Frequency noise stabilisation of a 1550 nm external cavity diode laser with hybrid feedback for next generation gravitational wave interferometry.

Longer wavelength lasers will be needed for future gravitational wave detectors that use cryogenic cooling of silicon based test-mass optics. Diode lasers with a 1550 nm wavelength output are potential seed light sources for such a detector, however diode laser devices have a different spectral profile and higher frequency noise than the solid state lasers used in current detectors. We present a frequency stabilisation system for a 1550 nm external cavity diode laser capable of reducing the laser frequency noise to a level of 0.1HzHz up to 1 kHz with a unity gain frequency of 535 kHz using a hybrid analogue-digital servo with in-loop cancellation of resonant features. In addition, a method of high speed digital filter optimisation and automated design is demonstrated.

Mirror Coating Solution for the Cryogenic Einstein Telescope.

Planned cryogenic gravitational-wave detectors will require improved coatings with a strain thermal noise reduced by a factor of 25 compared to Advanced LIGO. We present investigations of HfO_{2} doped with SiO_{2} as a new coating material for future detectors. Our measurements show an extinction coefficient of k=6×10^{-6} and a mechanical loss of ϕ=3.8×10^{-4} at 10 K, which is a factor of 2 below that of SiO_{2}, the currently used low refractive-index coating material. These properties make HfO_{2} doped with SiO_{2} ideally suited as a low-index partner material for use with a-Si in the lower part of a multimaterial coating. Based on these results, we present a multimaterial coating design which, for the first time, can simultaneously meet the strict requirements on optical absorption and thermal noise of the cryogenic Einstein Telescope.

Inhibition of ex vivo erythropoiesis by secreted and haemozoin-associated Plasmodium falciparum products.

It has been estimated that up to a third of global malaria deaths may be attributable to malarial anaemia, with children and pregnant women being those most severely affected. An inefficient erythropoietic response to the destruction of both infected and uninfected erythrocytes in infections with Plasmodium spp. contributes significantly to the development and persistence of such anaemia. The underlying mechanisms, which could involve both direct inhibition of erythropoiesis by parasite-derived factors and indirect inhibition as a result of modulation of the immune response, remain poorly understood. We found parasite-derived factors in conditioned medium (CM) of blood-stage Plasmodium falciparum and crude isolates of parasite haemozoin directly to inhibit erythropoiesis in an ex vivo model based on peripheral blood haematopoietic stem cells. Erythropoiesis-inhibiting activity was detected in a fraction of CM that was sensitive to heat inactivation and protease digestion. Erythropoiesis was also inhibited by crude parasite haemozoin but not by detergent-treated, heat-inactivated or protease-digested haemozoin. These results suggest that the erythropoiesis-inhibiting activity in both cases is mediated by proteins or protein-containing biomolecules and may offer new leads to the treatment of malarial anaemia.

The antimalarial action of FK506 and rapamycin: evidence for a direct effect on FK506-binding protein PfFKBP35.

FK506 and rapamycin (Rap) are immunosuppressive drugs that act principally on T-lymphocytes. The receptors for both drugs are FK506-binding proteins (FKBPs), but the molecular mechanisms of immunosuppression differ. An FK506-FKBP complex inhibits the protein phosphatase calcineurin, blocking a key step in T-cell activation, while the Rap -FKBP complex binds to the protein kinase target of rapamycin (TOR), which is involved in a subsequent signalling pathway. Both drugs, and certain non-immunosuppressive compounds related to FK506, have potent antimalarial activity. There is however conflicting evidence on the involvement of Plasmodium calcineurin in the action of FK506, and the parasite lacks an apparent TOR homologue. We therefore set out to establish whether inhibition of the Plasmodium falciparum FKBP PfFKBP35 itself might be responsible for the antimalarial effects of FK506 and Rap. Similarities in the antiparasitic actions of FK506 and Rap would constitute indirect evidence for this hypothesis. FK506 and Rap acted indistinguishably on: (i) specificity for different intra-erythrocytic stages in culture, (ii) kinetics of killing or irreversible growth arrest of parasites and (iii) interactions with other antimalarial agents. Furthermore, PfFKBP35's inhibitory effect on calcineurin was independent of FK506 under a range of conditions, suggesting that calcineurin is unlikely to be involved in the antimalarial action of FK506.

Two crystal structures of the FK506-binding domain of Plasmodium falciparum FKBP35 in complex with rapamycin at high resolution.

Antimalarial chemotherapy continues to be challenging in view of the emergence of drug resistance, especially artemisinin resistance in Southeast Asia. It is critical that novel antimalarial drugs are identified that inhibit new targets with unexplored mechanisms of action. It has been demonstrated that the immunosuppressive drug rapamycin, which is currently in clinical use to prevent organ-transplant rejection, has antimalarial effects. The Plasmodium falciparum target protein is PfFKBP35, a unique immunophilin FK506-binding protein (FKBP). This protein family binds rapamycin, FK506 and other immunosuppressive and non-immunosuppressive macrolactones. Here, two crystallographic structures of rapamycin in complex with the FK506-binding domain of PfFKBP35 at high resolution, in both its oxidized and reduced forms, are reported. In comparison with the human FKBP12-rapamycin complex reported previously, the structures reveal differences in the ß4-ß6 segment that lines the rapamycin binding site. Structural differences between the Plasmodium protein and human hFKBP12 include the replacement of Cys106 and Ser109 by His87 and Ile90, respectively. The proximity of Cys106 to the bound rapamycin molecule (4-5 Å) suggests possible routes for the rational design of analogues of rapamycin with specific antiparasitic activity. Comparison of the structures with the PfFKBD-FK506 complex shows that both drugs interact with the same binding-site residues. These two new structures highlight the structural differences and the specific interactions that must be kept in consideration for the rational design of rapamycin analogues with antimalarial activity that specifically bind to PfFKBP35 without immunosuppressive effects.

Immunophilin-protein interactions in Plasmodium falciparum.

Immunophilins comprise two protein families, cyclophilins (CYPs) and FK506-binding proteins (FKBPs), and are the major receptors for the immunosuppressive drugs cyclosporin A (CsA) and FK506 (tacrolimus), respectively. Most eukaryotic species have at least one immunophilin and some of them have been associated with pathogenesis of infectious or parasitic diseases or the action of antiparasitic drugs. The human malarial parasite Plasmodium falciparum has 13 immunophilin or immunophilin-like genes but the functions of their products are unknown. We set out to identify the parasite proteins that interact with the major CYPs, PfCYP19A and PfCYP19B, and the FKBP, PfFKBP35, using a combination of co-immunoprecipitation and yeast two-hybrid screening. We identified a cohort of putative interacting partners and further investigation of some of these revealed potentially novel roles in parasite biology. We demonstrated that (i) P. falciparum CYPs interacted with the heat shock protein 70, (ii) treatment of parasites with CYP ligands disrupted transport of the rhoptry-associated protein 1, and (iii) PfFKBP35 interacted with parasite histones in a way that might modulate gene expression. These findings begin to elucidate the functions of immunophilins in malaria. Furthermore, the known antimalarial effects of CsA, FK506 and non-immunosuppressive derivatives of these immunophilin ligands could be mediated through these partner proteins.

Synthesis and evaluation of phenoxyoxazaphospholidine, phenoxyoxazaphosphinane, and benzodioxaphosphininamine sulfides and related compounds as potential anti-malarial agents.

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 0.038 µM) and warrants further study.

Overexpression, purification and assessment of cyclosporin binding of a family of cyclophilins and cyclophilin-like proteins of the human malarial parasite Plasmodium falciparum.

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.

Synthesis and evaluation of phosphoramidate and phosphorothioamidate analogues of amiprophos methyl as potential antimalarial agents.

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.

Identification of plasmepsin inhibitors as selective anti-malarial agents using ligand based drug design.

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.

Chemical target validation studies of aminopeptidase in malaria parasites using alpha-aminoalkylphosphonate and phosphonopeptide inhibitors.

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.

Cellular and molecular actions of dinitroaniline and phosphorothioamidate herbicides on Plasmodium falciparum: tubulin as a specific antimalarial target.

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.

Peptidyl-prolyl cis-trans isomerases (immunophilins) and their roles in parasite biochemistry, host-parasite interaction and antiparasitic drug action.

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.

A Plasmodium falciparum FK506-binding protein (FKBP) with peptidyl-prolyl cis-trans isomerase and chaperone activities.

The immunosuppressive drugs FK506 and rapamycin have anti-malarial properties but their mechanisms of action against malaria parasites remain unknown. The pathway by which these drugs cause immunosuppression in humans is known to involve an FK506-binding protein (FKBP). Homologues of FKBPs have been identified in almost every organism in which they have been sought. Here, we describe the characterisation of the first member of the FKBP family identified in the human malarial parasite, Plasmodium falciparum. This 35-kDa protein, PfFKBP35, comprises a single, N-terminal, FKBP domain and a C-terminal tripartite tetratricopeptide repeat domain. A recombinant form of PfFKBP35, like most other FKBPs, displayed peptidyl-prolyl cis-trans isomerase activity that was inhibitable by FK506 and rapamycin. Unusually the phosphatase activity of calcineurin, the target of the FK506-FKBP complex in T-lymphocytes, was inhibited by PfFKBP35 independently of FK506 binding. PfFKBP35 also inhibited the thermal aggregation in vitro of two model substrates, suggesting that it has general chaperone properties. Analysis of the P. falciparum genome database suggested this to be the only FKBP present in the parasite. The function of this protein remains unknown but the presence of tetratricopeptide repeat motifs suggests a role in intracellular protein transport or modulation of protein function.

Antimalarial drug synergism and antagonism: mechanistic and clinical significance.

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.

Design and evaluation of antimalarial peptides derived from prediction of short linear motifs in proteins related to erythrocyte invasion.

The purpose of this study was to investigate the blood stage of the malaria causing parasite, Plasmodium falciparum, to predict potential protein interactions between the parasite merozoite and the host erythrocyte and design peptides that could interrupt these predicted interactions. We screened the P. falciparum and human proteomes for computationally predicted short linear motifs (SLiMs) in cytoplasmic portions of transmembrane proteins that could play roles in the invasion of the erythrocyte by the merozoite, an essential step in malarial pathogenesis. We tested thirteen peptides predicted to contain SLiMs, twelve of them palmitoylated to enhance membrane targeting, and found three that blocked parasite growth in culture by inhibiting the initiation of new infections in erythrocytes. Scrambled peptides for two of the most promising peptides suggested that their activity may be reflective of amino acid properties, in particular, positive charge. However, one peptide showed effects which were stronger than those of scrambled peptides. This was derived from human red blood cell glycophorin-B. We concluded that proteome-wide computational screening of the intracellular regions of both host and pathogen adhesion proteins provides potential lead peptides for the development of anti-malarial compounds.

Cyclosporin-binding proteins of Plasmodium falciparum.

A number of cyclosporins, including certain non-immunosuppressive ones, are potent inhibitors of the intraerythrocytic growth of the human malarial parasite Plasmodium falciparum. The major cyclosporin-binding proteins of P. falciparum were investigated by affinity chromatography on cyclosporin-Affigel followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting, and peptide mass fingerprinting. The two bands obtained on gels were shown to correspond to cyclophilins, PfCyP-19A (formerly PfCyP-19) and PfCyP-19B, whose genes had been characterised previously. PfCyP-19B was an abundant protein of intraerythrocytic P. falciparum (up to 0.5% of parasite protein) that was present in the highest amounts in schizont-stage parasites. Unexpectedly, given its apparent signal sequence, it was located primarily in the cytosol of the parasite. The peptidyl-prolyl cis-trans isomerase activity of recombinant PfCyP-19B had the same profile of susceptibility to cyclosporin derivatives as the bulk isomerase activity of crude P. falciparum extracts. The binding of cyclosporins to cyclophilins may be relevant to the mechanism of action of the drug in the parasite.

PEST sequences in the malaria parasite Plasmodium falciparum: a genomic study.

BACKGROUND: Inhibitors of the protease calpain are known to have selectively toxic effects on Plasmodium falciparum. The enzyme has a natural inhibitor calpastatin and in eukaryotes is responsible for turnover of proteins containing short sequences enriched in certain amino acids (PEST sequences). The genome of P. falciparum was searched for this protease, its natural inhibitor and putative substrates. METHODS: The publicly available P. falciparum genome was found to have too many errors to permit reliable analysis. An earlier annotation of chromosome 2 was instead examined. PEST scores were determined for all annotated proteins. The published genome was searched for calpain and calpastatin homologs. RESULTS: Typical PEST sequences were found in 13% of the proteins on chromosome 2, including a surprising number of cell-surface proteins. The annotated calpain gene has a non-biological "intron" that appears to have been created to avoid an unrecognized frameshift. Only the catalytic domain has significant similarity with the vertebrate calpains. No calpastatin homologs were found in the published annotation. CONCLUSION: A calpain gene is present in the genome and many putative substrates of this enzyme have been found. Calpastatin homologs may be found once the re-annotation is completed. Given the selective toxicity of calpain inhibitors, this enzyme may be worth exploring further as a potential drug target.

Simply red: A novel spectrophotometric erythroid proliferation assay as a tool for erythropoiesis and erythrotoxicity studies.

Most mammalian cell proliferation assays rely on manual or automated cell counting or the assessment of metabolic activity in colorimetric assays, with the former being either labor and time intensive or expensive and the latter being multistep procedures requiring the addition of several reagents. The proliferation of erythroid cells from hematopoietic stem cells and their differentiation into mature red blood cells is characterized by the accumulation of large amounts of hemoglobin. Hemoglobin concentrations are easily quantifiable using spectrophotometric methods due to the specific absorbance peak of the molecule's heme moiety between 400 and 420 nm. Erythroid proliferation can therefore be readily assessed using spectrophotometric measurement in this range. We have used this feature of erythroid cells to develop a simple erythroid proliferation assay that is minimally labor/time- and reagent-intensive and could easily be automated for use in high-throughput screening. Such an assay can be a valuable tool for investigations into hematological disorders where erythropoiesis is dysregulated, i.e., either inhibited or enhanced, into the development of anemia as a side-effect of primary diseases such as parasitic infections and into cyto-(particularly erythro-) toxicity of chemical agents or drugs.