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Ebselen : an antimalarial compound active against ferredoxin-NADP+ reductase of Plasmodium falciparum
No full textEbselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one)is a seleno-organic compound that has been extensively studied as an anti-inflammatory drug. It reacts with thiol groups forming selenylsulphide bonds and it has been reported to inhibit a variety of enzymes involved in different processes1. Moreover, ebselen has been shown possesse an antimalarial activity in vitro against P. falciparum strains in the asexual stages. Since this compounds displayed no cytotoxic effects on human epatocyte, it is of interest for the design of antimalarial drugs2. The effect of ebselen on ferredoxin-NADP+ reductase of Plasmodium falciparum (PfFNR), an enzyme located in the apicoplast of the parasite3, has been investigated. Ebselen was found to slowly and irreversibly inhibit PfFNR targeting one or more Cys residue of the enzyme. Spectrofluorimetric studies pointed out that ebselen promotes the release of prosthetic group FAD. PfFNR was found to be particularly sensitive to inactivation in comparison to similanr flavoenzymes. The compound is 9-fold less active against the omologue FNR from spinach leaf, while Mycobacterium tuberculosis FprA, a ferredoxin-reductase structurally unrelated to plant type FNRs, turned out to be completely insensitive to this inhibitor, These preliminary data suggest that PfFNR could be a potential target of the antiplasmodial activity of ebselen.1. Schewe T. (1995) Gen. Pharmac. 26 (6): 1153-1169 2. Huter A. M. et al. (1989) Parasitol Res. 75 (5): 353-60 3. Seeber F. et al. (2005) Curr. Pharm. Des. 11, 3159-317
The peculiar properties of the redox couple Ferredoxin-NADP+ Reductase/Ferredoxin of Plasmodium falciparum
No full textFERREDOXIN-NADP+ REDUCTASE OF PLASMODIUM FALCIPARUM (PFFNR): PROTEIN ENGINEERING AND INHIBITION STUDIES
Full text linkMalaria is recognised as one of the main health priorities by the World Health Organization. The most severe form of the disease, tropical malaria, is caused by Plasmodium falciparum. The diffusion of strains of this pathogen, resistant to traditional antimalarial treatments, urgently imposes the development of new therapies. P. falciparum belongs to the phylum Apicomplexa, which consists of unicellular, obligate intracellular parasites. These organisms possess an organelle of algal origin, the apicoplast, which has been shown to be required for pathogen survival and represents a known site of action of antimalarial compounds. In the apicoplast of P. falciparum a ferredoxin-NADP+ reductase (PfFNR) has been identified. This enzyme is directly involved in the electron transfer pathway from NADPH to LytB which catalyzes the last step of the mevalonate-independent isoprenoid biosynthesis. The crystallographic structure of PfFNR in complex with the substrate analogue 2’-P-AMP showed the presence of two basic residues, His286 and Lys249, in the NADP(H)-binding site, which are conserved within the Plasmodium genus but not in other plant-type homologues.
Aim of this work has been to clarify the role of His286 and Lys249 in substrate binding and catalysis, and to identify and characterize PfFNR inhibitors.
The study of His286 and Lys249 was carried out by site-directed mutagenesis. The replacement of His286 resulted in a multifaceted effect, which highly depended on the replacement made. Steady-state kinetics showed that the substitution with aliphatic residues, i.e. Ala and Leu, decreases both kcat/KmNADPH and kcat indicating that His286 plays a critical role not only in NADP(H) binding but also in catalysis. 2’-P-AMP inhibition studies and titrations with NADP+ also support this conclusions showing a decreasing in substrate affinity. Moreover rapid kinetic studies clearly demonstrated that the substitution His286Leu leads to a 3.4-fold decrease in the hydride-transfer (HT) rate between NADP(H) and FAD, with a destabilization of CT complexes between them. The His286Lys mutation resulted in the lowest kcat but surprisingly this enzyme form binds 2’-P-AMP and NADP+ with high affinity, indicating that this mutation destabilized the HT-competent conformation of the substrate NADP(H). Unexpectedly, steady-state kinetics and stopped-flow experiments showed that the mutation to Gln gave an enzyme more active than the wild-type, providing a better stabilization of the HT-competent conformation between nicotinamide and isoalloxazine ring. Thus, His286 has a role in modulating the enzyme affinity for NADP(H) and in the precise positioning of the nicotinamide ring in the active site. The replacement of Lys249 with an Ala resulted in an enzyme form with a 10-fold decrease in KmNADPH and Kd for NADP+ or 2’-P-AMP. In particular, as came out from the study of the NADH-K3Fe(CN)6 reductase reaction, this residue participates in substrate recognition by interacting with the 2’-phosphate of the pyridine dinucleotide.
By the in silico screening of two virtual libraries I have identified a pool of compounds potentially active against PfFNR. Five compounds, i.e. I8, I19, I21, I24 and I27, showed an IC50 in the micromolar range and I deeply characterized their inhibition mechanism by steady-state kinetic studies, also evaluating their specificity. Although in the in silico screening the substrate-binding site of PfFNR was targeted for the selection of enzyme ligands, all the compounds found turned out to inhibit PfFNR by a mixed-type mechanism, and did not show the expected pure competitive behavior towards NADPH. Some of the inhibitors turned out to be very specific for Apicomplexan FNRs.
Inhibition studies were carried out also to characterize the mechanism of ebselen, a compound selected within the experimental of a library of more than thousand diverse compounds. Steady-state kinetic studies and spectrofluorimetric analysis showed that ebselen is able to inhibit PfFNR with a two-phases mechanism consisting of a rapid enzyme inactivation followed by a slow FAD release. The inactivation process resulted to be irreversible without any formation of an initial reversible enzyme-inhibitor complex. Moreover the inactivation constant is higher at pH 8.2 rather than at pH 7 consistently with a covalent modification of PfFNR sulphydrylic groups. I also found that FAD release in PfFNR-C99A-C284S is markedly slowed down, suggesting a critical role of the active-site Cys284 in ebselen inhibition mechanism. Nevertheless the mild effect of mutation Cys284Ser on inactivation process suggests that FAD release and inactivation processes involve the modification of different Cys residues.
The need of large amount of purified enzyme for in-depth functional and structural characterization and inhibition studies prompted us to find a new expression system for PfFNR, able to decrease the cost of protein purification. Thus, I developed an expression system for PfFNR-C99A based on the fusion with the yeast SUMO protein. The partial in vivo degradation of poly-His-SUMO-PfFNR-C99A prevented the obtainment of high expression levels. However, a procedure for the rapid and cheap isolation of the recombinant protein was developed, representing an attractive alternative to the previous protocol.
The features of PfFNR highlighted by the studies here reported, point out that PfFNR could represent an attractive drug target, suitable for the development of novel antimalarial compounds
Expression of Plasmodium falciparum ferredoxin-NADP+ reductase in Escherichia coli as a cleavable fusion with the SUMO protein
No full textThe ferredoxin-NADP+ reductase of Plasmodium falciparum (PfFNR) is an enzyme similar to that found in non-photosynthetic plastids of plants (1). It is involved in the biosynthesis of isoprenoid precursors (2) and it has been proposed as a target for novel antimalarial drugs (3). In order to support in-depth inhibition studies, large amounts of recombinant PfFNR are required. To this aim, we have developed an E. coli expression system based on the fusion with the yeast SUMO protein (pET SUMO system, Invitrogen). Recombinant PfFNR has been purified by two successive steps of immobilized nickel ion affinity chromatography. Mature PfFNR has been generated during the purification procedure by treatment with recombinant SUMO protease. Although the total yield of the procedure is lowered by the tendency of the fusion product to undergo spontaneous splitting in vivo, this quick and cheap isolation protocol can provide about 30 mg highly purified PfFNR from each run.
1. Balconi, E., et al. (2009) FEBS J. 276, 3825–3836
2. Röhrich, R.C., et al. (2005) FEBS Lett. 579, 6433–6438
3. Seeber, F., et al. (2005) Curr. Pharm. Des. 11, 3159–3172
The elusive role played by the side chain of Lys249 of Plasmodium falciparum ferredoxin-NADP+ reductase in NADPH/NADH selectivity
No full textPlasmodium falciparum, the causative agent of tropical malaria, possesses plant-type ferredoxin-NADP+ reductase (PfFNR) and ferredoxin (PfFd), which have been proposed as targets for novel antimalarial drugs (1). The catalytic mechanism of PfFNR is under study in order to provide the bases for the design of effective enzyme inhibitors (2). Although PfFNR is quite specific for NADPH, no structural evidence was obtained for protein positive groups interacting with the 2’-phosphate of this substrate (3). We have found that change of Lys249 to Ala decreased the kcat/Km for NADPH by a factor of 10, without affecting the activity of the enzyme with NADH. In addition, PfFNR-K249A displayed an affinity for NADP+ 8-fold lower than those of the wild-type enzyme. We conclude that the side-chain of Lys249 actually interacts with the 2’-phosphate of NADPH during the catalytic cycle, and that it is part of a region flexible enough to adopt in the crystal form of the protein a conformation where such interaction is severed 1. Seeber, F., et al. (2005) Curr. Pharm. Des. 11, 3159-3172.
2. Crobu, D., et al. (2009) Biochemistry 48, 9525-9533.
3. Milani, M., et al. (2007) J. Mol. Biol. 367, 501-513
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Ferredoxin–NADP+ reductases of Apicomplexa: unique properties of protozoan plant–type enzymes
No full textPlant-type ferredoxin-NADP+ reductases (FNRs) are a family of flavin-dependent dehydrogenases/electron transferases ubiquitous in plant plastids, cyanobacteria and some eubacteria (1). Recently, FNRs have been identified in the apicoplast of apicomplexan parasites, which includes the causative agents of malaria and toxoplasmosis. FNRs from Toxoplasma gondii (TgFNR) and Plasmodium falciparum (PfFNR) have been cloned and characterized (2, 3). Whereas TgFNR failed to yield crystals suitable for X-ray analysis, the crystal structure of PfFNR has been determined (3). Compared to FNRs from other sources, PfFNR displays a significantly lower catalytic efficiency and poorer NADH/NADPH selectivity (3). These features of PfFNR have been interpreted on the basis of the peculiar structure of its NADP(H)-binding site. Furthermore, PfFNR undergoes an unprecedented NADP-triggered, redox-dependent homodimerization process leading to enzyme inactivation, which could represent a physiologic mechanism of enzyme regulation. PfFNR has been shown to be involved in the biosynthesis of isoprenoid precursors (4), which is the site of action of known antiplasmodial compounds. On this basis, apicomplexan FNRs have been proposed as a possible new target for novel antiparasitic drugs (2).
References
1. Aliverti, A., Pandini, V., Pennati, A., de Rosa, M., and Zanetti, G., Arch. Biochem. Biophys., 474, 283-291 (2008).
2. Seeber, F., Aliverti, A., and Zanetti, G., Curr. Farm. Des., 11, 3159-7312 (2005).
3. Milani, M., Balconi, E., Aliverti, A., Mastrangelo, E., Seeber, F., Bolognesi, M., and Zanetti, G., J. Mol. Biol., 367, 501-513 (2007).
4. Röhrich, R.C., et al., FEBS Lett., 579, 6433–6438 (2005)
Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis
No full textThe NADP-binding site of Plasmodium falciparum ferredoxin-NADP+ reductase contains two basic residues, His286 and Lys249, conserved within the Plasmodium genus, but not in other plant-type homologues. Previous crystal studies indicated that His286 interacts with the adenine ring and with the 5′-phosphate of 2′-P-AMP, a ligand that mimics the adenylate moiety of NADP(H). Here we show that replacement of His286 with aliphatic residues results both in a decrease in the affinity of the enzyme for NADPH and in a decrease in kcat, due to a lowered hydride-transfer rate. Unexpectedly, the mutation to Gln produces an enzyme more active than the wild-type one, whereas the change to Lys destabilizes the nicotinamide- isoalloxazine interaction, decreasing kcat. On the basis of the crystal structure of selected mutants complexed with 2′-P-AMP, we conclude that the His286 side chain plays a dual role in catalysis both by providing binding energy for NADPH and by favoring the catalytically competent orientation of its nicotinamide ring. For the latter function, the H-bonding potential rather than the positively charged state of the His286 imidazole seems sufficient. Furthermore, we show that the Lys249Ala mutation decreases K mNADPH and Kd for NADP+ or 2′-P-AMP by a factor of 10. We propose that the Lys249 side chain participates in substrate recognition by interacting with the 2′-phosphate of NADP(H) and that this interaction was not observed in the crystal form of the enzyme-2′-P-AMP complex due to a conformational perturbation of the substrate-binding loop induced by dimerization
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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