title: Characterization of the commercially-available fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a marker for chloroquine resistance and uptake in a 96-well plate assay.
abstract: Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Cheryl C Y
authors.initials: CC
authors.lastname: Loh
authors.affiliation: Singapore Immunology Network, Agency for Science Technology and Research, Biopolis, Singapore, Singapore.
authors.forename: Rossarin
authors.initials: R
authors.lastname: Suwanarusk
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore.
authors.forename: Yan Quan
authors.initials: YQ
authors.lastname: Lee
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Kitti W K
authors.initials: KW
authors.lastname: Chan
authors.affiliation: Department of Applied Biology and Chemical Technology, The State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, PR China.
authors.forename: Kit-Ying
authors.initials: KY
authors.lastname: Choy
authors.affiliation: Singapore Immunology Network, Agency for Science Technology and Research, Biopolis, Singapore, Singapore.
authors.forename: Laurent
authors.initials: L
authors.lastname: Rénia
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Bruce
authors.initials: B
authors.lastname: Russell
authors.affiliation: Department of Chemistry, Graduate School of Science, Tohoku University, Aza Aramaki, Aoba-ku, Sendai, Japan.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lear
authors.affiliation: Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom; Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand; Centre for Tropical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom.
authors.forename: François H
authors.initials: FH
authors.lastname: Nosten
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore.
authors.forename: Kevin S W
authors.initials: KS
authors.lastname: Tan
authors.affiliation: Department of Applied Biology and Chemical Technology, The State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, PR China.
authors.forename: Larry M C
authors.initials: LM
authors.lastname: Chow
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001896:Boron Compounds / Q000378:metabolism*; D002738:Chloroquine / Q000494:pharmacology*; D056915:DNA Copy Number Variations / Q000235:genetics; D004351:Drug Resistance / Q000187:drug effects* / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism*; D017125:Genes, Protozoan; D020128:Inhibitory Concentration 50; D020748:Mibefradil / Q000494:pharmacology; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D010271:Parasites / Q000187:drug effects / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D014700:Verapamil / Q000494:pharmacology
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D015415:Biomarkers; D001896:Boron Compounds; D005456:Fluorescent Dyes; D020748:Mibefradil; D002738:Chloroquine; D000255:Adenosine Triphosphate; D014700:Verapamil