The 8-amino and 9-hydroxy substituents of antimalarial cinchona alkaloids have the erythro orientation while their inactive 9-epimers are threo. From the X-ray structures a 90° difference in torsion angle between the N1-H1 and C9-O12 bonds in the two series is believed to be important. In order to kill the malaria parasite, alkaloids must cross the erythrocyte and parasite membranes to accumulate in the acid digestive vacuole where they prevent detoxication of haematin produced during haemoglobin breakdown.
Ionization constants, octanol/water distribution and haematin interaction are examined for eight alkaloids to explain the influence of small structural differences on activity.
Erythro isomers have a high distribution ratio of 55:1 from plasma to the erythrocyte membrane, while for the more basic threo epimers this is only 4.5:1. This gives an increased transfer rate of the erythro drugs into the erythrocyte and thence into the parasite vacuole where their favourable conformation allows interaction with haematin, inhibiting its dimerization strongly (90 ± 7%) and thereby killing the parasite. The threo compounds not only enter more slowly but are then severely restricted from binding to haematin by the gauche alignment of their N1-H1 and C9-O12 bonds. Confirmatory molecular models allowed measurement of angles and bond lengths and computation of the electronic spectrum of a quinine-haematin complex.
Differences in the antiplasmodial activity of the erythro and threo cinchona alkaloids may therefore be attributed to the cumulative effects of lipid/aqueous distribution ratio and drug-haematin interaction. Possible insights into the mechanism of chloroquine-resistance are discussed.
Aryl amino alcohol alkaloids from the bark of Cinchona spp. have played an invaluable role in the treatment of malaria since the 18th century when the extract became widely used. The bark contains laevorotatory (-) quinine (6'-methoxy cinchonan-9-ol) (Q), its diastereomer dextrorotatory (+) quinidine (QD), and their 9-epimers epiquinine (EQ) and epiquinidine (EQD). Also present are (-) cinchonidine (des methoxyquinine) (CD), its diastereomer (+) cinchonine (C) and their 9-epimers epicinchonidine (ECD) and epicinchonine (EC). With the purification of Q as the major alkaloid, it remains the main therapeutic drug for severe and complicated Plasmodium falciparum malaria . In addition to the above alkaloids, all of which contain the exocyclic unsaturated vinyl group attached to C3 of the quinuclidine ring, Cinchona bark produces the analogous dihydro compounds of which hydroquinine (HQ) and its diastereomer hydroquinidine (HQD) are examples, in which the exocyclic double-bond has been reduced to the saturated ethyl group (figure 1).
Q and other aryl amino alcohols competitively inhibit  morphological changes caused by chloroquine (CQ) [3, 6] in digestive vacuoles (lysosomes) of the erythrocytic parasite where haemoglobin is digested. All these agents become concentrated in infected erythrocytes , and, as weak membrane-soluble bases, are believed to accumulate in the acidic digestive vacuole contents where they undergo protonation . They are thought to cause the death of the intraerythrocytic malaria parasite by binding to toxic haematin [6, 7] released during haemoglobin digestion, preventing its dimerization to non-toxic malaria pigment (haemozoin, β-haematin ).
In chemical structure, cinchona alkaloids consist of a conjugated heterocyclic quinoline ring, joined to a bulky rigid bicyclo heterocyclic aliphatic quinuclidine ring by an alcoholic carbon, C9. This is one of four chiral centres in these molecules; only C8 and C9 vary in configuration, while C3 and C4 on the aliphatic ring (respectively rectus, R and sinister, S), are erythro and invariant. Centres C8 and C9 are S and R in Q and R and S in QD, both erythro. Their 9-epimers are 8 S and 9 S in EQ and 8 R and 9 R in EQD, both threo. (figure 1) In 1938 it was reported from the Wellcome Laboratories of Tropical Medicine, London, that EQ and EQD had little activity against experimental avian malaria . Further, a clinical study found in 1948 that while Q and QD effectively treated induced P. falciparum infections, QD was twice as potent as Q . Un-natural (8 R,9 S,3 S,4 R) HQ and (8 S,9 R,3 S,4 R) HQD synthesized at Hoffmann la Roche by Brossi's group were each as active in P. berghei infections in mice as natural HQ and HQD .
Before P. falciparum cultures  were available, EQ was confirmed inactive in short-term cultures of P. berghei. In agreement with the earlier clinical study, QD was four times more active than Q, and C three times more active than CD, emphasising the antiparasitic superiority of the dextrorotatory (+) alkaloids of the QD type .
In the synthetic aryl amino alcohols, however, erythro (1SR, 2RS)-mefloquine (WR 142,490) was only 2.5 times more active than the threo (1RS, 2RS)- racemate WR 177,602, and erythro WR 122,455 was 0.7 times as active as its threo racemate WR 165,355. This small difference in relative activities of erythro and threo mefloquine and WR 122,455, compared with the lack of activity of the threo epimers of the cinchona alkaloids, was attributed to the ease of rotation of the less bulky piperidine ring, which replaces the quinuclidine ring in these synthetic agents, around the link between the two relevant asymmetric carbon atoms (C-8 and C-9 in the cinchona alkaloids). As a result of these studies a drug receptor was proposed, comprising 2 planar areas accommodating the quinoline rings of Q and QD with, in between, an electropositive area in the plane and a raised electronegative area . The receptor concept was refined in 1981 on the basis of an experimental study of haematin interactions with aryl amino alcohol antimalarial drugs, and evidence for a co-ordination link between such drugs and haematin iron was obtained .
In 1990, using cultures of CQ-sensitive P. falciparum, Wesche & Black confirmed higher activity of (+)QD than (-) Q, and of (+) C than (-) CD . In addition, Karle et al.  found that, like (+) QD vs. (-) Q, (+) mefloquine was slightly more effective than (-) mefloquine against P. falciparum in vitro but this difference did not apply to enantiomers of aryl amino alcohols whose side-chains were more flexible than piperidine. They found in a further study that EQ and EQD were approximately 100 times less active than quinine and quinidine against chloroquine-sensitive P. falciparum in vitro while HQ and HQD were about 25% more active than Q and QD against CQ-sensitive P. falciparum in vitro .
One explanation for the much lower activity of EQ and EQD had been the suggested potential for intramolecular H-bonding between aliphatic N-1 and adjacent O-12 because of the shorter distance between these atoms , thereby reducing the ability of the drug to interact with a putative cellular receptor site. Crystallographic evaluation by Karle et al. revealed that the N-1 to O-12 distances in the epimers were similar to those of other active amino-alcohol antimalarials, and all showed potential for forming H-bonds with cellular constituents. However, the torsion angle between the N1-H1 and C9-O12 bonds showed a 90° difference between the erythro - and threo -alkaloids. These authors concluded that the main reason for the inactivity of EQ and EQD was that threo -alkaloids could not form intermolecular H-bonds with cellular receptor sites in the same direction as the active erythro -isomers . Recently Karle & Karle have compared the crystal structures of Q and QD with those of (-) and (+) mefloquine to derive a pharmacophore which defines the main features of the parasite's receptor .
Their findings are of considerable interest with respect to the proposed receptor for these drugs, haematin, which has a large planar area containing 4 N atoms co-ordinated with FeIII. The Q-haematin complex is formed by hydrophobic interaction between the conjugated rings and co-ordination between FeIII and the hydroxyl group on C-9 of Q . In water, solid haemin chloride (monomeric haematin) will readily dissolve in solutions of the antimalarially active erythro alkaloids Q, QD, CD and C to form benzene-soluble co-ordination complexes with α- and β-hemichrome absorption peaks around 600 and 490 nm. This does not occur with the inactive EQ . More recently, it has been shown that Q, but not EQ, inhibits spontaneous haematin dimerization to β-haematin in vitro .
We report below that haematin dimerization is also not inhibited by EQD and confirm the lack of activity of EQ and the activity of Q, QD, HQ, HQD, C and CD in this model system. We have also obtained reliable values for the octanol/water partition coefficients (Log P) and ionization constants (pKa values) of Q, QD, HQ, HQD, EQ, EQD, C and CD. This enables us to predict the log distribution ratio between water and lipid (logD) both at the physiological pH and at the acidic pH of the parasite lysosome, where haematin is released from haemoglobin, and to calculate the possible intralysosomal concentration ratios that would be expected, assuming the drugs are concentrated by hydrogen ion. These observations are discussed in the context of molecular modelling, in order to evaluate physicochemical and structural determinants of drug activity against chloroquine-sensitive and resistant P. falciparum. Our conclusions throw light on the prerequisites for activity in cinchona alkaloids, the nature of the haematin-quinine interaction, and the mechanism of chloroquine-resistance.
The accumulation of an ionizable drug in cells is determined (apart from special transport mechanisms) by two major parameters: the partition coefficient P between the aqueous and the lipid phase, usually expressed as log P (measured in the octanol-water system for the un-ionized compound), and its modification by the ionization constant(s) (pKa) of the drug. For partially ionized compounds, the partition coefficient between 2 phases at any fixed pH is called the distribution coefficient D, usually expressed as log D; this assumes that only the un-ionized species partitions from the aqueous to the organic phase . The difference between log P and log D is, therefore, determined by the percentage of the drug ionized at different pH values and is given by the standard equation for a base . When the difference between pKa and pH is more than 1.0 log unit, it has been shown  that one can merely subtract this difference from log P to obtain log D. This makes it possible to calculate log D  from a knowledge of log P, pKa and pH. The availability of the ClogP programme, developed by C. Hansch and A. Leo at Pomona College, California, as part of the Pomona College Medicinal Chemistry Project, has now provided a useful method of computing approximate log P values for compounds of known structure. Values may sometimes be unreliable since differences due to conformational isomerism are not detected .
In the case of a drug possessing two basic centres, as quinoline antimalarial drugs all do, the correction needed in log P to obtain log D at a given pH involves the additive contribution of both ionized species, and the base equation  must be modified: [26, 28].
Equation (1) for a diacidic base
Log D at the physiological pH of 7.4 was obtained experimentally from the plot of log D versus pH. This was compared with the calculated value of Log D, obtained from Log P, using equation (1). Log D at the probable pH of the digestive vacuole or lysosome was calculated from log P by substituting pH 5.2  for 7.4 in equation (1). The application of equation (1) provides a useful rapid method of checking measured values of log D, and of calculating these values from log P when unknown. It should be noted that for diacidic bases, pKa1 > pKa2.
Vacuolar accumulation ratios
To calculate the vacuolar accumulation ratios (VAR) that weakly basic lipophilic drugs might achieve by virtue of the pH difference between lysosome and external medium or plasma , the equation of Henderson & Hasselbach was used in the following form .
Dissociation constants and partition coefficients were determined by Robertson Microlit Laboratories (Madison, New Jersey, USA) at 25°C using the Sirius GLpK automated computerized potentiometric system . Titration employed water containing 0.15 N KCl in an argon atmosphere. The pKas were determined in triplicate with a S.E. of ± 0.20.
Partition coefficients between octan-1-ol and water were measured by dual-phase potentiometric titration using various amounts of water-saturated octanol. Titrant addition was carried out with vigorous stirring of the assay solution. Three different ratios of octanol/water were employed for each compound. The log P values were obtained from the difference between the aqueous pKa of the species and the apparent pKa determined from the dual phase titration . Measurements were carried out in triplicate with a S.E. of ± 0.40. The potentiometric method was validated by comparison with results obtained by the standard shake-flask technique .
The drug lipophilicity values at pH 7.4 and 5.2 (log D) are the octanol/water distribution ratios, and were obtained from the plot of log P versus pH. Calculated log D values for pH 5.2, representing the conditions in the lysosome, and for log D at pH 7.4, were obtained using equation (1).
β-Haematin Inhibitory Activity: BHIA
The method was slightly modified from Parapini et al. . The 8 M sodium acetate/acetic acid buffer (pH 5.0) was prepared as follows:
Make up solid sodium acetate to 8 M with warm water (continue to heat at 37°C in a water bath while mixing: it will not dissolve completely) and then adjust the pH to 5.0 with 8 M acetic acid at 37°C, which leads to the remaining solid dissolving).
One hundred μL was dispensed into wells of a microtitre plate, and 50 μL of alkaloid hydrochloride or sulphate solution in water, at 80 mM, or water alone, was added. Verapamil (VE) and promethazine (PMZ) (Sigma Chemicals, Poole, UK) were used as hydrochlorides. CQ (Sigma, UK) was used as the diphosphate. Addition of 50 μL of an 8 mM α-chlorohemin (Sigma, UK) solution in DMSO to each well started the experiment.
Experiments were allowed to run at 35°C for 24 hr., done in triplicate and repeated on three occasions. The final DMSO concentration in this system was 25% w/v, while final haematin concentration (0.4 μmol/well) was 2 mM, and the final drug concentration (4.0 μmol/well) was 20 mM. Drug/haematin molar ratio was 10:1.
Then insoluble β-haematin and haematin aggregate were pelleted by centrifugation at 3000 g for 20 min at room temperature (22°C) and the supernatant discarded. Two hundred μL of DMSO were added to each pellet to dissolve un-reacted haematin and centrifugation was repeated. After discarding the supernatant, 200 μL of 0.2 M NaOH were added to convert the β-haematin pellet to soluble alkaline haematin. For each well the alkaline haematin was quantified by serial twofold dilution in water and OD measurement at 414 nm. The effect of each agent on β-haematin production was recorded compared with the control (water + DMSO + acetate buffer alone).
Each mono-protonated cinchona alkaloid structure was built in HyperChem Release 7 for Windows (Hypercube Inc. Gainesville, Florida) using a molecular mechanics procedure under MM+  with bond-length restraints for N1-O12 and N1 to C5' distance  (bond dipoles, no cut offs employed). The geometry was optimised to an rms (root mean square) gradient of 0.001 in vacuo (Polak-Ribière method). A periodic box, 15 × 15 × 15Å around the drug was then set up, containing 112 water molecules. The system was optimised in MM+ using switched cut-offs (outer 7.5 and inner 3.5 Å), to an rms gradient of 0.5. Then a molecular dynamics programme was run for 1 ps, with 0.001 ps steps, relaxation time 0.1 ps, to a simulation temperature of 300 K. This was followed by MM+ geometry optimisation to an rms gradient of 0.2. The molecular dynamics run was repeated and a further MM+ protocol was carried out to a gradient of rms 0.004 on the selected drug.
Angles and bond-lengths were measured on the models, and the dipole moments were determined after removal of the N-1 proton, using the semi-empirical PM3 programme [35, 36] in singly-excited configuration interaction. (RHF [Restricted Hartree-Fock], charge 0, spin multiplicity 1, lowest state, orbital criterion, five occupied and five unoccupied orbitals.)
Models of the Q-haematin and EQ-haematin complexes were prepared in MM+ using the structures of Q and EQ optimised in water (see above), with a co-ordination link between haematin iron and drug O-12. The crystal structure of α-chlorohaemin  was used for the initial haematin monomer model.
The semi-empirical quantum mechanics program ZINDO/S  in singly excited configuration interaction was used to calculate the expected electronic spectrum of the optimised 1: 1 quinine/haematin complex in benzene. One molecule of singly protonated quinine, co-ordinated to one molecule of haematin, with carboxylic acid groups undissociated, was given an arbitrary charge of +1 and spin multiplicity of four. Fifteen occupied and 15 unoccupied orbitals were included in the calculation.
(EQ and EQD were kindly supplied by Dr. Reinecke of Buchler GmbH, D-38110 Braunschweig, Germany.)
Q and QD, C and CD are reported to have very similar pKa1 values (8.4 ± 0.4) for the quinuclidine aliphatic N (table 1 : footnotes). A value of 8.5 ± 0.15 (table 1) was found, with a slight increase in HQ and HQD due to the conversion of the electron-attracting vinyl group to an ethyl group. All the cinchona alkaloids in table 1 had very similar pKa2 values (4.25 ± 0.15) for the more weakly basic quinoline aromatic N. The only previous determinations  for the threo compounds EQ and EQD were carried out in 80% methyl-cellosolve/20% water and showed both 9-epimers to have higher pKa values than Q and QD in the same solvent. Results obtained in water (Table 1) confirmed this finding and revealed a consistent ΔpKa1 of 1.1 units between the two solvent systems, and of 1pKa unit between the erythro and the threo isomers. Experimental values for log P are shown in table 1 as are the figures calculated using the ClogP programme. The two sets are in generally good agreement, and show the expected slight increase (+0.4) with the dihydro compounds (HQ and HQD) and decrease (-0.3) with the loss of the 6-methoxy group (C, CD). Experimental measurement of log D vs pH over the range 3–11 permitted the determination of log D at pH 7.4. In addition knowledge of log P and pKa allowed the calculation of log D at pH 7.4 using equation (1). Measured and calculated figures (table 1) are in good agreement, while reported values (table 1 : footnotes) show a wider variation. Results for log D at pH 7.4 for the erythro compounds fall in the range 1.50–1.97 (mean 1.74 ± 0.24) while the threo isomers (range 0.55–0.76) show a mean of 0.66 ± 0.11. In view of the acidic milieu (pH 5.2) existing in the vacuole within the lysosomal membrane, log D at pH 5.2 was also measured and calculated and the figures are shown in table 1. The comparison of calculated results for these agents at pH 5.2 with those obtained for chloroquine (CQ) is of interest, since because of CQ's two high pKa values [43, 44], the drop in pH had a much larger influence than on Q, QD, EQ or EQD. The drop in log D between pH 7.4 and pH 5.2 for Q, QD, EQ and EQD was ~2 logs while for CQ it was > 4 logs.
Physicochemical parameters of Cinchona alkaloids. cpd. = compound, VAR = calculated vacuolar accumulation ratio. S = chloroquine-sensitive, R = chloroquine-resistant. S/R = ratio of IC50 values. BHIA = % inhibition in β-haematin inhibition test
Log D (pH7.4)
Log D (pH5.2)
IC50 nM S
IC50 nM R
BHIA % inhib.
84 ± 1
85 ± 9
93 ± 0.6
95 ± 0.4
94 ± 0.5
94 ± 0.4
2 ± 13
12 ± 12
81 ± 15
0 ± 21
15 ± 22.5
a : Lit 8.43, 8.4, 8.05, 8.59. In 80% methylcellosolve/20% water 7.73, 7.25; b : Lit. 4.34,4.32 , 4.33 . In 80% methylcellosolve 2.81, 2.38; c : Lit. 3.44, 2.64; d : Calc. from CLogP; e : Lit. 1.90, 2.14, 2.54, 1.73(pH7.0); f : Calc from eqn. (1).; g : Lit. 8.79, 7.95, 8.78. In 80% methylcellosolve 7.95 7.36; h : Lit. 4.34, in 80% methylcellosolve 2.66, 2.40; i : Lit: 2.88, 3.71; j : Lit. 2.03, 2.07,2.11, 2.22; k : Lit: -0.07; l : Lit. 8.87; m : Lit. 8.35, 8.32. In 80% methylcellosolve 7.36; n : Lit. 4.28, In 80% methylcellosolve 2.80, 2.15; o : Lit 2.82; p : Lit 1.85; q : Lit. 8.40, 8.30; r : Lit. 4.17. In 80% methylcellosolve 2.66, 2.12; s : Lit. 2.68; t : Lit. 1.73; u : Lit. In 80% methylcellosolve 8.44; v : Lit. in 80% methylcellosolve 8.32.
The parasite lysosome, which has been proposed as the main site of drug uptake, has an acidic pH. Assuming drug distribution reaches an equilibrium state, and only uncharged drug can pass through membranes, it is argued that the ratio between concentration of drug outside the erythrocyte and the concentration in the lysosome depends on the difference between [H+] of the medium in vitro or plasma in vivo (pH 7.4), and the lysosomal compartment, according to the Henderson-Hasselbach equation . Both the water/lipid partition (log P) and the acid dissociation constant(s) (pKa) of a drug influence the passage of drug into the lysosome and its trapping by protonation. Trapping of a drug in the acidic vacuole is enhanced if the base, like chloroquine (CQ), can accept more than one proton at the lysosomal pH . Calculated CQ concentrations within the infected erythrocyte approximate to measured uptake values, but the more lipophilic agents like amodiaquine (AQ) achieve significantly greater levels than calculated, indicating that other factors are involved in the concentrative uptake of the drug . Calculated lysosomal uptake ratios (vacuolar accumulation ratio: VAR, the ratio of calculated drug concentration in the lysosomal water to concentration in the external medium water at equilibrium) for cinchona alkaloids, for resistance-reversers VE and PMZ, and for CQ are given in table 1. The most interesting finding here was the small variation in calculated VAR seen among the eight alkaloids examined (mean: 167.8 ± 7.4 [SD]). The minor variation reflects slight differences in the protonation of the aromatic N at pH 5.2, while the aliphatic N is 100% protonated in all the alkaloids examined.
A 10 fold molar excess of Q, QD, HQ, HQD, C, CD, or CQ inhibited the BHIA haematin dimerization reaction. Significant inhibition was not seen with EQ, EQD, VE or PMZ. (Table 1, figure 2). These results fit in with the reported high activity of CQ  and the erythro cinchona alkaloids on P. falciparum and the low activity of EQ, EQD [15, 46], PMZ  and VE .
As pointed out by Karle and co-workers, [15, 46], the N1-H1 bond in 8, 9 threo -alkaloids consistently points in the same direction as the C9-O12 bond, while in the erythro -alkaloids they are at right angles (see the last column of table 2). The calculated dipole moment and proton affinity of each active alkaloid are lower than that of its 9-epimer, as first demonstrated by Karle & Bhattacharjee . This is confirmed by our finding of higher pKa1 values for the 9-epimers (table 1). Dipole moments and proton affinities calculated here consistently differ from values in ref. , probably because we used a semi-empirical configuration interaction calculation rather than an ab initio approach.
Electronic and structural parameters of cinchona alkaloids in aqueous solvent, based on the common pharmacophores described for (-) mefloquine and quinine and (+) mefloquine and quinidine by Karle & Karle . (*dipole moment calculated for free base).
drug in water
MM+ energy <0.004
Space-filling models of the cinchona alkaloids orientated to face a putative receptor are shown in figure 3.
A Q-haematin co-ordinated model was calculated using MM+, in comparison with EQ-haematin. The results of the ZINDO/S calculation of the electronic absorption spectrum of Q-haematin are compared with experimental observations in table 3 below. Differences in the orientation of the N1 proton with respect to the quinoline and porphyrin rings are shown in figure 4 below.
1:1 Quinine-haematin complex: Observed and calculated values of electronic absorption peaks and peak strengths. (* Peak strength normalised by taking γ peak strength as 1.0.)
α peak (nm)
β peak (nm)
γ peak (nm)
α peak * strength
β peak* strength
γ peak* strength
The cinchona alkaloids have an aromatic N with a pKa of 4.25 ± 0.15. At pH 7.4 the aromatic N is 100% un-ionized and makes its maximum contribution to lipophilicity. In the active erythro alkaloids, with partial protonation of the aliphatic N, this results in a log D at pH 7.4 of 1.74 ± 0.25 corresponding to a favourable mean membrane transfer ratio into the lipid phase of 55:1. This is applicable to the transfer of drug from blood plasma water to erythrocyte and subsequent membranes. For the (weakly active) threo epimers, the higher pKa of the aliphatic N, which is now 100% ionized and does not contribute to transfer into the lipid phase, reduces log D at pH 7.4 by one magnitude to 0.66 ± 0.1, corresponding to a decreased membrane transfer ratio of only 4.5: 1. It is known that CQ and other 4-aminoquinoline antimalarials need to reach a high concentration within the lysosome of the CQ-sensitive parasite in order to inhibit dimerization of the enclosed haematin , and this is also assumed to apply to cinchona alkaloids. The rise in pKa1 values observed for the inactive 9-epi alkaloids will therefore lower the rate at which that effective concentration is reached. The acidic environment of the vacuole (pH 5.2) results in substantial ionization (~40%) of the aromatic N of the cinchona alkaloids, with enhanced water solubility as shown by the marked drop in log D (table 1), so that all eight compounds are trapped there, causing their concentration to increase in the low pH milieu.
The absence of significant inhibition of β-haematin formation by both 9-epi alkaloids tested strengthens the link between 8, 9 erythro configuration in cinchona alkaloids and antiplasmodial activity, and indicates that reaching a high concentration in the lysosome, though necessary, is not sufficient in itself to confer antiplasmodial activity. Once a drug is in the lysosome, it must be able to interact with haematin, or with other vital functions like the proteolysis of haemoglobin , in order to exert a toxic effect.
Reported molecular electronic properties  of Q and QD and their epimers are in agreement with measured pKa values here. In Q and QD the positive electric field of the aliphatic N1 proton is directed towards the quinoline ring, and the dipole moment of the drug (unprotonated) is reported as 2.2 Debye or less, while in the 9-epimers EQ and EQD the field is directed towards the solvent and the dipole moment is higher. N1 in the erythro -alkaloids is in a position to interact with equatorial protons of the quinoline ring (C5' is only ~4 Å away). In the models the bulky quinuclidine group finds difficulty in rotating close to the quinoline ring in the threo -alkaloids; the O-12 is interposed, and the N1-C5' distance is up to 4.7 Å (table 2), see also [15, 46]. The probable reason for the higher proton affinity of the N1 and lower logD in the threo alkaloids is that the quinoline ring has less influence on N1 because of these steric factors, which lead to higher values of pKa (easier protonation) of N-1. This will also enhance interaction (H-bonding) with solvent water in the threo compounds.
The Q-haematin complex
Some features of the modelled drug-haematin complexes are of interest. The Fe-O12 bond length is 1.846 Å in Q-haematin and 1.842 Å in EQ-haematin which can be compared with 1.847 Å for the O-Fe distance in the high-spin co-ordination complex with a phenoxide . The minimum distance between the carbon atoms of the quinoline ring in the drug, and those in the porphyrin ring was 3.1 Å in both models. This would allow Van der Waals interactions between the conjugated rings for both Q-haematin and EQ-haematin.
The H1-Fe distance in Q-haematin is 4.6 Å, while in EQ-haematin it is 3.9 Å, Proximity of positive charges of N1 proton and iron in EQ-haematin, and the higher value of the proton charge  might make co-ordination complex formation more difficult than for Q-haematin. The more solvated, more positive, epimer N-1 proton may interfere with the initial hydrophobic interactions of the quinoline and porphyrin rings, affecting co-ordination of O-12 to haematin iron. The importance of solvent water interaction with protonated N1 in inhibiting hydrophobic binding to haematin is suggested by the observation that in 40% DMSO both Q and EQ interact similarly with haematin . The direction of the positive electric field of protonated N1 determines the higher pKa and solvent interaction and is likely to be, as suggested by Karle and colleagues [15, 48], the crucial difference between the inactive and active cinchona alkaloids, since this difference in direction, shown in figure 4, alters both the stereochemistry of interactions between Q or EQ and haematin and increases the solvation and charge of N1-H1 in EQ. These considerations are supported by the lesser effect of the threo/erythro distinction on the activity of mefloquine congeners and phenanthrene methanols (WR 122,455) where the piperidine ring is more easily able to rotate close to the quinoline ring.
The peak wavelengths and strengths of the calculated electronic absorption spectrum compare well with the observed spectrum of the aqueous complex, extracted into benzene, reported earlier  (table 3). Despite the fact that, in view of the discrepancy in wavelength and strength of the α-peak, the structure is not yet completely correct, the α- and β-peaks support the formation of a hydrophobic quinine-haematin co-ordination complex while the strong γ-peak at 408 nm indicates haematin iron in a high-spin state, as expected from the putative structure. This is similar to α-chlorohemin , where FeIII is 0.45 Å above the plane of the porphyrin ring. It appears unlikely that the quinoline ring interacts with the other side of the haematin ring structure, as has been proposed for the quinoline ring of CQ  since a high spin co-ordination complex could not then be formed.
In summary, positive correlations between antiplasmodial activity and drug lipophilicity have been reported [43, 45, 47] for a number of 4-aminoquinoline drugs related to CQ and amodiaquine (although not for the cinchona alkaloids) as well as for some aminothiol  and hydroxamate  chelators. In addition, a proportionality between activity and inhibition of formation of β-haematin has been recognized for 4-aminoquinolines  and also for Q and QD , The present report, however, suggests that the differences in the antiplasmodial potency of the erythro and threo cinchona alkaloids on CQ-sensitive isolates of P. falciparum may be accounted for as the cumulative consequences of their log D (pH7.4) and BHIA values. The availability of these two critical parameters within this highly homogeneous group of drugs (table 1) makes it possible to apply a simple test of their sequential and combined effects: The ratio of the mean log D (pH 7.4) for the erythro compounds (1.74 ± 0.24) to that of the threo compounds (0.66 ± 0.11) is 12:1. The BHIA values for the 2 series are 90 ± 7% and 7 ± 4%, with a similar ratio of 12.5:1. Since these effects are cumulative, their combined result would raise the mean IC50 value for the threo series to 150 (ie 12 × 12.5) times that of the erythro group. With an experimental mean IC50 of the erythro alkaloids of 26.7 nM, the calculated reduction of activity of the threo alkaloids leads to an expected mean IC50 of (26.7 × 150) ie 4.0 μM, while the observed experimental value is 3.1 ± 0.4 μM. Alternatively the enhancement in activity of the erythro series would lead to an IC50 of 3085/150 ie 20.6 nM; the experimental mean for the 6 erythro compounds is 26.7 nM.
The agreement between the calculated and experimental values of IC50 is noteworthy.
These observations may provide a useful platform for the further investigation of antiplasmodial agents, particularly in the climate of increased drug-resistance.
Lessons for the interpretation of CQ-resistance mechanisms
Our arguments so far have concentrated on the probable role of hydrophobicity (logD) and inhibition of haematin dimerization as the determinants of antiplasmodial activity among eight cinchona alkaloids. Clearly these factors are also important in activity against CQ-resistant isolates. However, a further parameter, interaction with the CQ-resistance mechanism, must also be considered. Entry to the cell, concentration in the acidic lysosomal vacuole, and interaction with haematin have all been postulated as the site of the resistance mechanism. Arguments [61, 77, 78] and evidence [51, 79, 85] for and against drug efflux have not yet reached a consensus. Nevertheless, the most important mutations associated with CQ-resistance are in the genes coding for two intrinsic proteins in the membrane of the lysosome [80, 56] where quinoline drugs are believed to be concentrated.
One of these, PGH-1, is a protein of the multidrug-resistance type , where over-expression is associated with resistance to highly hydrophobic drugs such as mefloquine, halofantrine and artemisinin [82–84]. The other, is PfCRT, where a Lys76Thr mutation is 100% associated with CQ-resistance in vitro . In clinical studies, inability to detect the mutant allele predicts treatment success . Replacement of the wild type pfcrt gene in sensitive clones by the resistance-associated allele from strains of Asian or South American origin confers CQ-resistance, accompanied by reduced uptake of the drug, and the acquisition of the characteristic chemosensitizing effect of VE . The detailed structure of the PfCRT protein is still under investigation. In its topology the sequence resembles the aqueous chloride channel of Salmonella typhimurium and a range of other organisms , though 3 of the 4 specific ion-binding motifs are modified. Modelling (work in progress) reveals that the side chain of residue 76 may project into an access pore leading from the lysosomal content. In the wild type (Lys76) this is positively charged, which would attract anions and repel cations. Mutated to neutral Thr76, cations would find access easier. PfCRT has recently been expressed efficiently in yeast Pichia pastoris by Roepe's research group , and functionally appears to be a membrane channel which, when mutated, may mediate chloride-dependent, VE-inhibitable drug transport. Thus PfCRT could indeed be an anion channel, and by varying the entrance or exit of charge-balancing chloride anion may modulate lysosomal pH [87, 88]. This might affect the trapping of basic drugs , or interfere with drug-haematin binding . Doubt has been cast  on the validity of pH determinations underlying the argument in refs 86–88. These pH effects are perhaps rendered unlikely by the stereospecific interactions of the physico-chemically similar diastereomers Q and QD with mutated PfCRT [58, 90]. It is more likely that CQ itself passes through modified PfCRT, since the high drug and proton concentration in the lysosome present a formidable pressure for efflux, given a suitable channel.
For 4-aminoquinolines, the positive correlation between hydrophobicity and relative activity in CQ-resistant isolates, where the more hydrophobic AQ and its metabolite DAQ show significant activity  is an important finding. This observation is paralleled by the CQ-sensitising effect, on CQ-resistant isolates, of lipophilic weak bases like VE and PM [52, 53]. We are investigating a link between polymorphism in PfCRT, resistance reversal by VE, and the clinical efficacy of AQ (in press).
VE and PM show no significant interaction with the haematin polymerisation process (table 1, figure 2) and they have little intrinsic antiplasmodial action. Combining our recent data on 4-aminoquinolines  and the erythro cinchona alkaloids studied here there is a significant correlation between hydrophobicity and log relative activity in CQ-resistant parasites, extending the observations of Bray et al.  (figure 5).
It has been proposed [61, 73] that 4-aminoquinolines which are more polar at lysosomal pH (such as CQ) are more likely to pass through the mutated PfCRT channel and escape from the lysosome into the parasite cytoplasm, while those which are more hydrophobic (AQ and its active metabolite DAQ) are likely to bind to the hydrophobic channel lining and stay inside. Their positive charges repel the further access of 4-aminoquinoline to the channel. The high hydrophobicity in acid of VE and PMZ (see table 1) would allow them to bind in a mutated PfCRT channel and their positive charge would prevent efflux of CQ and related drugs. Molecular computations on CQ-resistance reversing agents have indeed determined that their common features are hydrophobicity and electronegativity (i.e. proton attraction) .
Pathogen Molecular Biology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine
Department of Pharmaceutical Chemistry, University of California
Gilles HM: Management of severe and complicated malaria, a practical handbook.World Health Organisation GENEVA 1991, 56.
Warhurst DC, Thomas SC: Pharmacology of the malaria parasite.Biochem Pharmacol 1975, 24: 2047–56.View ArticlePubMed
Warhurst DC, Hockley DJ: The mode of action of chloroquine onPlasmodium bergheiandP. cynomolgi.Nature 1967, 214: 935–6.View ArticlePubMed
Fitch CD: Plasmodium falciparumin owl monkeys: drug resistance and chloroquine binding capacity.Science 1970, 169: 289–90.View ArticlePubMed
Homewood CA, Warhurst DC, Peters W, Baggaley VC: Lysosomes, pH and the anti-malarial action of chloroquine.Nature 1972, 235: 50–2.View ArticlePubMed
Chou AC, Chevli R, Fitch CD: Ferriprotoporphyrin IX fulfils the criteria for identification as the chloroquine receptor of malaria parasites.Biochemistry 1980, 19: 1543–1549.View ArticlePubMed
Pagola S, Stephens PW, Bohle DS, Kosar AD, Madsen SK: The structure of malaria pigment beta-haematin.Nature 2000, 404: 307–10.View ArticlePubMed
Buttle GAH, Henry TA, Solomon W, Trevan JW, Gibbs EM: The action of the cinchona and certain other alkaloids in bird malaria.Biochemistry 1938, 32: 47–58.
Taggart JV, Earle DP, Berliner RW, Zubrod CG, Welch WJ, Wise NB, Schroeder EF, London IM, Shannon JA: Studies on the chemotherapy of the human malarias. III. The physiological distribution and antimalarial activity of the cinchona alkaloids.J Clin Invest 1948, 27 (Suppl) : 80–86.View Article
Trager W, Jensen JB: Human malaria parasites in continuous culture.Science 1976, 193: 673–5.View ArticlePubMed
Warhurst DC: The quinine-haemin interaction and its relationship to antimalarial activity.Biochem Pharmacol 1981, 30: 3323–3327.View ArticlePubMed
Wesche DL, Black J: A comparison of the antimalarial activity of the cinchona alkaloids againstP. falciparumin vitro.J Trop Med Hyg 1990, 93: 153–159.PubMed
Karle JM, Olmeda R, Gerena L, Milhous WK: Plasmodium falciparum: Role of absolute stereochemistry in the antimalarial activity of synthetic amino alcohol antimalarial agents.Exp Parasitol 1993, 76: 345–351.View ArticlePubMed
Karle JM, Karle IL, Gerena L, Milhous WK: Stereochemical evaluation of the relative activities of the cinchona alkaloids againstPlasmodium falciparum.Antimicrob Agents Chemother 1992, 36: 1538–1544.PubMed
Oleksyn BJ, Suszko-Purzycka A, Dive G, Lamotte-Brasseur J: Molecular properties of Cinchona alkaloids: a theoretical approach.J Pharm Sci 1992, 81: 122–127.View ArticlePubMed
Karle JM, Karle IL: Crystal structure of (-)-mefloquine hydrochloride reveals consistency of configuration with biological activity.Antimicrob Agents Chemother 2002, 46: 1529–1534.View ArticlePubMed
Behere DV, Goff HM: High affinity binding of quinine to iron (III) porphyrin: novel formation of alkoxide complexes from alcohols and amines.J Am Chem Soc 1984, 106: 4945–4950.View Article
Egan TJ, Ross DC, Adams PA: Quinoline anti-malarial drugs inhibit spontaneous formation of beta-haematin (malaria pigment).FEBS Lett 1994, 352: 54–57.View ArticlePubMed
Kubinyi H: Lipophilicity and drug activity.in Progress in Drug Research(Edited by: Jucker E). 1979, 23: 97–198. Birkhauser (Basel)
Rekker RF: The hydrophobic fragmental constant. Its derivation and application.in The Hydrophobic Fragmental Constant(Edited by: Nauta WT, Rekker RF). 1977, 1: 1–24. Elsevier, (Amsterdam)
Hansch C, Leo A: Exploring QSAR: Fundamentals and Applications in Chemistry and Biology".American Chemical Society, Washington, DC 1995, 1: 120.
Scherrer RA, Howard SM: Use of distribution coefficients in quantitative structure-activity relationships.J Med Chem 1977, 20: 53–58.View ArticlePubMed
Brossi A, Uskokovic M, Gutzwiller J, Krettli AU, Brener Z: Antimalarial activity of natural, racemic and unnatural dihydroquinine, dihydroquinidine and their various racemic analogs in mice infected withPlasmodium berghei.Experientia 1971, 27: 1100–1101.View ArticlePubMed
Leo AJ, Hansch C: Role of hydrophobic effects in mechanistic QSAR.in Perspectives in Drug Discovery and Design(Edited by: Martin YC). 1999, 17: 1–25.
Van der Waterbeemd H, Testa B: The parametrization of lipophilicity and other structural properties in drug design.Advances in Drug Research 1987, 16: 85.
Yayon A, Cabantchik ZI, Ginsburg H: Identification of the acidic compartment ofPlasmodium falciparum-infected human erythrocytes as the target of the antimalarial drug chloroquine.EMBO J 1984, 3: 2695–700.PubMed
Krogstad DJ, Schlesinger PH: A perspective on antimalarial action: effects of weak bases onPlasmodium falciparum.Biochem Pharmacol 1986, 35: 547–552.View ArticlePubMed
Hawley SR, Bray PG, Park BK, Ward SA: Amodiaquine accumulation inPlasmodium falciparumas a possible explanation for its superior antimalarial activity over chloroquine.Mol Biochem Parasitol 1996, 80: 15–25.View ArticlePubMed
Avdeef A: pH-metric log P. 2: Refinement of partition coefficients and ionization constants of multiprotic substances.J Pharm Sci 1993, 82: 183–190.View ArticlePubMed
Chien YW, Lambert HJ, Karim A: Comparative binding of disopyramide phosphate and quinidine sulfate to human plasma proteins.J Pharm Sci 1974, 63: 1877–1879.View ArticlePubMed
Takacs-Novak K, Avdeef A: Interlaboratory study of log P determination by shake-flask and potentiometric methods.J Pharm Biomed Anal 1996, 14: 1405–13.View ArticlePubMed
Parapini S, Basilico N, Pasini E, Egan TJ, Olliaro P, Taramelli D, Monti D: Standardization of the physicochemical parameters to assess in vitrothe beta-haematin inhibitory activity of antimalarial drugs.Exp Parasitol 2000, 96: 249–56.View ArticlePubMed
Allinger NL: Conformational Analysis 130. MM2. A Hydrocarbon Force Field Utilizing V1 and V2 Torsional Terms.J Am Chem Soc 1977, 99: 8127–8134. (MM+ is a HyperChem modification and extension of Norman Allinger's Molecular Mechanics program MM2)View Article
Stewart JJP: Optimization of Parameters for Semi-empirical Methods. I. Method.J Comput Chem 1989, 10: 209. PM3 is JJP Stewart's reparametrization of the AMI neglect of diatomic differential overlap (NDDO) approximation described in the above publicationView Article
Stewart JJP: Optimization of Parameters for Semi-empirical Methods. II. Applications.J Comput Chem 1989, 10: 221.View Article
Koenig DF: The structure of β-chlorohemin.Acta Crystallogr 1965, 18: 663–673.View ArticlePubMed
Anderson WP, Edwards WD, Zerner MC: Calculated Spectra of Hydrated Ions of the First Transition Series.Inorg Chem 1986, 25: 2728–2732. ZINDO/S is Michael Zerner's intermediate neglect of differential overlap (INDO) quantum mechanical method for electronic spectra of molecular systems containing transition metals, discussed in the above article.View Article
Irwin JL, Irwin EM: Apparent dissociation exponents of quinine, pamaquine and a quinolylpiperidyl-carbinol; application of an extended pH scale.J Biol Chem 1948, 174: 577–587.
Perrin DD: Dissociation constants of organic bases in aqueous solution.Butterworth's London 1965.
Prelog V, Hafliger O: Über China-Alkaloide 9. Über den einflussder Konfiguration auf die Basizitat und über die relative Konfiguration an den Kohlenstoffatomen 8 und 9.Helv Chim Acta 1950, 33: 2021–2029.View Article
Mannhold R, Dross KP, Rekker RF: Drug lipophilicity in QSAR practice: I. A comparison of experimental with calculative approaches.Quant Struct-Act Relat 1990, 9: 21–28.View Article
Lullmann H, Wehling M: The binding of drugs to different polar lipids in vitro.Biochem Pharmacol 1979, 28: 3409–3415.View ArticlePubMed
Hansch C: in Comprehensive Medicinal Chemistry(Edited by: Hansch C). Pergamon press NY 1990, 2: 159.
Hawley SR, Bray PG, O'Neill PM, Park BK, Ward SA: The role of drug accumulation in 4-aminoquinoline antimalarial potency. The influence of structural substitution and physicochemical properties.Biochem Pharmacol 1996, 52: 723–733.View ArticlePubMed
Karle JM, Bhattacharjee AK: Stereoelectronic features of the cinchona alkaloids determine their differential antimalarial activity.Bioorg Med Chem 1999, 7: 1769–1774.View ArticlePubMed
Egan TJ, Hunter R, Kaschula CH, Marques HM, Misplon A, Walden J: Structure-function relationships in aminoquinolines: effect of amino and chloro groups on quinoline-haematin complex formation, inhibition of beta-haematin formation, and antiplasmodial activity.J Med Chem 2000, 43: 283–291.View ArticlePubMed
Moon RP, Bur D, Loetscher H, D'Arcy A, Tyas L, Oefner C, Grueninger-Leitch F, Mona D, Rupp K, Dorn A, Matile H, Certa U, Berry C, Kay J, Ridley RG: Studies on plasmepsins I and II from the malarial parasitePlasmodium falciparumand their exploitation as drug targets.Adv Exp Med Biol 1998, 436: 397–406.PubMed
Bominaar EL, Ding XQ, Gismelseed A, Bill E, Winkler H, Trautwein AX, Nasri H, Fischer J, Weiss R: Structural, Mossbauer and EPR investigations on 2 oxidation states of a five-co-ordinate, high spin synthetic heme. Quantitative interpretation of zero-field parameters and large quadrupole splitting.Inorg Chem 1992, 31: 1845–1854.View Article
Egan TJ, Mavuso WW, Ross DC, Marques HM: Thermodynamic factors controlling the interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX.J Inorg Biochem 1997, 68: 137–145.View ArticlePubMed
Krogstad DJ, Gluzman IY, Kyle DE, Oduola AM, Martin SK, Milhous WK, Schlesinger PH: Efflux of chloroquine fromPlasmodium falciparum: mechanism of chloroquine resistance.Science 1987, 238: 1283–1285.View ArticlePubMed
Oduola AM, Sowunmi A, Milhous W, Brewer TG, Kyle DE, Gerena L, Rossan RN, Salako LA, Schuster BG: In vitro and in vivo reversal of chloroquine-resistance inPlasmodium falciparumwith promethazine.Am J Trop Med Hyg 1998, 58: 625–629.PubMed
Martiney JA, Cerami A, Slater AFG: Verapamil reversal of chloroquine-resistance in the malaria parasitePlasmodium falciparumis specific for resistant parasites and independent of the weak base effect.J Biol Chem 1995, 270: 22393–22398.View ArticlePubMed
Bray PG, Hawley SR, Mungthin M, Ward SA: Physicochemical properties correlated with drug resistance and the reversal of drug resistance inPlasmodium falciparum.Mol Pharmacol 1996, 50: 1559–1566.PubMed
Bhattacharjee AK, Kyle DE, Vennerstrom JL, Milhous WK: A 3D QSAR pharmacophore model and quantum chemical structure-activity analysis of chloroquine-resistance reversal.J Chem Inf Comput Sci 2002, 45: 1212–20.
Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LMB, Sidhu ABS, Naude B, Deitsch KW, Su XZ, Wooton JC, Roepe PD, Wellems TE: Mutations in theP. falciparumdigestive vacuole transmembrane protein PFCRT and evidence for their role in chloroquine resistance.Molecular Cell 2000, 6: 861–71.View ArticlePubMed
Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, Coulibaly D: A molecular marker for chloroquine-resistant falciparum malaria.N Engl J Med 2001, 344: 257–63.View ArticlePubMed
Moreau S, Perly B, Chachaty C, Deleuze C: A nuclear magnetic resonance study of the interactions of antimalarial drugs with porphyrins.Biochim Biophys Acta 1985, 840: 107–16.PubMed
Schulman SG, Threatte RM, Capomachia AC, Paul WL: Fluorescence of 6-methoxyquinoline, quinine and quinidine in aqueous media.J Pharm Sci 1974, 63: 876–880.View ArticlePubMed
Tsai RS, Carrupt PA, Testa B, El Tayar N, Gruenewaald G, Casey A: Influence of stereochemical factors on the partition coefficient of diastereomers.J Chem Research 1993, 1901–1920.
Allen RI, Box KJ, Comer JEA, Peake C, Tam KY: Multiwavelength spectrophotometric determination of acid dissociation constants of ionizable drugs.J Pharm Biomed Anal 1998, 17: 699–712.View ArticlePubMed
Drzewiczak C, Suszko-Purzycka A, Skolik J: Correlation between structure and basicity of Cinchona alkaloids.Polish J Chem 1993, 67: 45–52.
Drzewiczak C, Zalewski RI, Skolik J, Suszko-Purzycka A: Spectrophotometric determination of the second dissociation constant of Cinchona alkaloids.Polish J Chem 1993, 67: 53–58.
Lepetit G: Die pH-abhängige Lipidlöslichkeit von Arzneistoffen.Pharmazie 1977, 32: 289–291.
Hansch C, Anderson SM: The effect of intramolecular hydrophobic bonding on partition coefficients.J Org Chem 1967, 32: 2583–2586.View Article
Cassidy SL, Lympany PA, Henry JA: Lipid solubility of a series of drugs.J Pharm Pharmacol 1988, 40: 130–132.PubMed
Unger SH, Chiang GH: Octanol-physiological buffer distribution coefficients of lipophilic amines and their correlation with biological activity.J Med Chem 1981, 24: 262–270.View ArticlePubMed
Warhurst DC, Steele JCP, Adagu IS, Craig JC, Cullander C: Hydroxychloroquine is much less active than chloroquine against chloroquine-resistantPlasmodium falciparum, in agreement with its physicochemical properties.J Antimicrob Chemother 2003, 52: 188–93.View ArticlePubMed
Loyevsky M, John C, Zaloujnyi I, Gordeuk V: Aminothiol multidentate chelators as antimalarials.Biochem Pharmacol 1997, 54: 451–8.View ArticlePubMed
Shanzer A, Libman J, Lytton SD, Glickstein H, Cabantchik ZI: Reversed siderophores act as antimalarial agents.Proc Natl Acad Sci U S A 1991, 88: 6585–6589.View ArticlePubMed
Kaschula CH, Egan TJ, Hunter R, Basilico N, Parapini S, Taramelli D, Pasini E, Monti D: Structure-activity relationships in 4-aminoquinoline antiplasmodials. The role of the group at the 7-position.J Med Chem 2002, 45: 3531–3539.View ArticlePubMed
Warhurst DC: Antimalarial schizontocides: why a permease is necessary.Parasitol Today 1986, 2: 331–334.View ArticlePubMed
Warhurst DC: Mechanism of chloroquine-resistance in malaria.Parasitol Today 1988, 4: 211–213.View ArticlePubMed
Bray PG, Howells RE, Ritchie GY, Ward SA: Rapid chloroquine efflux phenotype in both chloroquine-sensitive and chloroquine-resistantPlasmodium falciparum. A correlation of chloroquine sensitivity with energy-dependent drug accumulation.Biochem Pharmacol 1992, 44: 1317–1324.View ArticlePubMed
Cowman AF, Karcz S, Galatis D, Culvenor JG: A P-glycoprotein homologue ofPlasmodium falciparumis localized on the digestive vacuole.J Cell Biol 1991, 113: 1033–42.View ArticlePubMed
Karcz S, Cowman AF: Similarities and differences between the multidrug resistance phenotype of mammalian tumor cells and chloroquine resistance inPlasmodium falciparum.Exp Parasitol 1991, 73: 233–40.View ArticlePubMed
Duraisingh MT, Roper C, Walliker D, Warhurst DC: Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene ofPlasmodium falciparum.Mol Microbiol 2000, 36: 955–61.View ArticlePubMed
Price RN, Cassar C, Brockman A, Duraisingh M, van Vugt M, White NJ, Nosten F, Krishna S: Thepfmdr1gene is associated with a multidrug-resistant phenotype inPlasmodium falciparumfrom the western border of Thailand.Antimicrob Agents Chemother 1999, 43: 2943–9.PubMed
Waller KA, Muhle RA, Ursos LM, Horrocks P, Verdier-Pinard D, Sidhu AB, Fujioka H, Roepe PD, Fidock DA: Chloroquine resistance modulated in vitro by expression levels of thePlasmodium falciparumchloroquine resistance transporter (PfCRT).J Biol Chem 2003. June 17, 2003. [Epub ahead of print]
Ursos LM, DuBay KF, Roepe PD: Antimalarial drugs influence the pH dependent solubility of heme via apparent nucleation phenomena.Mol Biochem Parasitol 2001, 12: 11–17.View Article
Ursos LM, Dzekunov SM, Roepe PD: The effects of chloroquine and verapamil on digestive vacuolar pH ofP. falciparumeither sensitive or resistant to chloroquine.Mol Biochem Parasitol 2000, 110: 125–134.View ArticlePubMed
Dzekunov SM, Ursos LM, Roepe PD: Digestive vacuolar pH of intact intraerythrocyticP. falciparumeither sensitive or resistant to chloroquine.Mol Biochem Parasitol 2000, 110: 107–124.View ArticlePubMed
Bray PG, Saliba KJ, Davies JD, Spiller DG, White MR, Kirk K, Ward SA: Distribution of acridine orange fluorescence inPlasmodium falciparum-infected erythrocytes and its implications for the evaluation of digestive vacuole pH.Mol Biochem Parasitol 2002, 119: 301–304.View ArticlePubMed
Cooper RA, Ferdig MT, Su XZ, Ursos LM, Mu J, Nomura T, Fujioka H, Fidock DA, Roepe PD, Wellems TE: Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine resistance and unique stereospecific quinine and quinidine responses inPlasmodium falciparum.Mol Pharmacol 2002, 61: 35–42.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.