Boniface M Mailu

Center for Infectious Disease Research, Seattle, Washington, United States

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Publications (10)30.66 Total impact

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    ABSTRACT: The heme-containing enzymes indoleamine 2,3-dioxygenase-1 and -2 (IDO-1, -2) catalyze the conversion of the essential amino acid tryptophan into kynurenine. Metabolites of the kynurenine pathway and IDO itself are involved in immunity and the pathology of several diseases, having either immunoregulatory or antimicrobial effects. IDO-1 plays a central role in the pathogenesis of cerebral malaria, which is the most severe and often fatal neurological complication of infection with Plasmodium falciparum. Mouse models are usually used to study the underlying pathophysiology. In this study, we screened a natural compound library against mouse IDO-1 and identified the 8-aminobenzo[b]quinolizinium 2c as inhibitor of IDO-1 with nanomolar potency (IC50 164 nM). Twenty-one structurally modified derivatives of 2c were synthesized for structure activity relationship analyses. The compounds were found to be selective for IDO-1 over IDO-2. We therefore compared the role of prominent amino acids for the catalytic mechanisms of the two isoenzymes by homology modeling, site-directed mutagenesis, and kinetic analyses. Notably, methionine 385 of IDO-2 was identified to interfere with the entrance of L-tryptophan to the active site of the enzyme, which explains the selectivity of the inhibitors. Most interestingly, several benzo[b]quinolizinium derivatives were found to be highly effective against P. falciparum 3D7 blood stages in cell culture (6 compounds with EC50-values between 2.1 and 6.7 nM) with a mechanism independent from IDO-1 inhibition. We believe that the class of compounds presented here has unique characteristics: it combines the inhibition of mammalian IDO-1 with strong antiparasitic activity, two features that offer potential for drug development.
    No preview · Article · Oct 2015 · Antimicrobial Agents and Chemotherapy
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    ABSTRACT: The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl- tRNA synthetase to synthesize Glu-tRNAGln, and a glutaminyl-tRNA amidotransferase to convert Glu-tRNAGln to Gln-tRNAGln. Here we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyl-tRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNAGln to Gln-tRNAGln in vitro. We demonstrate that the apicoplast GatAB-catalyzed reaction is essential to the parasite blood stages because we could not delete the Plasmodium berghei gene encoding GatA in blood stage parasites in vivo. A phylogenetic analysis placed the split between Plasmodium GatB, archeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Moreover, Plasmodium GatA also appears to have emerged prior to the bacterial- archaeal phylogenetic divide. Thus, while GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Aug 2015 · Journal of Biological Chemistry
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    Full-text · Dataset · Nov 2014
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    ABSTRACT: The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNAGln biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNAGlu and tRNAGln, determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNAGln biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.
    Full-text · Article · Sep 2013 · Journal of Biological Chemistry
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    ABSTRACT: The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.
    Full-text · Article · Mar 2011 · Biochemical Journal
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    ABSTRACT: The first step in the kynurenine pathway of tryptophan catabolism is the cleavage of the 2,3-double bond of the indole ring of tryptophan. In mammals, this reaction is performed independently by indoleamine 2,3-dioxygenase-1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and the recently discovered indoleamine 2,3-dioxygenase-2 (IDO2). Here we describe characteristics of a purified recombinant mouse IDO2 enzyme, including its pH stability, thermal stability and structural features. An improved assay system for future studies of recombinant/isolated IDO2 has been developed using cytochrome b (5) as an electron donor. This, the first description of the interaction between IDO2 and cytochrome b (5), provides further evidence of the presence of a physiological electron carrier necessary for activity of enzymes in the "IDO family". Using this assay, the kinetic activity and substrate range of IDO2 were shown to be different to those of IDO1. 1-Methyl-D-tryptophan, a current lead IDO inhibitor used in clinical trials, was a poor inhibitor of both IDO1 and IDO2 activity. This suggests that its immunosuppressive effect may be independent of pharmacological inhibition of IDO enzymes, in the mouse at least. The different biochemical characteristics of the mouse IDO proteins suggest that they have evolved to have distinct biological roles.
    No preview · Article · Feb 2010 · Amino Acids
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    ABSTRACT: The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM) metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT) were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins, thereby contributing to our understanding of the redox interactome in malarial parasites.
    Full-text · Article · May 2009 · PLoS Pathogens
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    ABSTRACT: Adenylate kinases (AK; ATP+AMP double left right arrow 2 ADP; E.C. 2.7.4.3.) are enzymes essentially involved in energy metabolism and macromolecular biosynthesis. As we reported previously, the malarial parasite Plasmodium falciparum possesses one genuine AK and one GTP-AMP phosphotransferase. Analysis of the P. falciparum genome suggested the presence of one additional adenylate kinase, which we designated AK2. Recombinantly produced AK2 was found to be a monomeric protein of 33 kDa showing a specific activity of 10U/mg with ATP and AMP as a substrate pair and to interact with the AK-specific inhibitor P-1,P-5 -(diadenosine-5')-pentaphosphate (IC50 = 200 nM). At its N-terminus AK2 carries a predicted myristoylation sequence. This sequence is only present in AK2 of P. falciparum causing the severe tropical malaria and not in other malarial parasites. We heterologously coexpressed AK2 and P.falciparum N-myristoyltransferase (NMT) in the presence of myristate in Escherichia coli. As demonstrated by protein purification and mass spectrometry, AK2 is indeed myristoylated under catalysis of the parasites' transferase. The modification significantly enhances the stability of the kinase. Furthermore, AK2 and NMT were shown to interact strongly with each other forming a heterodimeric protein in vitro. To our knowledge this is the first direct evidence that P. falciparum NMT myristoylates an intact malarial protein.
    No preview · Article · Oct 2008 · Molecular and Biochemical Parasitology
  • Kathrin Buchholz · Boniface Mwongela Mailu · R. Heiner Schirmer · Katja Becker
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    ABSTRACT: The protozoon Plasmodium falciparum is the causative agent of tropical malaria which causes up to three million human deaths and up to 500 million episodes of clinical illness throughout the world annually. Children in African countries bear the largest part of this burden. Due to the rapid development of resistance to clinically used drugs like chloroquine and mefloquine and the increasing risk of resistance to artemisinins, novel effective and affordable antimalarial agents are urgently required. The progress made over the last years in the fields of genomics, proteomics, and clinical medicine coupled with improved facilities as well as technical progress in structural biology and high throughput screening methods are essential to support these drug development approaches. Furthermore concerted programs supported by governments, industry and academia contribute significantly to the progress in the field of antimalarial chemotherapy. Among the most interesting antimalarial target proteins currently studied are proteases, like plasmepsins, falcipains and falcilysin, but also protein kinases, glycolytic enzymes and enzymes involved in lipid metabolism and DNA replication. In addition, redox active proteins like glutathione reductase, thioredoxin reductase and glutathione S-transferase have become increasingly interesting. In this article we summarize the major current structure-based antimalarial drug development approaches. We briefly review the presently available three-dimensional structures of Plasmodium proteins together with their potential as drug targets. In parallel, we give an overview over inhibitors that have been developed on the basis of these known parasite protein structures or related structures of proteins from other organisms.
    No preview · Article · Feb 2007 · Frontiers in Drug Design & Discovery
  • Mailu BM · Katja Becker

    No preview · Article · Jan 2007

Publication Stats

116 Citations
30.66 Total Impact Points

Institutions

  • 2015
    • Center for Infectious Disease Research
      Seattle, Washington, United States
  • 2013
    • Seattle Institute for Biomedical and Clinical Research
      Seattle, Washington, United States
  • 2008-2011
    • Justus-Liebig-Universität Gießen
      • Research Centre for BioSystems, Land Use and Nutrition - IFZ
      Gieben, Hesse, Germany