Basic Issues and Recent Development in Science and Technology Policy in Thailand

Basic Issues and Recent Development in Science and Technology Policy in Thailan

Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

<p>Abstract</p> <p>Background</p> <p>The prevalence of drug resistance amongst the human malaria <it>Plasmodium </it>species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new <it>Plasmodium falciparum </it>dihydrofolate reductase (<it>Pf</it>DHFR) mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development.</p> <p>Methods</p> <p>A system to identify mutations in <it>Pfdhfr </it>gene that confer antifolate drug resistance using an animal <it>Plasmodium </it>parasite model was developed. By using error-prone PCR and <it>Plasmodium </it>transfection technologies, libraries of <it>Pfdhfr </it>mutant were generated and then episomally transfected to <it>Plasmodium berghei </it>parasites, from which pyrimethamine-resistant <it>Pf</it>DHFR mutants were selected.</p> <p>Results</p> <p>The principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic <it>P. berghei</it>, in which endogenous <it>Pbdhfr </it>allele was replaced with the mutant <it>Pfdhfr^S108N</it>, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine.</p> <p>Conclusion</p> <p>This study demonstrated the power of the transgenic <it>P. berghei </it>system to predict drug-resistant <it>Pfdhfr </it>mutations in an <it>in vivo </it>parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.</p

Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax </it>is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous <it>in vitro </it>culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-<it>P. vivax </it>drugs, bacterial and yeast surrogate models expressing the validated <it>P. vivax </it>target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and <it>in vivo </it>drug metabolism between the surrogate models and <it>P. vivax </it>parasites.</p> <p>Methods</p> <p><it>Plasmodium falciparum </it>and <it>Plasmodium berghei </it>parasites were transfected with DNA constructs bearing <it>P. vivax dhfr-ts </it>pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous <it>dhfr-ts </it>of <it>P. falciparum </it>and <it>P. berghei </it>parasites with <it>P. vivax </it>homologous genes. The integration of <it>Pvdhfr-ts </it>genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays.</p> <p>Results</p> <p>Transgenic <it>P. falciparum </it>and <it>P. berghei </it>lines stably expressing <it>Pv</it>DHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type <it>Pv</it>DHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant <it>Pv</it>DHFR-TS were pyrimethamine-resistant. The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.</p> <p>Conclusion</p> <p>With the permanent integration of <it>Pvdhfr-ts </it>gene in the genome, the transgenic <it>Plasmodium </it>lines expressing <it>Pv</it>DHFR-TS are genetically stable and will be useful for screening anti-<it>P. vivax </it>compounds targeting <it>Pv</it>DHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-<it>P. vivax </it>drugs in general.</p

Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I

BACKGROUND: Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. METHODS: Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ∆folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. RESULTS: Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. CONCLUSIONS: Functional assays for P. falciparum GCH1 based on enzymatic activity and genetic complementation were successfully developed. The assays in combination with a homology model characterized the enzymatic activity of P. falciparum GCH1 and the importance of its key amino acid residues. The potential to use the assay for inhibitor screening was validated by 8-oxo-GTP, a known GTP analogue inhibitor

Plasmodium parasites mount an arrest response to dihydroartemisinin, as revealed by whole transcriptome shotgun sequencing (RNA-seq) and microarray study

RNA-seq data analysis from DHA treatment of P. falciparum Limma results from 1 h treatments with 500 nM DHA in P. falciparum K1 rings, trophozoites and schizonts. (XLS 2040 kb

Anticancer properties of distinct antimalarial drug classes

We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, synthetic peroxides and DHFR (dihydrofolate reductase) inhibitors effected potent inhibition of proliferation with IC 50 s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase) and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor), emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC 50 s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings