Identification of ß-hematin inhibitors in the MMV Malaria Box.

The Malaria Box, assembled by the Medicines for Malaria Venture, is a set of 400 structurally diverse, commercially available compounds with demonstrated activity against blood-stage Plasmodium falciparum. The compounds are a representative subset of the 20,000 in vitro antimalarials identified from the high-throughput screening efforts of St. Jude Children's Research Hospital (TN, USA), Novartis and GlaxoSmithKline. In addition, a small set of active compounds from commercially available libraries was added to this group, but it has not previously been published. Elucidation of the biochemical pathways on which these compounds act is a major challenge; therefore, access to these compounds has been made available free of charge to the investigator community. Here, the Malaria Box compounds were tested for activity against the formation of ß-hematin, a synthetic form of the heme detoxification biomineral, hemozoin. Further, the mechanism of action of these compounds within the malaria parasite was explored. Ten of the Malaria Box compounds demonstrated significant inhibition of ß-hematin formation. In this assay, dose-response data revealed IC50 values ranging from 8.7 to 22.7 µM for these hits, each of which is more potent than chloroquine (a known inhibitor of hemozoin formation). The in vitro antimalarial activity of these ten hits was confirmed in cultures of the chloroquine sensitive D6 strain of the parasite resulting in IC50 values of 135-2165 nM, followed by testing in the multidrug resistant strain, C235. Cultures of P. falciparum (D6) were then examined for their heme distribution following treatment with nine of the commercially available confirmed compounds, seven of which disrupted the hemozoin pathway.

Evidence for redox cooperativity between c-type hemes of MauG which is likely coupled to oxygen activation during tryptophan tryptophylquinone biosynthesis.

MauG is a novel 42 kDa diheme protein which is required for the biosynthesis of tryptophan tryptophylquinone, the prosthetic group of methylamine dehydrogenase. The visible absorption and resonance Raman spectroscopic properties of each of the two c-type hemes and the overall redox properties of MauG are described. The absorption maxima for the Soret peaks of the oxidized and reduced hemes are 403 and 418 nm for the low-spin heme and 389 and 427 nm for the high-spin heme, respectively. The resonance Raman spectrum of oxidized MauG exhibits a set of marker bands at 1503 and 1588 cm(-1) which exhibit frequencies similar to those of the nu3 and nu2 bands of c-type heme proteins with bis-histidine coordination. Another set of marker bands at 1478 and 1570 cm(-1) is characteristic of a high-spin heme. Two distinct oxidation-reduction midpoint potential (E(m)) values of -159 and -244 mV are obtained from spectrochemical titration of MauG. However, the two nu3 bands located at 1478 and 1503 cm(-1) shift together to 1467 and 1492 cm(-1), respectively, upon reduction, as do the Soret peaks of the low- and high-spin hemes in the absorption spectrum. Thus, the two hemes with distinct spectral properties are reduced and oxidized to approximately the same extent during redox titrations. This indicates that the high- and low-spin hemes have similar intrinsic E(m) values but exhibit negative redox cooperativity. After the first one-electron reduction of MauG, the electron equilibrates between hemes. This makes the second one-electron reduction of MauG more difficult. Thus, the two E(m) values do not describe redox properties of distinct hemes, but the first and second one-electron reductions of a diheme system with two equivalent hemes. The structural and mechanistic implications of these findings are discussed.

Identification of heme macrocycle type by near-infrared magnetic circular dichroism spectroscopy at cryogenic temperatures.

The electron paramagnetic resonance (EPR) and near-infrared magnetic circular dichroism (MCD) spectra of the azide and cyanide adducts of nitrimyoglobin and hydroperoxidase II from Escherichia coli have been measured at cryogenic temperatures. For the first time, ligand-to-metal charge-transfer transitions in the near-infrared have been observed for an Fe(III)-chlorine system. It is shown that near-ultraviolet-to-visible region electronic spectra of 'green' hemes such as these are an unreliable indicator of macrocycle type. However, the combined application of EPR and near-infrared MCD spectroscopies clearly distinguishes between the porphyrin-containing nitrimyoglobin and the chlorine-containing hydroperoxidase II.

New developments in the regulation of heme metabolism and their implications.

Various endogenous and exogenous chemicals, such as hormones, drugs, and carcinogens and other environmental pollutants are enzymatically converted to polar metabolites as a result of their oxidative metabolism by the mixed-function oxidase system. This enzyme complex constitutes the major detoxifying system of man and utilizes the hemoprotein--cytochrome P-450--as the terminal oxidase. Recent studies with trace metals have revealed the potent ability of these elements to alter the synthesis and to enhance the degradation of heme moiety of cytochrome P-450. An important consequence of these metal actions is to greatly impair the ability of cells to oxidatively metabolize chemicals because of the heme dependence of this metabolic process. In this report the effects of exposure to trace metals on drug oxidations is reviewed within the framework of metal alterations of heme metabolism, including both its synthesis and degradation, since these newly discovered properties of metals have made it possible to define a major dimension of metal toxicity in terms of a unified cellular mechanism of action.

Individual assignments of the heme resonances in the 360 MHz 1H NMR spectra of cytochrome c-557 from Crithidia oncopelti.

With the use of nuclear Overhauser effects, spin decoupling and saturation transfer experiments individual assignments for numerous resonances of the heme group in the 360 MHz 1H NMR spectra of reduced and oxidized cytochrome c-557 from Crithidia oncopelti were obtained. These data provide direct evidence that the heme substituent in position 2 is a vinyl group. They further show that in spite of the different covalent structures of the heme groups the heme crevice and the electronic heme structure in the oxidized state are nearly identical in cytochrome c-557 and in mammalian cytochromes c.