Radioisotopic assays involve expense, multistep protocols, equipment, and radioactivity safety requirements which are problematic in high-throughput drug testing. This study reports an alternative, simple, robust, inexpensive, one-step fluorescence assay for use in antimalarial drug screening. Parasite growth is determined by using SYBR Green I, a dye with marked fluorescence enhancement upon contact with Plasmodium DNA. A side-by-side comparison of this fluorescence assay and a standard radioisotopic method was performed by testing known antimalarial agents against Plasmodium falciparum strain D6. Both assay methods were used to determine the effective concentration of drug that resulted in a 50% reduction in the observed counts (EC 50 ) after 48 h of parasite growth in the presence of each drug. The EC 50 s of chloroquine, quinine, mefloquine, artemisinin, and 3,6-bis--(N,N-diethylamino)-amyloxyxanthone were similar or identical by both techniques. The results obtained with this new fluorescence assay suggest that it may be an ideal method for highthroughput antimalarial drug screening.The global scope of malaria and the spread of drug-resistant Plasmodium falciparum make the need for improved therapy undeniable (4). Assessment of both existing drugs and new antimalarials, alone or in combination, requires reliable methods for high-throughput testing. For decades, antimalarial drug effects have been measured in vitro by quantifying parasite uptake of radioactive substrates as a measure of growth and viability in the presence of the test drug (2, 3). [3 H]hypoxanthine is the most widely used radiolabel, but because it requires purine starvation prior to the assay, the [ 3 H]ethanolamine assay is favored by our laboratory. While these methods are accurate and reliable, they rely on relatively expensive radioisotopes and multistep procedures that become increasingly problematic and impractical as the volume of testing increases. We report on the development of an alternative, fluorescencebased in vitro assay for use for the high-throughput screening of the activities of drugs against P. falciparum. The goals for the assay included simplicity, cost savings, robust performance, applicability to automated analysis, and speed.The principle behind the assay is the contrast between host erythrocytes, which lack DNA and RNA, and the malaria parasites, which do not and which are thus readily stained with dyes that show enhanced fluorescence in the presence of nucleic acids (8). Fluorescence-based in vitro antimalarial assays have been developed previously (1, 12) but have required complex, multistep protocols or additional equipment not amenable to rapid, high-throughput use. This report establishes and validates a facile, one-step method that uses the same principle. MATERIALS AND METHODSChloroquine diphosphate, quinine sulfate, artemisinin, and saponin were purchased from Sigma Aldrich Chemical Company (St. Louis, Mo.); mefloquine was obtained from Walter Reed Army Institute for Research (Silver Spring, Md.); and 3,6-b...

Chalcones with 2',3',4'-trimethoxy, 2',4'-dimethoxy, 4'-methoxy, 4'-ethoxy, 2',4'-dihydroxy, and 4'-hydroxy groups on ring B were synthesized and evaluated in vitro against Plasmodium falciparum (K1) in a [3H] hypoxanthine uptake assay. The other ring A was quinoline, pyridine, naphthalene, or phenyl rings with electron-donating or electron-withdrawing substituents of varying lipophilicities. Trimethoxy 6 and 27, dimethoxy 7, 8, 29, and methoxy 31 analogues had good in vitro activities (IC(50) < 5 microM). 3-Quinolinyl ring A derivatives were well represented among the active compounds. Hydroxylated chalcones were less active than the corresponding alkoxylated analogues. When evaluated in vivo, 8 and 208 were comparable to chloroquine in extending the lifespan of infected mice. Multivariate data analysis showed that in vitro activity was mainly determined by the properties of ring B. Quantitative structure-activity relationship models with satisfactory predictive ability were obtained for various B ring chalcones using projections to latent structures. A model with good predictability was proposed for 19 active chalcones. Size and hydrophobicity were identified as critical parameters.

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These findings suggest that compound heterozygosity of abnormal AE1 genes causes autosomal recessive dRTA in SAO.