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1.
Adv Biol Regul ; 64: 9-19, 2017 05.
Article in English | MEDLINE | ID: mdl-28342784

ABSTRACT

Production of lipid-derived inositol phosphates including IP4 and IP5 is an evolutionarily conserved process essential for cellular adaptive responses that is dependent on both phospholipase C and the inositol phosphate multikinase Ipk2 (also known as Arg82 and IPMK). Studies of Ipk2, along with Arg82 prior to demonstrating its IP kinase activity, have provided an important link between control of gene expression and IP metabolism as both kinase dependent and independent functions are required for proper transcriptional complex function that enables cellular adaptation in response to extracellular queues such as nutrient availability. Here we define a promoter sequence cis-element, 5'-CCCTAAAAGG-3', that mediates both kinase-dependent and independent functions of Ipk2. Using a synthetic biological strategy, we show that proper gene expression in cells lacking Ipk2 may be restored through add-back of two components: IP4/IP5 production and overproduction of the MADS box DNA binding protein, Mcm1. Our results are consistent with a mechanism by which Ipk2 harbors a dual functionality that stabilizes transcription factor levels and enzymatically produces a small molecule code, which together coordinate control of biological processes and gene expression.


Subject(s)
Gene Expression Regulation, Fungal , Inositol Phosphates/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Type C Phospholipases/genetics , Base Sequence , Cell Nucleus/genetics , Cell Nucleus/metabolism , Minichromosome Maintenance 1 Protein/genetics , Minichromosome Maintenance 1 Protein/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Promoter Regions, Genetic , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , Type C Phospholipases/metabolism
2.
Antimicrob Agents Chemother ; 60(12): 7407-7414, 2016 12.
Article in English | MEDLINE | ID: mdl-27736766

ABSTRACT

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z' factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC50) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening.


Subject(s)
Antimalarials/pharmacology , Fluorescence Resonance Energy Transfer/methods , Glucose/antagonists & inhibitors , High-Throughput Screening Assays , Monosaccharide Transport Proteins/antagonists & inhibitors , Plasmodium falciparum/drug effects , Protozoan Proteins/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Antimalarials/chemistry , Cells, Cultured , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythrocytes/parasitology , Gene Expression , Glucose/metabolism , Glucose Transporter Type 1/genetics , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 2/genetics , Glucose Transporter Type 2/metabolism , Glucose Transporter Type 3/genetics , Glucose Transporter Type 3/metabolism , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , HEK293 Cells , Humans , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Plasmodium falciparum/growth & development , Plasmodium falciparum/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Small Molecule Libraries/chemistry , Species Specificity , Structure-Activity Relationship , Tritium
3.
ACS Infect Dis ; 2(12): 923-935, 2016 12 09.
Article in English | MEDLINE | ID: mdl-27676224

ABSTRACT

Despite continued research efforts, the threat of drug resistance from a variety of bacteria continues to plague clinical communities. Discovery and validation of novel biochemical targets will facilitate development of new drugs to combat these organisms. The methylerythritol phosphate (MEP) pathway to make isoprene units is a biosynthetic pathway essential to many bacteria. We and others have explored inhibitors of the MEP pathway as novel antibacterial agents. Mycobacterium tuberculosis, the causative agent of tuberculosis, and Yersinia pestis, resulting in the plague or "black death", both rely on the MEP pathway for isoprene production. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr) catalyzes the first committed step in the MEP pathway. We examined two series of Dxr inhibitors based on the parent structure of the retrohydroxamate natural product FR900098. The compounds contain either an extended N-acyl or O-linked alkyl/aryl group and are designed to act as bisubstrate inhibitors of the enzyme. While nearly all of the compounds inhibited both Mtb and Yp Dxr to some extent, compounds generally displayed more potent inhibition against the Yp homologue, with the best analogs displaying nanomolar IC50 values. In bacterial growth inhibition assays, the phosphonic acids generally resulted in poor antibacterial activity, likely a reflection of inadequate permeability. Accordingly, diethyl and dipivaloyloxymethyl (POM) prodrug esters of these compounds were made. While the added lipophilicity did not enhance Yersinia activity, the compounds showed significantly improved antitubercular activities. The most potent compounds have Mtb MIC values of 3-12 µg/mL. Taken together, we have uncovered two series of analogs that potently inhibit Dxr homologues from Mtb and Yp. These inhibitors of the MEP pathway, termed MEPicides, serve as leads for future analog development.


Subject(s)
Aldose-Ketose Isomerases/antagonists & inhibitors , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/drug effects , Yersinia pestis/drug effects , Aldose-Ketose Isomerases/genetics , Aldose-Ketose Isomerases/metabolism , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biosynthetic Pathways , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Structure-Activity Relationship , Yersinia pestis/enzymology , Yersinia pestis/genetics , Yersinia pestis/metabolism
4.
J Infect Dis ; 214(7): 1085-91, 2016 10 01.
Article in English | MEDLINE | ID: mdl-27443612

ABSTRACT

Novel antimalarial therapies are needed in the face of emerging resistance to artemisinin combination therapies. A previous study found a high cure rate in Mozambican children with uncomplicated Plasmodium falciparum malaria 7 days after combination treatment with fosmidomycin-clindamycin. However, 28-day cure rates were low (45.9%), owing to parasite recrudescence. We sought to identify any genetic changes underlying parasite recrudescence. To this end, we used a selective whole-genome amplification method to amplify parasite genomes from blood spot DNA samples. Parasite genomes from pretreatment and postrecrudescence samples were subjected to whole-genome sequencing to identify nucleotide variants. Our data did not support the existence of a genetic change responsible for recrudescence following fosmidomycin-clindamycin treatment. Additionally, we found that previously described resistance alleles for these drugs do not represent biomarkers of recrudescence. Future studies should continue to optimize fosmidomycin combinations for use as antimalarial therapies.


Subject(s)
Antimalarials/therapeutic use , Clindamycin/therapeutic use , Drug Resistance , Fosfomycin/analogs & derivatives , Genomics/methods , Malaria, Falciparum/drug therapy , Plasmodium falciparum/drug effects , Antimalarials/pharmacology , Child, Preschool , Clindamycin/pharmacology , Clinical Trials as Topic , Fosfomycin/pharmacology , Fosfomycin/therapeutic use , Genome, Protozoan , Genotype , Humans , Infant , Malaria, Falciparum/parasitology , Mozambique , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Sequence Analysis, DNA/methods , Treatment Failure
5.
Am J Trop Med Hyg ; 93(5): 1092-8, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26350448

ABSTRACT

Anemia diminishes oxygen transport in the body, resulting in potentially irreversible growth and developmental consequences for children. Limited evidence for determinants of anemia exists for school-aged children. We conducted a cluster randomized controlled trial in Haiti from 2012 to 2013 to test the efficacy of a fortified school snack. Children (N = 1,047) aged 3-13 years were followed longitudinally at three time points for hemoglobin (Hb) concentrations, anthropometry, and bioelectrical impedance measures. Dietary intakes, infectious disease morbidities, and socioeconomic and demographic factors were collected at baseline and endline. Longitudinal regression modeling with generalized least squares and logit models with random effects identified anemia risk factors beyond the intervention effect. At baseline, 70.6% of children were anemic and 2.6% were severely anemic. Stunting increased the odds of developing anemia (adjusted odds ratio [OR]: 1.48, 95% confidence interval [CI]: 1.05-2.08) and severe anemia (adjusted OR: 2.47, 95% CI: 1.30-4.71). Parent-reported vitamin A supplementation and deworming were positively associated with Hb concentrations, whereas fever and poultry ownership showed a negative relationship with Hb concentration and increased odds of severe anemia, respectively. Further research should explore the full spectrum of anemia etiologies in school children, including genetic causes.


Subject(s)
Anemia/prevention & control , Dietary Supplements , Food, Fortified , Hemoglobins/analysis , Adolescent , Anemia/complications , Anemia/drug therapy , Anemia/epidemiology , Anthropometry , Body Composition , Body Weight , Child , Child, Preschool , Diet Therapy , Electric Impedance , Female , Growth Disorders/complications , Haiti/epidemiology , Hemoglobins/drug effects , Humans , Longitudinal Studies , Male , Nutritional Status , Risk Factors , Vitamin A/administration & dosage
6.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 9): 1824-34, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26327372

ABSTRACT

Haloacid dehalogenases (HADs) are a large enzyme superfamily of more than 500,000 members with roles in numerous metabolic pathways. Plasmodium falciparum HAD1 (PfHAD1) is a sugar phosphatase that regulates the methylerythritol phosphate (MEP) pathway for isoprenoid synthesis in malaria parasites. However, the structural determinants for diverse substrate recognition by HADs are unknown. Here, crystal structures were determined of PfHAD1 in complex with three sugar phosphates selected from a panel of diverse substrates that it utilizes. Cap-open and cap-closed conformations are observed, with cap closure facilitating substrate binding and ordering. These structural changes define the role of cap movement within the major subcategory of C2 HAD enzymes. The structures of an HAD bound to multiple substrates identifies binding and specificity-determining residues that define the structural basis for substrate recognition and catalysis within the HAD superfamily. While the substrate-binding region of the cap domain is flexible in the open conformations, this region becomes ordered and makes direct interactions with the substrate in the closed conformations. These studies further inform the structural and biochemical basis for catalysis within a large superfamily of HAD enzymes with diverse functions.


Subject(s)
Hydrolases/metabolism , Phosphoric Monoester Hydrolases/metabolism , Plasmodium falciparum/enzymology , Animals , Catalysis , Crystallography, X-Ray , Hydrolases/chemistry , Models, Molecular , Protein Conformation , Substrate Specificity
7.
Cell Host Microbe ; 18(2): 142-3, 2015 Aug 12.
Article in English | MEDLINE | ID: mdl-26269950

ABSTRACT

Toxoplasma gondii, the causative agent of toxoplasmosis, is an intracellular parasite that demonstrates a remarkable ability to adapt to nutrient availability. In this issue of Cell Host & Microbe, Blume et al. (2015) describe the unique role of a gluconeogenic enzyme in regulation of glucose catabolism in T. gondii.


Subject(s)
Carbon/metabolism , Fructose-Bisphosphatase/metabolism , Glucose/metabolism , Toxoplasma/enzymology , Toxoplasma/physiology , Animals
8.
Antimicrob Agents Chemother ; 59(10): 6203-9, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26248369

ABSTRACT

Malaria and HIV infection are coendemic in a large portion of the world and remain a major cause of morbidity and mortality. Growing resistance of Plasmodium species to existing therapies has increased the need for new therapeutic approaches. The Plasmodium glucose transporter PfHT is known to be essential for parasite growth and survival. We have previously shown that HIV protease inhibitors (PIs) act as antagonists of mammalian glucose transporters. While the PI lopinavir is known to have antimalarial activity, the mechanism of action is unknown. We report here that lopinavir blocks glucose uptake into isolated malaria parasites at therapeutically relevant drug levels. Malaria parasites depend on a constant supply of glucose as their primary source of energy, and decreasing the available concentration of glucose leads to parasite death. We identified the malarial glucose transporter PfHT as a target for inhibition by lopinavir that leads to parasite death. This discovery provides a mechanistic basis for the antimalarial effect of lopinavir and provides a direct target for novel drug design with utility beyond the HIV-infected population.


Subject(s)
Glucose/antagonists & inhibitors , HIV Protease Inhibitors/pharmacology , Lopinavir/pharmacology , Monosaccharide Transport Proteins/antagonists & inhibitors , Plasmodium falciparum/drug effects , Protozoan Proteins/antagonists & inhibitors , Antimalarials/chemistry , Antimalarials/pharmacology , Biological Transport , Drug Repositioning , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythrocytes/parasitology , Gene Expression , Glucose/metabolism , HEK293 Cells , HIV Protease Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Lopinavir/chemistry , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Plasmodium falciparum/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship
9.
Antimicrob Agents Chemother ; 59(9): 5511-9, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26124156

ABSTRACT

There is a pressing need for new antimicrobial therapies to combat globally important drug-resistant human pathogens, including Plasmodium falciparum malarial parasites, Mycobacterium tuberculosis, and Gram-negative bacteria, including Escherichia coli. These organisms all possess the essential methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, which is not found in humans. The first dedicated enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr), is inhibited by the phosphonic acid antibiotic fosmidomycin and its analogs, including the N-acetyl analog FR900098 and the phosphoryl analog fosfoxacin. In order to identify mutations in dxr that confer resistance to these drugs, a library of E. coli dxr mutants was screened at lethal fosmidomycin doses. The most resistant allele (with the S222T mutation) alters the fosmidomycin-binding site of Dxr. The expression of this resistant allele increases bacterial resistance to fosmidomycin and other fosmidomycin analogs by 10-fold. These observations confirm that the primary cellular target of fosmidomycin is Dxr. Furthermore, cell lines expressing Dxr-S222T will be a powerful tool to confirm the mechanisms of action of future fosmidomycin analogs.


Subject(s)
Aldose-Ketose Isomerases/genetics , Anti-Bacterial Agents/pharmacology , Fosfomycin/analogs & derivatives , Mycobacterium tuberculosis/enzymology , Prodrugs/pharmacology , Fosfomycin/pharmacology , Microbial Sensitivity Tests , Mutation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics
10.
mBio ; 6(2)2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25805727

ABSTRACT

UNLABELLED: The malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid organelle that possesses plant-like metabolic pathways. Plants use the plastidial isoprenoid biosynthesis pathway to produce volatile odorants, known as terpenes. In this work, we describe the volatile chemical profile of cultured malaria parasites. Among the identified compounds are several plant-like terpenes and terpene derivatives, including known mosquito attractants. We establish the molecular identity of the odorant receptors of the malaria mosquito vector Anopheles gambiae, which responds to these compounds. The malaria parasite produces volatile signals that are recognized by mosquitoes and may thereby mediate host attraction and facilitate transmission. IMPORTANCE: Malaria is a key global health concern. Mosquitoes that transmit malaria are more attracted to malaria parasite-infected mammalian hosts. These studies aimed to understand the chemical signals produced by malaria parasites; such an understanding may lead to new transmission-blocking strategies or noninvasive malaria diagnostics.


Subject(s)
Anopheles/drug effects , Anopheles/physiology , Pheromones/metabolism , Plasmodium/metabolism , Volatile Organic Compounds/metabolism , Animals , Terpenes/metabolism
11.
ACS Infect Dis ; 1(4): 157-167, 2015 Apr 10.
Article in English | MEDLINE | ID: mdl-26783558

ABSTRACT

As resistance to current therapies spreads, novel antimalarials are urgently needed. In this work, we examine the potential for therapeutic intervention via the targeting of Plasmodium IspD (2-C-methyl-D-erythritol 4-phosphate cytidyltransferase), the second dedicated enzyme of the essential methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis. Enzymes of this pathway represent promising therapeutic targets because the pathway is not present in humans. The Malaria Box compound, MMV008138, inhibits Plasmodium falciparum growth, and PfIspD has been proposed as a candidate intracellular target. We find that PfIspD is the sole intracellular target of MMV008138 and characterize the mode of inhibition and target-based resistance, providing chemical validation of this target. Additionally, we find that the Pf ISPD genetic locus is refractory to disruption in malaria parasites, providing independent genetic validation for efforts targeting this enzyme. This work provides compelling support for IspD as a druggable target for the development of additional, much-needed antimalarial agents.

12.
Eukaryot Cell ; 13(11): 1348-59, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25217461

ABSTRACT

Malaria kills nearly 1 million people each year, and the protozoan parasite Plasmodium falciparum has become increasingly resistant to current therapies. Isoprenoid synthesis via the methylerythritol phosphate (MEP) pathway represents an attractive target for the development of new antimalarials. The phosphonic acid antibiotic fosmidomycin is a specific inhibitor of isoprenoid synthesis and has been a helpful tool to outline the essential functions of isoprenoid biosynthesis in P. falciparum. Isoprenoids are a large, diverse class of hydrocarbons that function in a variety of essential cellular processes in eukaryotes. In P. falciparum, isoprenoids are used for tRNA isopentenylation and protein prenylation, as well as the synthesis of vitamin E, carotenoids, ubiquinone, and dolichols. Recently, isoprenoid synthesis in P. falciparum has been shown to be regulated by a sugar phosphatase. We outline what is known about isoprenoid function and the regulation of isoprenoid synthesis in P. falciparum, in order to identify valuable directions for future research.


Subject(s)
Antimalarials/pharmacology , Fosfomycin/analogs & derivatives , Plasmodium falciparum/metabolism , Terpenes/metabolism , Fosfomycin/pharmacology , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology
13.
Nat Commun ; 5: 4467, 2014 Jul 24.
Article in English | MEDLINE | ID: mdl-25058848

ABSTRACT

Isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway generates commercially important products and is a target for antimicrobial drug development. MEP pathway regulation is poorly understood in microorganisms. Here we employ a forward genetics approach to understand MEP pathway regulation in the malaria parasite, Plasmodium falciparum. The antimalarial fosmidomycin inhibits the MEP pathway enzyme deoxyxylulose 5-phosphate reductoisomerase (DXR). Fosmidomycin-resistant P. falciparum are enriched for changes in the PF3D7_1033400 locus (hereafter referred to as PfHAD1), encoding a homologue of haloacid dehalogenase (HAD)-like sugar phosphatases. We describe the structural basis for loss-of-function PfHAD1 alleles and find that PfHAD1 dephosphorylates a variety of sugar phosphates, including glycolytic intermediates. Loss of PfHAD1 is required for fosmidomycin resistance. Parasites lacking PfHAD1 have increased MEP pathway metabolites, particularly the DXR substrate, deoxyxylulose 5-phosphate. PfHAD1 therefore controls substrate availability to the MEP pathway. Because PfHAD1 has homologues in plants and bacteria, other HAD proteins may be MEP pathway regulators.


Subject(s)
Erythritol/analogs & derivatives , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/metabolism , Plasmodium falciparum/metabolism , Sugar Phosphates/metabolism , Aldose-Ketose Isomerases/antagonists & inhibitors , Aldose-Ketose Isomerases/metabolism , Antimalarials/pharmacology , Catalytic Domain , Cytoplasm/metabolism , Drug Resistance , Erythritol/metabolism , Fosfomycin/analogs & derivatives , Fosfomycin/pharmacology , Genetic Complementation Test , Phosphoric Monoester Hydrolases/genetics , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Protein Conformation , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Xylose/analogs & derivatives , Xylose/metabolism
14.
mBio ; 5(3): e01126-14, 2014 Apr 29.
Article in English | MEDLINE | ID: mdl-24781746

ABSTRACT

ABSTRACT New antifungals are needed, particularly in the developing world, to treat life-threatening fungal infections, such as cryptococcosis. Drug repurposing is one strategy to identify new drug-like compounds, but it is often difficult to identify a mechanism of action. Here we discuss the outstanding effort by Butts et al. to identify calmodulin as an antifungal target of repurposed estrogen receptor antagonists [A. Butts, K. Koselny, Y. Chabrier-Roselló, C. P. Semighini, Y. C. S. Brown, et al., mBio 5(1):e00765-13, 2014, doi:10.1128/mBio.00765-13]. The authors show that these compounds bind to and directly inhibit fungal calmodulin and also reduce fungal burden in an animal disease model. These studies thus establish both the key preclinical efficacy and the antifungal mechanism of action, which will allow these compounds to progress toward development of novel antifungal therapies.


Subject(s)
Antifungal Agents/pharmacology , Cryptococcus neoformans/drug effects , Drug Synergism , Fluconazole/pharmacology , Fungal Proteins/metabolism , Selective Estrogen Receptor Modulators/pharmacology
15.
Curr Clin Microbiol Rep ; 1(3-4): 37-50, 2014 Dec 01.
Article in English | MEDLINE | ID: mdl-25893156

ABSTRACT

Apicomplexan parasites include some of the most prevalent and deadly human pathogens. Novel antiparasitic drugs are urgently needed. Synthesis and metabolism of isoprenoids may present multiple targets for therapeutic intervention. The apicoplast-localized methylerythritol phosphate (MEP) pathway for isoprenoid precursor biosynthesis is distinct from the mevalonate (MVA) pathway used by the mammalian host, and this pathway is apparently essential in most Apicomplexa. In this review, we discuss the current field of research on production and metabolic fates of isoprenoids in apicomplexan parasites, including the acquisition of host isoprenoid precursors and downstream products. We describe recent work identifying the first MEP pathway regulator in apicomplexan parasites, and introduce several promising areas for ongoing research into this well-validated antiparasitic target.

16.
Eukaryot Cell ; 12(2): 215-23, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23223036

ABSTRACT

The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum. Since multiple classes of prenyltransferase inhibitors kill P. falciparum, we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors.


Subject(s)
Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Terpenes/metabolism , rab5 GTP-Binding Proteins/metabolism , Androstadienes/pharmacology , Antimalarials/pharmacology , Biosynthetic Pathways/drug effects , Cells, Cultured , Drug Resistance , Electron Transport/drug effects , Erythritol/analogs & derivatives , Erythritol/metabolism , Erythrocytes/parasitology , Fosfomycin/analogs & derivatives , Fosfomycin/pharmacology , Humans , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Plasmodium falciparum/drug effects , Plasmodium falciparum/growth & development , Protein Prenylation , Protein Transport/drug effects , Schizonts/drug effects , Schizonts/growth & development , Schizonts/metabolism , Sugar Phosphates/metabolism , Transport Vesicles/metabolism , Ubiquinone/metabolism , Vacuoles/drug effects , Vacuoles/metabolism , Vacuoles/ultrastructure , Wortmannin
17.
J Biol Chem ; 287(42): 35360-35369, 2012 Oct 12.
Article in English | MEDLINE | ID: mdl-22896696

ABSTRACT

Inositol phosphates (IPs) regulate vital processes in eukaryotes, and their production downstream of phospholipase C activation is controlled through a network of evolutionarily conserved kinases and phosphatases. Inositol phosphate multikinase (IPMK, also called Ipk2 and Arg82) accounts for phosphorylation of IP(3) to IP(5), as well as production of several other IP molecules. Here, we report the structure of Arabidopsis thaliana IPMKα at 2.9 Å and find it is similar to the yeast homolog Ipk2, despite 17% sequence identity, as well as the active site architecture of human IP(3) 3-kinase. Structural comparison and substrate modeling were used to identify a putative basis for IPMK selectivity. To test this model, we re-engineered binding site residues predicted to have restricted substrate specificity. Using steady-state kinetics and in vivo metabolic labeling studies in modified yeast strains, we observed that K117W and K117W:K121W mutants exhibited nearly normal 6-kinase function but harbored significantly reduced 3-kinase activity. These mutants complemented conditional nutritional growth defects observed in ipmk null yeast and, remarkably, suppressed lethality observed in ipmk null flies. Our data are consistent with the hypothesis that IPMK 6-kinase activity and production of Ins(1,4,5,6)P(4) are critical for cellular signaling. Overall, our studies provide new insights into the structure and function of IPMK and utilize a synthetic biological approach to redesign inositol phosphate signaling pathways.


Subject(s)
Amino Acid Substitution , Arabidopsis Proteins/chemistry , Arabidopsis/enzymology , Models, Molecular , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Crystallography, X-Ray , Humans , Mutation, Missense , Phosphatidylinositol Phosphates/genetics , Phosphatidylinositol Phosphates/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid , Signal Transduction/physiology , Structure-Activity Relationship , Substrate Specificity/genetics
18.
Biochemistry ; 50(17): 3570-7, 2011 May 03.
Article in English | MEDLINE | ID: mdl-21438569

ABSTRACT

Antimicrobial drug resistance is an urgent problem in the control and treatment of many of the world's most serious infections, including Plasmodium falciparum malaria, tuberculosis, and healthcare-associated infections with Gram-negative bacteria. Because the non-mevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum and this pathway is not present in humans, there is great interest in targeting the enzymes of non-mevalonate metabolism for antibacterial and antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent currently in clinical trials of combination therapies for the treatment of malaria. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. To define the in vivo metabolic response to fosmidomycin, we developed a novel mass spectrometry method to quantitate six metabolites of non-mevalonate isoprenoid metabolism from complex biological samples. Using this technique, we validate that the biological effects of fosmidomycin are mediated through blockade of de novo isoprenoid biosynthesis in both P. falciparum malaria parasites and Escherichia coli bacteria: in both organisms, metabolic profiling demonstrated a block of isoprenoid metabolism following fosmidomycin treatment, and growth inhibition due to fosmidomycin was rescued by media supplemented with isoprenoid metabolites. Isoprenoid metabolism proceeded through DXR even in the presence of fosmidomycin but was inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro. This work has validated fosmidomycin as a biological reagent for blocking non-mevalonate isoprenoid metabolism and suggests a second in vivo target for fosmidomycin within isoprenoid biosynthesis, in two evolutionarily diverse pathogens.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antimalarials/pharmacology , Escherichia coli/drug effects , Fosfomycin/analogs & derivatives , Plasmodium falciparum/drug effects , Terpenes/metabolism , Aldose-Ketose Isomerases/metabolism , Chromatography, Liquid , Culture Media , Drug Resistance, Microbial , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Fosfomycin/pharmacology , Mevalonic Acid/metabolism , Multienzyme Complexes/metabolism , Oxidoreductases/metabolism , Plasmodium falciparum/growth & development , Plasmodium falciparum/metabolism , Tandem Mass Spectrometry
20.
Mol Biochem Parasitol ; 170(2): 108-11, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20018214

ABSTRACT

Novel antimalarial drugs are urgently needed to treat severe malaria caused by Plasmodium falciparum. Isoprenoid biosynthesis is a promising target pathway, since the biosynthetic route in Plasmodia is biochemically distinct from the mevalonate pathway in humans. The small molecule fosmidomycin is an inhibitor of the enzyme responsible for the first dedicated step in isoprenoid biosynthesis, deoxyxylulose 5-phosphate reductoisomerase (DXR). However, the antimalarial effects of fosmidomycin might not be specific to DXR inhibition and further validation of DXR is warranted. We present the first functional genetic validation of P. falciparum DXR (PF14_0641). Using a single cross-over strategy, we show that plasmid integration occurs at the DXR locus but only when DXR gene function is preserved, but not when integration disrupts gene function. These data indicate that DXR is required for intraerythrocytic development of P. falciparum.


Subject(s)
Aldose-Ketose Isomerases/genetics , Erythrocytes/parasitology , Multienzyme Complexes/genetics , Oxidoreductases/genetics , Plasmodium falciparum/enzymology , Protozoan Proteins/genetics , Aldose-Ketose Isomerases/physiology , Animals , Genes, Essential , Genes, Protozoan , Humans , Multienzyme Complexes/physiology , Mutagenesis, Insertional , Oxidoreductases/physiology , Plasmids , Plasmodium falciparum/growth & development , Protozoan Proteins/physiology
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