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This review highlights the key role of fatty acid desaturases in the synthesis of naturally occurring, more common and not unsaturated fatty acids. The three major classes of fatty acid desaturases, such as acyl-lipid, acyl-acyl carrier protein and acyl-coenzyme A, are described in detail, with particular attention to the cellular localisation, the structure, the substrate and product specificity and the expression and regulation of desaturase genes. The review also gives an insight into the biocatalytic reaction of fatty acid desaturation by covering the general and more class-specific mechanistic studies around the synthesis of unsaturated fatty acids Finally, we conclude the review by looking at the numerous novel applications for desaturases in order to meet the very high demand for polyunsaturated fatty acids, taking into account the opportunity for the development of new, more efficient, easily reproducible, sustainable bioengineering advances in the field.

p67 is a type I transmembrane glycoprotein of the terminal lysosome of African trypanosomes. Its biosynthesis involves transport of an initial gp100 ER precursor to the lysosome, followed by cleavage to N-terminal (gp32) and C-terminal (gp42) subunits that remain non-covalently associated. p67 knockdown is lethal, but the only overt phenotype is an enlarged lysosome (~250 to >1000 nm). Orthologues have been characterized in Dictyostelium and mammals. These have processing pathways similar to p67, and are thought to have phospholipase B-like (PLBL) activity. The mouse PLBD2 crystal structure revealed that the PLBLs represent a subgroup of the larger N-terminal nucleophile (NTN) superfamily, all of which are hydrolases. NTNs activate by internal autocleavage mediated by a nucleophilic residue, i.e. Cys, Ser or Thr, on the upstream peptide bond to form N-terminal α (gp32) and C-terminal β (gp42) subunits that remain non-covalently associated. The N-terminal residue of the β subunit is then catalytic in subsequent hydrolysis reactions. All PLBLs have a conserved Cys/Ser dipeptide at the α/β junction (Cys241/Ser242 in p67), mutation of which renders p67 non-functional in RNAi rescue assays. p67 orthologues are found in many clades of parasitic protozoa, thus p67 is the founding member of a group of hydrolases that likely play a role broadly in the pathogenesis of parasitic infections.

This work was supported through funding from the EPSRC (grant number EP/J500549/1) and the University of St Andrews School of Chemistry. There is an urgent need for the development of new treatments against trypanosomatid parasites; the causative agents of some of the most debilitating diseases in the developing world. This work targets an interesting 6-5-6-6 fused carboline scaffold, accessing a range of substituted derivatives through stereospecific intramolecular Pictet-Spengler condensation. Modification of the cyclisation conditions allowed retention of the carbamate protecting group and gave insight into the reaction mechanism. Compounds' bioactivities were measured against T. brucei, T. cruzi, L. major and HeLa cells. We have identified promising pan-trypanocidal lead compounds based on the core scaffold, and highlight key SAR trends which will be useful for the future development of these compounds as potent trypanocidal agents. Postprint

This work was supported by the the University of Tours (to IDP and FDG), the University of Montpellier (to SD and EC), the Deutsche Forschungsgemeinschaft (to RTS), the Wellcome Trust project grant 093228 (to TKS) and the Campus France/DAAD PHC PROCOPE 24931RE (to RTS and EC). The funding source has no involvement in the conduct of the research and preparation of the article. Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs. Publisher PDF

Amino acid metabolism within Trypanosoma brucei, the causative agent of human African trypanosomiasis, is critical for parasite survival and virulence. Of these metabolic processes, the transamination of aromatic amino acids is one of the most important. In this study, a series of halogenated tryptophan analogues were investigated for their anti-parasitic potency. Several of these analogues showed significant trypanocidal activity. Metabolomics analysis of compound-treated parasites revealed key differences occurring within aromatic amino acid metabolism, particularly within the widely reported and essential transamination processes of this parasite., Author summary The uptake and metabolism of amino acids, particularly the aromatic amino acids tryptophan, phenylalanine and tyrosine, is greatly important to the survival of Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Substantial differences in amino acid metabolism between parasite and human host cells provide a potential route to selective chemotherapeutic agents, which has previously been exploited in the case of the frontline HAT drug eflornithine. We have investigated analogues of the amino acid tryptophan and found that several analogues displayed selective trypanocidal potency, which strongly correlated to a structural modification of halogenation at the 7-position of the indole ring. The trypanocidal activity of these potent compounds could be reversed by supplementing growth media with excess levels of natural tryptophan, suggesting competition and direct involvement of the compounds in tryptophan metabolism. Investigation of parasite metabolism when treated with potent analogues revealed large disturbances of the parasites’ metabolic function. Importantly, significant disruptions to the metabolism of aromatic amino acids were observed, giving evidence for the direct disruption of this pathway by these compounds. In the future, these analogues could serve as vital tools to gain a deeper understanding of the functional significance of these transamination processes within T. brucei parasites.

This work was funded by operating grants from the Canadian Institutes of Health Research (CIHR) and a Natural Sciences Engineering Research Council of Canada (NSERC) Discovery grant [Fonds de recherche du Québec — Nature et technologies (FRQNT) Regroupement Stratégique grant to the Centre for Host-Parasite Interactions (A.J.)]. N.C. was supported by a doctoral research scholarship from FRQNT. E.B. was supported by a Banting postdoctoral fellowship from CIHR. This work was also supported in part by Wellcome Trust grants [086658 and 093228] to T.K.S. C.S. recognizes the financial support from the Natural Sciences and Engineering Research Council of Canada and a Canada Foundation for Innovation grant [16299]. Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs. Postprint

Phenotypic assays are becoming increasingly more common among drug discovery practices, expanding drug target diversity as lead compounds identified through such screens are not limited to known targets. While increasing diversity is beneficial to the drug discovery process and the fight against disease, the unknown modes of action of new lead compounds can hamper drug discovery as, in most cases, the process of lead compound optimization is made difficult due to the unknown nature of the target; blindly changing substituents can prove fruitless due to the inexhaustible number of potential combinations, and it is therefore desirable to rapidly identify the targets of lead compounds developed through phenotypic screening. In addition, leads identified through target-based screening often have off-target effects that contribute towards drug toxicity, and by identifying those secondary targets, the drugs can be improved. However, the identification of a leads mode of action is far from trivial and now represents a major bottleneck in the drug discovery pipeline. This review looks at some of the recent developments in the identification of drug modes of action, focusing on phenotype-based methods using metabolomics, proteomics, transcriptomics, and genomics to detect changes in phenotype in response to the presence of the drug, and affinity-based methods using modified/unmodified drug as bait to capture and identify targets. © 2017 IUBMB Life, 70(1):9-22, 2018.

This research was funded by the Scottish Universities Life Science Alliance (SULSA) assay development fund. This research was also kindly supported by The Rosetrees Trust and The Alzheimer’s Society, specifically The Barcopel Foundation, and partly funded by the MSD Scottish Life Sciences fund. As part of an ongoing contribution to Scottish life sciences, MSD Limited, a global health care leader, has given substantial monetary funding to the Scottish Funding Council for distribution via SULSA to develop and deliver a high-quality drug discovery research and training program. A major hallmark of Alzheimer’s disease (AD) is the formation of neurotoxic aggregates composed of the amyloid-β peptide (Aβ). Aβ has been recognized to interact with numerous proteins, resulting in pathological changes to the metabolism of patients with AD. One such mitochondrial metabolic enzyme is amyloid-binding alcohol dehydrogenase (ABAD), where altered enzyme function caused by the Aβ-ABAD interaction is known to cause mitochondrial distress and cytotoxic effects, providing a feasible therapeutic target for AD drug development. Here we have established a high-throughput screening platform for the identification of modulators to the ABAD enzyme. A pilot screen with a total of 6759 compounds from the NIH Clinical Collections (NCC) and SelleckChem libraries and a selection of compounds from the BioAscent diversity collection have allowed validation and robustness to be optimized. The pilot screen revealed 16 potential inhibitors in the low µM range against ABAD with favorable physicochemical properties for blood-brain barrier penetration. Postprint

This work was supported primarily through the European Community’s Seventh Framework Programme under grant agreements no. 602773 (Project KINDRED), with additional support from Wellcome Trust Project grant (086658); Medical Research Council (MR/M020118/1) and the School of Chemistry (The University of St Andrews). The kinetoplastid parasite Trypanosoma brucei causes African trypanosomiasis in both humans and animals. Infections place a significant health and economic burden on developing nations in sub-Saharan Africa, but few effective anti-parasitic treatments are currently available. Hence, there is an urgent need to identify new leads for drug development. The T. brucei neutral sphingomyelinase (TbnSMase) was previously established as essential to parasite survival, consequently being identified as a potential drug target. This enzyme may catalyse the single route to sphingolipid catabolism outside the T. brucei lysosome. To obtain new insight into parasite sphingolipid catabolism, the substrate specificity of TbnSMase was investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Recombinant TbnSMase was shown to degrade sphingomyelin, inositol-phosphoceramide and ethanolamine-phosphoceramide sphingolipid substrates, consistent with the sphingolipid complement of the parasites. TbnSMase also catabolized ceramide-1-phosphate, but was inactive towards sphingosine-1-phosphate. The broad-range specificity of this enzyme towards sphingolipid species is a unique feature of TbnSMase. Additionally, ESI-MS/MS analysis revealed previously uncharacterized activity towards lyso-phosphatidylcholine despite the enzyme's inability to degrade phosphatidylcholine. Collectively, these data underline the enzyme's importance in choline homoeostasis and the turnover of sphingolipids in T. brucei. Postprint

Staphylococcus aureusis a major opportunistic pathogen that is exposed to antimicrobial innate immune effectors and antibiotics that can disrupt its cell membrane. An understanding ofS. aureuslipid composition and its role in defending the cell against membrane-disrupting agents is of fundamental importance. Common methods for characterising lipid profiles suffer shortcomings such as low sensitivity of detection and inferior resolution of the positional assignments of fatty acid chains in lipids. This present study developed a rapid and sensitive nano-electrospray ionisation tandem mass spectrometry (nESI-MS/MS) method to characterise the lipid composition of three commonly studiedS. aureusisolates: Newman, Mu50 and BB270. Confirming previous studies, nESI-MS/MS revealed that phosphatidylglycerols were most abundant inS. aureusmembranes, while diglucosyldiacylglycerols and lysyl-phosphatidylglycerols were also detected. Positional assignments for individual fatty acid chains within these lipids were also determined. Concomitantly, gas chromatography mass spectrometry of the fatty acids validated the molecular characterization and showed the principal species present in each strain were predominately anteiso- and iso-branched chain fatty acids. Though the fatty acid and lipid profiles were similar between theS. aureusstrains, this method was sufficiently sensitive to distinguish minor differences in lipid composition. In conclusion, this nESI-MS/MS methodology can characterise the role of lipids in antimicrobial resistance, and may even be applied to the rapid diagnosis of drug-resistant strains in the clinic.

Neglected tropical diseases caused by parasitic infections are an ongoing and increasing concern. They are a burden to human and animal health, having the most devastating effect on the world′s poorest countries. Building upon our previously reported triazole analogues, in this study we describe the synthesis and biological testing of other novel heterocyclic acetogenin‐inspired derivatives, namely 3,5‐isoxazoles, furoxans, and furazans. Several of these compounds maintain low‐micromolar levels of inhibition against Trypanosoma brucei, whilst having no observable inhibitory effect on mammalian cells, leading to the possibility of novel lead compounds for selective treatment.

This work is supported in part by the aquaculture company NIREUS SA and TKS's Wellcome Trust project grant 093228. Total lipids of olive pomace (OP), olive pomace diet (OP diet), fish oil diet (FO diet) and fish filets of farmed gilthead sea bream (fish fed with FO diet and OP diet respectively) were extracted and separated into polar (TPL) and neutral (TNL) lipids. All samples were assessed for their in vitro activity against washed rabbit platelets aggregation induced by platelet activating factor (PAF) and they were further analyzed by electrospray-mass spectrometry. The high levels of palmitic (16:0), oleic (18:1 cis ω − 9), linoleic (18:2 ω − 6) and docosapentaenoic acid (DPA 22:5 ω − 3) contained in both OP and FO diets are reflected to the gilthead sea breams fed with the individual diet respectively, while the gilthead sea bream fed with FO diet displays a decrease in DPA. All samples contained various glycerophospholipids species. Two PE species were identified in OP, OP diet and fish fed with OP diet and not in FO diet, while that might be an indication that these substances are likely to be the key polar phospholipids that have the ability to be in vitro PAF inhibitors, i.e. inhibit the formation of atherosclerotic plaques in blood arteries. Postprint

The cellular membranes of Trypanosoma cruzi, like all eukaryotes, contain varying amounts of phospholipids, sphingolipids, neutral lipids and sterols. A multitude of pathways exist for the de novo synthesis of these lipid families but Trypanosoma cruzi has also become adapted to scavenge some of these lipids from the host. Completion of the TriTryp genomes has led to the identification of many putative genes involved in lipid synthesis, revealing some interesting differences to higher eukaryotes. Although many enzymes involved in lipid synthesis have yet to be characterised, completed experiments have shown the indispensability of some lipid metabolic pathways. Furthermore, the bioactive lipids of Trypanosoma cruzi and their effects on the host are becoming increasingly studied. Further studies on lipid metabolism in Trypanosoma cruzi will no doubt reveal some attractive targets for therapeutic intervention as well as reveal the interplay between parasite lipids, host response and pathogenesis.

This work was supported by Grant‐in‐Aid for Young Scientists (B) (No. 26860025) and Grant‐in‐Aid for Scientific Research (C) (No. 17K08248) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), and partially supported by the Kurata Grant awarded by the Kurata Memorial Hitachi Science and Technology Foundation, the grant provided by the Ichiro Kanehara Foundation and the Platform for Drug Discovery, Informatics, and Structural Life Science from Japan Agency for Medical Research and Development (AMED). H.K. was supported by the Sasakawa Scientific Research Grant from the Japan Science Society and the SUNBOR Scholarship. We thank the Photon Factory for provision of synchrotron radiation facilities (Photon Factory Proposal No. 2014G532) and acknowledge the staff of the NW‐12A and BL‐17A beamlines. We thank our colleague Vu Linh Ly for preparing the hydroxysisomicin intermediate. The Montreal group thanks NSERC for financial support and a fellowship to J.P.M. from the Québec Research Fund: Nature and Technology. The T.K.S. group thanks the Medical Research Council (MR/M020118/1) for current financial support. Aminoglycosides (AG) are antibiotics that lower the accuracy of protein synthesis by targeting a highly conserved RNA helix of the ribosomal A-site. The discovery of AGs that selectively target the eukaryotic ribosome, but lack activity in prokaryotes, are promising as antiprotozoals for the treatment of neglected tropical diseases, and as therapies to read-through point-mutation genetic diseases. However, a single nucleobase change A1408G in the eukaryotic A-site leads to negligible affinity for most AGs. Herein we report the synthesis of 6-fluorosisomicin, the first 6-fluorinated aminoglycoside, which specifically interacts with the protozoal cytoplasmic rRNA A-site, but not the bacterial A-site, as evidenced by X-ray co-crystal structures. The respective dispositions of 6-fluorosisomicin within the bacterial and protozoal A-sites reveal that the fluorine atom acts only as a hydrogen-bond acceptor to favorably interact with G1408 of the protozoal A-site. Unlike aminoglycosides containing a 6-ammonium group, 6-fluorosisomicin cannot participate in the hydrogen-bonding pattern that characterizes stable pseudo-base-pairs with A1408 of the bacterial A-sites. Based on these structural observations it may be possible to shift the biological activity of aminoglycosides to act preferentially as antiprotozoal agents. These findings expand the repertoire of small molecules targeting the eukaryotic ribosome and demonstrate the usefulness of fluorine as a design element. Postprint

This work was funded by the Leverhulme Trust (GJF) and the European Community’s Seventh Framework Programme under grant agreement No. 602773 [Project KINDRED] (TKS). Neglected tropical diseases caused by parasitic infections are an ongoing and increasing concern that have a devastating effect on the developing world due to their burden on human and animal health. In this work, we detail the preparation of a focused library of substituted-tetrahydropyran derivatives and their evaluation as selective chemical tools for trypanosomatid inhibition and the follow-on development of photo-affinity probes capable of labeling target protein(s) in vitro. Several of these functionalised compounds maintain low micromolar activity against Trypanosoma brucei, Trypanosoma cruzi, Leishmania major and Leishmania donovani. In addition we demonstrate the utility of the photo-affinity probes for target identification through preliminary cellular localization studies. Postprint

myo -Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na + - or H + -linked myo -inositol transporters. While Na + -coupled myo -inositol transporters are found exclusively in the plasma membrane, H + -linked myo -inositol transporters are detected in intracellular organelles. In Trypanosoma brucei , the causative agent of human African sleeping sickness, myo -inositol metabolism is compartmentalized. De novo -synthesized myo -inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo -inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H + -linked myo -inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei . Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo -inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo -inositol.

Impaired Ectodysplasin A (EDA) - EDA receptor (EDAR) signaling affects ectodermally derived structures including teeth, hair follicles and cutaneous glands. X-linked hypohidrotic ectodermal dysplasia (XLHED), resulting from EDA deficiency, can be rescued with lifelong benefits in animal models by stimulation of ectodermal appendage development with EDAR agonists. Treatments initiated later in the developmental period restore progressively fewer of the affected structures. It is unknown whether EDAR stimulation in adults with XLHED might have beneficial effects. In adult Eda mutant mice treated for several weeks with agonist anti-EDAR antibodies, we find that sebaceous glands size and function can be restored to wild type levels. This effect is maintained upon chronic treatment but reverses slowly upon cessation of treatment. Sebaceous glands in all skin regions respond to treatment, though to varying degrees, and this is accompanied in both Eda mutant and wild type mice by sebum secretion to levels higher than those observed in untreated controls. Edar is expressed at the periphery of the glands, suggesting a direct homeostatic effect of Edar stimulation on the sebaceous gland. Sebaceous gland size and sebum production may serve as biomarkers for EDAR stimulation, and EDAR agonists may improve skin dryness and eczema frequently observed in XLHED.Journal of Investigative Dermatology accepted article preview online, 10 September 2014. doi:10.1038/jid.2014.382.

This work was supported through the Global Health Implementation programme at the University of St Andrews. Kinetoplastid parasites are responsible for a range of diseases with significant global impact. Trypanosoma brucei and Trypanosoma cruzi cause human African trypanosomiasis and Chagas disease, respectively, while various Leishmania species are responsible for cutaneous, mucocutaneous and visceral leishmaniasis. Understanding the biology of these organisms is key for effective diagnosis, prophylaxis and treatment. The insect parasite Crithidia fasciculata offers a safe and low-cost alternative for studies of kinetoplastid biology. C. fasciculata does not infect humans, can be cultured to high yields in inexpensive serum-free medium in a standard laboratory, and has a completely sequenced publically available genome. Taking advantage of these features, however, requires the adaptation of existing methods of analysis to C. fasciculata. Tandem affinity purification is a widely used method that allows for the rapid purification of intact protein complexes under native conditions. Here we report the application of tandem affinity purification to C. fasciculata for the first time, demonstrating the effectiveness of the technique by purifying both the intact exosome and replication factor C complexes. Adding tandem affinity purification to the C. fasciculata toolbox significantly enhances the utility of this excellent model system. Postprint

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model., Author summary Millions of people are at risk of developing African sleeping sickness through infection with the parasite Trypanosoma brucei, which is fatal if untreated. Current therapies are antiquated and inadequate, requiring hospitalization to deliver them through complex regimens, thus new effective, cheap and easy to administer therapies are urgently required. We recently reported the generation of a new compound that is selectively active against the parasite responsible for the disease, but its mechanism of action was unknown. This body of work identifies the specific target within the parasite as the essential FoF1-ATP synthase (mitochondrial complex V), first by analysing the proteins bound to a tagged analogue of our active compound (in a process known as photo-affinity labelling), and secondly by determining the effects of our active compound on cellular metabolism. With the target now identified work can begin to improve inhibitor potency and selectivity with the aid of rational drug design and structural optimisation towards future drug discovery.

The trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp, are causative agents of important human diseases such African sleeping sickness, Chagas' disease and Leishmaniasis, respectively. The high impact of these diseases on human health and economy worldwide, the unsatisfactory available chemotherapeutic options and the absence of human effective vaccines, strongly justifies the search for new drugs. The pentose phosphate pathway has been proposed to be a viable strategy to defeat several infectious diseases, including those from trypanosomatids, as it includes an oxidative branch, important in the maintenance of cell redox homeostasis, and a non-oxidative branch in which ribose 5-phosphate and erythrose 4-phosphate, precursors of nucleic acids and aromatic amino acids, are produced. This review provides an overview of the available chemotherapeutic options against these diseases and discusses the potential of genetically validated enzymes from the pentose phosphate pathway of trypanosomatids to be explored as potential drug targets. This article is a result of the projects NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and from the European Community's Seventh Framework Programme under grant agreements No. 602773 (Project KINDRED). JT is an Investigator FCT funded by National funds through FCT and co-funded through European Social Fund within the Human Potential Operating Programme.

This work was funded by Biotechnology and Biological Sciences Research Council Grant BB/L022087/1 (to T.K.S., N.C.O.T., and L.H.C.) and Wellcome Trust Grant 093228 (to T.K.S.). S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity. Postprint

This work was supported by the National Institutes of Health NIH R01AL105690. Phosphatidylserine (PS) synthase (Cho1p) and the PS decarboxylase enzymes (Psd1p and Psd2p), which synthesize PS and phosphatidylethanolamine (PE), respectively, are crucial for Candida albicans virulence. Mutations that disrupt these enzymes compromise virulence. These enzymes are part of the cytidine diphosphate-diacylglycerol pathway (i.e. de novo pathway) for phospholipid synthesis. Understanding how losses of PS and/or PE synthesis pathways affect the phospholipidome of Candida is important for fully understanding how these enzymes impact virulence. The cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutations cause similar changes in levels of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol and PS. However, only slight changes were seen in PE and phosphatidylcholine (PC). This finding suggests that the alternative mechanism for making PE and PC, the Kennedy pathway, can compensate for loss of the de novo synthesis pathway. Candida albicans Cho1p, the lipid biosynthetic enzyme with the most potential as a drug target, has been biochemically characterized, and analysis of its substrate specificity and kinetics reveal that these are similar to those previously published for Saccharomyces cerevisiae Cho1p. Postprint

This work was supported by the Ministry of Health of the Czech Republic (no. NV15-28967A), the Charles University in Prague (SVV 260 291), COST Action CM1103 (STSM 15879 and 17487) and CA15135, University of Hradec Kralove (Faculty of Informatics and Management, project Excellence 2015), University of St Andrews (undergraduate project funding to D.Z.), Biotechnology and Biological Sciences Research Council (BBSRC; no. BB/J01446X/1), the Alzheimer’s Society and the Barcopel Foundation. Alzheimer’s disease (AD) is a neurodegenerative disorder associated with an excessive accumulation of amyloid-beta peptide (Aβ). Based on the multifactorial nature of AD, preparation of multi-target-directed ligands presents a viable option to address more pathological events at one time. A novel class of asymmetrical disubstituted indolyl thioureas have been designed and synthesized to interact with monoamine oxidase (MAO) and/or amyloid-binding alcohol dehydrogenase (ABAD). The design combines the features of known MAO inhibitors scaffolds (e.g. rasagiline or ladostigil) and a frentizole moiety with potential to interact with ABAD. Evaluation against MAO identified several compounds that inhibited in the low to moderate micromolar range. The most promising compound ( 19 ) inhibited human MAO-A and MAOB with IC50 values of 6.34 μM and 0.30 μM, respectively. ABAD activity evaluation did not show any highly potent compound, but the compound series allowed identification of structural features to assist the future development of ABAD inhibitors. Finally, several of the compounds were found to be potent inhibitors of horseradish peroxidase (HRP), preventing the use of the Amplex™ Red assay to detect hydrogen peroxide produced by MAO, highlighting the need for serious precautions when using an enzyme-coupled assay. Postprint

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites’ genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme’s active site, consistent with the fact that the resistant line continues to produce the enzyme’s product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself., Author summary Antimicrobial resistance threatens to reverse many of the great strides made against pathogens responsible for disease. Understanding the molecular processes underlying resistance is crucial to quantifying and tackling the problem. Here we select resistance in Leishmania parasites to amphotericin B, an antileishmanial drug of increasing importance. We then combine genome sequencing with untargeted and targeted metabolomics analyses to identify a gene, sterol 14α-demethylase, mutation of which drives a change in sterol metabolism and loss of ergosterol, the molecular target of amphotericin B. Accumulation of a downstream intermediate of ergosterol biosynthesis indicated the enzyme itself retains activity, but the pathway to ergosterol is truncated. Expression of wild-type sterol 14α-demethylase in the resistant cells restored amphotericin B sensitivity and normal ergosterol production.

20 p.-3 fig.-4 tab., Leishmania infantum is an etiological agent of the life-threatening visceral form of leishmaniasis.Liposomal amphotericin B (AmB) followed by a short administration of miltefosine (MF) is a drug combination effective for treating visceral leishmaniasis in endemic regions of India. Resistance to MF can be due to point mutations in the miltefosine transporter (MT).Here we show that mutations in MT are also observed in Leishmania AmB-resistant mutants. The MF-induced MT mutations, but not the AmB induced mutations in MT, alter the translocation/uptake of MF. Moreover, mutations in the MT selected by AmB or MF have a major impact on lipid species that is linked to cross-resistance between both drugs. These alterations include changes of specific phospholipids, some of which are enriched with cyclopropanated fatty acids, as well as an increase in inositolphosphoceramide species. Collectively these results provide evidence of the risk of cross-resistance emergence derived from current AmB-MF sequential or co-treatments for visceral leishmaniasis, Work in TKS’s lab is supported by the Wellcome Trust grant 093228 and European Community’s Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED). LR is supported by FEDER ISCIII PI12-02706 and RETICS (Ricet RD12/0018/0007). MO holds a Canada research chair in antimicrobial resistance and work in his lab is supported by the Canadian Institutes of Health Research (grant 15501).

This work was funded by the Leverhulme Trust. The research data supporting this publication can be accessed at http://dx.doi.org/10.17630/1dd8b881-17c2-48e4-a72f-53af938f4537. The need for new treatments for the neglected tropical diseases African sleeping sickness, Chagas disease and Leishmaniasis remains urgent with the diseases widespread in tropical regions, affecting the worlds very poorest. We have previously reported bis-tetrahydropyran 1,4-triazole analogues designed as mimics of the annonaceous acetogenin natural product chamuvarinin, which maintained trypanocidal activity. Building upon these studies, we here report related triazole compounds with pendant heterocycles, mimicking the original butenolide of the natural product. Analogues were active against T. brucei, with a nitrofuran compound displaying nanomolar trypanocidal activity- several analogues also showed strong activity against T. cruzi and L. major. Importantly, select compounds gave excellent selectivity over mammalian cells with a furan-based analogue highly selective while remaining active against all three cell lines, thus representing a potential lead for a new broad spectrum kinetoplastid inhibitor. Postprint

© 2016 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/), Trypanosoma brucei is an extracellular parasite that causes sleeping sickness. In mammalian hosts, trypanosomes are thought to exist in two major niches: early in infection, they populate the blood; later, they breach the blood-brain barrier. Working with a well-established mouse model, we discovered that adipose tissue constitutes a third major reservoir for T. brucei. Parasites from adipose tissue, here termed adipose tissue forms (ATFs), can replicate and were capable of infecting a naive animal. ATFs were transcriptionally distinct from bloodstream forms, and the genes upregulated included putative fatty acid β-oxidation enzymes. Consistent with this, ATFs were able to utilize exogenous myristate and form β-oxidation intermediates, suggesting that ATF parasites can use fatty acids as an external carbon source. These findings identify the adipose tissue as a niche for T. brucei during its mammalian life cycle and could potentially explain the weight loss associated with sleeping sickness., This work was supported by 55007419 (HHMI) and 2151 (EMBO) to L.M.F., D.P.-N., F.B., and F.G.; FCT fellowships to S.T., F.R.-F., and F.A.-B. (SFRH/BPD/ 89833/2012, SFRH/BD/51286/2010, and SFRH/BD/80718/2011, respectively); Wellcome Trust grant (093228), MRC MR/M020118/1, and European Community Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED) to S.A.Y. and T.K.S.; and PAI 7/41 (Belspo) and ERC-NANOSYM to J.V.D.A.

The fluid mosaic model has recently been amended to account for the existence of membrane domains enriched in certain phospholipids. In rod-shaped bacteria, the anionic phospholipid cardiolipin is enriched at the cell poles but its role in the morphogenesis of the filamentous bacterium Streptomyces coelicolor is unknown. It was impossible to delete clsA (cardiolipin synthase; SCO1389) unless complemented by a second copy of clsA elsewhere in the chromosome. When placed under the control of an inducible promoter, clsA expression, phospholipid profile and morphogenesis became inducer dependent. TLC analysis of phospholipid showed altered profiles upon depletion of clsA expression. Analysis of cardiolipin by mass spectrometry showed two distinct cardiolipin envelopes that reflected differences in acyl chain length; the level of the larger cardiolipin envelope was reduced in concert with clsA expression. ClsA-EGFP did not localize to specific locations, but cardiolipin itself showed enrichment at hyphal tips, branch points and anucleate regions. Quantitative analysis of hyphal dimensions showed that the mycelial architecture and the erection of aerial hyphae were affected by the expression of clsA. Overexpression of clsA resulted in weakened hyphal tips, misshaped aerial hyphae and anucleate spores and demonstrates that cardiolipin synthesis is a requirement for morphogenesis in Streptomyces.

Trypanosoma brucei membranes consist of all major eukaryotic glycerophospholipid and sphingolipid classes. These are de novo synthesized from precursors obtained either from the host or from catabolised endocytosed lipids. In recent years, substantial progress has been made in the molecular and biochemical characterisation of several of these lipid biosynthetic pathways, using gene knockout or RNA interference strategies or by enzymatic characterization of individual reactions. Together with the completed genome, these studies have highlighted several possible differences between mammalian and trypanosome lipid biosynthesis that could be exploited for the development of drugs against the diseases caused by these parasites.

Summary Sphingomyelin is the main sphingolipid in Trypanosoma brucei, the causative agent of African sleeping sickness. In vitro and in vivo characterization of the T. brucei neutral sphingomyelinase demonstrates that it is directly involved in sphingomyelin catabolism. Gene knockout studies in the bloodstream form of the parasite indicate that the neutral sphingomyelinase is essential for growth and survival, thus highlighting that the de novo biosynthesis of ceramide is unable to compensate for the loss of sphingomyelin catabolism. The phenotype of the conditional knockout has given new insights into the highly active endocytic and exocytic pathways in the bloodstream form of T. brucei. Hence, the formation of ceramide in the endoplasmic reticulum affects post-Golgi sorting and rate of deposition of newly synthesized GPI-anchored variant surface glycoprotein on the cell surface. This directly influences the corresponding rate of endocytosis, via the recycling endosomes, of pre-existing cell surface variant surface glycoprotein. The trypanosomes use this coupled endocytic and exocytic mechanism to maintain the cell density of its crucial variant surface glycoprotein protective coat. TbnSMase is therefore genetically validated as a drug target against African trypanosomes, and suggests that interfering with the endocytic transport of variant surface glycoprotein is a highly desirable strategy for drug development against African trypanosomasis.

A putative GDP-Man PP (guanidine diphosphomannose pyrophosphorylase) gene from Trypanosoma brucei (TbGDP-Man PP) was identified in the genome and subsequently cloned, sequenced and recombinantly expressed, and shown to be a catalytically active dimer. Kinetic analysis revealed a Vmax of 0.34 μmol/min per mg of protein and Km values of 67 μM and 12 μM for GTP and mannose 1-phosphate respectively. Further kinetic studies showed GDP-Man was a potent product feedback inhibitor. RNAi (RNA interference) of the cytosolic TbGDP-Man PP showed that mRNA levels were reduced to ~20% of wild-type levels, causing the cells to die after 3–4 days, demonstrating that TbGDP-Man PP is essential in the bloodstream form of T. brucei and thus a potential drug target. The RNAi-induced parasites have a greatly reduced capability to form GDP-Man, leading ultimately to a reduction in their ability to synthesize their essential GPI (glycosylphosphatidylinositol) anchors. The RNAi-induced parasites also showed aberrant N-glycosylation of their major cell-surface glycoprotein, variant surface glycoprotein, with loss of the high-mannose Man9GlcNAc2 N-glycosylation at Asn428 and formation of complex N-glycans at Asn263.

Phosphatidylethanolamine (GPEtn), a major phospholipid component of trypanosome membranes, is synthesized de novo from ethanolamine through the Kennedy pathway. Here the composition of the GPEtn molecular species in the bloodstream form of Trypanosoma brucei is determined, along with new insights into phospholipid metabolism, by in vitro and in vivo characterization of a key enzyme of the Kennedy pathway, the cytosolic ethanolamine-phosphate cytidylyltransferase (TbECT). Gene knockout indicates that TbECT is essential for growth and survival, thus highlighting the importance of the Kennedy pathway for the pathogenic stage of the African trypanosome. Phosphatiylserine decarboxylation, a potential salvage pathway, does not appear to be active in cultured bloodstream form T. brucei, and it is not upregulated even when the Kennedy pathway is disrupted. In vivo metabolic labelling and phospholipid composition analysis by ESI-MS/MS of the knockout cells confirmed a significant decrease in GPEtn species, as well as changes in the relative abundance of other phospholipid species. Reduction in GPEtn levels had a profound influence on the morphology of the mutants and it compromised mitochondrial structure and function, as well as glycosylphosphatidylinositol anchor biosynthesis. TbECT is therefore genetically validated as a potential drug target against the African trypanosome.

Drug-like molecules with activity against Trypanosoma brucei are urgently required as potential therapeutics for the treatment of African sleeping sickness. Starting from known inhibitors of other glycosyltransferases, we have developed the first small molecular inhibitors of dolicholphosphate mannose synthase (DPMS), a mannosyltransferase critically involved in glycoconjugate biosynthesis in T. brucei. We show that these DPMS inhibitors prevent the biosynthesis of glycosylphosphatidylinositol (GPI) anchors, and possess trypanocidal activity against live trypanosomes.

Note related output below contains correction of this paper. Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (glycero-phosphoethanolamine) and GPCho (glycerophosphocholine). Ethanolamine is also found as an integral component of the GPI (glycosylpliosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK 1 (T brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The K values for ethanolamine and ATP were found to be 18.4 +/- 0.9 and 219 29 mu M respectively. TbC/EK2 (T brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a K-m 80 times lower than that of ethanolamine. The K. values for choline, ethanolamine and ATP were 31.4 +/- 2.6 mu M, 2.56 +/- 0.31 mu M and 20.6 +/- 1.96 mu M respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents oil C-2, but substitutions oil C-1 and elongations of the carbon chain were not well tolerated. Publisher PDF

Summary In bloodstream-form Trypanosoma brucei (the causative agent of African sleeping sickness) the glycosylphosphatidylinositol (GPI) anchor biosynthetic pathway has been validated genetically and chemically as a drug target. The conundrum that GPI anchors could not be in vivo labelled with [3H]-inositol led us to hypothesize that de novo synthesis was responsible for supplying myo-inositol for phosphatidylinositol (PI) destined for GPI synthesis. The rate-limiting step of the de novo synthesis is the isomerization of glucose 6-phosphate to 1-d-myo-inositol-3-phosphate, catalysed by a 1-d-myo-inositol-3-phosphate synthase (INO1). When grown under non-permissive conditions, a conditional double knockout demonstrated that INO1 is an essential gene in bloodstream-form T. brucei. It also showed that the de novo synthesized myo-inositol is utilized to form PI, which is preferentially used in GPI biosynthesis. We also show for the first time that extracellular myo-inositol can in fact be used in GPI formation although to a limited extent. Despite this, extracellular inositol cannot compensate for the deletion of INO1. Supporting these results, there was no change in PI levels in the conditional double knockout cells grown under non-permissive conditions, showing that perturbation of growth is due to a specific lack of de novo synthesized myo-inositol and not a general inositol-less death. These results suggest that there is a distinction between de novo synthesized myo-inositol and that from the extracellular environment.

High-resolution crystallographic structures of recombinant human peroxisome proliferator-activated receptor ligand-binding domain (isotype beta/delta) reveal a fatty acid in the binding site. Mass spectrometry confirmed the presence of C16:0, C16:1, C18:0 and C18:1 in a ratio of approximately 3:2:1:4 with 11, Z-octadecenoic acid (cis-vaccenic acid) identified as the predominant species. These are endogenous fatty acids acquired from the bacterial expression system, and serve to lock the ligand-binding domain into the activated conformation. A requirement for crystal growth, the additive n-heptyl-beta-d-glucopyranoside, binds near the activation function helix where recognition of co-activator proteins occurs. Our observations suggest potential physiological ligands for human PPAR-beta/delta and highlight that reported binding studies must be treated with caution unless endogenous fatty acids have been removed from the sample prior to analysis.

Glycosylphosphatidylinositol (GPI) represents a mechanism for the attachment of proteins to the plasma membrane found in all eukaryotic cells. GPI biosynthesis has been mainly studied in parasites, yeast, and mammalian cells. Aspergillus fumigatus, a filamentous fungus, produces GPI-anchored molecules, some of them being essential in the construction of the cell wall. An in vitro assay was used to study the GPI biosynthesis in the mycelium form of this organism. In the presence of UDP-GlcNAc and coenzyme A, the cell-free system produces the initial intermediates of the GPI biosynthesis: GlcNAc-PI, GlcN-PI, and GlcN-(acyl)PI. Using GDP-Man, two types of mannosylation are observed. First, one or two mannose residues are added to GlcN-PI. This mannosylation, never described in fungi, does not require dolichol phosphomannoside (Dol-P-Man) as the monosaccharide donor. Second, one to five mannose residues are added to GlcN-(acyl)PI using Dol-P-Man as the mannose donor. The addition of ethanolamine phosphate groups to the first, second, and third mannose residue is also observed. This latter series of GPI intermediates identified in the A. fumigatus cell-free system indicates that GPI biosynthesis in this filamentous fungus is similar to the mammalian or yeast systems. Thus, these biochemical data are in agreement with a comparative genome analysis that shows that all but 3 of the 21 genes described in the Saccharomyces cerevisiae GPI pathways are found in A. fumigatus.

Glycosyl-phosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa such as Trypanosoma brucei, Leishmania major, Plasmodium falciparum and Toxoplasma gondii, and represent the major carbohydrate modification of many cell-surface parasite proteins. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. Therefore, the characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. In vitro and in vivo studies using sugars and substrate analogues as well as natural compounds have shown that it is possible to interfere with GPI biosynthesis at different steps in a species-specific manner. Here we review the recent and promising progress in the field of GPI inhibition.

A series of synthetic analogues of d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, consisting of 22 variants of the d-GlcN or lipid components, were tested in trypanosomal and human (HeLa) cell-free systems. The assays measured the abilities of the analogues to act as substrates or inhibitors of the enzymes of glycosylphosphatidylinositol biosynthesis downstream of GlcNAc-phosphatidylinositol (GlcNAc-PI) de-N-acetylase. One compound, 4-deoxy-d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, proved to be an inhibitor of both the trypanosomal and HeLa pathways, whereas 4-O-methyl-d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol and the 4'-epimer, d-GalN-alpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, were neither substrates nor inhibitors. The results with other analogues showed that the 6-OH of the alpha-d-GlcN residue is not required for substrate recognition in the trypanosomal and human pathways, whereas the 3-OH group is essential for both. Parasite-specific recognition of the beta-linked analogue d-GlcN beta 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol is striking. This suggests that, like the GlcNAc-PI de-N-acetylase, the trypanosomal glycosylphosphatidylinositol alpha-mannosyltransferases, inositol acyltransferse and ethanolamine phosphate transferase, do not recognize the 2-, 3-, 4-, and 5-OH groups of the d-myo-inositol residue, whereas the human inositol acyltransferase and/or first alpha-mannosyltransferase recognizes one or more of these groups. All of the various lipid analogues tested served as substrates in both the trypanosomal and HeLa cell-free systems, suggesting that a precise lipid structure and stereochemistry are not essential for substrate recognition. However, an analogue containing a single C18:0 alkyl chain in place of sn-1,2-dipalmitoylglycerol proved to be a better substrate in the trypanosomal than in the HeLa cell-free system. These findings should have a bearing on the design of future generations of specific inhibitors of the trypanosomal glycosylphosphatidylinositol biosynthetic pathway.

The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D-GlcNR1-6-D-myo-inositol-1-HPO4-sn-1,2- dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de- N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN- (L)-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or ‚GlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor (Smith, T. K., et al. (2001) EMBO J. 20, 3322- 3332), GalNCONH2-PI and GlcNCONH2-‚-PI were designed and found to be potent (IC50 0.2 IM), Plasmodium-specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs. Plasmodium falciparum is a virulent human parasite, causing malaria in approximately 400 million people, result- ing in over 2 million deaths worldwide annually, mainly in tropical and subtropical countries. These protozoa have been shown to synthesize glycosylphosphatidylinositol (GPI) 1

Neglected tropical diseases remain a serious global health concern. Here, a series of novel bis-tetrahydropyran 1,4-triazole analogues based on the framework of chamuvarinin, a polyketide natural product isolated from the annonaceae plant species are detailed. The analogues synthesized display low micromolar trypanocidal activities towards both bloodstream and insect forms of Trypanosoma brucei, the causative agent of African sleeping sickness, also known as Human African Trypanosomiasis (HAT). A divergent synthetic strategy was adopted for the synthesis of the key tetrahydropyran intermediates to enable rapid access to diastereochemical variation either side of the 1,4-triazole core. The resulting diastereomeric analogues displayed varying degrees of trypanocidal activity and selectivity in structure–activity relationship studies. Together, the biological potency and calculated lipophilicity values indicate that while there is room for improvement, these derivatives may represent a promising novel class of anti-HAT agents. Postprint

Chemical genetics and a global comparative analysis of phosphorylation and phospholipids in vivo shows that PKG is the upstream regulator that induces calcium signals that enables Plasmodium to progress through its complex life cycle., Many critical events in the Plasmodium life cycle rely on the controlled release of Ca2+ from intracellular stores to activate stage-specific Ca2+-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca2+ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca2+ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca2+ effectors, PKG emerges as a unifying factor to control multiple cellular Ca2+ signals essential for malaria parasite development and transmission., Author Summary Malaria, caused by Plasmodium spp. parasites, is a profound human health problem. Plasmodium parasites progress through a complex life cycle as they move between infected humans and blood-feeding mosquitoes. We know that tight regulation of calcium ion levels within the cytosol of the parasite is critical to control multiple signalling events in their life cycle. However, how these calcium levels are controlled remains a mystery. Here, we show that a single protein kinase, the cGMP-dependent protein kinase G (PKG), controls the calcium signals that are critical at three different points of the life cycle: (1) for the exit of the merozoite form of the parasite from human erythrocytes (red blood cells), (2) for the cellular activation that happens when Plasmodium sexual transmission stages are ingested by a blood-feeding mosquito, and (3) for the productive gliding of the ookinete, which is the parasite stage that invades the mosquito midgut. We provide initial evidence that the universal role of PKG relies on the production of lipid precursors which then give rise to inositol (1,4,5)-trisphosphate (IP3), a messenger molecule that serves as a signal for the release of calcium from stores within the parasite. This signalling pathway provides a potential target to block both malaria development in the human host and transmission to the mosquito vector.

Spermidine is a natural polyamine involved in many important cellular functions, whose supplementation in food or water increases life span and stress resistance in several model organisms. In this work, we expand spermidine's range of age-related beneficial effects by demonstrating that it is also able to improve locomotor performance in aged flies. Spermidine's mechanism of action on aging has been primarily related to general protein hypoacetylation that subsequently induces autophagy. Here, we suggest that the molecular targets of spermidine also include lipid metabolism: Spermidine-fed flies contain more triglycerides and show altered fatty acid and phospholipid profiles. We further determine that most of these metabolic changes are regulated through autophagy. Collectively, our data suggests an additional and novel lipid-mediated mechanism of action for spermidine-induced autophagy. Publisher PDF

The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6'-OH group is dispensable and the 4'-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing betaGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite-specific suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs.

Synthetic analogues of D-GlcNalpha1-6D-myo-inositol-1-HPO(4)-3(sn-1, 2-diacylglycerol) (GlcN-PI), with the 2-position of the inositol residue substituted with an O-octyl ether [D-GlcNalpha1-6D-(2-O-octyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-octyl) PI] or O-hexadecyl ether [D-GlcNalpha1-6D-(2-O-hexadecyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-hexadecyl)PI], were tested as substrates or inhibitors of glycosylphosphatidylinositol (GPI) biosynthetic pathways using cell-free systems of the protozoan parasite Trypanosoma brucei (the causative agent of human African sleeping sickness) and human HeLa cells. Neither these compounds nor their N-acetyl derivatives are substrates or inhibitors of GPI biosynthetic enzymes in the HeLa cell-free system but are potent inhibitors of GPI biosynthesis in the T.brucei cell-free system. GlcN-(2-O-hexadecyl)PI was shown to inhibit the first alpha-mannosyltransferase of the trypanosomal GPI pathway. The N-acetylated derivative GlcNAc-(2-O-octyl)PI is a substrate for the trypanosomal GlcNAc-PI de-N-acetylase and this compound, like GlcN-(2-O-octyl)PI, is processed predominantly to Man(2)GlcN-(2-O-octyl)PI by the T.brucei cell-free system. Both GlcN-(2-O-octyl)PI and GlcNAc(2-O-octyl)PI also inhibit inositol acylation of Man(1-3)GlcN-PI and, consequently, the addition of the ethanolamine phosphate bridge in the T.brucei cell-free system. The data establish these substrate analogues as the first generation of in vitro parasite GPI pathway-specific inhibitors.

Glycosylphosphatidylinositols (GPIs) are synthesized in the endoplasmic reticulum (ER) via the sequential addition of monosaccharides, fatty acid, and phosphoethanolamine(s) to phosphatidylinositol (PI). While attempting to establish a mammalian cell-free system for GPI biosynthesis, we found that the assembly of mannosylated GPI species was impaired when purified ER preparations were substituted for unfractionated cell lysates as the enzyme source. To explore this problem we analyzed the distribution of the various GPI biosynthetic reactions in subcellular fractions prepared from homogenates of mammalian cells. The results indicate the following: (i) the initial reaction of GPI assembly, i.e. the transfer of GlcNAc to PI to form GlcNAc-PI, is uniformly distributed in the ER; (ii) the second step of the pathway, i.e. de-N-acetylation of GlcNAc-PI to yield GlcN-PI, is largely confined to a subcompartment of the ER that appears to be associated with mitochondria; (iii) the mitochondria-associated ER subcompartment is enriched in enzymatic activities involved in the conversion of GlcN-PI to H5 (a singly mannosylated GPI structure containing one phosphoethanolamine side chain; and (iv) the mitochondria-associated ER subcompartment, unlike bulk ER, is capable of the de novo synthesis of H5 from UDP-GlcNAc and PI. The confinement of these GPI biosynthetic reactions to a domain of the ER provides another example of the compositional and functional heterogeneity of the ER. The implications of these findings for GPI assembly are discussed.

The t(14;18) translocation, which involves the bcl-2oncogene, occurs in follicular lymphomas (FL) at two common sites: the major breakpoint region (MBR) and the minor cluster region (mcr). The biological and clinical significance of these breakpoints is unknown. The bcl-2 breakpoint site was determined in 247 previously untreated patients (49% men; median age 52 years) with indolent FL (155 grade I, 83 grade II, and 8 grade III) to correlate it with pretreatment characteristics, response, and outcome. The bcl-2 breakpoint site was determined by a polymerase chain reaction method of peripheral blood (all cases), bone marrows (149 cases), and fresh lymph node biopsy specimens (68 cases). The breakpoint site occurred at MBR in 175 cases (71%) and atmcr in 27 (11%). In 45 cases (18%), no breakpoint was detected (germline). No significant relationship was found between the rearrangements and the expression of BLC-2 and BAXproteins. Patients’ germline for MBR and mcr tended to present more frequently with stage IV disease and higher β2-microglobulin (β2M) levels, whereas mcr-rearranged patients presented more frequently with early stage and normal β2M. The complete response rate of germline patients was significantly lower than that of MBR and mcr patients. An estimated 3-year failure-free survival (FFS) for mcr, MBR, and germline cases was 95%, 76%, and 57%, respectively (P

Most patients with follicular lymphoma (FL) achieve a complete response (CR) after treatment, but eventually most of them, particularly those with stage IV, relapse due to minimal residual disease (MRD). The t(14;18) gives rise to a rearrangement of the bcl-2 oncogene that constitutes an excellent target for detection of MRD by polymerase chain reaction (PCR). One hundred ninety-four previously untreated patients with indolent FL and detectable bcl-2 rearrangement were studied. The PCR assay was used to detect bcl-2–rearranged cells in blood and marrow before and after treatment. Molecular response rate was 37%, 53%, 56%, and 66% at 3 to 5, 6 to 8, 9 to 14, and 15 to 18 months from the start of therapy, respectively. Although molecular response was higher among clinical CRs, one third of partial responders at 3 to 5 months also achieved a molecular response. Patients who achieved a molecular response during the first year of treatment had a significantly longer failure-free survival (FFS) than those who did not (4-year FFS: 76% v 38%, respectively; P < .001). Similar results were also observed in the subset of patients in clinical CR 1 year after treatment. By multivariate analysis, β2-microglobulin (β2-M; P < .01), and molecular response (P < .001) were the most important variables associated with outcome. When we combined β2-M and molecular response, three prognostic groups emerged: (1) low β2-M and molecular responders, (2) low β2-M and nonresponders or high β2-M and responders, and (3) high β2-M and nonresponders. The 4-year FFS of these 3 groups were 86%, 65%, and 23%, respectively. Finally, patients who achieved molecular response and sustained it had better FFS than those who either reverted back to PCR-positive or who never achieved molecular response. Serial PCR analysis to determine the molecular response in FL correlates well with outcome especially when combined with pretreatment β2-M.