Parasitology

Malaria

1. The Structure of Malaria Pigment and its importance in malaria biology

For hundreds of years it has been know that people infected with malaria accumulate a black pigment in their livers and spleens. Over the years various suggestions were made about what this material was. One of the unusual properties of the pigment was that it was completely insoluble in water or organic solvents- the only way to solubilize it was in concentrated sodium hydroxide. In this paper the structure of the pigment was determined to be polymerized heme in which the carboxylate of one heme was attached to the iron of another to form a polymerized structure, hemozoin. In this article we proposed that this mechanism allowed the parasite to avoid the toxicity associated with heme.

Proc. Nati. Acad. Sci. USA Vol. 88, pp. 325-329, January 1991 An iron-carboxylate bond links the heme units of malaria pigment Andrew F.G. Slater, William J. Swiggard, Brian R. Orton, William D. Flitter, Daniel E. Goldberg, Anthony Cerami, and Graeme B. Henderson

ABSTRACT The intraerythrocytic malaria parasite uses hemoglobin as a major nutrient source. Digestion of hemoglobin releases heme, which the parasite converts into an insoluble microcrystalline material called hemozoin or malaria pigment. We have purified hemozoin from the human malaria organism Plasmodium falciparum and have used infrared spectroscopy, x-ray absorption spectroscopy, and chemical synthesis to determine its structure. The molecule consists of an unusual polymer of hemes linked between the central ferric ion of one heme and a carboxylate side-group oxygen of another. The hemes are sequestered via this linkage into an insoluble product, providing a unique way for the malaria parasite to avoid the toxicity associated with soluble heme.

2. Mechanism of action of chloroquine

The drug chloroquine to treat malaria had been know for fifty years but its mechanism of action was not understood. In this paper we showed that chloroquine could inhibit the polymerization of heme to form hemozoin. The free heme was then toxic to the parasite causing its death.

Slater AFG, Cerami A. (1992) Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites. Nature 355:167-169.

The incidence of human malaria has increased during the past 20 years; 270 million people are now estimated to be infected with the parasite1. An important contribution to this increase has been the appearance of malaria organisms resistant to quinoline-containing antimalarials such as chloroquine and quinine2. These drugs accumulate in the acid food vacuoles of the intraerythrocytic-stage malaria parasite3-5, although the mechanism of their specific toxicity in this organelle is uncertain. The primary function of the food vacuole is the proteolysis of ingested red cell haemoglobin6-7 to provide the growing parasite with essential amino acids. Haemoglobin breakdown in the food vacuole releases haem, which if soluble can damage biological membranes8 and inhibit a variety of enzymes9-10. Rather than degrading or excreting the haem, the parasite has evolved a novel pathway for its detoxification by incorporating it into an insoluble crystalline material called haemozoin or malaria pigment11. These crystals form in the food vacuole of the parasite concomitant with haemoglobin degradation, where they remain until the infected red cell bursts. The structure of haemozoin comprises a polymer of haems linked between the central ferric ion of one haem and a carboxylate side-group oxygen of another12. This structure does not form spontaneously form either free haem or haemoglobin under physiological conditions13-14, and the biochemistry of its formation is unclear. Here we report the identification and characterization of a haem polymerase enzyme activity from extracts of Plasmodium falciparum trophozoites, and show that this enzyme is inhibited by quinoline-containing drugs such as chloroquine and quinine. This provides a possible explanation for the highly stage-specific anti-malarial properties of these drugs.

Trypanosomiasis

In the early part of the twentieth century Paul Erhlich synthesized a number of organic arsenicals which could kill trypanosomes without harming the mammalian host. He proposed that these arsenicals were binding to two adjacent sulfhydryl groups in a critical pathway in the parasite that was responsible for the selective killing of the parasite. In this paper we confirmed his suspicion was indeed correct. Trpanosomoatids have a unique co-factor, trypanothione, which has two adjacent sulphydryls for a critical enzyme trypanothione reductase.

1. Fairlamb AH, Blackburn P, Ulrich P, Chait BT, Cerami A. (1985). Trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. Science 227: 1485‑1487.

Trypanothione

A Novel Bis(glutathionyl)spermidine Cofactor for Glutathione Reductase in Trypanosomatids Abstract. Glutathione reductase from trypanosomes and leishmanias, unlike glutathione reductase from other organisms, requires an unusual low molecular weight cofactor for activity. The cofactor was purifiedfrom the insect trypanosomatid Crithidia fasciculata and identified as a novel glutathione-spermidine conjugate, N’ ,N8-bis(L–y-glutamyl-L-hemicystinyl-glycyl)spermidine, for which the trivial name trypanothione is proposed. This discovery may open a new chemotherapeutic approach to trypanosomiasis and leishmaniasis.

2. A subsequent study showed that organic arsenicals bound specifically to trypanothione.

Trypanothione is the primary target for arsenical drugs against African trypanosomes (chemotherapy) Alan H. Fairlamb*, Graeme B. Henderson, and Anthony Cerami

ABSTRACT The trypanosomatid metabolite N’,N5-bis- (glutathionyl)spermidine (trypanothione) has been demonstrated to form a stable adduct with the aromatic arsenical drug melarsen oxide [p-(4,6-diamino-s-triazinyl-2-yl)aminophenyl arsenoxide]. The stability constant of the melarsen-trypanothione adduct (Mel T) has been determined to be 1.05 x 107 M-1. When bloodstream Trypanosoma brucei are incubated with either melarsen oxide or the 2,3-dimercaptopropanol adduct of melarsen oxide (melarsoprol), Mel T is the only arsenical derivative detectable in acid-soluble extracts of the cells. Trypanothione may therefore be regarded as a primary target for aromatic arsenical derivatives against African trypanosomes. The selective toxic action of these compounds might arise through sequestration of intracellular trypanothione in the form ofMel T, or Mel T itselfmay be toxic within the cell. The latter possibility is illustrated by the finding that Mel T is an inhibitor of trypanothione reductase from T. brucei (K; = 9.0 ,uM)-an enzyme that is central to the regulation of the thiol/disulfide redox balance in the parasite and absent from the host.

3. The structure of trypanothione reductase

Proc. Nati. Acad. Sci. USA Vol. 88, pp. 8764-8768, October 1991 Biochemistry X-ray structure of trypanothione reductase from Crithidia fasciculata at 2.4-A resolution (glutathione reductase/oxidative stress/trypanosomiasls/protein crystaoraphy/drug design) John Kuriyan*, Xiang-Peng Kong, T. S. R. Krishna*, Robert M. Sweet, Nicholas J. Murgolo, Helen Field, Anthony Cerami, and Graeme B. Henderson

4. Changed the substrate specificity of glutathione reductase so that it could carry out the reduction of trypanothione

Henderson GB, Murgolo NJ, Kuriyan J, Osapay K, Kominos D, Berry A, Scrutton NS, Hinchliffe NW, Perham RN, Cerami A. (1991) Engineering the substrate specificity of glutathione reductase towards trypanothione reduction. Proc Natl Acad Sci, USA 88:8769-8773.

ABSTRACT Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CAS registry number 102210-35-5), which are related flavoprotein disulfide oxidoreductases, have marked specificities for glutathione and trypanothione, respectively. A combination of primary sequence alignments and molecular modeling, together with the high-resolution crystal structure of human glutathione reductase, identified certain residues as potentially being responsible for substrate discrimination. Site-directed mutagenesis ofEscherichia coli glutathione reductase was used to test these predictions. The mutation of Asn-21 to Arg demonstrated that this single change was insufficient to generate the greater discrimination against trypanothione shown by human glutathione reductase compared with the E. coli enzyme. However, the mutation of Ala-18, Asn-21, and Arg-22 to the amino acid residues (Glu, Trp, and Asn, respectively) in corresponding positions in Trypanosoma congolense trypanothione reductase confirmed that this region of polypeptide chain is intimately involved in substrate recognition. It led to a mutant form of E. coli glutathione reductase that possessed essentially no activity with glutathione but that was able to catalyze trypanothione reduction with a k.t/Klm value that was 10% of that measured for natural trypanothione reductases. These results should be of considerable importance in the design of trypanocidal drugs targeted at the differences between glutathione and trypanothione metabolism in trypanosomatids and their hosts.

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