Publications
241 results found
Yusuf NA, Green JL, Wall RJ, et al., 2015, The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain, Journal of Biological Chemistry, Vol: 290, Pages: 12147-12164, ISSN: 1083-351X
Myosin B (MyoB) is one of the two short class XIV myosinsencoded in the Plasmodium genome. Class XIV myosins arecharacterized by a catalytic “head,” a modified “neck,” and theabsence of a “tail” region. Myosin A (MyoA), the other class XIVmyosin in Plasmodium, has been established as a component ofthe glideosome complex important in motility and cell invasion,but MyoB is not well characterized. We analyzed the propertiesof MyoB using three parasite species as follows: Plasmodiumfalciparum, Plasmodium berghei, and Plasmodium knowlesi.MyoB is expressed in all invasive stages (merozoites, ookinetes,and sporozoites) of the life cycle, and the protein is found in adiscrete apical location in these polarized cells. In P. falciparum,MyoB is synthesized very late in schizogony/merogony, and itslocation in merozoites is distinct from, and anterior to, that of arange of known proteins present in the rhoptries, rhoptry neckor micronemes. Unlike MyoA, MyoB is not associated withglideosome complex proteins, including the MyoA light chain,myosin A tail domain-interacting protein (MTIP). A uniqueMyoB light chain (MLC-B) was identified that contains a calmodulin-likedomain at the C terminus and an extended N-terminalregion. MLC-B localizes to the same extreme apical polein the cell as MyoB, and the two proteins form a complex. Wepropose that MLC-B is a MyoB-specific light chain, and for theshort class XIV myosins that lack a tail region, the atypical myosinlight chains may fulfill that role.
Masumoto N, Lanyon-Hogg T, Rodgers UR, et al., 2015, Membrane bound O-acyltransferases and their inhibitors, Biochemical Society Transactions, Vol: 43, Pages: 246-252, ISSN: 1470-8752
Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin Oacyltransferase(GOAT)] have been shown to acylate specific proteins or peptides. In this review, topologydetermination, development of assays to measure enzymatic activities and discovery of small moleculeinhibitors are compared and discussed for each of these enzymes.
Ciepla P, Magee AI, Tate EW, 2015, Cholesterylation: a tail of hedgehog, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 43, Pages: 262-267, ISSN: 0300-5127
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- Citations: 12
Wright MH, Paape D, Storck EM, et al., 2015, Global Analysis of Protein <i>N</i>-Myristoylation and Exploration of <i>N</i>-Myristoyltransferase as a Drug Target in the Neglected Human Pathogen <i>Leishmania donovani</i>, CHEMISTRY & BIOLOGY, Vol: 22, Pages: 342-354, ISSN: 1074-5521
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- Citations: 63
Lanyon-Hogg T, Ritzefeld M, Masumoto N, et al., 2015, Modulation of Amide Bond Rotamers in 5-Acyl-6,7-dihydrothieno[3,2-c]pyridines, Journal of Organic Chemistry, Vol: 80, Pages: 4370-4377, ISSN: 1520-6904
2-Substituted N-acyl-piperidine is a widespread and important structuralmotif, found in approximately 500 currently available structures, and present in nearly30 pharmaceutically active compounds. Restricted rotation of the acyl substituent insuch molecules can give rise to two distinct chemical environments. Here wedemonstrate, using NMR studies and density functional theory modeling of the lowestenergy structures of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine derivatives, that the amideE:Z equilibrium is affected by non-covalent interactions between the amide oxygen andadjacent aromatic protons. Structural predictions were used to design molecules that promote either the E- or Z-amideconformation, enabling preparation of compounds with a tailored conformational ratio, as proven by NMR studies. Analysis ofthe available X-ray data of a variety of published N-acyl-piperidine-containing compounds further indicates that these moleculesare also clustered in the two observed conformations. This finding emphasizes that directed conformational isomerism hassignificant implications for the design of both small molecules and larger amide-containing molecular architectures.
Konitsiotis AD, Jovanovic B, Ciepla P, et al., 2015, Topological Analysis of Hedgehog Acyltransferase, a Multipalmitoylated Transmembrane Protein, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 290, Pages: 3293-3307
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- Citations: 46
Tate EW, Kalesh KA, Lanyon-Hogg T, et al., 2015, Global profiling of protein lipidation using chemical proteomic technologies, CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 24, Pages: 48-57, ISSN: 1367-5931
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- Citations: 80
Douse CH, Vrielink N, Zhang W, et al., 2015, Targeting a Dynamic Protein-Protein Interaction: Fragment Screening against the Malaria Myosin A Motor Complex, CHEMMEDCHEM, Vol: 10, Pages: 134-143, ISSN: 1860-7179
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- Citations: 13
Kelly DJ, Warren SC, Alibhai D, et al., 2015, Automated multiwell fluorescence lifetime imaging for Forster resonance energy transfer assays and high content analysis, ANALYTICAL METHODS, Vol: 7, Pages: 4071-4089, ISSN: 1759-9660
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- Citations: 10
Kalesh KA, Clulow JA, Tate EW, 2015, Target profiling of zerumbone using a novel cell-permeable clickable probe and quantitative chemical proteomics, CHEMICAL COMMUNICATIONS, Vol: 51, Pages: 5497-5500, ISSN: 1359-7345
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- Citations: 27
Yu Z, Brannigan JA, Rangachari K, et al., 2015, Discovery of pyridyl-based inhibitors of <i>Plasmodium falciparum N</i>-myristoyltransferase, MEDCHEMCOMM, Vol: 6, Pages: 1767-1772, ISSN: 2040-2503
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- Citations: 10
Furse S, Mak L, Tate EW, et al., 2015, Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on <i>Sop</i>B phosphatase activity, ORGANIC & BIOMOLECULAR CHEMISTRY, Vol: 13, Pages: 2001-2011, ISSN: 1477-0520
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- Citations: 7
Bunney TD, Cole AR, Broncel M, et al., 2014, Crystal Structure of the Human, FIC-Domain Containing Protein HYPE and Implications for Its Functions, STRUCTURE, Vol: 22, Pages: 1831-1843, ISSN: 0969-2126
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- Citations: 36
Paape D, Bell AS, Heal WP, et al., 2014, Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting <i>N</i>-myristoylation in Intracellular <i>Leishmania</i> Amastigotes, PLOS NEGLECTED TROPICAL DISEASES, Vol: 8, ISSN: 1935-2735
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- Citations: 12
Hutton JA, Goncalves V, Brannigan JA, et al., 2014, Structure-Based Design of Potent and Selective Leishmania <i>N</i>-Myristoyltransferase Inhibitors, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 57, Pages: 8664-8670, ISSN: 0022-2623
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- Citations: 45
Douse CH, Maas SJ, Thomas JC, et al., 2014, Crystal Structures of Stapled and Hydrogen Bond Surrogate Peptides Targeting a Fully Buried Protein-Helix Interaction, ACS CHEMICAL BIOLOGY, Vol: 9, Pages: 2204-2209, ISSN: 1554-8929
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- Citations: 38
Olaleye TO, Brannigan JA, Roberts SM, et al., 2014, Peptidomimetic inhibitors of N-myristoyltransferase from human malaria and leishmaniasis parasites, Organic & Biomolecular Chemistry, Vol: 12, Pages: 8132-8137, ISSN: 1477-0539
N-Myristoyltransferase (NMT) has been shown to be essential in Leishmania and subsequently validated as a drug target in Plasmodium. Herein, we discuss the use of antifungal NMT inhibitors as a basis for inhibitor development resulting in the first sub-micromolar peptidomimetic inhibitors of Plasmodium and Leishmania NMTs. High-resolution structures of these inhibitors with Plasmodium and Leishmania NMTs permit a comparative analysis of binding modes, and provide the first crystal structure evidence for a ternary NMT-Coenzyme A/myristoylated peptide product complex.
Thinon E, Serwa RA, Broncel M, et al., 2014, Global profiling of co- and post-translationally N-myristoylated proteomes in human cells, Nature Communications, Vol: 5, Pages: 1-13, ISSN: 2041-1723
Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells.
Ciepla P, Konitsiotis AD, Serwa RA, et al., 2014, New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish, Chemical Science, Vol: 5, Pages: 4249-4259, ISSN: 2041-6520
Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in theprogression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requiresattachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidatedShh has been challenging due to the limited array of tools available, and the roles of theseposttranslational modifications are poorly understood. Herein, we describe the development andvalidation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable itsvisualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown tobe an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shhfrom signalling cells, formation of multimeric transport complexes and signalling. We have used thisprobe to determine the size of transport complexes of lipidated Shh in culture medium and expressionlevels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitativechemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy anddetection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probesprovide a set of novel and well-validated tools that can be used to investigate the role of lipidation onactivity of Shh, and potentially other members of the Hedgehog protein family
Guttery DS, Poulin B, Ramaprasad A, et al., 2014, Genome-wide Functional Analysis of Plasmodium Protein Phosphatases Reveals Key Regulators of Parasite Development and Differentiation, Cell Host & Microbe, Vol: 16, Pages: 128-140, ISSN: 1934-6069
Reversible protein phosphorylation regulated by kinasesand phosphatases controls many cellular processes.Although essential functions for the malariaparasite kinome have been reported, the roles ofmost protein phosphatases (PPs) during Plasmodiumdevelopment are unknown. We report a functionalanalysis of the Plasmodium berghei protein phosphatome,which exhibits high conservation with theP. falciparum phosphatome and comprises 30 predictedPPs with differential and distinct expressionpatterns during various stages of the life cycle. Genedisruption analysis of P. berghei PPs reveals thathalf of the genes are likely essential for asexualblood stage development, whereas six are requiredfor sexual development/sporogony in mosquitoes.Phenotypic screening coupled with transcriptomesequencing unveiled morphological changes andaltered gene expression in deletion mutants of twoN-myristoylated PPs. These findings provide systematicfunctional analyses of PPs in Plasmodium, identifyhow phosphatases regulate parasite developmentand differentiation, and can inform the identification ofdrug targets for malaria.
Kalesh KA, Tate EW, 2014, A succinyl lysine-based photo-cross-linking peptide probe for Sirtuin 5, ORGANIC & BIOMOLECULAR CHEMISTRY, Vol: 12, Pages: 4310-4313, ISSN: 1477-0520
Brannigan JA, Roberts SM, Bell AS, et al., 2014, Diverse modes of binding in structures of Leishmania major N-myristoyltransferase with selective inhibitors, IUCrJ, Vol: 1, Pages: 250-260, ISSN: 2052-2525
The leishmaniases are a spectrum of global diseases of poverty associated withimmune dysfunction and are the cause of high morbidity. Despite the longhistory of these diseases, no effective vaccine is available and the currently useddrugs are variously compromised by moderate efficacy, complex side effects andthe emergence of resistance. It is therefore widely accepted that new therapiesare needed. N-Myristoyltransferase (NMT) has been validated pre-clinically asa target for the treatment of fungal and parasitic infections. In a previouslyreported high-throughput screening program, a number of hit compounds withactivity against NMT from Leishmania donovani have been identified. Here,high-resolution crystal structures of representative compounds from four hitseries in ternary complexes with myristoyl-CoA and NMT from the closelyrelated L. major are reported. The structures reveal that the inhibitors associatewith the peptide-binding groove at a site adjacent to the bound myristoyl-CoAand the catalytic -carboxylate of Leu421. Each inhibitor makes extensiveapolar contacts as well as a small number of polar contacts with the protein.Remarkably, the compounds exploit different features of the peptide-bindinggroove and collectively occupy a substantial volume of this pocket, suggestingthat there is potential for the design of chimaeric inhibitors with significantlyenhanced binding. Despite the high conservation of the active sites of theparasite and human NMTs, the inhibitors act selectively over the host enzyme.The role of conformational flexibility in the side chain of Tyr217 in conferringselectivity is discussed.
Gray K, Elghadban S, Thongyoo P, et al., 2014, Potent and specific inhibition of the biological activity of the type-II transmembrane serine protease matriptase by the cyclic microprotein MCoTI-II, Thrombosis and Haemostasis, Vol: 112, Pages: 402-411, ISSN: 0340-6245
Matriptase is a type-II transmembrane serine protease involved in epithelial homeostasis in both health and disease, and is implicated in the development and progression of a variety of cancers. Matriptase mediates its biological effects both via as yet undefined substrates and pathways, and also by proteolytic cleavage of a variety of well-defined protein substrates, several of which it shares with the closely-related protease hepsin. Development of targeted therapeutic strategies will require discrimination between these proteases. Here we have investigated cyclic microproteins of the squash Momordica cochinchinensis trypsin-inhibitor family (generated by total chemical synthesis) and found MCoTI-II to be a high-affinity (Ki 9 nM) and highly selective (> 1,000-fold) inhibitor of matriptase. MCoTI-II efficiently inhibited the proteolytic activation of pro-hepatocyte growth factor (HGF) by matriptase but not by hepsin, in both purified and cell-based systems, and inhibited HGF-dependent cell scattering. MCoTI-II also selectively inhibited the invasion of matriptase-expressing prostate cancer cells. Using a model of epithelial cell tight junction assembly, we also found that MCoTI-II could effectively inhibit the re-establishment of tight junctions and epithelial barrier function in MDCK-I cells after disruption, consistent with the role of matriptase in regulating epithelial integrity. Surprisingly, MCoTI-II was unable to inhibit matriptase-dependent proteolytic activation of prostasin, a GPI-anchored serine protease also implicated in epithelial homeostasis. These observations suggest that the unusually high selectivity afforded by MCoTI-II and its biological effectiveness might represent a useful starting point for the development of therapeutic inhibitors, and further highlight the role of matriptase in epithelial maintenance.
Bell AS, Goncalves V, Hutton JA, et al., 2014, <i>N</i>-Myristoyltransferase inhibitors as anti-leishmanial agents, 247th National Spring Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
Konitsiotis AD, Chang S-C, Jovanovic B, et al., 2014, Attenuation of Hedgehog Acyltransferase-Catalyzed Sonic Hedgehog Palmitoylation Causes Reduced Signaling, Proliferation and Invasiveness of Human Carcinoma Cells, PLOS ONE, Vol: 9, ISSN: 1932-6203
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- Citations: 31
Rackham MD, Brannigan JA, Rangachari K, et al., 2014, Design and Synthesis of High Affinity Inhibitors of Plasmodium falciparum and Plasmodium vivax N-Myristoyltransferases Directed by Ligand Efficiency Dependent Lipophilicity (LELP), Journal of Medicinal Chemistry, Vol: 57, Pages: 2773-2788, ISSN: 0022-2623
N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme and an attractive drug target in parasiticinfections such as malaria. We have previously reported that 2-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimethyl-1H-pyrazol-4-yl)methyl)-1,3,4-oxadiazole (34c) is a high affinity inhibitor of both Plasmodium falciparum and P. vivax NMT anddisplays activity in vivo against a rodent malaria model. Here we describe the discovery of 34c through optimization of apreviously described series. Development, guided by targeting a ligand efficiency dependent lipophilicity (LELP) score of lessthan 10, yielded a 100-fold increase in enzyme affinity and a 100-fold drop in lipophilicity with the addition of only two heavyatoms. 34c was found to be equipotent on chloroquine-sensitive and -resistant cell lines and on both blood and liver stage formsof the parasite. These data further validate NMT as an exciting drug target in malaria and support 34c as an attractive tool forfurther optimization.
Wright MH, Clough B, Rackham MD, et al., 2013, Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach, Nature Chemistry, Vol: 6, Pages: 112-121, ISSN: 1755-4349
Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase.
Poulin B, Patzewitz E-M, Brady D, et al., 2013, Unique apicomplexan IMC sub-compartment proteins are early markers for apical polarity in the malaria parasite, Biology Open, Vol: 2, Pages: 1160-1170, ISSN: 2046-6390
The phylum Apicomplexa comprises over 5000 intracellularprotozoan parasites, including Plasmodium and Toxoplasma,that are clinically important pathogens affecting humans andlivestock. Malaria parasites belonging to the genusPlasmodium possess a pellicle comprised of a plasmalemmaand inner membrane complex (IMC), which is implicated inparasite motility and invasion. Using live cell imaging andreverse genetics in the rodent malaria model P. berghei, welocalise two unique IMC sub-compartment proteins (ISPs)and examine their role in defining apical polarity duringzygote (ookinete) development. We show that these proteinslocalise to the anterior apical end of the parasite where IMCorganisation is initiated, and are expressed at alldevelopmental stages, especially those that are invasive.Both ISP proteins are N-myristoylated, phosphorylated andmembrane-bound. Gene disruption studies suggest that ISP1is likely essential for parasite development, whereas ISP3 isnot. However, an absence of ISP3 alters the apical localisationof ISP1 in all invasive stages including ookinetes andsporozoites, suggesting a coordinated function for theseproteins in the organisation of apical polarity in the parasite.
Alibhai D, Kelly DJ, Warren S, et al., 2013, Automated fluorescence lifetime imaging plate reader and its application to Forster resonant energy transfer readout of Gag protein aggregation, Journal of Biophotonics, Vol: 6, Pages: 398-408, ISSN: 1864-0648
Fluorescence lifetime measurements can provide quantitativereadouts of local fluorophore environment andcan be applied to biomolecular interactions via Fo¨ rsterresonant energy transfer (FRET). Fluorescence lifetimeimaging (FLIM) can therefore provide a high contentanalysis (HCA) modality to map protein-protein interactions(PPIs) with applications in drug discovery, systemsbiology and basic research. We present here an automatedmultiwell plate reader able to perform rapid unsupervisedoptically sectioned FLIM of fixed and livebiological samples and illustrate its potential to assayPPIs through application to Gag protein aggregationduring the HIV life cycle. We demonstrate both heteroFRETand homo-FRET readouts of protein aggregationand report the first quantitative evaluation of a FLIMHCA assay by generating dose response curves throughaddition of an inhibitor of Gag myristoylation. Z0 factorsexceeding 0.6 are realised for this FLIM FRET assay.Fluorescence lifetime plate map with representativeimages of high and low FRET cells and correspondingdose response plot.
Tate EW, Bell AS, Rackham MD, et al., 2013, N- Myristoyltransferase as a potential drug target in malaria and leishmaniasis, Parasitology, Vol: 141, Pages: 37-49, ISSN: 1469-8161
Infections caused by protozoan parasites are among the most widespread and intractable transmissible diseases affecting the developing world, with malaria and leishmaniasis being the most costly in terms of morbidity and mortality. Although new drugs are urgently required against both diseases in the face of ever-rising resistance to frontline therapies, very few candidates passing through development pipelines possess a known and novel mode of action. Set in the context of drugs currently in use and under development, we present the evidence for N-myristoyltransferase (NMT), an enzyme that N-terminally lipidates a wide range of specific target proteins through post-translational modification, as a potential drug target in malaria and the leishmaniases. We discuss the limitations of current knowledge regarding the downstream targets of this enzyme in protozoa, and our recent progress towards potent cell-active NMT inhibitors against the most clinically-relevant species of parasite. Finally, we outline the next steps required in terms of both tools to understand N-myristoylation in protozoan parasites, and the generation of potential development candidates based on the output of our recently-reported high-throughput screens.
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