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

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

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Accumulation of CQ_GREEN and ³H-CQ in PfCRT-Dd2 microsomes.

<p>Microsomes were incubated with 10 µM of chemosensitizers before addition of CQ_GREEN or ³H-CQ. Ctrl: negative control; Ver: verapamil; Mtp: methiothepin; Mgl: metergoline; Lop: loperamide; Oct: octoclothepin; Mib: mibefradil; L703: L703,606; Chl: chlorprothixene. *: p<0.05, comparing CQ_GREEN uptake against control. ^‡: p<0.05, comparing ³H-CQ uptake against control. Data presented are means ± S.E.M.; n≥3.</p

CQ_GREEN uptake by resistant-type PfCRT is inhibited by mibefradil in a dose-dependent manner.

<p>Microsomes were preincubated with varying concentrations of the PfCRT inhibitor mibefradil prior to addition of CQ_GREEN. At the highest concentration of 10 µM, mibefradil drastically suppressed CQ_GREEN uptake in PfCRT-Dd2 microsomes but had no significant effect on uptake in PfCRT-3D7 microsomes. *, ***: p<0.05 and p<0.001 respectively, against no mibefradil control (Ctrl). Data presented are means ± S.E.M.; n≥3.</p

CQ_GREEN uptake by Dd2 PfCRT microsomes.

<p>Total uptake of CQ_GREEN was measured at various CQ_GREEN concentrations in microsomes carrying Dd2 PfCRT. Non-specific uptake of CQ_GREEN was measured with pre-treatment of excess unlabelled CQ. Specific uptake was estimated as the difference between total and non-specific uptake. V_max and K_m of the specific uptake was 938.5 nmol/mg PfCRT/min and 105.1 µM respectively. Data are means ± S.E.M.; n≥3.</p

CQ_GREEN uptake correlates with CQ resistance.

<p>(A) CQ_GREEN fluorescence is inversely correlated with CQ logIC₅₀. Total data set shows moderate <i>R</i>² of 0.53. (B) When split into subpopulations on the basis of Pgh1 residue 1226, <i>R</i>² is improved. Data shown are means of at least 3 experiments. Error bars represent S.E.M.</p

CQ_GREEN localization in <i>P. falciparum</i> 3D7.

<p>Parasites were stained with CQ_GREEN and Hoechst and visualized via confocal microscopy under a 100× objective. CQ_GREEN accumulates in the DV but also slightly stains parasite cytosol; erythrocyte cytosol is not stained. Arrowheads denote the DV. Scale bars represent 5 µm.</p

CQ IC₅₀s, PfCRT and Pgh1 polymorphisms, and <i>pfmdr1</i> copy number.

<p>CQ IC₅₀s, PfCRT and Pgh1 polymorphisms, and <i>pfmdr1</i> copy number.</p

ATP-dependent, verapamil-sensitive uptake of CQ_GREEN in microsomes.

<p>Yeast microsomes expressing CQ-sensitive or -resistant PfCRT (“PfCRT-3D7” and “PfCRT-Dd2” respectively), or microsomes from plasmid vector control (“No PfCRT”), were incubated with CQ_GREEN under different conditions. Preincubation with 150 µM verapamil abrogated CQ_GREEN uptake from PfCRT-Dd2 but did not affect uptake in PfCRT-3D7 microsomes. Removal of ATP from buffer abolished CQ_GREEN uptake entirely. **, ***: p<0.005 and p<0.001 respectively, against untreated control. ###: p<0.001. N.s.: not significant. Data presented are means ± S.E.M.; n≥3.</p