Quinoline antimalarials decrease the rate of β-hematin formation

https://doi.org/10.1016/j.jinorgbio.2005.04.013Get rights and content

Abstract

The strength of inhibition of β-hematin (synthetic hemozoin or malaria pigment) formation by the quinoline antimalarial drugs chloroquine, amodiaquine, quinidine and quinine has been investigated as a function of incubation time. In the assay used, β-hematin formation was brought about using 4.5 M acetate, pH 4.5 at 60 °C. Unreacted hematin was detected by formation of a spectroscopically distinct low spin pyridine complex. Although, these drugs inhibit β-hematin formation when relatively short incubation times are used, it was found that β-hematin eventually forms with longer incubation periods (<8 h for chloroquine and >8 h for quinine). This conclusion was supported by both infrared and X-ray powder diffraction observations. It was further found that the IC50 for inhibition of β-hematin formation increases markedly with increasing incubation times in the case of the 4-aminoquinolines chloroquine and amodiaquine. By contrast, in the presence of the quinoline methanols quinine and quinidine the IC50 values increase much more slowly. This results in a partial reversal of the order of inhibition strengths at longer incubation times. Scanning electron microscopy indicates that β-hematin crystals formed in the presence of chloroquine are more uniform in both size and shape than those formed in the absence of the drug, with the external morphology of these crystallites being markedly altered. The findings suggest that these drugs act by decreasing the rate of hemozoin formation, rather than irreversibly blocking its formation. This model can also explain the observation of a sigmoidal dependence of β-hematin inhibition on drug concentration.

Introduction

During its pathogenic blood stage, the intraerythrocytic malaria parasite digests large quantities of host hemoglobin [1]. The protein component of hemoglobin is digested by proteolytic enzymes [2], [3], [4]. Simultaneously, heme is released and oxidized to produce hematin (aqua/hydroxyferriprotoporphyrin IX). This hematin is the source of hemozoin or malaria pigment [5]. Recent studies on cultured Plasmodium falciparum have shown that at least 95% of the hematin is incorporated into hemozoin [6].

Hemozoin is chemically and structurally identical to a synthetic hematin product known as β-hematin [7], [8], a cyclic dimer of ferriprotoporphyrin IX [9]. This substance is readily synthesized [7], [10], [11]. Numerous studies have shown that chloroquine and related antimalarial compounds inhibit β-hematin formation under various conditions [11], [12], [13], [14], [15]. There is now considerable evidence that this may be the mechanism of antimalarial action of chloroquine and other 4-aminoquinolines [11], [15], [16], [17], [18], [19], [20], [21]. Nonetheless, the mechanism by which β-hematin formation is inhibited is poorly understood.

The formation of β-hematin from hematin appears to be a thermodynamically spontaneous process under acidic conditions, probably requiring only the presence of a suitable catalyst (for example acetic acid when prepared synthetically in aqueous medium) [11], [22], [23]. There is no evidence that this product spontaneously converts back to hematin under acidic conditions to any measurable extent. Thus, formation of β-hematin from hematin is probably to all intents and purposes thermodynamically irreversible. By contrast, the interaction of chloroquine and related compounds with hematin (and possibly also with the surface of β-hematin [24]) is a reversible equilibrium process [25]. This suggests that these drugs are unlikely to be able to thermodynamically block β-hematin formation. Rather, their effects are probably kinetic, decreasing the rate of its formation. In this study, we demonstrate that this is indeed the case.

Section snippets

Materials

N-[2-hydroxyethyl]piperazine-N′-[2-ethanesufonic acid] (HEPES), bovine hemin, chloroquine diphosphate, quinidine sulfate, quinine sulfate, amodiaquine dihydrochloride and sodium acetate (NaOAc) were purchased from Sigma–Aldrich South Africa. Pyridine (AR grade) was purchased from BDH laboratory suppliers and glacial acetic acid (AcOH) from Saarchem laboratory suppliers. All water was distilled in glass.

Phiβ assay of β-hematin inhibition

This assay is based on the ability of hematin, but not β-hematin to form a low spin complex

Results

Fig. 1(a) shows the dose–response curve for inhibition of β-hematin formation by chloroquine using a new pyridine based assay called the Phiβ assay. This assay has been fully described and validated elsewhere [26]. Briefly, however, the assay is based on the ability of a 5% (v/v) aqueous pyridine solution (pH 7.5) to react with hematin, but not β-hematin. This results in the formation of a monomeric, low spin Fe(III)PPIX-pyridine complex (probably a bis-pyridyl complex) with a distinctive

Discussion

Chong and Sullivan [30] have recently reported for the first time that inhibition of β-hematin formation by chloroquine and quinidine is reversible. Our study has not only confirmed their observation, but also demonstrated that these compounds in effect act by slowing the rate of β-hematin formation, rather than inhibiting the process via a competitive thermodynamic equilibrium. This behavior is consistent with expectation, as β-hematin formation in acidic medium appears to be essentially

Conclusions

The 4-aminoquinoline and quinoline methanol antimalarials appear to act by slowing rather than irreversibly blocking hemozoin formation. The IC50 values for inhibition of β-hematin formation by the 4-aminoquinolines in particular depend strongly on incubation time. This must now be an important consideration when investigating β-hematin inhibitors. It remains to be seen whether the fact that β-hematin formation can occur slowly in the presence of quinoline drugs has any relevance in vivo. We

Acknowledgments

This material is based upon work supported in part by the National Research Foundation under Grant No. 2061833. Any opinion, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Research Foundation. We also acknowledge the Medical Research Council of South Africa and the University of Cape Town for financial support.

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