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Executive system dysfunction and impaired prospective memory (PM) are widely documented in schizophrenia. However, it is not yet clarified which components of PM function are impaired in this disorder. Two plausible target components are the maintenance of delayed intentions and the execution of PM responses. Furthermore, it is debated whether the impaired performance on frequently used executive tasks is associated with deficit in PM functions. The aim of our study was twofold. First, we aimed to investigate the specific processes involved in event-based PM function, mainly focusing on difference between maintenance of intention and execution of PM responses. Second, we aimed to unfold the possible connections between executive functions, clinical symptoms, and PM performance. An event-based PM paradigm was applied with three main conditions: baseline (with no expectation of PM stimuli, and without PM stimuli), expectation condition (participants were told that PM stimuli might occur, though none actually did), and execution condition (participants were told that PM stimuli might occur, and PM stimuli did occur). This procedure allowed us to separately investigate performances associated with intention maintenance and execution of PM responses. We assessed working memory and set-shifting executive functions by memory span tasks and by the Wisconsin Card Sorting Test (WCST), respectively. Twenty patients diagnosed with schizophrenia and 20 healthy control subjects (matched according to age and education) took part in the study. It was hypothesized that patients would manifest different levels of performance in the expectation and execution conditions of the PM task. Our results confirmed that the difference between baseline performance and performance in the execution condition (execution cost) was significantly larger for participants diagnosed with schizophrenia in comparison with matched healthy control group. However, this difference was not observed in the expectation condition. The PM performance in the execution condition was correlated with impaired executive functions in schizophrenia. Specifically, the size of execution cost positively correlated with percent of perseverative errors committed on WCST by the patient group. Our results suggest that maintenance of delayed intentions is unimpaired in schizophrenia, whereas the impairment in execution of PM responses is associated with set-shifting deficit.

We investigated whether visual orientation anisotropies (known as oblique effect) exist in non-attended visual changes using event-related potentials (ERP). We recorded visual mismatch negativity (vMMN) which signals violation of sequential regularities. In the visual periphery unattended, task-irrelevant Gábor patches were displayed in an oddball sequence while subjects performed a tracking task in the central field. A moderate change (50°) in the orientation of stimuli revealed no consistent change-related components. However we found orientation-related differences around 170 ms in occipito-temporal areas in the amplitude of the ERPs evoked by standard stimuli. In a supplementary experiment we determined the amount of orientation difference that is needed for change detection in an active, attended paradigm. Results exhibited the classical oblique effect; subjects detected 10° deviations from cardinal directions, while threshold from oblique directions was 17°. These results provide evidence that perception of change could be accomplished at significantly smaller thresholds, than what elicits vMMN. In Experiment2 we increased the orientation change to 90°. Deviant-minus-standard difference was negative in occipito-parietal areas, between 120-200 ms after stimulus onset. VMMNs to changes from cardinal angles were larger and more sustained than vMMNs evoked by changes from oblique angles. Changes from cardinal orientations represent a more detectable signal for the automatic change detection system than changes from oblique angles, thus increased vMMN to these larger deviances might be considered a variant of the magnitude of deviance effect rarely observed in vMMN studies.

The present study investigated whether gender information for human faces was represented by the predictive mechanism indexed by the visual mismatch negativity (vMMN) event – related brain potential (ERP). While participants performed a continuous size-change-detection task, random sequences of cropped faces were presented in the background, in an oddball setting: either various female faces were presented infrequently among various male faces, or vice versa. In Experiment 1 the inter-stimulus- interval (ISI) was 400 ms, while in Experiment 2 the ISI was 2250 ms. The ISI difference had only a small effect on the P1 component, however the subsequent negativity (N1/N170) was larger and more widely distributed at longer ISI, showing different aspects of stimulus processing.As deviant -minus- standard ERP difference, a parieto – occipital negativity (vMMN) emerged in the 200 – 500 ms latency range (~ 350 ms peak latency in both experiments). We argue that regularity of gender on the photographs is automatically registered, and the violation of the gender category is reflected by the vMMN. In conclusion the results can be interpreted as evidence for the automatic activity of a predictive brain mechanism, in case of an ecologically valid category.

Malaria, a devastating parasitic infection, is the leading cause of death in many developing countries. Unfortunately, the most deadliest causative agent of malaria, Plasmodium falciparum , has developed resistance to nearly all currently available antimalarial drugs. The P. falciparum Niemann-Pick type C1-related (PfNCR1) transporter has been identified as a druggable target, but its structure and detailed molecular mechanism are not yet available. Here, we present three structures of PfNCR1 with and without the functional inhibitor MMV009108 at resolutions between 2.98 and 3.81 Å using single-particle cryo-electron microscopy (cryo-EM), suggesting that PfNCR1 binds cholesterol and forms a cholesterol transport tunnel to modulate the composition of the parasite plasma membrane. Cholesterol efflux assays show that PfNCR1 is an exporter capable of extruding cholesterol from the membrane. Additionally, the inhibition mechanism of MMV009108 appears to be due to a direct blockage of PfNCR1, preventing this transporter from shuttling cholesterol.

Surveillance of drug resistance and the discovery of novel targets-key objectives in the fight against malaria-rely on identifying resistance-conferring mutations in Plasmodium parasites. Current approaches, while successful, require laborious experimentation or large sample sizes. To elucidate shared determinants of antimalarial resistance that can empower in silico inference, we examined the genomes of 724 Plasmodium falciparum clones, each selected in vitro for resistance to one of 118 compounds. We identified 1448 variants in 128 recurrently mutated genes, including drivers of antimalarial multidrug resistance. In contrast to naturally occurring variants, those selected in vitro are more likely to be missense or frameshift, involve bulky substitutions, and occur in conserved, ordered protein domains. Collectively, our dataset reveals mutation features that predict drug resistance in eukaryotic pathogens.

The identification of novel drug targets for the purpose of designing small molecule inhibitors is key component to modern drug discovery. In malaria parasites, discoveries of antimalarial targets have primarily occurred retroactively by investigating the mode of action of compounds found through phenotypic screens. Although this method has yielded many promising candidates, it is time- and resource-consuming and misses targets not captured by existing antimalarial compound libraries and phenotypic assay conditions. Leveraging recent advances in protein structure prediction and data mining, we systematically assessed the Plasmodium falciparum genome for proteins amenable to target-based drug discovery, identifying 867 candidate targets with evidence of small molecule binding and blood stage essentiality. Of these, 540 proteins showed strong essentiality evidence and lack inhibitors that have progressed to clinical trials. Expert review and rubric-based scoring of this subset based on additional criteria such as selectivity, structural information, and assay developability yielded 67 high priority candidates. This study also provides a genome-wide data resource and implements a generalizable framework for systematically evaluating and prioritizing novel pathogenic disease targets., Competing Interests: Declarations Competing Interest Statement MKG has an equity interest in and is a cofounder and scientific advisor of VeraChem LLC, and is on the SABs of InCerebro Inc, Denovicon Therapeutics, and Beren Therapeutics. ELF and SPSR are employees of Novartis Pharma AG and may own shares in Novartis Pharma AG. KD holds stock in TropIQ Health Sciences. The rest of authors declare no competing interests.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism., (© 2024. The Author(s).)

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl tRNA synthetase ( Pf AsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of Pf AsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound Pf AsnRS provide insights into the structure activity relationship and the selectivity mechanism., Competing Interests: Competing interests. The authors have no competing interests to declare.

Protein kinases have proven to be a very productive class of therapeutic targets, and over 90 inhibitors are currently in clinical use primarily for the treatment of cancer. Repurposing these inhibitors as antimalarials could provide an accelerated path to drug development. In this study, we identified BI-2536, a known potent human polo-like kinase 1 inhibitor, with low nanomolar antiplasmodial activity. Screening of additional PLK1 inhibitors revealed further antiplasmodial candidates despite the lack of an obvious orthologue of PLKs in Plasmodium . A subset of these inhibitors was profiled for their in vitro killing profile, and commonalities between the killing rate and inhibition of nuclear replication were noted. A kinase panel screen identified Pf NEK3 as a shared target of these PLK1 inhibitors; however, phosphoproteome analysis confirmed distinct signaling pathways were disrupted by two structurally distinct inhibitors, suggesting Pf NEK3 may not be the sole target. Genomic analysis of BI-2536-resistant parasites revealed mutations in genes associated with the starvation-induced stress response, suggesting BI-2536 may also inhibit an aminoacyl-tRNA synthetase.

Development of antimalarial compounds into clinical candidates remains costly and arduous without detailed knowledge of the target. As resistance increases and treatment options at various stages of disease are limited, it is critical to identify multistage drug targets that are readily interrogated in biochemical assays. Whole-genome sequencing of 18 parasite clones evolved using thienopyrimidine compounds with submicromolar, rapid-killing, pan-life cycle antiparasitic activity showed that all had acquired mutations in the P. falciparum cytoplasmic isoleucyl tRNA synthetase (cIRS). Engineering two of the mutations into drug-naïve parasites recapitulated the resistance phenotype, and parasites with conditional knockdowns of cIRS became hypersensitive to two thienopyrimidines. Purified recombinant P. vivax cIRS inhibition, cross-resistance, and biochemical assays indicated a noncompetitive, allosteric binding site that is distinct from that of known cIRS inhibitors mupirocin and reveromycin A. Our data show that Plasmodium cIRS is an important chemically and genetically validated target for next-generation medicines for malaria.

Widespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains., Competing Interests: Declaration of interests B.L. and M.D. are employees of MMV; M.G.G.-L. and F.-J.G. are employees of GSK., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

There is a shift in antimalarial drug discovery from phenotypic screening toward target-based approaches, as more potential drug targets are being validated in Plasmodium species. Given the high attrition rate and high cost of drug discovery, it is important to select the targets most likely to deliver progressible drug candidates. In this paper, we describe the criteria that we consider important for selecting targets for antimalarial drug discovery. We describe the analysis of a number of drug targets in the Malaria Drug Accelerator (MalDA) pipeline, which has allowed us to prioritize targets that are ready to enter the drug discovery process. This selection process has also highlighted where additional data are required to inform target progression or deprioritization of other targets. Finally, we comment on how additional drug targets may be identified.

The Malaria Drug Accelerator (MalDA) is a consortium of 15 leading scientific laboratories. The aim of MalDA is to improve and accelerate the early antimalarial drug discovery process by identifying new, essential, druggable targets. In addition, it seeks to produce early lead inhibitors that may be advanced into drug candidates suitable for preclinical development and subsequent clinical testing in humans. By sharing resources, including expertise, knowledge, materials, and reagents, the consortium strives to eliminate the structural barriers often encountered in the drug discovery process. Here we discuss the mission of the consortium and its scientific achievements, including the identification of new chemically and biologically validated targets, as well as future scientific directions., Competing Interests: Declaration of Interests K.J.D. holds stock in TropIQ Health Sciences. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stage P. falciparum parasites in the low nanomolar to low micromolar range. In order to understand their mechanism of action, parasites from two different genetic backgrounds were exposed to sublethal concentrations of MMV085203 and GNF-Pf-3600 until resistance emerged. Whole genome sequencing revealed all 17 resistant clones acquired nonsynonymous mutations in the gene encoding the orphan apicomplexan transporter PF3D7_0312500 ( pfmfr3 ) predicted to encode a member of the major facilitator superfamily (MFS). Disruption of pfmfr3 and testing against a panel of antimalarial compounds showed decreased sensitivity to MMV085203 and GNF-Pf-3600 as well as other compounds that have a mitochondrial mechanism of action. In contrast, mutations in pfmfr3 provided no protection against compounds that act in the food vacuole or the cytosol. A dihydroorotate dehydrogenase rescue assay using transgenic parasite lines, however, indicated a different mechanism of action for both MMV085203 and GNF-Pf-3600 than the direct inhibition of cytochrome bc1. Green fluorescent protein (GFP) tagging of PfMFR3 revealed that it localizes to the parasite mitochondrion. Our data are consistent with PfMFR3 playing roles in mitochondrial transport as well as drug resistance for clinically relevant antimalarials that target the mitochondria. Furthermore, given that pfmfr3 is naturally polymorphic, naturally occurring mutations may lead to differential sensitivity to clinically relevant compounds such as atovaquone.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H + -ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.

Plasmepsins are a group of diverse aspartic proteases in the malaria parasite Plasmodium Their functions are strikingly multifaceted, ranging from hemoglobin degradation to secretory organelle protein processing for egress, invasion, and effector export. Some, particularly the digestive vacuole plasmepsins, have been extensively characterized, whereas others, such as the transmission-stage plasmepsins, are minimally understood. Some ( e.g. plasmepsin V) have exquisite cleavage sequence specificity; others are fairly promiscuous. Some have canonical pepsin-like aspartic protease features, whereas others have unusual attributes, including the nepenthesin loop of plasmepsin V and a histidine in place of a catalytic aspartate in plasmepsin III. We have learned much about the functioning of these enzymes, but more remains to be discovered about their cellular roles and even their mechanisms of action. Their importance in many key aspects of parasite biology makes them intriguing targets for antimalarial chemotherapy. Further consideration of their characteristics suggests that some are more viable drug targets than others. Indeed, inhibitors of invasion and egress offer hope for a desperately needed new drug to combat this nefarious organism., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Nasamu et al.)

We report detailed susceptibility profiling of asexual blood stages of the malaria parasite Plasmodium falciparum to clinical and experimental antimalarials, combined with metabolomic fingerprinting. Results revealed a variety of stage-specific and metabolic profiles that differentiated the modes of action of clinical antimalarials including chloroquine, piperaquine, lumefantrine, and mefloquine, and identified late trophozoite-specific peak activity and stage-specific biphasic dose-responses for the mitochondrial inhibitors DSM265 and atovaquone. We also identified experimental antimalarials hitting previously unexplored druggable pathways as reflected by their unique stage specificity and/or metabolic profiles. These included several ring-active compounds, ones affecting hemoglobin catabolism through distinct pathways, and mitochondrial inhibitors with lower propensities for resistance than either DSM265 or atovaquone. This approach, also applicable to other microbes that undergo multiple differentiation steps, provides an effective tool to prioritize compounds for further development within the context of combination therapies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Plasmodium parasites possess a protein with homology to Niemann-Pick Type C1 proteins (Niemann-Pick Type C1-Related protein, NCR1). We isolated parasites with resistance-conferring mutations in Plasmodium falciparum NCR1 (PfNCR1) during selections with three diverse small-molecule antimalarial compounds and show that the mutations are causative for compound resistance. PfNCR1 protein knockdown results in severely attenuated growth and confers hypersensitivity to the compounds. Compound treatment or protein knockdown leads to increased sensitivity of the parasite plasma membrane (PPM) to the amphipathic glycoside saponin and engenders digestive vacuoles (DVs) that are small and malformed. Immuno-electron microscopy and split-GFP experiments localize PfNCR1 to the PPM. Our experiments show that PfNCR1 activity is critically important for the composition of the PPM and is required for DV biogenesis, suggesting PfNCR1 as a novel antimalarial drug target., Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., Competing Interests: EI, SD, SB, JB, EO, ML, SG, JN, EW, AV, DG No competing interests declared, (© 2019, Istvan et al.)

The human parasite Plasmodium falciparum kills an estimated 445,000 people a year, with the most fatalities occurring in African children. Previous studies identified falcilysin (FLN) as a malarial metalloprotease essential for parasite development in the human host. Despite its essentiality, the biological roles of this protease are not well understood. Here we describe the optimization of a piperazine-based hydroxamic acid scaffold to develop the first reported inhibitors of FLN. Inhibitors were tested against cultured parasites, and parasiticidal activity correlated with potency against FLN. This suggests these compounds kill P. falciparum by blocking FLN, and that FLN is a druggable target. These compounds represent an important step towards validating FLN as a therapeutic target and towards the development of chemical tools to investigate the function of this protease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium falciparum is highly variable depending on the commercial source. Here we identify a minor contaminant (pepstatin butyl ester) as the active anti-parasitic principle. We synthesize a series of derivatives and characterize an analogue (pepstatin hexyl ester) with low nanomolar activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. Recombinant PfPARE hydrolyses pepstatin esters and de-esterifies MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance.

Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance.

The human malaria parasite Plasmodium falciparum is auxotrophic for most amino acids. Its amino acid needs are met largely through the degradation of host erythrocyte hemoglobin; however the parasite must acquire isoleucine exogenously, because this amino acid is not present in adult human hemoglobin. We report that when isoleucine is withdrawn from the culture medium of intraerythrocytic P. falciparum, the parasite slows its metabolism and progresses through its developmental cycle at a reduced rate. Isoleucine-starved parasites remain viable for 72 h and resume rapid growth upon resupplementation. Protein degradation during starvation is important for maintenance of this hibernatory state. Microarray analysis of starved parasites revealed a 60% decrease in the rate of progression through the normal transcriptional program but no other apparent stress response. Plasmodium parasites do not possess a TOR nutrient-sensing pathway and have only a rudimentary amino acid starvation-sensing eukaryotic initiation factor 2α (eIF2α) stress response. Isoleucine deprivation results in GCN2-mediated phosphorylation of eIF2α, but kinase-knockout clones still are able to hibernate and recover, indicating that this pathway does not directly promote survival during isoleucine starvation. We conclude that P. falciparum, in the absence of canonical eukaryotic nutrient stress-response pathways, can cope with an inconsistent bloodstream amino acid supply by hibernating and waiting for more nutrient to be provided.

Intraerythrocytic malaria parasites can obtain nearly their entire amino acid requirement by degrading host cell hemoglobin. The sole exception is isoleucine, which is not present in adult human hemoglobin and must be obtained exogenously. We evaluated two compounds for their potential to interfere with isoleucine utilization. Mupirocin, a clinically used antibacterial, kills Plasmodium falciparum parasites at nanomolar concentrations. Thiaisoleucine, an isoleucine analog, also has antimalarial activity. To identify targets of the two compounds, we selected parasites resistant to either mupirocin or thiaisoleucine. Mutants were analyzed by genome-wide high-density tiling microarrays, DNA sequencing, and copy number variation analysis. The genomes of three independent mupirocin-resistant parasite clones had all acquired either amplifications encompassing or SNPs within the chromosomally encoded organellar (apicoplast) isoleucyl-tRNA synthetase. Thiaisoleucine-resistant parasites had a mutation in the cytoplasmic isoleucyl-tRNA synthetase. The role of this mutation in thiaisoleucine resistance was confirmed by allelic replacement. This approach is generally useful for elucidation of new targets in P. falciparum. Our study shows that isoleucine utilization is an essential pathway that can be targeted for antimalarial drug development.

A family of aspartic proteases called plasmepsins is important for hemoglobin degradation in intraerythrocytic Plasmodium parasites. Plasmepsin II (PM II) is the best studied member of this family. PM II and its close orthologs and paralogs form homodimers with extensive interfaces in all known crystal structures. This raised the question whether the homodimer is the functional subunit of plasmepsins in solution. We have used gel filtration chromatography, site-directed mutagenesis, and analytical ultracentrifugation to study the oligomeric status of PM II in solution. Our results reveal that PM II exists mainly as a monomer in solution and that the monomer is fully functional for catalysis. A hydrophobic loop at the PM II monomer surface, which would be buried in a PM II dimer, is shown to be essential for the hemoglobin degradation capability of PM II.

Degradation of host hemoglobin by the human malaria parasite Plasmodium falciparum is a massive metabolic process. What role this degradation plays and whether it is essential for parasite survival have not been established, nor have the roles of the various degradative enzymes been clearly defined. We report that P. falciparum can grow in medium containing a single amino acid (isoleucine, the only amino acid missing from human hemoglobin). In this medium, growth of hemoglobin-degrading enzyme gene knockout lines (missing falcipain-2 and plasmepsins alone or in combination) is impaired. Blockade of plasmepsins with the potent inhibitor pepstatin A has a minimal effect on WT parasite growth but kills falcipain-2 knockout parasites at low concentrations and is even more potent on falcipain-2, plasmepsin I and IV triple knockout parasites. We conclude that: (i) hemoglobin degradation is necessary for parasite survival; (ii) hemoglobin degradation is sufficient to supply most of the parasite's amino acid requirements; (iii) external amino acid acquisition and hemoglobin digestion are partially redundant nutrient pathways; (iv) hemoglobin degradation uses dual protease families with overlapping function; and (v) hemoglobin-degrading plasmepsins are not promising drug targets.

Plasmepsin II (PM II) is an aspartic protease active in hemoglobin (Hb) degradation in the protozoan parasite Plasmodium falciparum. A fluorescence-quenched octapeptide substrate based on the initial hemoglobin cleavage site is recognized well by PM II. C-terminal extension of this peptide has little effect, but N-terminal extension results in higher maximal velocity and dramatic concentration-dependent substrate inhibition. This inhibition, but not the rate stimulation, depends on the presence of a DABCYL group on the peptide N terminus. Using site-directed mutagenesis, we have identified PM II residues that interact with N-terminal amino acids of peptide substrates. The same residues influence degradation of Hb by PM II. Cathepsin E (CatE), a related mammalian aspartic protease, is also stimulated by N-terminally extended substrates. This suggests that distal substrate interactions as far out as P6 may be a general property of aspartic proteases and may be important in substrate and inhibitor recognition. Although PM II and CatE are similar in their ability to cleave Hb-based peptides and Hb alpha-chains, CatE is not able to degrade native Hb, which is a substrate for PM II. Based on these results, we propose that PM II may have the special feature of being a Hb denaturase.

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. HMGR is the target of compounds (HMGR inhibitors, commonly referred to as statins) that are very effective in lowering serum cholesterol levels. These inhibitors have K(i) values in the nanomolar range and are widely prescribed in the treatment of hypercholesterolemia. We have determined structures of this enzyme in complexes with 6 different statins (compactin, simvastatin, fluvastatin, cerivastatin, atorvastatin, and rosuvastatin). The statins occupy a portion of the binding site of HMG-CoA, thus blocking access of this substrate to the active site. The nicotinamide binding pocket of NADP(H) (the second substrate of the enzyme) is unoccupied by the inhibitor molecules. Near the C-terminus of HMGR, several catalytically relevant residues are disordered in the enzyme-statin complexes. The flexibility of these residues is critical for binding with statins; if ordered, they would sterically hinder such binding.

The committed step in the biosynthesis of isoprenoids is catalyzed by HMG-CoA reductase. Recently determined structures of the distantly related bacterial and mammalian HMG-CoA reductases have revealed two distinct classes of the protein with differently formed active sites and oligomeric states. An appreciation of the remarkable differences in the active site architecture may ultimately lead to the development of novel antibacterial agents.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. Statins are HMGR inhibitors with inhibition constant values in the nanomolar range that effectively lower serum cholesterol levels and are widely prescribed in the treatment of hypercholesterolemia. We have determined structures of the catalytic portion of human HMGR complexed with six different statins. The statins occupy a portion of the binding site of HMG-CoA, thus blocking access of this substrate to the active site. Near the carboxyl terminus of HMGR, several catalytically relevant residues are disordered in the enzyme-statin complexes. If these residues were not flexible, they would sterically hinder statin binding.

In higher plants, fungi, and animals isoprenoids are derived from the mevalonate pathway. The carboxylic acid mevalonate is formed from acetyl-CoA and acetoacetyl-CoA via the intermediate 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). The four-electron reduction of HMG-CoA to mevalonate, which utilizes two molecules of NADPH, is the committed step in the biosynthesis of isoprenoids. This reaction is catalyzed by HMG-CoA reductase (HMGR). The activity of HMGR is controlled through synthesis, degradation and phosphorylation. The human enzyme has also been targeted successfully by drugs, known as statins, in the clinical treatment of high serum cholesterol levels. The crystal structure of the catalytic portion of HMGR has been determined recently with bound reaction substrates and products. The structure illustrates how HMG-CoA and NADPH are recognized and suggests a catalytic mechanism. Catalytic portions of human HMGR form tight tetramers, explaining the influence of the enzyme's oligomeric state on the activity and suggesting a mechanism for cholesterol sensing.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the formation of mevalonate, the committed step in the biosynthesis of sterols and isoprenoids. The activity of HMGR is controlled through synthesis, degradation and phosphorylation to maintain the concentration of mevalonate-derived products. In addition to the physiological regulation of HMGR, the human enzyme has been targeted successfully by drugs in the clinical treatment of high serum cholesterol levels. Three crystal structures of the catalytic portion of human HMGR in complexes with HMG-CoA, with HMG and CoA, and with HMG, CoA and NADP(+), provide a detailed view of the enzyme active site. Catalytic portions of human HMGR form tight tetramers. The crystal structure explains the influence of the enzyme's oligomeric state on the activity and suggests a mechanism for cholesterol sensing. The active site architecture of human HMGR is different from that of bacterial HMGR; this may explain why binding of HMGR inhibitors to bacterial HMGRs has not been reported.

Background: Glucose homeostasis is maintained by the processes of glycolysis and gluconeogenesis. The importance of these pathways is demonstrated by the severe and life threatening effects observed in various forms of diabetes. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate, a potent regulator of glycolysis. Thus this bifunctional enzyme plays an indirect yet key role in the regulation of glucose metabolism., Results: We have determined the 2.0 A crystal structure of the rat testis isozyme of this bifunctional enzyme. The enzyme is a homodimer of 55 kDa subunits arranged in a head-to-head fashion, with each monomer consisting of independent kinase and phosphatase domains. The location of ATPgammaS and inorganic phosphate in the kinase and phosphatase domains, respectively, allow us to locate and describe the active sites of both domains., Conclusions: The kinase domain is clearly related to the superfamily of mononucleotide binding proteins, with a particularly close relationship to the adenylate kinases and the nucleotide-binding portion of the G proteins. This is in disagreement with the broad speculation that this domain would resemble phosphofructokinase. The phosphatase domain is structurally related to a family of proteins which includes the cofactor independent phosphoglycerate mutases and acid phosphatases.

Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.