ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 312 (2003) 258–260 www.elsevier.com/locate/yabio
Notes & Tips
Applying malaria parasiteÕs heme detoxification system for screening potential antimalarial drugsq Dinkar Sahal,* R. Kannan, and V.S. Chauhan International Center for Genetic Engineering and Biotechnology, New Delhi 110 067, India Received 9 September 2002
Plasmodium’s ancient origin predisposes this parasite to have a wide spectrum of species variants, each with its own idiosyncrasies, compounding the problem of tackling it through drugs or vaccines [1]. Widespread resistance to chloroquine [2] and the diminishing prospects of one single drug vanquishing the parasite call for discovering novel drugs to tackle the menace of malaria. Methods reported for screening antimalarial drugs include microscopic procedures of scoring for parasitemia and radioisotopic hypoxanthine incorporation assay [3] in in vitro parasitzed red blood cells. These methods suffer from the use of animals, low throughput and labor intensiveness. The malaria parasite in its blood stage is a voracious consumer of the globin component of hemoglobin, which causes the release of copious amounts (>400 mM) of toxic heme [4]. However, the parasite withstands heme toxicity by employing efficient mechanisms of heme detoxification. The conversion of heme to the relatively benign heme polymer, hemozoin, is achieved by a process akin to template-mediated biomineralization [5]. Templates for heme polymerization include preformed hemozoin, histidine-rich proteins, and lipids [6]. Owing to the strategic need of heme detoxification steps for the survival of the parasite, it is not surprising that classical antimalarial drugs such as chloroquine and artemisinin, discovered much before the dawn of modern biology, also antagonize the heme polymerization pathway [7,8]. The nonenzymatic nature of the heme polymerization pathways is also probably a reason that development of resistance against drugs such as chloroquine and artemisinin has been relatively slow [9,10].
It is therefore expected that drug discovery methods that focus on the heme polymerization niche can identify those antimalarials that are both potent and likely to remain useful for a long time. However, there is virtually no method in use for screening drugs in the context of heme polymerization. A high-throughput method that claims to measure antimalarial potency in terms of a moleculeÕs ability to inhibit hemozoin formation suffers from the fact that, by not using the biological templates of heme polymerization, it relies on the slow unassisted heme polymerization [11]. As a result, the signal to noise ratio of the method is low. Here we report a simple in vitro, one-step, one-pot, microtiter-plate-based, high-throughput antimalaria drug screening method based on the fact that the red color of heme–PfHRP II1 (Plasmodium falciparum histidine-rich protein II) complex changes to green upon dissociation of the bound heme by a candidate antimalarial drug. As shown in Fig. 1A, this method involves the use of a mixture of heme and a recombinantly produced HRP II, to which a candidate drug is added and the change in color following a short incubation period is monitored. We have examined the potency of over a dozen compounds, which include quinoline, xanthone, imidazole, and pyrazine derivatives. The most potent antimalarials such as quinacrine and chloroquine also showed the highest potencies in the present assay (Fig. 1B). It is interesting to note that clotrimazole, an antifungal antibiotic that has been found to be anti1
q
The subject matter of this work has been filed as Patent No.705/ DEL/2002 on 1 July 2002 in the Indian Patent Office. * Corresponding author. Fax: +91-11-686-2316. E-mail address:
[email protected] (D. Sahal).
Abbreviations used: Amil, amiloride; Aq, amodiaquine; Cq, chloroquine, Clt, clotrimazole; Mq, mefloquine, PfHRP II, Plasmodium falciparum histidine-rich protein II; Pq, primaquine; Qc, quinacrine; Qn, quinine; Qnd, quinidine; Qnld, quinalidine; X2, 1,3 dihydroxyxanthone.
0003-2697/02/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 2 6 9 7 ( 0 2 ) 0 0 5 1 2 - 2
Notes & Tips / Analytical Biochemistry 312 (2003) 258–260
Fig. 1. Microtiter-plate-based screening of molecules for antimalarial drug potential by heme–HRP II-based colorimetric system: Different concentrations of each drug in a total volume of 50 lL of 100 mM Hepes buffer, pH 7 (buffer A), were first transferred to the respective wells (xanthone, clotrimazole, and amiloride were taken in 50 lL dimethyl sulfoxide). Then 80 l g HRP II in 100 lL of 20 mM acetate buffer, pH 5.5, was mixed with 400 lL of 1 mM heme in buffer A. The mixture was incubated (37 °C, 1 h) before diluting to 10 mL in buffer A. Aliquots (5.2 lg heme + 1.6 lg HRP II/200 lL) were transferred to the respective wells using a multipipette. The plate, covered with a lid, was incubated on a shaker (120 rpm, 37 °C, 1 h). Absorbance was read at 415 nm on a microtiter plate reader. (A) Shows a plate in which the wells contain heme (green bar) or Heme + HRP II (red bar) with varying amounts of test substances. Unlike the greenish color of heme; the color of heme–HRP II solution is red. Drugs such as chloroquine, which interferes with heme binding, cause a reversal in color from red to green. A plot of the colorimetric data from the plate in (A) is shown in (B). These data have been plotted after subtracting the corresponding heme + drug absorbances from the respective data points. Standard deviations of triplicate data points are within the points. (C) Shows results of a similar experiment with three different seventh position substituted derivatives of chloroquine.
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malarial also [12], has shown strong potency in the present assay. Our present knowledge of the antimalarial activity of this antifungal agent is that clotrimazole, by complexing with heme, enhances heme-dependent hemolysis. However, our results suggest that clotrimazole, by way of mimicking chloroquine in successfully removing heme from HRP II, may also kill the parasite by blocking template-mediated heme polymerization. Quinine and quinidine represent stereospecific isomers and the latter shows higher in vivo antimalarial potency than the former [13]. It is interesting to note that in the present assay also the potency of quinidine is significantly higher than that of quinine. A recent study has demonstrated sterospecific differences between quinine and quinidine binding to heme [14]. This indicates that in the shuffling of heme from an HRP II-bound state to a state of complexation with an externally added drug, the stereospecificity of the latter may play a key role. Using the present assay we tested three derivatives with different substitutions (H, F, and OCH3 ) at the seventh position of the chloroquine molecule (Fig. 1C). We observed the hydrogen-substituted derivative to be the least potent, the methoxy derivative to be intermediate, and the halogenated derivatives to be the most potent. Of the two halogen derivatives, chloroquine seemed to be slightly more effective than floroquine. In summary, a combination of electronegativity and bulkiness of the substituent at the seventh position seems to influence the efficacy of quinolines as disruptors of heme–HRP II interactions. Several xanthone derivatives studied for their antimalarial efficacy have been shown to be promising and their efficacy has been found to be directly correlated with the degree of hydroxylation. Thus hexahydroxy, pentahydroxy, and tetrahydroxyxanthones showed antimalarial IC50 values of 0.1, 0.4, and 5.0 lM, respectively [15]. Further, it has been reported that in unassisted heme polymerization experiments, these derivatives were inhibitors of heme polymerization [15]. However, when we examined these derivatives in the present assay, it was found that all the xanthone derivatives, irrespective of their degree of hydroxylation, were ineffective in removing heme from HRP II. A representative result seen with dihydroxy xanthone is shown in Fig. 1B. The inability of xanthone derivatives to dissociate heme from HRP II suggests that heme has stronger affinity for HRP II than for xanthones. Therefore, it is unlikely that the antimalarial effect of these xanthone derivatives is mediated by interception of protein template-mediated heme polymerization. Likewise amiloride, which has been suggested to bind heme [16], was found to be incapable of removing heme from HRP II in the present assay. The IC50 values of the drugs tested in the present assay are shown in Table 1. The results obtained suggest that the present method allow us to classify antimalarial drugs based on their site of action. Since
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Notes & Tips / Analytical Biochemistry 312 (2003) 258–260
Table 1 IC50 values of tested drugs in heme–HRP II colorimetric microtiter assay Drug 1. Clotrimazole 2. Quinacrine 3. Chloroquine 4. Quinidine 5. Quinine 6. Seven-substituted chloroquine derivatives (a) F (b) OCH3 (c) H 7. Primaquine, quinalidine, amodiaquine, mefloquine, amiloride, and 1,3-dihydroxyxanthone
[3]
IC50 ðlMÞ 34 37 43 195 400 64 108 243 >400
the heme–HRP II interaction site has been validated as a promising target of antimalarial drug action, the microtiter format of the method described here is expected to facilitate novel drug discovery by way of the screening of natural products and synthetic molecular libraries for their ability to disrupt heme–HRP interactions.
[4]
[5]
[6]
[7]
[8]
[9] [10] [11]
Acknowledgments We thank Dr. T.J. Egan, University of Cape Town, South Africa for providing the chloroquine derivatives and Dr. Michael K. Riscoe, Veterans Affairs Medical Center, Portland, Oregon for providing the xanthone derivatives. We thank Dr. D.E. Goldberg, Washington University School of Medicine, St. Louis for a gift of the plasmid harboring the PfHRP II gene. Our thanks to the Indian Council of Medical Research, New Delhi for financial support
[12]
[13]
[14]
[15]
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