Genome-scale modeling drives 70-fold improvement of intracellular heme production in Saccharomyces cerevisiae.

Heme is an oxygen carrier and a cofactor of both industrial enzymes and food additives. The intracellular level of free heme is low, which limits the synthesis of heme proteins. Therefore, increasing heme synthesis allows an increased production of heme proteins. Using the genome-scale metabolic model (GEM) Yeast8 for the yeast Saccharomyces cerevisiae, we identified fluxes potentially important to heme synthesis. With this model, in silico simulations highlighted 84 gene targets for balancing biomass and increasing heme production. Of those identified, 76 genes were individually deleted or overexpressed in experiments. Empirically, 40 genes individually increased heme production (up to threefold). Heme was increased by modifying target genes, which not only included the genes involved in heme biosynthesis, but also those involved in glycolysis, pyruvate, Fe-S clusters, glycine, and succinyl-coenzyme A (CoA) metabolism. Next, we developed an algorithmic method for predicting an optimal combination of these genes by using the enzyme-constrained extension of the Yeast8 model, ecYeast8. The computationally identified combination for enhanced heme production was evaluated using the heme ligand-binding biosensor (Heme-LBB). The positive targets were combined using CRISPR-Cas9 in the yeast strain (IMX581-HEM15-HEM14-HEM3-Δshm1-HEM2-Δhmx1-FET4-Δgcv2-HEM1-Δgcv1-HEM13), which produces 70-fold-higher levels of intracellular heme.

Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme-dependent proteins.

BACKGROUND: Heme proteins and heme-derived molecules are essential in numerous cellular processes. Research into their in vitro functionality requires the production of large amounts of protein. Unfortunately, high yield expression is hampered by the lack of E. coli strains naturally capable of taking up heme from the medium. We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme containing proteins, as it encodes the outer membrane heme receptor, ChuA, which allows for natural uptake of heme. The EcN strain however lacks the gene for T7 RNA polymerase, which is necessary for the expression of genes under the control of the T7-promotor, widely used in expression vectors like the pET or pDuet series. RESULTS: A new T7-promoter compatible EcN strain was constructed by integrating the gene for T7-RNA polymerase under the control of a lacUV5 promoter into the malEFG operon of EcN. Test expressions of genes via T7 promoter-based vectors in the new EcN(T7) strain were successful. Expression in EcN(T7) resulted in the efficient production of recombinant heme proteins in which the heme cofactor was incorporated during protein production. In addition, the new EcN(T7) strain can be used to co-express genes for the production of heme-derived molecules like biliverdin or other linear tetrapyrroles. We demonstrate the successful recombinant production of the phytochromes BphP, from Pseudomonas aeruginosa, and Cph1, from Synechocystis sp. PCC6803, loaded with their linear tetrapyrrole cofactors, biliverdin and phycocyanobilin, respectively. CONCLUSION: We present a new E. coli strain for efficient production of heme proteins and heme-derived molecules using T7-promoter based expression vectors. The new EcN(T7) strain enables the use of a broader spectrum of expression vectors, as well as the co-expression of genes using the pDuet expression vectors, for expressing heme containing proteins. By utilizing E. coli strains EcN and EcN(T7), capable of being fed heme, the rate limiting step of heme biosynthesis in E. coli is eliminated, thereby permitting higher heme saturation of heme proteins and also higher yields of heme-derived molecules.

Lapatinib, Nilotinib and Lomitapide Inhibit Haemozoin Formation in Malaria Parasites.

With the continued loss of antimalarials to resistance, drug repositioning may have a role in maximising efficiency and accelerating the discovery of new antimalarial drugs. Bayesian statistics was previously used as a tool to virtually screen USFDA approved drugs for predicted ß-haematin (synthetic haemozoin) inhibition and in vitro antimalarial activity. Here, we report the experimental evaluation of nine of the highest ranked drugs, confirming the accuracy of the model by showing an overall 93% hit rate. Lapatinib, nilotinib, and lomitapide showed the best activity for inhibition of ß-haematin formation and parasite growth and were found to inhibit haemozoin formation in the parasite, providing mechanistic insights into their mode of antimalarial action. We then screened the USFDA approved drugs for binding to the ß-haematin crystal, applying a docking method in order to evaluate its performance. The docking method correctly identified imatinib, lapatinib, nilotinib, and lomitapide. Experimental evaluation of 22 of the highest ranked purchasable drugs showed a 24% hit rate. Lapatinib and nilotinib were chosen as templates for shape and electrostatic similarity screening for lead hopping using the in-stock ChemDiv compound catalogue. The actives were novel structures worthy of future investigation. This study presents a comparison of different in silico methods to identify new haemozoin-inhibiting chemotherapeutic alternatives for malaria that proved to be useful in different ways when taking into consideration their strengths and limitations.

Monocyte phagocytosis of malaria ß-haematin in the presence of artemisinin, amodiaquine, chloroquine, doxycycline, primaquine, pyrimethamine and quinine.

The intraerythrocytic malaria parasite digests haemoglobin to provide amino acids for metabolism and releases toxic haem that is sequestered into haemozoin, a non-toxic, insoluble, crystalline pigment. Following erythrocyte rupture, haemozoin is released into circulation and phagocytosed by monocytes. Phagocytosed haemozoin and antimalarial drugs have both been reported to modulate monocyte functions. This study determined the effects of therapeutic concentrations of seven antimalarial drugs; amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of ß-haematin (synthetic haemozoin) by two monocytic cell lines, J774A.1 and U937, and human peripheral blood mononuclear cells. A novel spectrophotometric method based on the absorbance (O.D 400 nm) of alkali/SDS treated monocytes containing ß-haematin was developed to complement counting phagocytosis with microscopy. The method has potential use for the large scale screening of monocyte phagocytic activity. Artemisinin, quinine, primaquine and pyrimethamine activated ß-haematin phagocytosis by 12% or more, whereas amodiaquine, chloroquine and doxycyline inhibited ß-haematin phagocytosis. In contrast, antimalarial drugs had minimal inhibitory effects on the phagocytosis of latex beads with only quinine resulting in more than 20% inhibition. Antimalarial drugs appear to alter monocyte phagocytic activity which has implications for the treatment, pathogenicity and adjunct therapies for malaria.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of Chiral Chloroquine Analogues as Antimalarial Agents.

In a focused exploration, we designed, synthesized, and biologically evaluated chiral conjugated new chloroquine (CQ) analogues with substituted piperazines as antimalarial agents. In vitro as well as in vivo studies revealed that compound 7c showed potent activity (in vitro 50% inhibitory concentration, 56.98 nM for strain 3D7 and 97.76 nM for strain K1; selectivity index in vivo [up to at a dose of 12.5 mg/kg of body weight], 3,510) as a new lead antimalarial agent. Other compounds (compounds 6b, 6d, 7d, 7h, 8c, 8d, 9a, and 9c) also showed moderate activity against a CQ-sensitive strain (3D7) and superior activity against a CQ-resistant strain (K1) of Plasmodium falciparum Furthermore, we carried out docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of all in-house data sets (168 molecules) of chiral CQ analogues to explain the structure-activity relationships (SAR). Our new findings specify the significance of the H-bond interaction with the side chain of heme for biological activity. In addition, the 3D-QSAR study against the 3D7 strain indicated the favorable and unfavorable sites of CQ analogues for incorporating steric, hydrophobic, and electropositive groups to improve the antimalarial activity.

Synthesis of novel benzimidazole acrylonitriles for inhibition of Plasmodium falciparum growth by dual target inhibition.

Antimalarial drug resistance has emerged as a threat for treating malaria, generating a need to design and develop newer, more efficient antimalarial agents. This research aimed to identify novel leads as antimalarials. Dual receptor mechanism could be a good strategy to combat developing drug resistance. A series of benzimidazole acrylonitriles containing 18 compounds were designed, synthesized and evaluated for cytotoxicity, heme binding, ferriprotoporphyrin IX biomineralisation inhibition, and falcipain-2 enzyme assay. Furthermore, in silico docking and MD simulation studies were also performed.The tests revealed quite encouraging results. Three compounds, viz. R-01 (0.69 µM), R-04 (1.60 µM), and R-08 (1.61 µM), were found to have high antimalarial activity. These compounds were found to be in bearable cytotoxicity limits and their biological assay suggested that they had inhibitory activity against falcipain-2 and hemozoin formation. The docking revealed the binding mode of benzimidazole acrylonitrile derivatives and MD simulation studies revealed that the protein-ligand complex was stable. The agents exhibit good hemozoin formation inhibition activity and, hence, may be utilized as leads to design a newer drug class to overcome the drug resistance of hemozoin formation inhibitors such as chloroquine.

Simplified Reversed Chloroquines To Overcome Malaria Resistance to Quinoline-Based Drugs.

Building on our earlier work of attaching a chemosensitizer (reversal agent) to a known drug pharmacophore, we have now expanded the structure-activity relationship study to include simplified versions of the chemosensitizer. The change from two aromatic rings in this head group to a single ring does not appear to detrimentally affect the antimalarial activity of the compounds. Data from in vitro heme binding and ß-hematin inhibition assays suggest that the single aromatic RCQ compounds retain activities against Plasmodium falciparum similar to those of CQ, although other mechanisms of action may be relevant to their activities.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry.

Enhancing nosiheptide production in Streptomyces actuosus by heterologous expression of haemoprotein from Sinorhizobium meliloti.

UNLABELLED: Oxygen deficiency is a critical limiting factor for nosiheptide production in Streptomyces actuosus during fermentation. To alleviate oxygen limitation and enhance the yield of nosiheptide, haemoprotein from Sinorhizobium meliloti (SmHb) was overexpressed in S. actuosus with overexpression of haemoglobin from Vitreoscilla (VHb) as a positive control. The expression of SmHb and VHb in S. actuosus was confirmed by SDS-PAGE and CO-difference spectra analysis. The results showed that S. actuosus recombinant strain with SmHb expression had higher nosiheptide production (increased by 151%) than the wild strain (WT) under the low aeration conditions, which was similar with S. actuosus mutant strain with VHb expression. Furthermore, two copies of SmfHb gene were integrated in S. actuosus, which further increased the nosiheptide production by approx. 1·9-fold compared with original strain, and final nosiheptide yield was up to 2352 µg ml(-1) . These results suggested that engineering of SmHb expression could be used as an efficient method for constructing a high nosiheptide-accumulating strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant improvement of nosiheptide production was found in recombinant strain with overexpressed sm gene. And further improvement was obtained in the two copies of sm overexpressing strain. These results suggested that engineering of SmHb expression could be used as an efficient method for constructing a high nosiheptide-accumulating strain.

Protein complex directs hemoglobin-to-hemozoin formation in Plasmodium falciparum.

Malaria parasites use hemoglobin (Hb) as a major nutrient source in the intraerythrocytic stage, during which heme is converted to hemozoin (Hz). The formation of Hz is essential for parasite survival, but to date, the underlying mechanisms of Hb degradation and Hz formation are poorly understood. We report the presence of a ∼200-kDa protein complex in the food vacuole that is required for Hb degradation and Hz formation. This complex contains several parasite proteins, including falcipain 2/2', plasmepsin II, plasmepsin IV, histo aspartic protease, and heme detoxification protein. The association of these proteins is evident from coimmunoprecipitation followed by mass spectrometry, coelution from a gel filtration column, cosedimentation on a glycerol gradient, and in vitro protein interaction analyses. To functionally characterize this complex, we developed an in vitro assay using two of the proteins present in the complex. Our results show that falcipain 2 and heme detoxification protein associate with each other to efficiently convert Hb to Hz. We also used this in vitro assay to elucidate the modes of action of chloroquine and artemisinin. Our results reveal that both chloroquine and artemisinin act during the heme polymerization step, and chloroquine also acts at the Hb degradation step. These results may have important implications in the development of previously undefined antimalarials.

Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14)C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

Glycosyl hydroperoxides: a new class of potential antimalarial agents.

Motivated by the antimalarial properties observed in organic peroxides, an extensive series of glycosyl hydroperoxides was prepared with the aim of identifying new bioactive molecules. Selected compounds were tested against a Plasmodium falciparum culture (chloroquine-susceptible strain D10 and chloroquine-resistant strain W2). Screening results indicated that the factors critical for antimalarial activity were the presence of a hydroperoxide moiety and solubility in water at pH 5.0. Moreover, the ability to inhibit ß-hematin formation in vitro has been evaluated (BHIA Assay).

Expression, purification, and solid-state NMR characterization of the membrane binding heme protein nitrophorin 7 in two electronic spin states.

The nitrophorins (NPs) comprise a group of NO transporting ferriheme b proteins found in the saliva of the blood sucking insect Rhodnius prolixus . In contrast to other nitrophorins (NP1-4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of (13)C,(15)N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with S = 1/2 and S = 0 at the ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center.

A pivotal heme-transfer reaction intermediate in cytochrome c biogenesis.

c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway.

One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans.

The appearance of heme, an organic ring surrounding an iron atom, in evolution forever changed the efficiency with which organisms were able to generate energy, utilize gasses and catalyze numerous reactions. Because of this, heme has become a near ubiquitous compound among living organisms. In this review we have attempted to assess the current state of heme synthesis and trafficking with a goal of identifying crucial missing information, and propose hypotheses related to trafficking that may generate discussion and research. The possibilities of spatially organized supramolecular enzyme complexes and organelle structures that facilitate efficient heme synthesis and subsequent trafficking are discussed and evaluated. Recently identified players in heme transport and trafficking are reviewed and placed in an organismal context. Additionally, older, well established data are reexamined in light of more recent studies on cellular organization and data available from newer model organisms. This article is part of a Special Issue entitled: Cell Biology of Metals.

Processing of HEBP1 by cathepsin D gives rise to F2L, the agonist of formyl peptide receptor 3.

The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure-function analysis of F2L identified three amino acids, G(3), N(7), and S(8), as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo.

Differences in anti-malarial activity of 4-aminoalcohol quinoline enantiomers and investigation of the presumed underlying mechanism of action.

BACKGROUND: A better anti-malarial efficiency and lower neurotoxicity have been reported for mefloquine (MQ) (+)- enantiomer. However, the importance of stereoselectivity remains poorly understood as the anti-malarial activity of pure enantiomer MQ analogues has never been described. Building on these observations, a series of enantiopure 4-aminoalcohol quinoline derivatives has previously been synthesized to optimize the efficiency and reduce possible adverse effects. Their in vitro activity on Plasmodium falciparum W2 and 3D7 strains is reported here along with their inhibition of ß-haematin formation and peroxidative degradation of haemin, two possible mechanisms of action of anti-malarial drugs. RESULTS: The (S)-enantiomers of this series of 4-aminoalcohol quinoline derivatives were found to be at least as effective as both chloroquine (CQ) and MQ. The derivative with a 5-carbon side-chain length was the more efficient on both P. falciparum strains. (R )-enantiomers displayed an activity decreased by 2 to 15-fold as compared to their (S) counterparts. The inhibition of ß-haematin formation was significantly stronger with all tested compounds than with MQ, irrespective of the stereochemistry. Similarly, the inhibition of haemin peroxidation was significantly higher for both (S) and (R)-enantiomers of derivatives with a side-chain length of five or six carbons than for MQ and CQ. CONCLUSIONS: The prominence of stereochemistry in the anti-malarial activity of 4-aminoalcohol quinoline derivatives is confirmed. The inhibition of ß-haematin formation and haemin peroxidation can be put forward as presumed mechanisms of action but do not account for the stereoselectivity of action witnessed in vitro.

Biological activities of nitidine, a potential anti-malarial lead compound.

BACKGROUND: Nitidine is thought to be the main active ingredient in several traditional anti-malarial remedies used in different parts of the world. The widespread use of these therapies stresses the importance of studying this molecule in the context of malaria control. However, little is known about its potential as an anti-plasmodial drug, as well as its mechanism of action. METHODS: In this study, the anti-malarial potential of nitidine was evaluated in vitro on CQ-sensitive and -resistant strains. The nitidine's selectivity index compared with cancerous and non-cancerous cell lines was then determined. In vivo assays were then performed, using the four-day Peter's test methodology. To gain information about nitidine's possible mode of action, its moment of action on the parasite cell cycle was studied, and its localization inside the parasite was determined using confocal microscopy. The in vitro abilities of nitidine to bind haem and to inhibit ß-haematin formation were also demonstrated. RESULTS: Nitidine showed similar in vitro activity in CQ-sensitive and resistant strains, and also a satisfying selectivity index (> 10) when compared with a non-cancerous cells line. Its in vivo activity was moderate; however, no sign of acute toxicity was observed during treatment. Nitidine's moment of action on the parasite cycle showed that it could not interfere with DNA replication; this was consistent with the observation that nitidine did not localize in the nucleus, but rather in the cytoplasm of the parasite. Nitidine was able to form a 1-1 complex with haem in vitro and also inhibited ß-haematin formation with the same potency as chloroquine. CONCLUSION: Nitidine can be considered a potential anti-malarial lead compound. Its ability to complex haem and inhibit ß-haematin formation suggests a mechanism of action similar to that of chloroquine. The anti-malarial activity of nitidine could therefore be improved by structural modification of this molecule to increase its penetration of the digestive vacuole in the parasite, where haemoglobin metabolization takes place.

Role of nitric oxide and flavohemoglobin homolog genes in Aspergillus nidulans sexual development and mycotoxin production.

Flavohemoglobins are widely distributed in both prokaryotes and eukaryotes. These proteins are involved in reducing nitric oxide levels. Deletion of the Aspergillus nidulans flavohemoglobin gene fhbA induced sexual development and decreased sterigmatocystin production. Supplementation with a nitric oxide-releasing compound promoted cleistothecial formation and increased nsdD and steA expression, indicating that nitric oxide induces sexual development. This is the first study on the effect of nitric oxide on morphogenesis and secondary metabolism in fungi.

In vitro antimalarial activity of ICL670: a further proof of the correlation between inhibition of ß-hematin formation and of peroxidative degradation of hemin.

Iron chelators such as deferiprone, deferoxamine (DFO) and ICL670 (deferasirox) have previously been shown to display in vitro and/or in vivo antimalarial activities. To gain further insight in their antimalarial mechanism of action, their activities on inhibition of ß-hematin formation and on both peroxidative and glutathione (GSH)-mediated degradation of hemin were investigated. Neither deferiprone nor DFO were able to inhibit ß-hematin formation while ICL670 activity nearly matched that of chloroquine (CQ). Peroxidative degradation of hemin was also only strongly inhibited by both CQ and ICL670, the latter being significantly more efficient at pH 5.2. All iron chelators displayed minor, if any, inhibitory activity on GSH-mediated degradation of hemin. Discrepancies in the results obtained for the three iron chelators show that iron chelation is not the main driving force behind interference with heme degradation. Deferiprone, DFO and ICL670 share little structural community but both ICL670 and antimalarial ursolic acid derivatives (previously shown to block ß-hematin formation and the peroxidative degradation of hemin) have hydrophobic groups and hydroxyphenyl moieties. These similarities in structures and activities further back up a possible two-step mechanism of action previously proposed for ursolic acid derivatives (Mullié et al., 2010) implying (1) stacking of an hydrophobic structure to hemin and (2) additive protection of hemin ferric iron from H(2)O(2) by hydroxyphenyl groups through steric hindrance and/or trapping of oxygen reactive species in the direct neighborhood of ferric iron. These peculiar antimalarial mechanisms of action for ICL670 warrant further investigations and development.