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Journal articleOlaleye 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: 1477-0539N-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.
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Journal articleThinon 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: 2041-1723Protein 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.
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Journal articleCiepla 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: 2041-6520Sonic 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
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Journal articleGuttery 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: 1934-6069Reversible 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.
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Journal articleKalesh 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 -
Journal articleBrannigan 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: 2052-2525The 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.
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Journal articleGray 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: 0340-6245Matriptase 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.
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Conference paperBell 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 -
Journal articleKonitsiotis 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- Author Web Link
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Journal articleRackham 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: 0022-2623N-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.
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