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

as a Marker for Chloroquine Resistance and Uptake in a 96-Well Plate Assay. PLoS ONE 9(10): e110800. doi:10.1371/journal.pone. 0110800 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 Cheryl C. Y. Loh Rossarin Suwanarusk Yan Quan Lee Kitti W. K. Chan Kit-Ying Choy Laurent Re nia Bruce Russell Martin J. Lear Franc ois H. Nosten Kevin S. W. Tan Larry M. C. Chow Georges Snounou, Universite Pierre et Marie Curie, France 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 sensitiveor 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. - Funding: As part of the Oxford Tropical Medicine Research Program of Wellcome TrustMahidol University, Shoklo Malaria Research Unit (SMRU) is funded by the Wellcome Trust of Great Britain. The authors further thank the National Research Foundation (NRF2009NRF-POC002102), the National Medical Research Council (NMRC/1310/2011; NMRC/EDG/1038/2011), and the Agency for Science, Technology and Research (A*STAR, Singapore) for their generous support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: KSWT and MJL are founding directors of BioLynx Technologies (Singapore), a private company that specializes in fluorophore-conjugated drug surrogates including LynxTag-CQGREEN. Other authors declare no competing interests. KSWT and MJL own minority shares in BioLynx Technologies (Singapore). LR and BR are PLOS ONE Editorial Board Members. This does not alter the authors adherence to PLOS ONE Editorial policies and criteria. . These authors contributed equally to this work. Despite years of intense global effort to eradicate it, malaria is still one of the deadliest infectious diseases, killing more than 600 000 people in 2010 alone [1,2]. The severest form of malaria is caused by the protozoan parasite Plasmodium falciparum. Chloroquine (CQ), once a spectacularly successful antimalarial drug, was first discovered by the German chemist Johann Andersag but was mistakenly thought to be too toxic for therapeutic purposes, an incident which became known as the resochin error (resochin being the name given to the compound by Andersag) [3,4]. CQ was so effective that it inspired optimism for the eradication of malaria. However, resistance soon arose, first appearing along the Thai-Cambodian border in the 1950s. By the 1970s, CQ resistance had spread throughout the world [5,6]. This resistance is generally attributed to mutations in the pfcrt (P. falciparum chloroquine resistance transporter) gene, which codes for a transporter situated on the membrane of the parasite digestive vacuole (DV). During parasite development in the intraerythrocytic cycle, haemoglobin is digested in the DV and the toxic heme moiety is released, which the parasite crystalizes into non-toxic hemozoin [7]. CQ is generally thought to kill the parasite by inhibiting the formation of hemozoin and thus preventing the detoxification of free heme [810]. In wild-type parasites CQ diffuses through the DV membrane and is diprotonated in the acidic environment of the DV, acquiring a net positive charge which prevents it from escaping the DV; however, mutant PfCRT found in CQ-resistant parasites effluxes this charged CQ out of the DV, removing it from its site of action [11]. Although the current first line artemisinincombination therapies are effective in clearing parasitaemia, resistance against artemisinins has emerged [1217]. There is therefore an urgent need to develop novel antimalarial strategies. Several research groups, including our own, have tried different approaches to tackle the problem of CQ resistance by either reversing CQ resistance with a PfCRT inhibitor or synthesizing reversed CQ analogues that cannot be effluxed by PfCRT [18 24]. The ultimate goal is to reintroduce CQ as a viable treatment for malaria. Both development of PfCRT inhibitors and synthesis of reversed CQ analogues require a sensitive assay for CQ uptake which is typically performed by the use of radiolabelled CQ [22,2528]. Such methods are difficult to adopt in a highthroughput screen and may raise concerns of safety. To overcome this technical difficulty, fluorescent derivatives of chloroquine have recently been developed and used for this purpose; fluorophores used include 6-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoic acid (NBD) [29], coumarin [21,30], and 4, 4-difluoro-4bora-3a, 4a-diaza-s-indacene (BODIPY) [31]. BODIPY derivatives typically exhibit strong fluorescence and are relatively inert in biological conditions [32]. Furthermore, their maximum emission wavelengths are in the green-red region [32], allowing them to be used with many DNA dyes that fluoresce blue, such as the DAPI and Hoechst stains. These properties make BODIPY a promising candidate as a marker for CQ uptake in P. falciparum. We therefore present here the characterization of a commercially-available BODIPY-CQ conjugate, LynxTagCQGREEN, in several laboratory strains and clinical isolates. Parasite culture and synchronization P. falciparum laboratory strains 3D7 (MRA-102), K1 (MRA159), 7G8 (MRA-154), HB3 (MRA-155), CS2 (MRA-96), T9-94 (MRA-153), and Dd2 (MRA-156) were obtained from MR4, ATCC Manassas Virginia. Strain T9/96 was obtained from The European Malaria Reagent Repository. A further twelve clini (...truncated)