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      In Vitro Resistance Selections for Plasmodium falciparum Dihydroorotate Dehydrogenase Inhibitors Give Mutants with Multiple Point Mutations in the Drug-binding Site and Altered Growth*

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          Abstract

          Background: Inhibiting PfDHODH kills malaria parasites, but the potential for drug resistance is unknown.

          Results: Selections gave several categories of resistance mutations. Several mutants were hypersensitive to other drugs.

          Conclusion: Resistance to PfDHODH inhibitors is largely though mutations in or amplification of the target gene, PfDHODH.

          Significance: Resistance to PfDHODH inhibitors is possible but often increases sensitivity to other compounds.

          Abstract

          Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.

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          Most cited references23

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          Return of chloroquine antimalarial efficacy in Malawi.

          In 1993, Malawi became the first country in Africa to replace chloroquine with the combination of sulfadoxine and pyrimethamine for the treatment of malaria. At that time, the clinical efficacy of chloroquine was less than 50%. The molecular marker of chloroquine-resistant falciparum malaria subsequently declined in prevalence and was undetectable by 2001, suggesting that chloroquine might once again be effective in Malawi. We conducted a randomized clinical trial involving 210 children with uncomplicated Plasmodium falciparum malaria in Blantyre, Malawi. The children were treated with either chloroquine or sulfadoxine\#8211;pyrimethamine and followed for 28 days to assess the antimalarial efficacy of the drug. In analyses conducted according to the study protocol, treatment failure occurred in 1 of 80 participants assigned to chloroquine, as compared with 71 of 87 participants assigned to sulfadoxine\#8211;pyrimethamine. The cumulative efficacy of chloroquine was 99% (95% confidence interval [CI], 93 to 100), and the efficacy of sulfadoxine\#8211;pyrimethamine was 21% (95% CI, 13 to 30). Among children treated with chloroquine, the mean time to parasite clearance was 2.6 days (95% CI, 2.5 to 2.8) and the mean time to the resolution of fever was 10.3 hours (95% CI, 8.1 to 12.6). No unexpected adverse events related to the study drugs occurred. Chloroquine is again an efficacious treatment for malaria, 12 years after it was withdrawn from use in Malawi. (ClinicalTrials.gov number, NCT00125489 [ClinicalTrials.gov].). Copyright 2006 Massachusetts Medical Society.
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            In vivo selection of Plasmodium falciparum pfmdr1 86N coding alleles by artemether-lumefantrine (Coartem).

            Artemisinin derivative-based combination therapy is expected to suppress the development of Plasmodium falciparum drug resistance in Africa. We have performed an artemether-lumefantrine (Coartem; Novartis) follow-up clinical trial in Zanzibar, in which pfcrt K76T and pfmdr1 N86Y frequencies were determined before drug administration and in all recurrent parasites during a follow-up period of 42 days. A significant increase in pfmdr1 86N was observed after exposure to the drug. This points to 86N as a potential marker of lumefantrine resistance in vivo, while suggesting that Coartem is not robust enough to avoid selection of resistance-associated mutations in some malarial settings.
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              Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening.

              Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examined. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and approximately 0.5% parasitemia, respectively. The MSF assay quality was significantly robust, displaying a Z' range of 0.73 to 0.95. The 50% inhibitory concentrations for each drug and culture condition combination were determined by using the MSF assay. Compared to the standard [(3)H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r(2) >/= 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                27 June 2014
                29 April 2014
                29 April 2014
                : 289
                : 26
                : 17980-17995
                Affiliations
                From the []Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115,
                the [§ ]Tres Cantos Medicines Development Campus, GlaxoSmithKline, 28760 Tres Cantos, Madrid, Spain,
                the []Computational and Structural Chemistry, GlaxoSmithKline, Stevenage, Hertfordshire SG1 2NY, United Kingdom, and
                the []Infectious Disease Initiative, Broad Institute, Cambridge, Massachusetts 02142
                Author notes
                [1 ] To whom correspondence should be addressed: Dept. of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave., SPH1 Rm. 705, Boston, MA 02115. Tel.: 617-432-1563; Fax: 617-432-4766; E-mail: dfwirth@ 123456hsph.harvard.edu .
                Article
                M114.558353
                10.1074/jbc.M114.558353
                4140291
                24782313
                09f0d5f2-6790-4e28-b6b6-d9e528c0b17f
                © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Unported License applies to Author Choice Articles

                History
                : 18 February 2014
                : 20 April 2014
                Funding
                Funded by: National Institutes of Health
                Award ID: R01 AI093716-01A1
                Categories
                Microbiology

                Biochemistry
                drug resistance,evolution,infectious disease,malaria,nucleotide,pyrimidine
                Biochemistry
                drug resistance, evolution, infectious disease, malaria, nucleotide, pyrimidine

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