Prapon Wilairat

Simple and Inexpensive Fluorescence-Based Technique for High-Throughput Antimalarial Drug Screening

ABSTRACT 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 (EC50) after 48 h of parasite growth in the presence of each drug. The EC50s of chloroquine, quinine, mefloquine, artemisinin, and 3,6-bis-ε-(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 high-throughput antimalarial drug screening.

Antimalarial activity of ferrocenyl chalcones.

A series of ferrocenyl chalcones were synthesized and evaluated for in vitro antimalarial activity against a chloroquine-resistant strain of Plasmodium falciparum. The most active compounds were 1-(3-pyridyl)-3-ferrocenyl-2-propen-1-one (6) and 1-ferrocenyl-3-(4-nitrophenyl)-2-propen-1-one (28) with IC(50) of 4.5 and 5.1 microM, respectively. Differences in activity were not readily explained by the size and lipophilicity characteristics of these compounds.

Antiplasmodial Chalcones Inhibit Sorbitol-Induced Hemolysis of Plasmodium falciparum -Infected Erythrocytes

ABSTRACT A series of alkoxylated and hydroxylated chalcones previously reported to have antiplasmodial activities in vitro were investigated for their effects on the new permeation pathways induced by the malaria parasite in the host erythrocyte membrane. Of 21 compounds with good antiplasmodial activities (50% inhibitory concentrations [IC50s], ≤20 μM), 8 members were found to inhibit sorbitol-induced lysis of parasitized erythrocytes to a significant extent (≤40% of control values) at a concentration (10 μM) that was close to their antiplasmodial IC50s. Qualitative structure-activity analysis suggested that activity was governed to a greater extent by a substitution on ring B than on ring A of the chalcone template. Most of the active compounds had methoxy or dimethoxy groups on ring B. Considerable variety was permitted on ring A in terms of the electron-donating or -withdrawing property. Lipophilicity did not appear to be an important determinant for activity. Although they are not exceptionally potent as inhibitors (lowest IC50, 1.9 μM), the chalcones compare favorably with other more potent inhibitors in terms of their selective toxicities against plasmodia and their neutral character.

Severity differences in β‐thalassaemia/haemoglobin E syndromes: implication of genetic factors

Summary. Genetic factors determining the difference in severity of anaemia in β‐thalassaemia/HbE disease were studied in 90 patients who had haemoglobin levels, at steady state, ranging from 4.2 to 12.6 g/dl. Co‐inheritance of α‐thalassaemia 2 and haemoglobin Constant Spring could significantly decrease the severity of the disease. Inheritance of a β‐thalassaemia chromosome with Xmn I cleavage site at position — 158 of the Gγ‐globin gene which was linked to the haplotype ‐ + ‐ ++ or ++ ‐ ++, was associated with a milder anaemia. Two copies of these alleles were necessary to produce a significant clinical effect. Increased expression of the Gγ‐globin gene and higher production of haemoglobin F. which could reduce the overall globin chain imbalance, were also associated with homozygosity for the Xmn I cleavage site and thus with less severe anaemia. However, this effect was not seen in Xmn I site heterozygotes. Whether the effects of the Xmn I polymorphism, HbF concentration and Gγ/Aγ ratio act separately or through common mechanisms in reducing anaemia remains to be ascertained.

A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates

Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites.

The sensitivity of Plasmodium protein synthesis to prokaryotic ribosomal inhibitors

The prokaryotic protein synthesis inhibitors tetracycline and doxycycline are used in the treatment of malaria, as a way to overcome the problem of drug resistance to more conventional antimalarial drugs. It has been suggested that the antimalarial activity is mediated through the inhibition of the protein synthetic machinery of the mitochondria [1], as suggested by its sensitivity in a wide range of eukaryotic organisms to inhibitors of the 70S prokaryotic ribosomes; these inhibitors do not normally inhibit the main protein synthesizing machinery in the cytosol [2,3]. Several recent findings, however, have prompted us to examine the sensitivity of the Plasmodium protein synthesis to these inhibitors and to question the mechanism of action of the

Pyrimethamine resistant mutations in Plasmodium falciparum

Three mutations in Plasmodium falciparum yielding increased resistance to pyrimethamine were obtained following treatment with chemical mutagens and selection in presence of pyrimethamine. From parasite clone TM4/8.2 a mutant, TM4/8.2/4.1, was produced which raised pyrimethamine resistance about 500 times and was found to involve an amino acid change in the DHFR-TS enzyme molecule from Ser108 to Asn108. A clone of another isolate, T9/94, yielded a mutant, T9/94/300.300, raising pyrimethamine resistance about 10 times and involving an amino acid change from Ile164 to Met164. However, another mutant from T9/94, T9/94/M1-1(b3), although it raised the pyrimethamine resistance 100 times, did not involve any changes in the coding sequence of the DHFR-TS gene, but resulted in the production of about twice as much DHFR-TS enzyme as the original clone T9/94. No amplification of the DHFR-TS gene was detected. It is concluded that changes in pyrimethamine resistance of malaria parasites may arise in at least 2 ways: (1) by structural changes in the DHFR domain of the DHFR-TS gene (as previously found by other workers); (2) by other changes, possibly affecting the expression of the DHFR-TS gene. The relative importance of these 2 mechanisms in causing resistance in wild populations of P. falciparum is discussed.

A novel detection of a single plasmodium falciparum in infected blood

Detection of Plasmodium falciparum malaria by a specific DNA probe is a highly promising means for epidemiological surveillance of human malaria. However, none of presently available DNA probe methods could detect as little as a few parasites in infected blood. By amplification of a specific 206 base pairs P. falciparum DNA sequence using the polymerase chain reaction (PCR), as little as 0.01 picogram DNA or one-half of a parasite was sufficient for a specific detection. A PCR procedure for detection of P. falciparum in infected blood without prior DNA extraction was also developed which was sensitive for a single parasite. The procedure was simple and should be applicable for a large scale epidemiological study involving a very low parasitemia situation.

Targeting of Hematin by the Antimalarial Pyronaridine

ABSTRACT Pyronaridine, 2-methoxy-7-chloro-10[3′,5′-bis(pyrrolidinyl-1-methyl-)4′hydroxyphenyl]aminobenzyl-(b)-1,5-naphthyridine, a new Mannich base schizontocide originally developed in China and structurally related to the aminoacridine drug quinacrine, is currently undergoing clinical testing. We now show that pyronaridine targets hematin, as demonstrated by its ability to inhibit in vitro β-hematin formation (at a concentration equal to that of chloroquine), to form a complex with hematin with a stoichiometry of 1:2, to enhance hematin-induced red blood cell lysis (but at 1/100 of the chloroquine concentration), and to inhibit glutathione-dependent degradation of hematin. Our observations that pyronaridine exerted this mechanism of action in situ, based on growth studies of Plasmodium falciparum K1 in culture showing antagonism of pyronaridine in combination with antimalarials (chloroquine, mefloquine, and quinine) that inhibit β-hematin formation, were equivocal.

Cloning and expression of 130-kd mosquito-larvicidal δ-endotoxin gene of Bacillus thuringiensis var. Israelensis in Escherichia coli

SummaryFive recombinant E. coli clones exhibiting toxicity to Aedes aegypti larvae were obtained from a library of 800 clones containing XbaI DNA fragments of 110 kb plasmid from B. thuringiensis var. israelensis. All the five clones (pMU 14/258/303/388/679) had the same 3.8-kb insert and encoded a major protein of 130 kDa which was highly toxic to A. aegypti larvae. Three clones (pMU 258/303/388) transcribed the 130 kD a gene in the same direction as that of lac Z promoter of pUC12 vector whereas the transcription of the other two (pMU 14/679) was in the opposite direction. A 1.9-kb fragment of the 3.8 kb insert coded for a protein of 65 kDa. Partial DNA sequence of the 3.8 kb insert, corresponding to the 5′-terminal of the 130 kDa gene, revealed a continuous reading frame, a Shine-Dalgarno sequence and a tentative 5′-regulatory region. These results demonstrated that the 3.8 kb insert is a minimal DNA fragment containing a regulatory region plus the coding sequence of the 130 kDa protein that is highly toxic to mosquito larvae.