On the Mechanism of Chloroquine Resistance in Plasmodium falciparum

Resistance to chloroquine of malaria strains is known to be associated with a parasite protein named PfCRT, the mutated form of which is able to reduce chloroquine accumulation in the digestive vacuole of the pathogen. Whether the protein mediates extrusion of the drug acting as a channel or as a carrier and which is the protonation state of its chloroquine substrate is the subject of a scientific debate. We present here an analytical approach that explores which combination of hypotheses on the mechanism of transport and the protonation state of chloroquine are consistent with available equilibrium experimental data. We show that the available experimental data are not, by themselves, sufficient to conclude whether the protein acts as a channel or as a transporter, which explains the origin of their different interpretation by different authors. Interestingly, though, each of the two models is only consistent with a subset of hypotheses on the protonation state of the transported molecule. The combination of these results with a sequence and structure analysis of PfCRT, which strongly suggests that the molecule is a carrier, indicates that the transported species is either or both the mono and di-protonated forms of chloroquine. We believe that our results, besides shedding light on the mechanism of chloroquine resistance in P. falciparum, have implications for the development of novel therapies against resistant malaria strains and demonstrate the usefulness of an approach combining systems biology strategies with structural bioinformatics and experimental data.


Table of Contents
, cells C3-5,5) S9 -Detailed numerical calculation showing that chloroquine-HM binding in experiment C is not in the saturation region S10 -Proof that eq. (18) (main text) is an increasing function of [H + ] DV in the interval 0 < pH DV < 9.15 S12 -Calculation of CAR D in saturated condition for chloroquine-HM binding and J PfCRT = λ[CQ ++ ] DV (cells C5,1-4, Figure 1 of the main text]) S13 -Calculations relating to each single hypothesis for the chloroquine:HM binding in saturated condition and J PfCRT = λ [CQ + ] DV (cells C4,1-4, Figure 1 of the main text]) S14 -Prediction for CAR D in saturated condition for chloroquine-HM binding (H2) and J PfCRT = const (J2b) (cells C2,1-4, Figure 1, main text) In the hypothesis that PfCRT is a passive channel for CQ, both J mem and J PfCRT are only functions of the difference of CQ concentrations on the two sides of the vacuolar membrane, i.e.  the average chloroquine concentration in the infected erythrocyte is The total concentration of chloroquine in each compartment of the infected erythrocyte only depends on the pH of the compartment and on the concentration of the un-protonated chloroquine CQ. Recent measurements [1,2,3] have shown that the pH of the infected erythrocyte cytoplasm and of the plasmodium cytoplasm are very close to physiological pH. Thus, it is reasonable to assume that the pHs of the external medium, of the erythrocyte and of the plasmodium cytoplasm are equal. We indicate this value as pH e . Furthermore, due to the permeability of the membrane to CQ, at equilibrium the CQ concentration is equal in all compartments. The CQ concentration and pH being equal inside the erythrocyte and in the external buffer, we have [C] e = [C] out (as more rigorously shown below in section S4). Therefore, the cellular accumulation ratio CAR can be expressed as: CAR expressions corresponding to equations (8-10) of the main text are: (eq-S1) holds, where J mem is a function of the chloroquine difference between the plasmodium cytoplasm and the vacuole, i.e. J mem = g([CQ] e -[CQ] DV ). In particular we have that J mem has the following properties: In experiment D, pH e = pH DV , hence (eq-S12a), (eq-S12b) and (eq-S12c) imply Combining (eq-S11a) with (eq-S13a), it is apparent that [CQ] DV = [CQ] e is a solution of equation (eq-S1).
In the following we will show that this solution is unique, i. while equation (eq-S13b) implies J PfCRT > 0, i.e. J mem ≠ J PfCRT, which is not consistent with (eq-S1). Therefore, no solution of eq (eq-S1) exists if we assume [CQ] DV > [CQ] e . A similar argument holds in the hypothesis [CQ] DV < [CQ] e : Equation (eq-S11b) implies J mem > 0 while equation (eq-S13c) implies J PfCRT < 0, i.e. J mem ≠ J PfCRT , that is not consistent with (eq-S1). Therefore no solution of eq (eq-S1) exists if we (eq-S6) (eq-S8) and (eq-S18), gives Since the third term of (eq-S19) is always positive, CAR C > CAR A -1, which is not compatible with the experimental data.

S10 -Proof that eq. (18) (main text) is an increasing function of [H + ] DV in the
interval 0 < pH DV < 9.15 In this section we demonstrate that eq. (18) of the main text is an increasing function of [H + ] DV in the interval 0 < pH DV < 9.15 for any value of λ and P cq . For the sake of clarity we report here eq. (18) Being the denominator of (eq-S20) always positive, the sign of the derivative will be determined by the sign of its numerator that could be rewritten as The   (5) and (6), main text), we can write  and, therefore, a conclusionon about the relationship between CAR D and CAR C cannot be drawn. This implies that the current hypothesis cannot be excluded and cell C 5,2 cannot be shaded in Figure 1 of the main text.
The expression in brackets on the right side of inequality (eq-S27) corresponds to CAR C ; therefore: CAR D < 1.2 CAR C . Since this result is not compatible with the experimental data, we shaded the cell C 5,3 in Figure 1 of the main text. whether CAR C is larger or smaller that CAR D . Accordingly, the corresponding cell in Figure 1 of the main text cannot be shaded.

2) [CQ:HM] DV = f([CQ + ]), corresponding to cell C 4,2 (Figure 1, main text)
Using the chloroquine dissociation equilibrium (5) (18) is an increasing function of [H + ] DV (see section S10) implies that CAR C is larger than CAR D , which is not consistent with experimental data. Accordingly, the corresponding cell in Figure 1 of the main text is shaded.

3) [CQ:HM] DV = f([CQ ++ ]), corresponding to cell C 4,3 (Figure 1, main text)
Using the chloroquine dissociation equilibrium (6) (18) is an increasing function of [H + ] DV (see section S10) implies that CAR C is larger than CAR D , which is not consistent with experimental data. Accordingly, the corresponding cell in Figure 1 of the main text is shaded.

4) [CQ:HM] DV = f([CQ TOT ]), corresponding to cell C 4,4 (Figure 1, main text)
In section S10 we showed that implies that CAR C is larger than CAR D , which is not consistent with experimental data. Accordingly, the corresponding cell in Figure 1 of the main text is shaded.  Figure 1 of the main text.