Evidences for the formation of bisbenzamidine–heme complexes in cell-free systems

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Abstract

Infrared and colorimetry data suggest that bisbenzamidines connected by various rigid or flexible linkers are able to interact with heme in cell-free systems. At pH 5.0 the inhibition of formation of β-hematin could be ascertained by infrared spectroscopy whereas at pH 7.0 the interaction yielded insoluble complexes for which a sandwich-type structure of stoichiometry 2:1, heme–drug, is tentatively proposed.

IR spectra and colorimetry data indicated that bisbenzamidines readily interact with heme in cell-free systems at pH 5.0 and 7.0.

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Introduction

Malaria1 represents the most widespread and deadly parasitic disease in man, killing more than one million people each year essentially in the regions located between the tropics. Among the plethora of antimalarial drugs, chloroquine (1) is recognized as an exceptionally safe drug that has been successfully used for more than 50 years. However, its intensive use has led to the emergence of resistant strains of Plasmodium falciparum, the most predominant and dangerous of the four species of the parasite.

Plasmodium falciparum has a life cycle divided into three overall stages (mosquito, liver, and blood stages) that can be selectively targeted by chemotherapeutic agents. Chloroquine and most structurally related antimalarials (quinine, mefloquine) are generally thought to act during the blood stage, when the parasite digests hemoglobin to obtain the amino acids it requires. This reaction produces free heme (ferriprotoporphyrin IX, FPIX; hemin and hematin are the terms used to characterized FPIX chloride and FPIX hydroxide respectively), which is toxic to Plasmodium falciparum. Detoxication of heme can occur in the lysosomal vacuole of the parasite via formation of an insoluble polymer called hemozoin, or malaria pigment (β-hematin is the term used to characterize the synthetic form of hemozoin2). Chloroquine is believed to exert its antimalarial action primarily by inhibiting hemozoin formation either by binding to the free heme or by capping the end of the growing polymer.1 Other modes of action that have been proposed for chloroquine and structurally related antimalarials include the inhibition of detoxication routes that are dependent on the peroxidative degradation of heme within the food vacuole3 or on the glutathione-mediated reaction outside the vacuole.4

As part of our research program5 aimed at the design of novel pentamidine (2) congeners as improved antimicrobial agents, we recently focused our attention on their antiplasmodial activity. Pentamidine is an important therapeutic agent that is clinically used in the treatment of Pneumocystis carinii pneumonia, African trypanosomiasis, and leishmaniasis. However, there are only limited reports on the antiplasmodial effects of pentamidine. Recently Stead et al.6 suggested that the antiplasmodial action of pentamidine is based on its selective transport through a specific pore in the parasite and the subsequent binding to FPIX and inhibition of hemozoin formation. In order to assess whether other aromatic diamidines might act in a manner similar to pentamidine, we analyzed their behavior in the presence of FPIX in cell-free systems. These ex vivo experiments were conducted with the series of derivatives depicted in Figure 1.

Section snippets

Infrared study

Conversion of hemin into β-hematin can be effected in non-biological media as reported by several authors.6, 7, 8, 9, 10 In particular, Egan9 demonstrated that the reaction readily proceeded under acidic conditions (acetate buffer, pH 5.0) yielding to the precipitation of β-hematin. However, this reaction can be inhibited by addition of antimalarials (quinine, chloroquine, amodiaquin) that are known to bind to heme and function as intra-erythrocytically active agents.

Inhibition of β-hematin

Colorimetry study

Formation of a complex between pentamidine and ferriprotoporphyrin at micromolar concentrations in a neutral cell-free system could be observed by visible spectroscopy as reported by Stead et al.6 The authors showed that the absorbance around 400 nm of a solution of FPIX (3 μM) decreased from 0.18 to 0.13 when one equivalent of pentamidine (3 μM) is present in a solution of HEPES buffer, pH 7.0.

In the first set of experiments, we reproduced that reaction and we noticed a comparable decrease of

Conclusion

Numerous studies indicated that chloroquine and other antimalarials, for example, xanthones,21 exert their antiplasmodial activities by complexation to heme and inhibition of hemozoin formation. Such a reaction can be mimicked in cell-free systems and our data22, 23, 24 revealed that bisbenzamidines25 structurally related to pentamidine also behaved in a similar manner. It is noteworthy that a good correlation between the binding of these compounds to heme in the cell-free experiments and their

Acknowledgements

This study was financially supported by NIH grants DA 13546, 2S06GM08008 and by a Cooperative Agreement for Antimalarial Development from the Emerging Infections Program of the Centers for Disease Control (CCU/UR3 418652). The authors are grateful to Dr. F. M. Krogstad and Dr. D. J. Krogstad for the in vitro antiplasmodial analyses.

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