidine. TbAT1-null mutants had lost almost all capacity for [(3)H]diminazene transport. However, this cell line still displayed a small but detectable rate of [(3)H]diminazene accumulation, in a nonsaturable manner. We conclude that TbAT1 mediates [(3)H]diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1-null mutants and field isolates.

Adenosine↗
PubMed2004

Trypanocidal activity of melamine-based nitroheterocycles.

A series of nitroheterocyclic compounds were designed with linkages to melamine or benzamidine groups that are known substrates of the P2 aminopurine and other transporters in African trypanosomes of the brucei group. Several compounds showed in vitro trypanotoxicity with 50% inhibitory concentrations in the submicromolar range. Although most compounds interacted with the P2 transporter, as judged by their ability to inhibit adenosine transport via this carrier, uptake through this route was not necessary for activity since TbAT1-null mutant parasites, deficient in this transporter, retained sensitivity to these drugs. One compound, a melamine-linked nitrofuran, also showed pronounced activity against parasites in mice. Studies into the mode of action of this compound indicated that neither reductive, nor oxidative, stress were related to its trypanocidal activity ruling out a genotoxic effect in T. brucei, distinguishing it from some other, mammalian cell toxic, trypanocidal nitroheterocycles.

Acetylglucosamine↗