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Malaria is a potentially fatal disease caused by Plasmodium parasites and poses a major medical risk in large parts of the world. The development of new, affordable antimalarial drugs is of vital importance as there are increasing reports of resistance to the currently available therapeutics. In addition, most of the current drugs used for chemoprophylaxis merely act on parasites already replicating in the blood. At this point, a patient might already be suffering from the symptoms associated with the disease and could additionally be infectious to an Anopheles mosquito. These insects act as a vector, subsequently spreading the disease to other humans. In order to cure not only malaria but prevent transmission as well, a drug must target both the blood- and pre-erythrocytic liver stages of the parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) reductase (ENR) is a key enzyme of plasmodial type II fatty acid biosynthesis (FAS II). It has been shown to be essential for liver-stage development of Plasmodium berghei and is therefore qualified as a target for true causal chemoprophylaxis. Using virtual screening based on two crystal structures of PfENR, we identified a structurally novel class of FAS inhibitors. Subsequent chemical optimization yielded two compounds that are effective against multiple stages of the malaria parasite. These two most promising derivatives were found to inhibit blood-stage parasite growth with IC(50) values of 1.7 and 3.0 μM and lead to a more prominent developmental attenuation of liver-stage parasites than the gold-standard drug, primaquine.

Plasmodium falciparum lipoate protein ligase 1 (PfLipL1) is an ATP-dependent ligase that belongs to the biotin/lipoate A/B protein ligase family (PFAM PF03099). PfLipL1 is the only known canonical lipoate ligase in Pf and functions as a redox switch between two lipoylation routes in the parasite mitochondrion. Here, we report the crystal structure of a deletion construct of PfLipL1 (PfLipL1Δ243–279) bound to lipoate, and validate the lipoylation activity of this construct in both an in vitro lipoylation assay and a cell based lipoylation assay. This characterization represents the first step in understanding the redox dependence of the lipoylation mechanism in malaria parasites.

Chloroquine, long the default first-line treatment against malaria, is now abandoned in large parts of the world because of widespread drug-resistance in Plasmodium falciparum. In spite of its importance as a cost-effective and efficient drug, a coherent understanding of the cellular mechanisms affected by chloroquine and how they influence the fitness and survival of the parasite remains elusive. Here, we used a systems biology approach to integrate genome-scale transcriptomics to map out the effects of chloroquine, identify targeted metabolic pathways, and translate these findings into mechanistic insights. Specifically, we first developed a method that integrates transcriptomic and metabolomic data, which we independently validated against a recently published set of such data for Krebs-cycle mutants of P. falciparum. We then used the method to calculate the effect of chloroquine treatment on the metabolic flux profiles of P. falciparum during the intraerythrocytic developmental cycle. The model predicted dose-dependent inhibition of DNA replication, in agreement with earlier experimental results for both drug-sensitive and drug-resistant P. falciparum strains. Our simulations also corroborated experimental findings that suggest differences in chloroquine sensitivity between ring- and schizont-stage P. falciparum. Our analysis also suggests that metabolic fluxes that govern reduced thioredoxin and phosphoenolpyruvate synthesis are significantly decreased and are pivotal to chloroquine-based inhibition of P. falciparum DNA replication. The consequences of impaired phosphoenolpyruvate synthesis and redox metabolism are reduced carbon fixation and increased oxidative stress, respectively, both of which eventually facilitate killing of the parasite. Our analysis suggests that a combination of chloroquine (or an analogue) and another drug, which inhibits carbon fixation and/or increases oxidative stress, should increase the clearance of P. falciparum from the host system., Graphical abstract Image 1

Trafficking of soluble proteins to the apicoplast in Plasmodium falciparum is determined by an N-terminal transit peptide (TP) which is necessary and sufficient for apicoplast import. Apicoplast precursor proteins are synthesized at the rough endoplasmic reticulum, but are then specifically sorted from other proteins in the secretory pathway. The mechanism of TP recognition is presently unknown. Apicoplast TPs do not contain a conserved sequence motif; therefore, we asked whether they contain an essential structural motif. Using nuclear magnetic resonance to study a model TP from acyl carrier protein, we found a short, low-occupancy helix, but the TP was otherwise disordered. Using an in vivo localization assay, we blocked TP secondary structure by proline mutagenesis, but found robust apicoplast localization. Alternatively, we increased the helical content of the TP through mutation while maintaining established TP characteristics. Apicoplast import was disrupted in a helical mutant TP, but import was then restored by the further addition of a single proline. We conclude that structure in the TP interferes with apicoplast import, and therefore TPs are functionally disordered. These results provide an explanation for the amino acid bias observed in apicoplast TPs.

Phytochemical investigation of the figs of Ficus mucuso led to the isolation of three new isoflavone dimer derivatives, mucusisoflavones A-C (1-3), together with 16 known compounds. Some of the isolates were tested in vitro for their inhibitory properties toward β-glucuronidase and Plasmodium falciparum enoyl-ACP reductase (PfENR) enzymes. Compound 1 (IC₅₀) 0.68 μM) showed inhibitory activity on β-glucuronidase enzyme, while 3 (IC₅₀) 7.69 μM) exhibited a weak inhibitory activity against P. falciparum enoyl-ACP reductase (PfENR).

Plasmodium falciparum , in addition to scavenging essential fatty acids from its intra- and intercellular environments, possesses a functional complement of type II fatty acid synthase (FAS) enzymes targeted to the apicoplast organelle. Recent evidence suggests that products of the plasmodial FAS II system may be critical for the parasite's liver-to-blood cycle transition, and it has been speculated that endogenously generated fatty acids may be precursors for essential cofactors, such as lipoate, in the apicoplast. β-Ketoacyl-acyl carrier protein (ACP) synthase III (pfKASIII or FabH) is one of the key enzymes in the initiating steps of the FAS II pathway, possessing two functions in P. falciparum : the decarboxylative thio-Claisen condensation of malonyl-ACP and various acyl coenzymes A (acyl-CoAs; KAS activity) and the acetyl-CoA:ACP transacylase reaction (ACAT). Here, we report the generation and characterization of a hybrid Lactococcus lactis strain that translates pfKASIII instead of L. lactis f abH to initiate fatty acid biosynthesis. The L. lactis expression vector pMG36e was modified for the efficient overexpression of the plasmodial gene in L. lactis . Transcriptional analysis indicated high-efficiency overexpression, and biochemical KAS and ACAT assays confirm these activities in cell extracts. Phenotypically, the L. lactis strain expressing pfKASIII has a growth rate and fatty acid profiles that are comparable to those of the strain complemented with its endogenous gene, suggesting that pfKASIII can use L. lactis ACP as substrate and perform near-normal function in L. lactis cells. This strain may have potential application as a bacterial model for pfKASIII inhibitor prescreening.

SUMMARY Lipoic acid [( R )-5-(1,2-dithiolan-3-yl)pentanoic acid] is an enzyme cofactor required for intermediate metabolism in free-living cells. Lipoic acid was discovered nearly 60 years ago and was shown to be covalently attached to proteins in several multicomponent dehydrogenases. Cells can acquire lipoate (the deprotonated charge form of lipoic acid that dominates at physiological pH) through either scavenging or de novo synthesis. Microbial pathogens implement these basic lipoylation strategies with a surprising variety of adaptations which can affect pathogenesis and virulence. Similarly, lipoylated proteins are responsible for effects beyond their classical roles in catalysis. These include roles in oxidative defense, bacterial sporulation, and gene expression. This review surveys the role of lipoate metabolism in bacterial, fungal, and protozoan pathogens and how these organisms have employed this metabolism to adapt to niche environments.

Two new sphingolipids mucusamide (1) and mucusoside (2) have been isolated from methanol soluble part of the stem bark of Ficus mucuso WELW., together with fifteen known secondary metabolites including cellobiosylsterol (3), β-sitosterol (4), stigmasterol (5), β-sitosterol 3-O-β-D-glucopyranoside (6), lupeol acetate (7), ursolic acid (8), procatechuic acid (9), 2-methyl-5,7-dihydroxychromone 8-C-β-D-glucoside (10), apigenin (11), (-)-epicatechin (12), (+)-catechin (13), N-benzoyl-L-phenylalanilol (14), α-acetylamino-phenylpropyl α-benzoylamino-phenylpropionate (15), asperphenamate (16) and bejaminamide (17). Structures of compounds 1 and 2 were elucidated by spectroscopic analysis and chemical methods.

Acyl Carrier Protein (ACP) has a single reactive sulfhydryl necessary for function in covalently binding nascent fatty acids during biosynthesis. In Plasmodium falciparum, the causative agent of the most lethal form of malaria, fatty acid biosynthesis occurs in the apicoplast organelle during the liver stage of the parasite life cycle. During the blood stage, fatty acid biosynthesis is inactive and the redox state of the apicoplast has not been determined. We solved the crystal structure of ACP from P. falciparum in reduced and disulfide-linked forms, and observe the surprising result that the disulfide in the PfACP cross-linked dimer is sequestered from bulk solvent in a tight molecular interface. We assessed solvent accessibility of the disulfide with small molecule reducing agents and found that the disulfide is protected from BME but less so for other common reducing agents. We examined cultured P. falciparum parasites to determine which form of PfACP is prevalent during the blood stages. We readily detected monomeric PfACP in parasite lysate, but do not observe the disulfide-linked form, even under conditions of oxidative stress. To demonstrate that PfACP contains a free sulfhydryl and is not acylated or in the apo state, we treated blood stage parasites with the disulfide forming reagent diamide. We found that the effects of diamide are reversed with reducing agent. Together, these results suggest that the apicoplast is a reducing compartment, as suggested by models of P. falciparum metabolism, and that PfACP is maintained in a reduced state during blood stage growth. Proteins 2010. © 2009 Wiley-Liss, Inc.

The importance of fatty acids to the human malaria parasite, Plasmodium falciparum, and differences due to a type I fatty acid synthesis (FAS) pathway in the parasite, make it an attractive drug target. In the present study, we developed and a utilized a pharmacophore to select compounds for testing against PfKASIII, the initiating enzyme of FAS. This effort identified several PfKASIII inhibitors that grouped into various chemical classes of sulfides, sulfonamides, and sulfonyls. Approximately 60% of the submicromolar inhibitors of PfKASIII inhibited in vitro growth of the malaria parasite. These compounds inhibited both drug sensitive and resistant parasites and testing against a mammalian cell line revealed an encouraging in vitro therapeutic index for the most active compounds. Docking studies into the active site of PfKASIII suggest a potential binding mode that exploits amino acid residues at the mouth of the substrate tunnel.

Lipoate is an essential cofactor for key enzymes of oxidative metabolism. Plasmodium falciparum possesses genes for lipoate biosynthesis and scavenging, but it is not known if these pathways are functional, nor what their relative contribution to the survival of intraerythrocytic parasites might be. We detected in parasite extracts four lipoylated proteins, one of which cross-reacted with antibodies against the E2 subunit of apicoplast-localized pyruvate dehydrogenase (PDH). Two highly divergent parasite lipoate ligase A homologues (LplA), LipL1 (previously identified as LplA) and LipL2, restored lipoate scavenging in lipoylation-deficient bacteria, indicating that Plasmodium has functional lipoate-scavenging enzymes. Accordingly, intraerythrocytic parasites scavenged radiolabelled lipoate and incorporated it into three proteins likely to be mitochondrial. Scavenged lipoate was not attached to the PDH E2 subunit, implying that lipoate scavenging drives mitochondrial lipoylation, while apicoplast lipoylation relies on biosynthesis. The lipoate analogue 8-bromo-octanoate inhibited LipL1 activity and arrested P. falciparum in vitro growth, decreasing the incorporation of radiolabelled lipoate into parasite proteins. Furthermore, growth inhibition was prevented by lipoate addition in the medium. These results are consistent with 8-bromo-octanoate specifically interfering with lipoate scavenging. Our study suggests that lipoate metabolic pathways are not redundant, and that lipoate scavenging is critical for Plasmodium intraerythrocytic survival.

The crystal structures of T. gondii and P. falciparum ENR in complex with NAD+ and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively., Recent studies have demonstrated that submicromolar concentrations of the biocide triclosan arrest the growth of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii and inhibit the activity of the apicomplexan enoyl acyl carrier protein reductase (ENR). The crystal structures of T. gondii and P. falciparum ENR in complex with NAD+ and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively. The structures of T. gondii ENR have revealed that, as in its bacterial and plant homologues, a loop region which flanks the active site becomes ordered upon inhibitor binding, resulting in the slow tight binding of triclosan. In addition, the T. gondii ENR–triclosan complex reveals the folding of a hydrophilic insert common to the apicomplexan family that flanks the substrate-binding domain and is disordered in all other reported apicomplexan ENR structures. Structural comparison of the apicomplexan ENR structures with their bacterial and plant counterparts has revealed that although the active sites of the parasite enzymes are broadly similar to those of their bacterial counterparts, there are a number of important differences within the drug-binding pocket that reduce the packing interactions formed with several inhibitors in the apicomplexan ENR enzymes. Together with other significant structural differences, this provides a possible explanation of the lower affinity of the parasite ENR enzyme family for aminopyridine-based inhibitors, suggesting that an effective antiparasitic agent may well be distinct from equivalent antimicrobials.

Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a “LAMPole”) that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections.

Lipoate is an organosulfur cofactor that participates in several, well-conserved metabolic pathways in malaria parasites. The mitochondrial proteins of Plasmodium are lipoylated through the uptake ...

Cyclin-dependent protein kinases (CDKs) are attractive targets for drug discovery and efforts have led to the identification of novel CDK selective inhibitors in the development of treatments for cancers, neurological disorders, and infectious diseases. More recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. CDKs are valid targets as they function as essential regulators of cell growth and differentiation. To date, several CDKs have been characterized from the genome of the malaria-causing protozoan Plasmodium falciparum . Our approach employs experimental and virtual screening methodologies to identify and refine chemical inhibitors of the parasite CDK Pfmrk, a sequence homologue of human CDK7. Chemotypes of Pfmrk inhibitors include the purines, quinolinones, oxindoles, and chalcones, which have sub-micromolar IC 50 values against the parasite enzyme, but not the human CDKs. Additionally, we have developed and validated a pharmacophore, based on Pfmrk inhibitors, which contains two hydrogen bond acceptor functions and two hydrophobic sites, including one aromatic ring hydrophobic site. This pharmacophore has been exploited to identify additional compounds that demonstrate significant inhibitory activity against Pfmrk. A molecular model of Pfmrk designed using the crystal structure of human CDK7 highlights key amino acid substitutions in the ATP binding pocket. Molecular modeling and docking of the active site pocket with selective inhibitors has identified possible receptor–ligand interactions that may be responsible for inhibitor specificity. Overall, the unique biochemical characteristics associated with this protein, to include distinctive active site amino acid residues and variable inhibitor profiles, distinguishes the Pfmrk drug screen as a paradigm for CDK inhibitor analysis in the parasite.

S-adenosylhomocysteine hydrolase (SAHH) is a key regulator of S-adenosylmethionine-dependent methylation reactions and an interesting pharmacologic target. We cloned the SAHH gene from Plasmodium falciparum (PfSAHH), with an amino acid sequence agreeing with that of the PlasmoDB genomic database. Even though the expressed recombinant enzyme, PfSAHH, could use 3-deaza-adenosine (DZA) as an alternative substrate in contrast to the human SAHH, it has a unique inability to substitute 3-deaza-(+/-)aristeromycin (DZAri) for adenosine. Among the analogs of DZA, including neplanocin A, DZAri was the most potent inhibitor of the PfSAHH enzyme activity, with a K(i) of about 150 nM, whether Ado or DZA was used as a substrate. When the same DZA analogs were tested for their antimalarial activity, they also inhibited the in vitro growth of P. falciparum parasites potently. Homology-modeling analysis revealed that a single substitution (Thr60-Cys59) between the human and malarial PfSAHH, in an otherwise similar SAH-binding pocket, might account for the differential interactions with the nucleoside analogs. This subtle difference in the active site may be exploited in the development of novel drugs that selectively inhibit PfSAHH. We performed a comprehensive phylogenetic analysis of the SAHH superfamily and inferred that SAHH evolved in the common ancestor of Archaea and Eukaryota, and was subsequently horizontally transferred to Bacteria. Additionally, an analysis of the unusual and uncharacterized AHCYL1 family of the SAHH paralogs extant only in animals reveals striking divergence of its SAH-binding pocket and the loss of key conserved residues, thus suggesting an evolution of novel function(s).

Cyclin dependent protein kinases (CDKs) have become attractive drug targets in an effort to identify effective inhibitors of the parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria. We tested known CDK inhibitors for their ability to inhibit two malarial CDKs: Pfmrk and PfPK5. Many broad spectrum CDK inhibitors failed to inhibit Pfmrk suggesting that the active site differs from other CDKs in important ways. By screening compounds in the Walter Reed chemical database, we identified oxindole-based compounds as effective inhibitors of Pfmrk (IC(50) = 1.5 microM). These compounds have low cross-reactivity against PfPK5 and human CDK1 demonstrating selectivity for Pfmrk. Amino acid comparison of the active sites of Pfmrk and PfPK5 identified unique amino acid differences that may explain this selectivity and be exploited for further drug development efforts.

Malaria, a disease caused by protozoan parasites of the genus Plasmodium, is one of the most dangerous infectious diseases, claiming millions of lives and infecting hundreds of millions of people annually. The pressing need for new antimalarials has been answered by the discovery of new drug targets from the malaria genome project. One of the early findings was the discovery of two genes encoding Type II fatty acid biosynthesis proteins: ACP (acyl carrier protein) and KASIII (beta-ketoacyl-ACP synthase III). The initiating steps of a Type II system require a third protein: malonyl-coenzyme A:ACP transacylase (MCAT). Here we report the identification of a single gene from P. falciparum encoding pfMCAT and the functional characterization of this enzyme. Pure recombinant pfMCAT catalyzes malonyl transfer from malonyl-coenzyme A (malonyl-CoA) to pfACP. In contrast, pfACP(trans), a construct of pfACP containing an amino-terminal apicoplast transit peptide, was not a substrate for pfMCAT. The product of the pfMCAT reaction, malonyl-pfACP, is a substrate for pfKASIII, which catalyzes the decarboxylative condensation of malonyl-pfACP and various acyl-CoAs. Consistent with a role in de novo fatty acid biosynthesis, pfKASIII exhibited typical KAS (beta-ketoacyl ACP synthase) activity using acetyl-CoA as substrate (k(cat) 230 min(-1), K(M) 17.9 +/- 3.4 microM). The pfKASIII can also catalyze the condensation of malonyl-pfACP and butyryl-CoA (k(cat) 200 min(-1), K(M) 35.7 +/- 4.4 microM) with similar efficiency, whereas isobutyryl-CoA is a poor substrate and displayed 13-fold less activity than that observed for acetyl-CoA. The pfKASIII has little preference for malonyl-pfACP (k(cat)/K(M) 64.9 min(-1)microM(-1)) over E. coli malonyl-ACP (k(cat)/K(M) 44.8 min(-1)microM(-1)). The pfKASIII also catalyzes the acyl-CoA:ACP transacylase (ACAT) reaction typically exhibited by KASIII enzymes, but does so almost 700-fold slower than the KAS reaction. Thiolactomycin did not inhbit pfKASIII (IC(50)330 microM), but three structurally similar substituted 1,2-dithiole-3-one compounds did inhibit pfKASIII with IC(50) values between 0.53 microM and 10.4 microM. These compounds also inhibited the growth of P. falciparum in culture.

Lipoate scavenging from the human host is essential for malaria parasite survival. Scavenged lipoate is covalently attached to three parasite proteins: the H-protein and the E2 subunits of branched chain amino acid dehydrogenase (BCDH) and α-ketoglutarate dehydrogenase (KDH). We show mitochondrial localization for the E2 subunits of BCDH and KDH, similar to previously localized H-protein, demonstrating that all three lipoylated proteins reside in the parasite mitochondrion. The lipoate ligase 1, LipL1, has been shown to reside in the mitochondrion and it catalyzes the lipoylation of the H-protein; however, we show that LipL1 alone cannot lipoylate BCDH or KDH. A second mitochondrial protein with homology to lipoate ligases, LipL2, does not show ligase activity and is not capable of lipoylating any of the mitochondrial substrates. Instead, BCDH and KDH are lipoylated through a novel mechanism requiring both LipL1 and LipL2. This mechanism is sensitive to redox conditions where BCDH and KDH are exclusively lipoylated under strong reducing conditions in contrast to the H-protein which is preferentially lipoylated under less reducing conditions. Thus, malaria parasites contain two different routes of mitochondrial lipoylation, an arrangement that has not been described for any other organism.

The regulatory mechanisms of most cyclin dependent protein kinases (CDKs) are well understood and are highly conserved in eukaryotes. CDKs from the malaria parasite, Plasmodium falciparum, appear to be regulated in a similar manner with regard to cyclin binding and phosphorylation. In order to further understand their regulatory mechanisms, we examined two classes of cyclin dependent kinase inhibitors (CDIs) to inhibit a panel of plasmodial CDKs. We find that Pfmrk and PfPK5 are inhibited by heterologous p21(CIP1) with varying degrees of inhibition. In contrast, PfPK6, a kinase with sequence features characteristic of both a CDK and MAP kinase, is unaffected by this CDI. Furthermore, the CDK4/6 specific CDI, p16(INK4), fails to inhibit these plasmodial CDKs. Taken together, these results suggest that plasmodial CDKs may be regulated by the binding of inhibitory proteins in vivo.

Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from Plasmodium falciparum and Plasmodium vivax have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. We developed an automated assay of P. falciparum plasmepsin II and P. vivax plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) diphenylurea derivative (WR268961) was found to inhibit plasmepsins with a K i of 1 to 6 μM. This compound appears to be selective for plasmepsin, since it is a poor inhibitor of the human aspartic protease cathepsin D ( K i greater than 280 μM). WR268961 inhibited the growth of P. falciparum strains W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 μg/ml, but was much less toxic to mammalian cells. The Walter Reed chemical database contains over 1,500 compounds with a diphenylurea core structure, 9 of which inhibit the plasmepsins, with K i values ranging from 0.05 to 0.68 μM. These nine compounds show specificity for the plasmepsins over human cathepsin D, but they are poor inhibitors of P. falciparum growth in vitro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.

The eukaryotic cell cycle is regulated by a group of highly conserved cyclin dependent protein kinases (CDKs). Several CDKs have been identified in Plasmodium falciparum, however, their regulatory mechanisms as well as their role in parasite growth and differentiation are not understood fully. To further our understanding of Plasmodium CDK regulation, we have characterized Pfmrk kinase activity. Pfmrk was expressed and purified as a 6xHis tagged recombinant protein from Escherichia coli and assayed for histone H1 kinase activity. Pfmrk has significant histone H1 kinase activity and is autophosphorylated in vitro. Human cyclin H forms a stable complex with Pfmrk and stimulates kinase activity. This is the first indication that Plasmodial CDKs are partially regulated by cyclin subunits, as are human CDKs. CDKs are attractive drug targets due to their role in cellular proliferation. Specific CDK inhibitors were selected to evaluate Pfmrk as a potential drug target. Olomoucine and roscovitine failed to inhibit Pfmrk kinase activity which places Pfmrk with a class of CDKs that are insensitive to these compounds. A molecular model of Pfmrk provides a structural explanation for the failure of these compounds to inhibit Pfmrk.

S-Adenosylmethionine (AdoMet) synthetase (SAMS: EC 2.5.1.6) catalyses the formation of AdoMet from methionine and ATP. We have cloned a gene for Plasmodium falciparum AdoMet synthetase (PfSAMS) (GenBank accession no. AF097923), consisting of 1209 base pairs with no introns. The gene encodes a polypeptide (PfSAMS) of 402 amino acids with a molecular mass of 44844 Da, and has an overall base composition of 67% A+T. PfSAMS is probably a single copy gene, and was mapped to chromosome 9. The PfSAMS protein is highly homologous to all other SAMS, including a conserved motif for the phosphate-binding P-loop, HGGGAFSGKD, and the signature hexapeptide, GAGDQG. All the active-site amino acids for the binding of ADP, Pi and metal ions are similarly preserved, matching entirely those of human hepatic SAMS and Escherichia coli SAMS. Molecular modelling of PfSAMS guided by the X-ray crystal structure of E. coli SAMS indicates that PfSAMS binds ATP/Mg2+ in a manner similar to that seen in the E. coli SAMS structure. However, the PfSAMS model shows that it can not form tetramers as does E. coli SAMS, and is probably a dimer instead. There was a differential sensitivity towards the inhibition by cycloleucine between the expressed PfSAMS and the human hepatic SAMS with Ki values of 17 and 10 mM, respectively. Based on phylogenetic analysis using protein parsimony and neighbour-joining algorithms, the malarial PfSAMS is closely related to SAMS of other protozoans and plants.

Peptide amidation is a ubiquitous posttranslational modification of bioactive peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first step of this reaction, is composed of two domains, each of which binds one copper atom. The coppers are held 11 A apart on either side of a solvent-filled interdomain cleft, and the PHM reaction requires electron transfer between these sites. A plausible mechanism for electron transfer might involve interdomain motion to decrease the distance between the copper atoms. Our experiments show that PHM catalytic core (PHMcc) is enzymatically active in the crystal phase, where interdomain motion is not possible. Instead, structures of two states relevant to catalysis indicate that water, substrate and active site residues may provide an electron transfer pathway that exists only during the PHM catalytic cycle.

Fatty acid biosynthesis has been viewed as an important biological function of and therapeutic target for Plasmodium falciparum asexual blood stage infection. This apicoplast-resident type II pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of the bacterial FabI inhibitor triclosan. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood stage growth. In contrast, mosquito-derived fabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver stage development in vitro. This is characterized by an inability to form intra-hepatic merosomes that normally initiate blood stage infections. These data illuminate key differences between liver and blood stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Crystals of T. gondii ENR in complex with NAD+ and triclosan were grown using the hanging-drop vapour-diffusion method with PEG 8000 as precipitant., The protozoan parasite Toxoplasma gondii is the causative agent of one of the most widespread parasitic infections of man and is a leading cause of congenital neurological birth defects and the third most common cause of food-borne deaths in the United States. Despite this, to date no drugs are available that provide a fully effective treatment. Recently, the antibacterial agent triclosan was shown to inhibit the fatty-acid biosynthesis pathway in T. gondii and to interact with the enoyl reductase (ENR). In order to analyse the potential of triclosan as a lead compound targeting T. gondii ENR and to explore unique features of the apicomplexan enzyme that could be exploited in future drug development, structural studies have been initiated on T. gondii ENR. Crystals of T. gondii ENR in complex with NAD+ and triclosan were grown using the hanging-drop vapour-diffusion method with PEG 8000 as precipitant. The crystals belong to space group P3221, with approximate unit-cell parameters a = 78.1, b = 78.1, c = 188.5 Å, α = β = 90, γ = 120° and a dimer in the asymmetric unit. Test data were collected to beyond 2.6 Å on cryocooled crystals (100 K) using a Rigaku MM007 rotating-anode X-ray source, revealing that the crystals are suitable for a full structural determination.

Many microbial pathogens rely on a type II fatty acid synthesis (FASII) pathway that is distinct from the type I pathway found in humans. Enoyl-acyl carrier protein reductase (ENR) is an essential FASII pathway enzyme and the target of a number of antimicrobial drug discovery efforts. The biocide triclosan is established as a potent inhibitor of ENR and has been the starting point for medicinal chemistry studies. We evaluated a series of triclosan analogues for their ability to inhibit the growth of Toxoplasma gondii, a pervasive human pathogen, and its ENR enzyme (TgENR). Several compounds that inhibited TgENR at low nanomolar concentrations were identified but could not be further differentiated because of the limited dynamic range of the TgENR activity assay. Thus, we adapted a thermal shift assay (TSA) to directly measure the dissociation constant (Kd) of the most potent inhibitors identified in this study as well as inhibitors from previous studies. Furthermore, the TSA allowed us to determine the mode of action of these compounds in the presence of the reduced nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide (NAD⁺) cofactor. We found that all of the inhibitors bind to a TgENR-NAD⁺ complex but that they differed in their dependence on NAD⁺ concentration. Ultimately, we were able to identify compounds that bind to the TgENR-NAD⁺ complex in the low femtomolar range. This shows how TSA data combined with enzyme inhibition, parasite growth inhibition data, and ADMET predictions allow for better discrimination between potent ENR inhibitors for the future development of medicine.

Lipoxygenases are a class of non-heme iron dioxygenases which catalyze the hydroperoxidation of fatty acids for the biosynthesis of leukotrienes and lipoxins. The structure of the 839-residue soybean lipoxygenase-1 was used as a template to model human 5-, 12-, and 15-lipoxygenases. A distance-based algorithm for placing side chains in a low homology environment (only the four iron ligands were fixed during side chain placement) was devised. Twenty-six of the 56 conserved lipoxygenase residues were grouped in four distinct regions of the enzyme. These regions were analyzed to discern whether the side chain interactions could be duplicated in the models or whether alternate conformers should be considered. The effects of site directed mutagenesis variants were rationalized using the models of the human lipoxygenases. In particular, variants which shifted positional specificity between 12- and 15-lipoxygenase activity were analyzed. Analysis of active site residues produced a model which accounts for observed lipoxygenase positional specificity and stereospecificity.

New hope has been gained in the control of the malaria parasite Plasmodium falciparum (pf) with the discovery that the parasite contains a prokaryotic type II fatty-acid synthase (FAS). Since enzymes of this type are absent in humans, they are potential targets for the development of new drugs. The enoyl reductase enzyme (ENR) belonging to this pathway is of particular interest because it has been shown to be inhibited by submicromolar concentrations of the antimicrobial agent triclosan. Here, the development of an efficient overexpression system for pfENR as a fusion protein with maltose-binding protein, its simple one-step purification and cleavage from its fusion protein and crystallization under new conditions with bound NAD(+) cofactor and triclosan are reported. The crystals belong to the space group P2(1), with approximate unit-cell parameters a = 88.2, b = 82.4, c = 94.8 A, beta = 90.77 degrees, and contain a tetramer in the asymmetric unit. Cryocooled crystals (100 K) diffracted to beyond 2.2 A resolution at the Daresbury Synchrotron Radiation Source.

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis., Author Summary Iron is essential for the survival of blood stage P. falciparum and is used primarily in the synthesis of iron-sulfur (FeS) cluster cofactors. We investigated the role that (FeS) clusters play in malaria parasites. We demonstrated that the synthesis of FeS clusters is partitioned between two organelles: the Isc pathway is mitochondrial while the Suf pathway is found exclusively in the apicoplast organelle. Attempts to interfere with the Suf pathway through a dominant negative approach were only successful when parasite cultures were supplemented with an isoprenoid product. This result demonstrates that isoprenoid biosynthesis depends on a functional Suf pathway. Unexpectedly, we also observed the complete loss of the apicoplast organelle when we disrupted the Suf pathway. This phenotype does not result from inhibition of isoprenoid biosynthesis; we treated parasites with high levels of the isoprenoid inhibitor fosmidomycin without any loss of the apicoplast organelle. These results demonstrate that the Suf pathway has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis. Inhibition of the Suf pathway, which is not found in humans, will block the growth of malaria parasites.

The successful navigation of malaria parasites through their life cycle, which alternates between vertebrate hosts and mosquito vectors, requires a complex interplay of metabolite synthesis and salvage pathways. Using the rodent parasite Plasmodium berghei, we have explored the synthesis and scavenging pathways for lipoic acid, a short-chain fatty acid derivative that regulates the activity of α-ketoacid dehydrogenases including pyruvate dehydrogenase. In Plasmodium, lipoic acid is either synthesized de novo in the apicoplast or is scavenged from the host into the mitochondrion. Our data show that sporozoites lacking the apicoplast lipoic acid protein ligase LipB are markedly attenuated in their infectivity for mice, and in vitro studies document a very late liver stage arrest shortly before the final phase of intra-hepaticparasite maturation. LipB-deficient asexual blood stage parasites show unimpaired rates of growth in normal in vitro or in vivo conditions. However, these parasites showed reduced growth in lipid-restricted conditions induced by treatment with the lipoic acid analogue 8-bromo-octanoate or with the lipid-reducing agent clofibrate. This finding has implications for understanding Plasmodium pathogenesis in malnourished children that bear the brunt of malarial disease. This study also highlights the potential of exploiting lipid metabolism pathways for the design of genetically attenuated sporozoite vaccines.

Background Anti-malarial drug resistance in Kenya prompted two drug policy changes within a decade: sulphadoxine-pyrimethamine (SP) replaced chloroquine (CQ) as the first-line anti-malarial in 1998 and artemether-lumefantrine (AL) replaced SP in 2004. Two cross-sectional studies were conducted to monitor changes in the prevalence of molecular markers of drug resistance over the period in which SP was used as the first-line anti-malarial. The baseline study was carried out from 1999-2000, shortly after implementation of SP, and the follow-up study occurred from 2003-2005, during the transition to AL. Materials and methods Blood was collected from malaria smear-positive, symptomatic patients presenting to outpatient centers in Kisumu, Kenya, during the baseline and follow-up studies. Isolates were genotyped at codons associated with SP and CQ resistance. In vitro IC50 values for antifolates and quinolones were determined for isolates from the follow-up study. Results The prevalence of isolates containing the pfdhfr N51I/C59R/S108N/pfdhps A437G/K540E quintuple mutant associated with SP-resistance rose from 21% in the baseline study to 53% in the follow-up study (p < 0.001). Isolates containing the pfdhfr I164L mutation were absent from both studies. The pfdhps mutations A581G and A613S/T were absent from the baseline study but were present in 85% and 61%, respectively, of isolates from the follow-up study. At follow-up, parasites with mutations at five pfdhps codons, 436, 437, 540, 581, and 613, accounted for 39% of isolates. The CQ resistance-associated mutations pfcrt K76T and pfmdr1 N86Y rose from 82% to 97% (p = 0.001) and 44% to 76% (p < 0.001), respectively, from baseline to follow-up. Conclusions During the period in which SP was the first-line anti-malarial in Kenya, highly SP-resistant parasites emerged, including isolates harboring pfdhps mutations not previously observed there. SP continues to be widely used in Kenya; however, given the highly resistant genotypes observed in this study, its use as a first-line anti-malarial should be discouraged, particularly for populations without acquired immunity to malaria. The increase in the pfcrt K76T prevalence, despite efforts to reduce CQ use, suggests that either these efforts are not adequate to alleviate CQ pressure in Kisumu, or that drug pressure is derived from another source, such as the second-line anti-malarial amodiaquine.

Background Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin. Results In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. Conclusions The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.

Toxoplasmosis causes significant morbidity and mortality, and yet available medicines are limited by toxicities and hypersensitivity. Because improved medicines are needed urgently, rational approaches were used to identify novel lead compounds effective against Toxoplasma gondii enoyl reductase (TgENR), a type II fatty acid synthase enzyme essential in parasites but not present in animals. Fifty-three compounds, including three classes that inhibit ENRs, were tested. Six compounds have antiparasite MIC(90)sor = 6 microM without toxicity to host cells, three compounds have IC(90)s45 nM against recombinant TgENR, and two protect mice. To further understand the mode of inhibition, the cocrystal structure of one of the most promising candidate compounds in complex with TgENR has been determined to 2.7 A. The crystal structure reveals that the aliphatic side chain of compound 19 occupies, as predicted, space made available by replacement of a bulky hydrophobic residue in homologous bacterial ENRs by Ala in TgENR. This provides a paradigm, conceptual foundation, reagents, and lead compounds for future rational development and discovery of improved inhibitors of T. gondii.

The glycine cleavage complex (GCV) is a potential source of the one carbon donor 5,10-methylene-tetrahydrofolate (5,10-CH 2 -THF) in the malaria parasite Plasmodium falciparum . One carbon (C1) donor units are necessary for amino acid and nucleotide biosynthesis, and for the initiation of mitochondrial and plastid translation. In other organisms, GCV activity is closely coordinated with the activity of serine hydroxymethyltransferase (SHMT) enzymes. P. falciparum contains cytosolic and mitochondrial SHMT isoforms, and thus, the subcellular location of the GCV is an important indicator of its role in malaria metabolism. To determine the subcellular localization of the GCV, we used a modified version of the published method for mycobacteriophage integrase-mediated recombination in P. falciparum to generate cell lines containing one of the component proteins of the GCV, the H-protein, fused to GFP. Here, we demonstrate that this modification results in rapid generation of chromosomally integrated transgenic parasites, and we show that the H-protein localizes to the mitochondrion.

Recent years have seen rapid advances in our understanding of malaria parasite cell biology. Some of this progress has been the result of developments in genetic techniques, advances in imaging technology, and new molecular tools. Herein, three aspects of parasite cell biology will be the topic of our focus: (i) plastid metabolism, (ii) sporozoite biology, and (iii) protein transport to and from the host erythrocyte. In each case, recent work has led to a deeper understanding of parasite biology, often at the expense of previously accepted paradigms. These studies also highlight the impediments, technical and otherwise, which will have to be overcome for continued rapid progress in these fields.

The control and eventual eradication of human malaria is considered one of the most important global public health goals of the 21st Century. Malaria, caused by intraerythrocytic protozoan parasites of the genus Plasmodium, is by far the most lethal and among the most prevalent of the infectious diseases. Four species of Plasmodium (P. falciparum, P. malariae, P. ovale, and P. vivax) are known to be infectious to humans, and more recent cases of infection due to P. knowlesi also have been reported. These species cause approximately 300 million annual cases of clinical malaria resulting in around one million deaths mostly caused by P. falciparum. The rapid emergence of drug-resistant Plasmodium strains has severely reduced the potency of medicines commonly used to treat and block the transmission of malaria and threatens the effectiveness of combination therapy in the field. New drugs that target important parasite functions, which are not the target of current antimalarial drugs, and have the potential to act against multi-drug-resistant Plasmodium strains are urgently needed. Recent studies in P. falciparum have unraveled new metabolic pathways for the synthesis of the parasite phospholipids and fatty acids. The present review summarizes our current understanding of these pathways in Plasmodium development and pathogenesis, and provides an update on the efforts underway to characterize their importance using genetic means and to develop antimalarial therapies targeting lipid metabolic pathways.

Acyl Carrier Protein (ACP) has a single reactive sulfhydryl necessary for function in covalently binding nascent fatty acids during biosynthesis. In Plasmodium falciparum, the causative agent of the most lethal form of malaria, fatty acid biosynthesis occurs in the apicoplast organelle during the liver stage of the parasite life cycle. During the blood stage, fatty acid biosynthesis is inactive and the redox state of the apicoplast has not been determined. We solved the crystal structure of ACP from P. falciparum in reduced and disulfide-linked forms, and observe the surprising result that the disulfide in the PfACP cross-linked dimer is sequestered from bulk solvent in a tight molecular interface. We assessed solvent accessibility of the disulfide with small molecule reducing agents and found that the disulfide is protected from BME but less so for other common reducing agents. We examined cultured P. falciparum parasites to determine which form of PfACP is prevalent during the blood stages. We readily detected monomeric PfACP in parasite lysate, but do not observe the disulfide-linked form, even under conditions of oxidative stress. To demonstrate that PfACP contains a free sulfhydryl and is not acylated or in the apo state, we treated blood stage parasites with the disulfide forming reagent diamide. We found that the effects of diamide are reversed with reducing agent. Together, these results suggest that the apicoplast is a reducing compartment, as suggested by models of P. falciparum metabolism, and that PfACP is maintained in a reduced state during blood stage growth.

Three metabolic pathways can be used by protozoan parasites for the de novo synthesis of fatty acids. Parasites employ diverse strategies involving these three pathways, as well as scavenging from the host, to obtain fatty acids required for growth. Keywords: type I fatty acid synthase; type II fatty acid synthase; fatty acid elongase pathway; apicomplexa; trypanosomatids

SUMMARYApicomplexan parasites of the genusEimeriaare the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show thatEimeria tenellaharbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form ofE. tenellaenoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed theE. tenellagenomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting thatE. tenellacontains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed inToxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

Cyclin-dependent protein kinases (CDKs) are key regulators of cell cycle control. In humans, CDK7 performs dual roles as the CDK activating kinase (CAK) responsible for regulating numerous CDKs and as the RNA polymerase II carboxyl-terminal domain (CTD) kinase involved in the regulation of transcription. Binding of an effector protein, human MAT1, stimulates CDK7 kinase activity and influences substrate specificity. In Plasmodium falciparum, CDKs and their roles in regulating growth and development are poorly understood. In this study, we characterized the regulatory mechanisms of Pfmrk, a putative homolog of human CDK7. We identified an effector, PfMAT1, which stimulates Pfmrk kinase activity in a cyclin-dependent manner. The addition of PfMAT1 stimulated RNA polymerase II CTD phosphorylation and had no effect on the inability of Pfmrk to phosphorylate PfPK5, a putative CDK1 homolog, which suggests that Pfmrk may be a CTD kinase rather than a CAK. In an attempt to abrogate the requirement for PfMAT1 stimulation, we mutated amino acids within the active site of Pfmrk. We found that two independent mutants, S138K and F143L, yielded a 4-10-fold increase in Pfmrk activity. Significant kinase activity of these mutants was observed in the absence of either cyclin or PfMAT1. Finally, we observed autophosphorylation of Pfmrk that is unaffected by the addition of either cyclin or PfMAT1.

New chemical classes of compounds must be introduced into the malaria drug development pipeline in an effort to develop new chemotherapy options for the fight against malaria. In this review we describe an iterative approach designed to identify potent inhibitors of a kinase family that collectively functions as key regulators of the cell cycle. Cyclin-dependent protein kinases (CDKs) are attractive drug targets in numerous diseases and, most recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. Our approach uses experimental and virtual screening methodologies to identify and refine chemical inhibitors and increase the success rate of discovering potent and selective inhibitors. The active pockets of the plasmodial CDKs are unique in terms of size, shape and amino acid composition compared with those of the mammalian orthologues. These differences exemplified through the use of screening assays, molecular modeling, and crystallography can be exploited for inhibitor design. To date, several classes of compounds including quinolines and oxindoles have been identified as selective inhibitors of the plasmodial CDK7 homologue, Pfmrk. From these initial studies and through the iterative rational drug design process, more potent, selective, and most importantly, chemically unique compound classes have been identified as effective inhibitors of the plasmodial CDKs and the malarial parasite.

In biological systems, fatty acids can be synthesized by two related, but distinct de novo fatty acid synthase (FAS) pathways. Human cells rely on a type I FAS whereas plants, bacteria and other microorganisms contain type II FAS pathways. This difference exposes the type II FAS enzymes as potential targets for anti-microbial drugs that have little to no side effects in the human host. A number of inhibitors of type II FAS enzymes have been discovered - many of which have anti-bacterial activity. Extensive biochemical and structural studies have shed light on how these compounds inhibit their target enzymes, laying the foundation for the design of inhibitors with increased potency. Recent work has shown that malaria parasites do not contain a type I FAS and rely solely on a type II FAS for the de novo biosynthesis of fatty acids. The malaria FAS enzymes are therefore an exciting source of new drug targets, and are being actively exploited by several drug discovery efforts. Rapid progress has been made, largely due to the vast body of mechanistic and structural information about type II FAS enzymes from bacteria and the availability of inhibitors. Ongoing antimalarial drug discovery projects will be described in this review as well as background information about the well-studied bacterial type II FAS enzymes.

The cell division cycle is regulated by a family of cyclin-dependent protein kinases (CDKs) that are functionally conserved among many eukaryotic species. The characterization of plasmodial CDKs has identified them as a leading antimalarial drug target in our laboratory. We have developed a three-dimensional QSAR pharmacophore model for inhibition of a Plasmodium falciparum CDK, known as Pfmrk, from a set of fifteen structurally diverse kinase inhibitors with a wide range of activity. The model was found to contain two hydrogen bond acceptor functions and two hydrophobic sites including one aromatic-ring hydrophobic site. Although the model was not developed from X-ray structural analysis of the known CDK2 structure, it is consistent with the structure-functional requirements for binding of the CDK inhibitors in the ATP binding pocket. Using the model as a template, a search of the in-house three-dimensional multiconformer database resulted in the discovery of sixteen potent Pfmrk inhibitors. The predicted inhibitory activities of some of these Pfmrk inhibitors from the molecular model agree exceptionally well with the experimental inhibitory values from the in vitro CDK assay.

Copper active sites play a major role in enzymatic activation of dioxygen. We trapped the copper-dioxygen complex in the enzyme peptidylglycine-alphahydroxylating monooxygenase (PHM) by freezing protein crystals that had been soaked with a slow substrate and ascorbate in the presence of oxygen. The x-ray crystal structure of this precatalytic complex, determined to 1.85-angstrom resolution, shows that oxygen binds to one of the coppers in the enzyme with an end-on geometry. Given this structure, it is likely that dioxygen is directly involved in the electron transfer and hydrogen abstraction steps of the PHM reaction. These insights may apply to other copper oxygen-activating enzymes, such as dopamine beta-monooxygenase, and to the design of biomimetic complexes.

The genome of the malaria parasite, Plasmodium falciparum, appears to contain the proteins necessary for a Type II dissociated fatty acid biosynthetic system. Here we report the functional characterization of two proteins from this system. Purified recombinant acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase III (KASIII) from P. falciparum are soluble and active in a truncated form. Malarial ACP is activated by the addition of a 4'-phosphopantetheine prosthetic group derived from coenzyme A, generating holo-PfACP. Holo-PfACP is an effective substrate for the transacylase activity of PfKASIII, but substitution of a key active site cysteine in PfKASIII to alanine or serine abolishes enzymatic activity. During the schizont stage of parasite development, there is a significant up-regulation of the mRNAs corresponding to these proteins, indicating an important metabolic requirement for fatty acids during this stage.