Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite’s Chloroquine Resistance Transporter

<div><p>Mutations in the <i>Plasmodium falciparum</i> ‘chloroquine resistance transporter’ (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite’s digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRT^CQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRT^CQR that suppress the parasite’s hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRT^CQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in <i>Xenopus</i> oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRT^CQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite’s survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite’s hypersensitivity to this drug arises from the PfCRT^CQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRT^CQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite’s sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite.</p></div

CQ, quinine, and quinidine transport activities differ significantly between isoforms of PfCRT^K1.

<p>Noninjected oocytes accumulate low levels of CQ, quinine, and quinidine via simple diffusion of the neutral species of the drug [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005725#ppat.1005725.ref029" target="_blank">29</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005725#ppat.1005725.ref030" target="_blank">30</a>]. This represents the background level of drug accumulation. In all cases, the concentration of the [³H]drug under study was 0.25 μM. (A) The uptake of [³H]CQ was measured at pH 6.0 and in the presence of 15 μM unlabeled CQ. The rates of CQ uptake (pmol per oocyte/h) in noninjected oocytes and oocytes expressing PfCRT^K1 were 0.98 ± 0.13 and 6.75 ± 0.94, respectively. (B, C) The uptake of [³H]quinine or [³H]quinidine was measured at pH 5.0 and in the presence of 1 μM of the respective unlabeled drug. The rates of uptake (pmol per oocyte/h) in noninjected oocytes and oocytes expressing PfCRT^K1 were 0.10 ± 0.01 and 0.55 ± 0.03, respectively, for quinine and 0.063 ± 0.010 and 0.38 ± 0.08, respectively, for quinidine. Note that the relatively low total concentration of quinine and quinidine (1.25 μM) used in these assays enabled the detection of [³H]quinine transport via the 76I-PfCRT^K1 isoform. However, this low drug concentration also resulted in rates of quinine and quinidine transport that were 10–15 times lower than those measured for CQ (a difference which corresponds well with the ~12-fold reduction in the total concentration of quinine or quinidine relative to the total concentration of CQ). In all panels, drug uptake is expressed relative to that measured in oocytes expressing PfCRT^K1 and the data are the mean + SEM of at least five independent experiments (performed using oocytes from different frogs), within which measurements were made from 10 oocytes per treatment. The asterisks denote a significant difference in drug uptake between the noninjected treatment and that measured in oocytes expressing a variant of PfCRT: *<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.001 (one-way ANOVA).</p

Isoforms of PfCRT^K1 localize to the surface of <i>Xenopus</i> oocytes.

<p>(A) Immunofluorescence microscopy was used to localize PfCRT in the oocyte. In each case, the expression of the PfCRT variant resulted in a fluorescent band external to the pigment layer, indicating that the protein was expressed in the oocyte plasma membrane. The band was not present in noninjected oocytes. The images are representative of at least two independent experiments (performed using oocytes from different frogs), within which images were obtained from a minimum of three oocytes per oocyte type. (B) The level of PfCRT protein in the oocyte membrane was semiquantified using a western blot method [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005725#ppat.1005725.ref042" target="_blank">42</a>]. The analysis included PfCRT^K1 as a positive control, to which the other band intensity values were normalized. The data are the mean + SEM of at least five independent experiments (performed using oocytes from different frogs), within which measurements were averaged from two independent replicates. There were no significant differences in expression levels between constructs (<i>P</i> > 0.05; one-way ANOVA); hence, all of the PfCRT variants were present at similar levels in the oocyte membrane.</p

Kinetic parameters for the transport of CQ, quinine, and quinidine via variants of PfCRT^K1.

<p>Kinetic parameters for the transport of CQ, quinine, and quinidine via variants of PfCRT^K1.</p

<i>In vitro</i> antiplasmodial activities of CQ and amantadine against CQ-sensitive and CQ-resistant <i>P</i>. <i>falciparum</i> parasites.

<p><i>In vitro</i> antiplasmodial activities of CQ and amantadine against CQ-sensitive and CQ-resistant <i>P</i>. <i>falciparum</i> parasites.</p

Isoforms of PfCRT^K1 from amantadine-hypersensitive parasites transport amantadine.

<p>(A) Amantadine inhibits the transport of [³H]CQ via 76I-PfCRT^K1 and 76I,369F-PfCRT^K1 in a concentration-dependent manner (IC₅₀s of 186 ± 16 and 174 ± 26 μM, respectively). (B) The uptake of [³H]amantadine (0.146 μM) in the absence and presence of verapamil or saquinavir. The measurements were undertaken at pH 5.0 and in the presence of 50 μM unlabeled amantadine. (C) The PfCRT-mediated transport of [³H]amantadine. Using the data shown for the solvent control in panel B, the component of [³H]amantadine transport attributable to PfCRT was calculated by subtracting the background level of accumulation (i.e., the average of the uptake measured in noninjected oocytes and oocytes expressing 76K-PfCRT^K1) from that measured for each of the oocyte types. The rates of amantadine uptake (nmol per oocyte/h) in noninjected oocytes and PfCRT^K1-expressing oocytes were 5.4 ± 0.9 and 12 ± 2.8, respectively. The asterisks denote a significant difference from the noninjected control: *<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.001 (one-way ANOVA). (D) <i>Trans</i>-stimulation of PfCRT-mediated [³H]CQ transport by unlabeled amantadine. The oocytes were microinjected with a buffer control or with buffer containing amantadine, spermine, or histidine. The estimated intracellular concentrations ([compound]_i) are indicated. The asterisks denote a significant difference from the relevant buffer-injected control. (E) Concentration-dependence of the <i>trans</i>-stimulation of [³H]CQ uptake by amantadine. The oocytes were microinjected with buffer containing amantadine to achieve an estimated [amantadine]_i of 1 to 20 mM. The app K_m and app V_max values are the apparent kinetic parameters for the <i>trans</i>-stimulatory effect of amantadine. The data show CQ uptake above that measured in the relevant buffer-injected control; the total rates of CQ uptake are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005725#ppat.1005725.s006" target="_blank">S6 Fig</a>. The noninjected data overlays the data obtained with oocytes expressing PfNT1, PfCRT^3D7, or 76K-PfCRT^K1. (F) A magnified plot of the 76I-PfCRT^K1 and 76I,369F-PfCRT^K1 data from panel E. In all panels, the data are the mean ± SEM of five independent experiments (performed using oocytes from different frogs), within which measurements were made from 10 oocytes per treatment. Where not shown, error bars fall within the symbols.</p

Amantadine is a substrate of PfCRT^Dd2 and PfCRT^7G8, and not of wild-type PfCRT, <i>in situ</i>.

<p>The rate of concanamycin A-induced DV alkalinization (expressed as the inverse of the half-time for DV alkalinization) in the C2^GC03 (CQ-sensitive, expressing PfCRT^3D7), C4^Dd2 (CQ-resistant, expressing PfCRT^Dd2), and C6^7G8 (CQ-resistant, expressing PfCRT^7G8) transfectant lines was measured in the absence and presence of amantadine (AMT), CQ, or verapamil (VP). The effect of amantadine on the DV alkalinization rate was also measured in the presence of verapamil (final concentration of 50 μM). The drugs were added to suspensions of saponin-isolated trophozoite-stage parasites containing fluorescein-dextran in their DVs 4 min before the addition of concanamycin A (100 nM). CQ was added at a concentration of 2.5 μM and the concentration of amantadine was 10 μM. Consistent with previous studies, the addition of CQ elevated the rate of alkalinization in both of the CQ-resistant lines and had a small buffering effect in the C2^GC03 parasites [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005725#ppat.1005725.ref034" target="_blank">34</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005725#ppat.1005725.ref036" target="_blank">36</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005725#ppat.1005725.ref046" target="_blank">46</a>]. The biological basis for the difference in the rate of CQ-dependent alkalinization between the two CQ-resistant lines is currently unclear, but could be due to differences in (1) the expression of PfCRT, (2) the transport properties of the two PfCRT isoforms, (3) the volume of the DV, and/or (4) the concentration of PfCRT’s natural substrates within the DV. The data are the mean + SEM of five independent experiments (performed on different days). The asterisks denote a significant difference from the relevant solvent control: ***<i>P</i> < 0.001 (one-way ANOVA).</p

Molecular mechanisms for the drug hypersensitivities induced by PfCRT isoforms in the malaria parasite.

<p>(A) The variants of PfCRT^K1 that contain 72R, 76K, 163R, 352K, or 352R (R/K) do not possess significant quinine (QN) transport activity. The drug would therefore remain in the parasite’s DV where it exerts an anti-hemozoin effect that kills the parasite, which is consistent with the QN-sensitive (S) status of the respective lines. PfCRT^K1 (76T) is able to transport QN out of the DV and thereby imparts low-level resistance (<i>low</i>-R) to QN. By contrast, 76I-PfCRT^K1 has an extremely high affinity for QN coupled with an extremely low maximum rate of transport. This causes QN to clog the binding site of 76I-PfCRT^K1, thereby blocking the transport of the natural substrate. Hence, the QN-hypersensitivity (<i>hyper</i>-S) observed in 106/1^76I parasites results from QN exerting two killing effects—anti-hemozoin and anti-PfCRT^CQR. The gain of a positively-charged residue at position 72 or 352 (76I R/K) prevents the interaction of the transporter with QN and returns the parasites to QN-sensitive status. (B) Amantadine (AMT) is a relatively poor inhibitor of both 76I-PfCRT^K1 and 76I,369F-PfCRT^K1, making it unlikely that the AMT-hypersensitivity of CQ-resistant parasites is due to an anti-PfCRT^CQR effect. The isoforms of PfCRT from AMT-hypersensitive parasites (PfCRT^K1 and 76I-PfCRT^K1) have the ability to transport this weak-base drug out of the DV (where it accumulates) whereas those from AMT-sensitive parasites either do not possess significant AMT transport activity (e.g., 76K-PfCRT^K1 and 163R,356V-PfCRT^K1; R/K) or transport AMT with low affinity and low capacity (76I,369F-PfCRT^K1). The data therefore converge on a scenario in which AMT exerts its main antimalarial activity in the cytosol and AMT-hypersensitivity arises from the redistribution of the drug from the DV into the cytosol via a PfCRT^CQR variant (e.g., PfCRT^K1 or 76I-PfCRT^K1).</p

The origins, drug susceptibilities, and PfCRT haplotypes of different 106/1 and K1 <i>P</i>. <i>falciparum</i> lines.

<p>The origins, drug susceptibilities, and PfCRT haplotypes of different 106/1 and K1 <i>P</i>. <i>falciparum</i> lines.</p

NMR solution structure of FOG-PR.

<p>(a) Overlay of backbone traces of the 20 lowest energy structures following RECOORD refinement. NMR data indicated that residues 100–102 and 207–254 are disordered, and these are not shown. (b) Ribbon diagram of the lowest energy structure of FOG-PR. The N/C-termini, residue numbers and the position of the pseudoknot are indicated. (c) Comparison of the DIM5 SET domain structure (PDB: 1PEG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106011#pone.0106011-Zhang1" target="_blank">[36]</a>, <i>left</i>) with FOG-PR (<i>right</i>). Corresponding elements of structure are coloured similarly in a pattern ranging from red to orange to yellow to green to blue to purple (N- to C-terminal end). The regions in gray are a small pre-SET domain and the SET-I variable region that lies between the yellow and green regions. The pseudoknot is apparent as the purple C-terminal β-strand that passes through the green helix/loop. Zinc ions are shown as grey spheres. (d) Comparison of the PRDM4 PR domain structure (<i>left</i>, PDB 3DB5) with FOG-PR (<i>right</i>). Corresponding elements of structure are coloured as in part (b). Diagrams were produced using the programs PYMOL and MOLMOL.</p