Infectious diseases
Heme detoxification and antimalarial drugs – Known mechanisms and future prospects

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Malaria parasite degrades hemoglobin and releases toxic heme, which is quickly detoxified by the parasite. Whereas several important antimalarials like artemisinin and chloroquine disrupt this process, the mechanism of heme detoxification is not fully understood and is a subject of intense debate. This review summarizes the current state of research in heme detoxification, its future directions and reasons why this process has been and will remain one of the most attractive targets for developing antimalarials.

Section editors:

Gary Woodnutt – CovX, San Diego, USA

Paul-Henri Lambert – Centre of Vaccinology, University of Geneva, Switzerland

Introduction

Malaria is the most lethal parasitic disease that kills more than one million individuals each year. Whereas malaria infection begins with the invasion of hepatocytes by the Plasmodium sporozoites inoculated by an infected mosquito, clinical symptoms of malaria, which includes high fever, chills and anemia, are due to the subsequent infection and rapid multiplication of the parasite inside the red blood cells (RBCs). To sustain its rapid pace of development, the parasite cannibalizes hemoglobin (Hb), which represents 90% of the total protein present inside an RBC [1]; approximately 75% of which is degraded during the erythrocytic stage of development [2]. Degradation of hemoglobin releases heme, which is effectively neutralized by the parasite, primarily by its conversion into an insoluble crystalline material called hemozoin, by utilizing a mechanism that is not fully understood.

Section snippets

Mechanism of hemoglobin transport

The process starts with the formation of a double membrane cytostome, an invagination in the plasma and parasitophorous membrane of the parasite, which intakes host cytoplasm (primarily containing Hb) in a pinocytic fashion and subsequently buds off as a transport vesicle (Tv) (Fig. 1). This Hb-laden Tv undertakes a retrograde trip to the food vacuole (Fv), an oxygen-rich, lysosome-like organelle in the parasite to which it fuses and delivers its contents (Fig. 1). Inside the Fv, several

Heme detoxification is a unique drug target

Several ancient antimalarials known to traditional healers as well as some of the most potent drugs developed during the pre-genomic era, exert their antimalarial activity by blocking heme detoxification. In fact, prevention of heme detoxification is the most widely used strategy for controlling malaria (Table 1). Chloroquine, the most widely used antimalarial in history, binds to heme [6, 7] with a 1:2 stoichiometry [8] and prevents its detoxification, leading to heme toxicity and death of the

Known mechanisms of heme detoxification

In the past two decades, several mechanisms for heme detoxification have been identified in vitro, primarily by utilizing parasite extracts from human and rodent parasites. Based on outcomes, all the known mechanisms can be broadly classified into two major types. One involves accumulation of heme by its dimerzation and stacking into a nontoxic crystalline product called hemozoin (Hz) (Fig. 2) whereas the other suggests degradation of heme by glutathione (GSH) and hydrogen peroxide. Although,

Chemical structure of Hz

In a parasite-infected RBC, Hz is visible under the microscope as a dark pigmented body and was one of the key factors in the discovery of the parasite. The chemical structure of Hz has been deciphered utilizing several spectroscopic techniques [11, 12]. The Hz crystal is composed of unit cells of ferriprotoporphyrin (FP) dimers (also known as Fe1–O41 dimers) coordinated by reciprocal iron to side chain carboxylate bonds (Fig. 3). These individual head to tail dimers form a hydrogen bond

How is Hz made?

In the past 15 years, several processes that lead to Hz formation under in vitro conditions have been identified. Hz formation has been shown to be possible utilizing parasite proteins, lipid extracts, preformed Hz crystals, along with theories that its formation is chemically driven, independent of any parasite or host factors. A summary of each of the known processes along with their limitations is depicted in Table 2.

Hydrogen peroxide and glutathione mediated heme degradation

Along with Hz formation, possibility of a hydrogen peroxide-mediated decomposition of heme in the food vacuole has also been demonstrated. Under acidic conditions in vitro, hydrogen peroxide has been shown to degrade heme [24, 31]. Likewise, a cytosolic, glutathione-mediated model of heme degradation has also been proposed. This model suggests that heme that is not converted into Hz, escapes from the Fv to the parasite cytosol where it is degraded by glutathione (GSH) [26]. Although, parasite

Heme targeted drug discovery

Whereas several antimalarials from the pre-genomic era that fortuitously target this pathway are already in the market, opportunities for developing new drugs that inhibit this pathway have not been fully explored. With the advent of high throughput screening (HTS), attempts are now focused on screening compound libraries for identification of new inhibitors of this pathway. These attempts have been greatly facilitated with the miniaturization of assays that until recently were primarily

Conclusions

Protective mechanisms employed by the parasite to defend itself from the toxic effects of heme are poorly understood. One of the major reasons behind our limited understanding is the inability to study these mechanisms in a live parasite, which has made their in vivo validation a major challenge. Nonetheless, the available information regarding these mechanisms has provided vital clues about one of the most novel strategies employed by the parasite for its development and survival inside the

Acknowledgements

This work was supported by a JHU-VBI Infectious disease research partnership and the university research funds to D.R. We thank June Mullins for technical support and Wandy Beatty for the electron micrograph of the parasite. The views expressed herein are of the authors and do not represent the official position of the US Food and Drug Administration.

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