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.

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.

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.

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.

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.

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.

Antimitotic herbicides bind to an unidentified site on malarial parasite tubulin and block development of liver-stage Plasmodium parasites.

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.

Anti-disease therapy for malaria - 'resistance proof'?

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.

A family of cyclophilin-like molecular chaperones in Plasmodium falciparum.

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.

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.

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.

Downstream effects of haemoglobinase inhibition in Plasmodium falciparum-infected erythrocytes.

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.

Susceptibility of Plasmodium falciparum to glutamate dehydrogenase inhibitors--a possible new antimalarial target.

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.

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.