Identification of novel membrane structures in Plasmodium falciparum infected erythrocytes

Phosphorylation sites were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting synthetic fragments encoding two and six phosphorylation sites into an expression vector, pdHL7. The phosphorylation sites were created by using the predicted consensus sequences for phosphorylation by the cAMP-dependent protein kinase to the carboxyl terminus of the heavy chain constant region of the MAb-chCC49. The resultant modified antibodies (MAb-chCC49K1 and MAb-chCC49-6P) were expressed in NS0 cells and purified. The MAb-chCC49K1 protein contains two phosphorylation sites per heavy chain whereas the MAb-chCC49-6P protein contains six sites per heavy chain. Both MAb-chCC49K1 and MAb-chCC49-6P proteins can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity. The 32P-labeled MAb-chCC49K1 and MAb-chCC49-6P proteins bind to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into a monoclonal antibody provides a reagent for the diagnosis and treatment of cancer. The use of multiple phosphorylation sites provides antibodies with very high specific radioactivity and demonstrates that cassettes of phosphorylation sites can be introduced into proteins without altering their functional activity.     

Inhibition of invasion and intraerythrocytic development of Plasmodium falciparum by kinase inhibitors

We have examined the effects of seven protein kinase inhibitors (staurosporine, genistein, methyl 2,5-dihydroxycinnamate, tyrphostins B44 and B46, lavendustin A and R03) on the erythrocytic cycle of the malaria parasite, Plasmodium falciparum. One (staurosporine) strongly inhibits serine/threonine kinases, but the remainder all exhibit a strong preference for tyrosine kinases. We have been able to discriminate between effects on invasion and on intraerythrocytic development. All reagents impeded development of intraerythrocytic parasites, though at widely differing concentrations, from the sub-micromolar to the millimolar. Several inhibitors, including staurosporine, also reduced invasion. The phosphatase inhibitor, okadaic acid, had a strong inhibitory effect both on invasion and development. The regulation of malaria development by phosphorylation or dephosphorylation reactions at several points in the blood-stage cycle is implied.     

Highly potent irreversible inhibitors of neutrophil elastase generated by selection from a randomized DNA-valine phosphonate library

We incorporated a phosphonate irreversible inhibitor of neutrophil elastase into a randomized DNA library and, using the SELEX process, iteratively selected these assemblies for the most potent elastase inhibitors. The inhibitors were selected against purified elastase and against secreted elastase in the presence of activated neutrophils. Very active aptamer inhibitors were obtained by both methods, with second-order rate constants for inactivation of human neutrophil elastase ranging (1-3) x 10(8) M(-1) min(-1). These rates exceed those of any reported irreversible inhibitor of elastase and exceed the previous best phosphonate inhibitors by 80-fold. The selected inhibitors are also significantly more potent than alpha-1 proteinase inhibitor in blocking degradation of elastin by activated neutrophils. In contrast to a previous experiment [Smith et al. (1995) Chem. Biol. 2, 741-750], a single-enantiomer form of the valyl phosphonate was used rather than a racemic mixture. Our analysis shows that this use of a chirally resolved valyl phosphonate results in selection of much more potent inhibitors and that these inhibitors specifically potentiate a single enantiomeric form of the phosphonate.     

Identification of a potent analogue of Nazumamide A through iteration of combinatorial tetrapeptide libraries

Five sets of N-acylated tetrapeptide libraries and sublibraries related to Nazumamide A have been prepared using 25 natural and unnatural amino acids. They were evaluated in antithrombin assay, in order to quantify inhibition at each step of the tetrapeptide sublibrary iteration. The studies led to the identification of 2,5-dihydroxybenzoyl-lysyl-isoleucyl-phenylalanyl-arginine as a novel inhibitor of thrombin and was found to be at least 25 times more potent than the natural tetrapeptide 2,5-dihydroxybenzoyl-arginyl-prolyl-isoleucyl-alpha-aminobutyric acid (NAZA).     

Lead discovery of inhibitors of the dihydrofolate reductase domain of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase

A three-dimensional structure model of the dihydrofolate reductase (DHFR) domain of the bifunctional DHFR-thymidylate synthase of Plasmodium falciparum was used as a basis for computational screening of commercially available compounds for candidate inhibitors. Compounds which can stably dock to the model with strong ionic hydrogen bonds via protonation by an aspartic acid residue at the bottom of the active site were identified through docking simulation. Among compounds thus identified, 21 were assayed for inhibitory activity towards the recombinant DHFR domain. Two compounds, 2-amino-1,4-dihydro-4,4,7,8-tetramethyl-s-triazino(1,2-a)benzimida zole and Trp-P-2, inhibited the recombinant P. falciparum DHFR domain with Ki values of 0.54 and 8.7 microM, respectively. Kinetic analysis showed that these compounds competitively inhibited the enzyme with respect to the substrate dihydrofolate. These findings support the validity of both the modeled structure and the docking results. Furthermore, these compounds serve as leads for developing new DHFR inhibitors, since their skeletal structures are different from any of known DHFR inhibitors. This paper also reveals a new biological activity of Trp-P-2, a potent mutagen.     

Discovery of a novel tetrahydroacridine acetylcholinesterase inhibitor through an indexed combinatorial library

BACKGROUND: Methods for the rapid and efficient preparation of drug candidates through combinatorial chemistry are of increasing interest. We have previously reported an indexed combinatorial library method that allows both the preparation and testing of compounds in solution. We set out to apply this method to develop more effective analogs of the known, marketed drug tacrine, an acetylcholinesterase inhibitor. RESULTS: A one-step condensation of cyclohexanones with cyanoanilines to generate tetrahydroacridine pools was developed. The resulting library of (formally) 72 tetrahydroacridines was screened against acetylcholinesterase, and a compound 10-fold more potent than tacrine, 7-nitrotacrine, was discovered. Its increased potency could be readily explained by examining the known structure of the complex of acetylcholinesterase with tetrahydroacridine. CONCLUSIONS: In this work, we have provided a relatively rare example of carbon-carbon bond formation in a pool synthesis and have discovered a potentially useful acetylcholinesterase inhibitor.     

Thermodynamic analysis of an RNA combinatorial library contained in a short hairpin

Prediction of nucleic acid structure from sequence requires thermodynamic parameters for a variety of motifs, many of which are complex and consist of a large number of possible sequence combinations. Here we report an experimental approach for identifying the stable and unstable members of an RNA combinatorial library. Short model RNA hairpins consisting of 13 base pairs (bp) flanked by primer binding sites are constructed and separated according to their relative thermodynamic stabilities using temperature gradient gel electrophoresis (TGGE). Partially denaturing TGGE is carried out with potassium chloride, sodium chloride, or magnesium chloride salts in the gel. The TMs of model hairpins can be tuned by adjusting the concentration of urea in the gel while maintaining the correct order of stabilities for the hairpins. Mixtures of RNAs differing by a single Watson-Crick base pair are resolved according to their relative thermodynamic stabilities, as are mixtures of GC or AU base pair transversions differing in DeltaG degrees37 by only 0.3-0.5 kcal/mol. In addition, a simple combinatorial library with one position of randomization opposite a guanosine is prepared and separated into its four members by parallel and perpendicular TGGE. The order of thermodynamic stabilities for the library determined by TGGE is shown to be the same when assayed by UV-melting experiments. Analysis of the thermodynamics of folding of combinatorial libraries is general and may be applied to a wide variety of complex nucleic acid secondary and tertiary motifs in order to identify the stable and unstable members.     

tRNA genes transcribed from the plastid-like DNA of Plasmodium falciparum

Besides their mitochondrial genome, malarial parasites contain a second organellar DNA. This 35 kb circular molecule has a number of features reminiscent of plastid DNAs. Sequence analysis shows that along with other genes the circle codes for 25 different tRNAs all of which are transcribed. Six of the tRNAs have some unusual features, and one has an intron, the only one found so far on the circle. Comparison of codon and anticodon usage indicates that the 25 tRNAs are sufficient to decode all the protein genes present on the circle. The maintenance of such a parsimonious but complete translation system is further evidence for the functionality of the circle.     

Identification and characterization of an unusual double serine/threonine protein phosphatase 2C in the malaria parasite Plasmodium falciparum

We have cloned a gene from Plasmodium falciparum with homology to the Mg2+-dependent serine/threonine protein phosphatase 2C (PP2C) family. The predicted coding region is 920 amino acids long, twice the size of other members of this family. We show that this protein can be divided into two halves (Pf2C-1 and Pf2C-2), each a complete phosphatase unit with homology to other phosphatases of this class. To study the function of this PP2C, we have tested the ability of different constructs to complement conditional null mutants of yeast. Our results show that expression of the full-length protein, the first half alone, the second half alone, or a hybrid with the N terminus of the first half and the C terminus of the second half was able to complement the heat shock response defect of a Schizosaccharomyces pombe strain with a PP2C (PTC1) deletion. Recombinant P. falciparum PP2C expressed in Escherichia coli was active in dephosphorylating 32P-labeled casein in an Mg2+- or Mn2+-dependent reaction. Each half alone was also active in recombinant form. Using the two-hybrid system, we have shown that the two halves can interact. Gel filtration assay of P. falciparum protein extracts suggests that full-length PfPP2C is a dimer, and phosphatase activity competition experiments indicate that dimerization of PfPP2C is required for its optimal activity. This unusual phosphatase molecule appears to be composed of four catalytic units on two polypeptide chains.     

Gamma delta T cells in the peripheral blood of individuals from an area of holoendemic Plasmodium falciparum transmission

gamma delta T cells bearing V gamma 9 T cell receptors from unexposed Caucasian donors make large responses to Plasmodium falciparum in vitro. This finding, together with observations of others showing high levels of V gamma 9+ T cells in the blood of infected non-immune individuals, led us to hypothesize that the response of these cells might contribute to the pathology of P. falciparum malaria. Acquisition of immunity to disease in people naturally exposed to infection may therefore be due in part to down-regulation or alteration of the function of gamma delta T cells. Supporting this view, and in contrast to infection in non-immune individuals, V gamma 9+ T cells are not elevated in peripheral blood of children or adults living in an endemic area despite constant exposure to P. falciparum. After in vitro stimulation with P. falciparum, however, the expansion of V gamma 9+ cells from the African donors is of similar magnitude to that observed for non-exposed Europeans. Thus, although these cells are not elevated in peripheral blood, they are still able to respond to P. falciparum antigens. In adult European donors the major gamma delta T cell population in peripheral blood is V gamma 9+ (approximately 70% of all gamma delta cells), whereas in the majority of adult Africans V delta 1+ V gamma 9- T cells predominated (approximately 70% of total gamma delta cells).     

4-Aminoquinoline resistance of Plasmodium falciparum: insights from the study of amodiaquine uptake

The relationship between antimalarial activity and drug accumulation of chloroquine and amodiaquine was evaluated with four chloroquine-resistant and two chloroquine-susceptible isolates of Plasmodium falciparum. Susceptibility of the strains to amodiaquine was correlated with susceptibility to chloroquine (r2 = 0.96). Similarly, accumulation of amodiaquine was correlated with accumulation of chloroquine (r2 = 0.94). Accumulation of both chloroquine and amodiaquine was significantly reduced in chloroquine-resistant isolates (p < 0.005). For the panel of isolates, the accumulation ratio of both drugs was inversely proportional to drug susceptibility (r2 = 0.963 and 0.994 for amodiaquine and chloroquine, respectively). Time course studies highlighted a reduced initial rate of amodiaquine accumulation in chloroquine-resistant isolates compared with chloroquine-susceptible isolates, with no evidence of an enhanced drug efflux rate. Daunomycin, a modulator of parasite chloroquine transport, significantly increased steady state accumulation of both drugs in chloroquine-resistant isolates and, to a lesser extent, in chloroquine-susceptible isolates. Furthermore, daunomycin increased the initial rate of accumulation of amodiaquine in both chloroquine-resistant and chloroquine-susceptible isolates. Resistance to 4-aminoquinoline drugs is associated with reduced drug permeability rather than enhanced cellular exit of preaccumulated drug, and daunomycin seems to increase the permeability of parasites to aminoquinolines. A new model of 4-aminoquinoline resistance is proposed to take account of these and earlier observations.     

Identification of a subpopulation of immune Nigerian adult volunteers by antibodies to the circumsporozoite protein of Plasmodium falciparum

Collections of human sera from malaria-endemic areas would be valuable for identifying and characterizing antigens as malaria vaccine candidates if the contributing serum donors' ability to resist infection were fully characterized. We prepared such a serum collection from 26 apparently immune Nigerian adults who failed to develop patent parasitemia for at least 20 weeks following a documented increase in antibodies to the circumsporozoite protein (CSP) from Plasmodium falciparum. Volunteers were evaluated five times per week for malaria symptoms and bimonthly for parasites by examining thick blood smears. The incidence rate over 13 months for the cohort was 42% (47 malaria-confirmed volunteers) and the risk of infection was 1.3 infections/year. Responses to CSP did not correlate with protection. Because antibody responses to antigens other than CSP may be associated with protection, the sera from these immune individuals may be useful for identifying and characterizing other potential malaria vaccine candidates.     

Crystal structure of fructose-1,6-bisphosphate aldolase from the human malaria parasite Plasmodium falciparum

The structure of the glycolytic enzyme class I fructose-1, 6-bisphosphate aldolase from the human malaria parasite Plasmodium falciparum has been determined by X-ray crystallography. Homotetrameric P. falciparum aldolase (PfALDO) crystallizes in space group P3221 with one 80 kDa dimer per asymmetric unit. The final refined PfALDO model has an R-factor of 0.239 and an R-free of 0.329 with respect to data from 8 to 3.0 A resolution. PfALDO is potentially a target for antimalarial drug design as the intraerythrocytic merozoite lifestage of P. falciparum is completely dependent upon glycolysis for its ATP production. Thus, inhibitors directed against the glycolytic enzymes in P. falciparum may be effective in killing the parasite. The structure of PfALDO is compared with the previously determined structure of human aldolase in order to determine possible targets for the structure-based design of selective PfALDO ligands. The salient structural differences include a hydrophobic pocket on the surface of PfALDO, which results from some amino acid changes and a single residue deletion compared with human aldolase, and the overall quaternary structure of the PfALDO tetramer, which buries less surface area than human aldolase.     

Minimal variation in the Pfs28 ookinete antigen from Philippine field isolates of Plasmodium falciparum

The study examined therapists' accuracy in predicting the length of individual outpatient psychotherapy for 109 clients and attempted to identify variables associated with predicted and actual treatment lengths. The mean predicted treatment length (9.7 months) was significantly longer than the mean length of actual treatment (6.6 months). Therapists correctly predicted treatment length to the nearest month in 26 percent of the cases. Predictions were more accurate for older clients. Treatment tended to be shorter for clients with less than a high school education. Therapists more often predicted shorter treatments for clients with an adjustment disorder and those with less education. Predicting treatment length appears to be difficult.     

Characterization of Plasmodium falciparum isolated from the Amazon region of Brazil: evidence for quinine resistance

The prevalence and severity of drug-resistant malaria is emerging rapidly in the Amazon basin of Brazil. In support of clinical trials using the new antimalarial drug combination of atovaquone and proguanil, we performed in vitro drug sensitivities, molecular characterization of parasite populations using the circumsporozoite protein, merozoite surface antigen-1 (MSA-1), and MSA-2 markers, and an analysis of the Plasmodium falciparum multidrug resistance (pfmdr1) gene sequence and copy number in 26 isolates of P. falciparum obtained in a gold-mining endemic area in Peixoto de Azevedo, Mato Grosso State. All 26 isolates were found to be resistant to chloroquine (50% inhibitory concentration [IC50] = 100-620 nM) and sensitive to mefloquine (IC50 < 23 nM) and halofantrine (IC50 < 6 nM). The isolates also show reduced susceptibility to quinine (IC50 = 48-280 nM). Sequence analysis of the pfmdr1 gene revealed Asn, Phe, Cys, Asp, and Tyr in positions 86, 184, 1034, 1042, and 1246, respectively. These point mutations were similar to that previously described in other Brazilian isolates. Southern blot analysis revealed no amplification of the pfmdr1 gene. These results suggest that three different mechanisms for drug resistance exist for chloroquine, mefloquine, and quinine.     

Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from subsaharan Africa

In this study, we examined the effect of systemic administration of SSG, a soluble highly branched (1-->3)-beta-D-glucan obtained from a fungus Sclerotinia sclerotiorum IFO 9395, on pulmonary immune responses in mice. SSG (10 mg/kg) administered intravenously (i.v.) rapidly leaked into the alveolar space and enhanced several functions of alveolar macrophages (AMs), such as phagocytic activity, lysosomal enzyme activity, active oxygen secretion and cytokine production, on day 1 post-administration. However, kinetic changes of influx of SSG into alveoli and AM activation after SSG treatment were different. The enhanced AM functions decreased to control value on day 2 when SSG still existed at the alveolar space. Additionally, a high dose (500 micrograms/ml) of SSG was needed to activate AMs in vitro. These data imply that the stimulation by SSG alone is not effective on AM activation. SSG administered i.v. also augmented interferon gamma (IFN gamma) mRNA expression in the lung tissue, and the kinetic change of the expression was similar to that of AM activation. Additionally, a synergistic effect of SSG and IFN gamma was observed on AM activation in vitro. It may be possible that IFN gamma produced by pulmonary T cells is one of the important factors for AM activation in vivo by SSG injection. Furthermore, SSG administered i.v. enhanced candidacidal activity and cytolytic activity against pulmonary metastatic Lewis lung carcinoma (3LL) cells of AMs, and inhibited significantly the experimental pulmonary metastasis of 3LL cells. These observations are very useful for the clinical application of SSG as a biological response modifier (BRM).     

Identification of troponin C antagonists from a phage-displayed random peptide library

Affinity purification of a phage-displayed library, expressing random peptide 12-mers at the N terminus of protein III, has identified 10 distinct novel sequences which bind troponin C specifically. The troponin C-selected peptides yield a consensus binding sequence of (V/L)(D/E)XLKXXLXXLA. Sequence comparison revealed as much as a 62.5% similarity between phiT5, the peptide sequence of the phage clone with the highest level of binding to troponin C, and the N-terminal region of troponin I isoforms. Biotinylated peptides corresponding to library-derived sequences and similar sequences from various isoforms of troponin I were synthesized shown to bind troponin C specifically. Alkaline phosphatase fusion proteins of two of the phage clone sequences bound troponin C specifically, and were specifically competed by both library-derived and native troponin I peptides. Measurement of equilibrium dissociation constants of the peptides by surface plasmon resonance yielded dissociation constants for troponin C as low as 0.43 microM for pT5; in contrast, dissociation constants for calmodulin were greater than 6 microM for all peptides studied. Nondenaturing polyacrylamide gel electrophoresis demonstrated that pT5 formed a stable complex with troponin C in the presence of calcium. We also found that the pT5 peptide inhibited the maximal calcium-activated tension of rabbit psoas muscle fibers.