A series of chalcones and their derivatives have been synthesized and identified as novel potential antimalarials using both molecular modeling and in vitro testing against the intact parasite. A large number of chalcones and their derivatives were prepared using one-step Claisen-Schmidt condensations of aldehydes with methyl ketones. These condensates were screened in vitro against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and shown to be active at concentrations in the nanomolar range. The most active chalcone derivative, 1-(2,5-dichlorophenyl)-3-(4-quinolinyl)-2-propen-1-one (7), had an IC50 value of 200 nM against both a chloroquine-resistant strain (W2) and a chloroquine-sensitive strain (D6). The resistance indexes for all compounds were substantially lower than for chloroquine, suggesting that this series will be active against chloroquine-resistant malaria. Structure-activity relationships (SAR) of the chalcones in the context of a homology-based model structure of the malaria trophozoite cysteine protease, the most likely target enzyme, are presented.

We report on the development of a new SYBR Green I-based plate assay for analyzing the activities of antimalarial drugs against intraerythrocytic Plasmodium falciparum. This assay is considerably faster, less labor-intensive, and less expensive than conventional radiotracer (e.g., [3 H]hypoxanthine and [ 3 H]ethanolamine)-based assays or P. falciparum lactate dehydrogenase activity-based assays. The assay significantly improves the pace at which antimalarial drug discovery efforts may proceed.The continued emergence and spread of multidrug-resistant strains of Plasmodium falciparum and P. vivax are arguably the most pressing problems in the area of infectious diseases today. Also, although the recent deciphering of the P. falciparum genome reveals many promising new drug targets, the financial cost of bringing drugs to the clinic is a major obstacle in the development of new antimalarials (6). A faster, less expensive, high-throughput means of screening the activities of drugs against a variety of malarial parasite strains would greatly assist preclinical drug development.Quantitative assessment of the effects of drugs on parasite growth and development can be achieved by direct (but extremely tedious) microscopic examination of blood smears. An alternative assay is measurement of the effect of drug exposure by determination of the level of incorporation of radiolabeled hypoxanthine. While the latter method can be automated, it requires radioactive materials and is not convenient for detection of parasite stage-specific effects. Another assay measures parasite lactate dehydrogenase activity by methods that do not require radioisotopes. However, this assay requires multiple processing steps and expensive reagents and is not particularly cost-effective for large-scale drug screening efforts.We have thus endeavored to develop more rapid and convenient cell-based assays for quantifying antimalarial drug activities. We have strived to enhance simplicity and reduce cost. In this paper, we report on the development of one such assay that relies on the fluorophore SYBR Green I. MATERIALS AND METHODSCell culture. Asexual culture is routinely performed. Parasite cultures are initiated from stabilates preserved in liquid nitrogen (the level of parasitemia during storage is Ն10%). Following the initiation of a fresh culture, at least two full life cycles (96 h) are completed before parasites are used for assays. In general, cultures are synchronized in the laboratory, and assays are initiated when the parasites are at the ring stage. However, we find that this assay is equally applicable to asynchronous culture and that similar 50% inhibitory concentrations (IC 50 s) are calculated from data with asynchronous and synchronous cultures (data not shown). Prior to assay initiation, the level of parasitemia of an aliquot of a stock culture is measured by light microscopy following Giemsa staining or by fluorescence-activated cell sorter analysis after staining with propidium iodide. In general, stock cultures with 5 to 10% paras...

No abstract

No abstract

Induction of proinflammatory cytokine responses by glycosylphosphatidylinositols (GPIs) of intraerythrocytic Plasmodium falciparum is believed to contribute to malaria pathogenesis. In this study, we purified the GPIs of P. falciparum to homogeneity and determined their structures by biochemical degradations and mass spectrometry. The parasite GPIs differ from those of the host in that they contain palmitic (major) and myristic (minor) acids at C-2 of inositol, predominantly C18:0 and C18:1 at sn-1 and sn -2, respectively, and do not contain additional phosphoethanolamine substitution in their core glycan structures. The purified parasite GPIs can induce tumor necrosis factor ␣ release from macrophages. We also report a new finding that adults who have resistance to clinical malaria contain high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. Absence of a persistent anti-GPI antibody response correlated with malaria-specific anemia and fever, suggesting that anti-GPI antibodies provide protection against clinical malaria. The antibodies are mainly directed against the acylated phosphoinositol portion of GPIs. These results are likely to be valuable in studies aimed at the evaluation of chemically defined structures for toxicity versus immunogenicity with implications for the development of GPI-based therapies or vaccines.

The nature and extent of carbohydrate modification in intraerythrocytic stage Plasmodium falciparum proteins have been controversial. This study describes the characterization of the carbohydrates in intraerythrocytic P. falciparum proteins and provides an overall picture of the nature of carbohydrate modification in the parasite proteins. P. falciparum strains were metabolically labeled with radioactive sugar precursors and ethanolamine at different developmental stages. The individual parasite proteins separated on SDS-polyacrylamide gels and whole parasite cell lysates were analyzed for the carbohydrate moieties. The results established the following: 1) glycosylphosphatidylinositol (GPI) anchors represent the major carbohydrate modification in the intraerythrocytic stage P. falciparum proteins; 2) in contrast to previous reports, O-linked carbohydrates are either absent or present only at very low levels in the parasite; and 3) P. falciparum contains low levels of N-glycosylation capability. The amount of N-linked carbohydrates in whole parasite proteins is ϳ6% compared with the GPI anchors attached to proteins based on radioactive GlcN incorporated into the proteins.The glycan cores of multiple parasite protein GPI anchors are all similar, consisting of protein-ethanolamine-phosphate-(Man␣1-2)6Man␣1-2Man␣1-6Man␣1-4GlcN. The fourth Man residues distal to GlcN of the GPI anchor glycan cores contain unidentified substituents that are susceptible to conditions of nitrous acid deamination. This unusual structural feature may contribute to the reported pathogenic properties of the P. falciparum GPI anchors.Malaria, a life-threatening disease caused by parasitic protozoa of the genus Plasmodium, is a major health problem throughout the tropical and subtropical regions of the world. Among the four species that infect humans, Plasmodium falciparum is the most virulent. New approaches such as vaccine development and novel therapeutic agents are urgently needed due to the emergence of parasite strains resistant to chloroquine and other commonly used drugs (1).Vaccines based on antigens of the blood stage parasite are under intensive study. A major focus has been on synthetic peptides or recombinant proteins of cell surface antigens (2-6). However, this approach has not been highly effective, although immunization with native cell surface proteins purified from the erythrocytic stage parasite is known to confer significant protective immunity (7-10). It is plausible that post-translational modifications play an important role in antigenicity. Accordingly, a basic understanding of such modifications may assist in the development of effective vaccines.Glycosylation is an often extensive post-translational modification of eukaryotic proteins. Carbohydrate moieties of glycoproteins perform a variety of functions including modulation of immunological properties, receptor-ligand interactions, sorting and localization of proteins, cell adhesion, and cell-cell communication (11). In addition, they contribute to protein conform...

No abstract

Though highly desirable, neither a single experimental technique nor a computational approach can be sufficient enough to rationalize a protein structure. The incorporation of biophysical constraints, which can be rationalized based on conventional biophysical measurements, might lead to considerable improvement of the simulation procedures. In this regard, our analysis of 180 proteins in different conformational states allows prediction of the overall protein dimension based on the chain length, i.e., the protein molecular weight, with an accuracy of 10%.