Edward W. Tate

Protein myristoylation in health and disease

N-myristoylation is the attachment of a 14-carbon fatty acid, myristate, onto the N-terminal glycine residue of target proteins, catalysed by N-myristoyltransferase (NMT), a ubiquitous and essential enzyme in eukaryotes. Many of the target proteins of NMT are crucial components of signalling pathways, and myristoylation typically promotes membrane binding that is essential for proper protein localisation or biological function. NMT is a validated therapeutic target in opportunistic infections of humans by fungi or parasitic protozoa. Additionally, NMT is implicated in carcinogenesis, particularly colon cancer, where there is evidence for its upregulation in the early stages of tumour formation. However, the study of myristoylation in all organisms has until recently been hindered by a lack of techniques for detection and identification of myristoylated proteins. Here we introduce the chemistry and biology of N-myristoylation and NMT, and discuss new developments in chemical proteomic technologies that are meeting the challenge of studying this important co-translational modification in living systems.

Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides

The naturally-occurring cyclic cystine-knot microprotein trypsin inhibitors MCoTI-I and MCoTI-II have been synthesised using both thia-zip native chemical ligation and a biomimetic strategy featuring chemoenzymatic cyclisation by an immobilised protease. Engineered analogues have been produced containing a range of substitutions at the P1 position that redirect specificity towards alternative protease targets whilst retaining excellent to moderate affinity. Furthermore, we report an MCoTI analogue that is a selective low-microM inhibitor of foot-and-mouth-disease virus (FMDV) 3C protease, the first reported peptide-based inhibitor of this important viral enzyme.

Potent Inhibitors of β-Tryptase and Human Leukocyte Elastase Based on the MCoTI-II Scaffold

MCoTI-II is a member of a class of microproteins known as cyclotides that possess a macrolactam-cystine knot scaffold imparting exceptional physiological stability and structural rigidity. Modification of residues in the active loop and engineered truncations have resulted in MCoTI-II analogues that possess potent activity against two therapeutically significant serine proteases: beta-tryptase and human leukocyte elastase. These results suggest that MCoTI-II is a versatile scaffold for the development of protease inhibitors against targets in inflammatory disease.

Validation of N -myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach

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.

Global Profiling of Co- and Post-Translationally N-Myristoylated Proteomes in Human Cells.

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.

N-myristoyltransferase from Leishmania donovani: structural and functional characterisation of a potential drug target for visceral leishmaniasis.

N-Myristoyltransferase (NMT) catalyses the attachment of the 14-carbon saturated fatty acid, myristate, to the amino-terminal glycine residue of a subset of eukaryotic proteins that function in multiple cellular processes, including vesicular protein trafficking and signal transduction. In these pathways, N-myristoylation facilitates association of substrate proteins with membranes or the hydrophobic domains of other partner peptides. NMT function is essential for viability in all cell types tested to date, demonstrating that this enzyme has potential as a target for drug development. Here, we provide genetic evidence that NMT is likely to be essential for viability in insect stages of the pathogenic protozoan parasite, Leishmania donovani, causative agent of the tropical infectious disease, visceral leishmaniasis. The open reading frame of L. donovaniNMT has been amplified and used to overproduce active recombinant enzyme in Escherichia coli, as demonstrated by gel mobility shift assays of ligand binding and peptide-myristoylation activity in scintillation proximity assays. The purified protein has been crystallized in complex with the non-hydrolysable substrate analogue S-(2-oxo)pentadecyl-CoA, and its structure was solved by molecular replacement at 1.4 Å resolution. The structure has as its defining feature a 14-stranded twisted β-sheet on which helices are packed so as to form an extended and curved substrate-binding groove running across two protein lobes. The fatty acyl-CoA is largely buried in the N-terminal lobe, its binding leading to the loosening of a flap, which in unliganded NMT structures, occludes the protein substrate binding site in the carboxy-terminal lobe. These studies validate L. donovani NMT as a potential target for development of new therapeutic agents against visceral leishmaniasis.

Global profiling of protein lipidation using chemical proteomic technologies

Highlights • Protein lipidation is an essential modification (PTM) in all forms of life.• Key modifications include acylation, prenylation, cholesterylation and GPI anchors.• Global profiling of this class of PTM is beyond the scope of standard technologies.• Metabolic chemical tagging can reveal the full scope of protein lipidation in vivo.• Chemical proteomics will have a deep impact on understanding of protein lipidation.

N-myristoyltransferase: a prospective drug target for protozoan parasites.

The development of new drugs targeted at parasite pathogens represents an important step towards decreasing the morbidity and mortality caused by these organisms, which affect hundreds of millions of people worldwide. Many of the drugs used today to treat diseases such as malaria, leishmaniasis, and African sleeping sickness were discovered decades ago and are often ineffective against resistant organisms, or possess toxic side effects. As a result an increasing amount of fundamental research is being directed towards identifying and validating new drug targets. Identification of suitable macromolecular targets that can be exploited for therapeutic development is challenging, as physiological knowledge and tools for genetically manipulating these organisms are somewhat limited. Numerous biochemical pathways are being assessed as drug targets and include post-translational modification mechanisms such as phosphorylation in kinase pathways and lipidation. For the latter category, the inhibition of prenylation processes with farnesyltransferase inhibitors has shown potential for treating malaria and African trypanosomiasis. However, as discussed below, more recent data are emerging on the suitability of another type of lipid-modification pathway involving the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). NMT is a monomeric enzyme, ubiquitous in eukaryotes, that catalyses the co-translational transfer of the saturated 14carbon fatty acid, myristate, from myristoyl-CoA (14:0 CoA) to the N-terminal glycine residue of susceptible proteins by an amide bond. The co-translational nature of this modification was demonstrated by showing that the nascent polypeptide is labelled whilst still attached to the ribosome, and this is thought to be the normal sequence of events for the majority of the N-myristoylation modifications of a range of proteins with a variety of cellular roles. One exception is a subset of proteins that have been shown to be post-translationally modified by NMT after caspase-mediated cleavage of a mature protein. Post-translational modification of proteins from the bacterial type III secretion system by NMT has also been predicted and confirmed. NMTs have been identified and characterised from a range of eukaryotic organisms including human, mouse, rat, and cow, as well as plant and insect species. NMT was first purified from Saccharomyces cerevisiae (ScNMT), yielding a protein of ~50 kDa, and extensive studies have also been conducted on fungal NMTs, from Candida albicans, Cryptococcus neoformans, and Histoplasma capsulatum. More recently, NMTs have also been characterised from the parasitic protozoa Leishmania major, Trypanosoma brucei, and Plasmodium falciparum. Alignments of NMT sequences from many species can be obtained from http://mendel.imp.ac.at/myristate/SUPLalignment.htm (last access: February 1, 2008). There are two sequence motifs characteristic for all NMTs: EINFLCxHK and KFGxGDG; these are thus presumably of functional necessity to this enzyme. N-Myristoylation by NMT has been demonstrated, by product inhibition profiles, to proceed via an ordered Bi–Bi reaction mechanism in which binding of myristoyl-CoA generates a second binding pocket for the docking of the substrate protein, as summarised in Figure 1. The myristate group from myristoyl-CoA is then transferred to the N-terminal glycine of the bound protein in a nucleophilic addition–elimination reaction. This is followed by stepwise release, first of the free CoA and then the N-myristoylated protein. The catalytic mechanism requires the presence of an N-terminal glycine on the substrate protein exposed by the action of methionine aminopeptidase. Initial studies indicated the presence of an acyl intermediate formed as a result of physical transfer of the myristate group to the enzyme, and have led to the proposal of an acyl intermediate in the reaction mechanism, although this has yet to be observed. Crystallographic studies of ScNMT in binary complex with myristoyl-CoA and ternary complex with S-(2-oxo)pentadecyl-CoA and the peptide substrate GLYASKLA has allowed the residues involved in the active site to be identified. The availability of this X-ray crystal structure

Selective Inhibitors of Protozoan Protein N-myristoyltransferases as Starting Points for Tropical Disease Medicinal Chemistry Programs

Inhibition of N-myristoyltransferase has been validated pre-clinically as a target for the treatment of fungal and trypanosome infections, using species-specific inhibitors. In order to identify inhibitors of protozoan NMTs, we chose to screen a diverse subset of the Pfizer corporate collection against Plasmodium falciparum and Leishmania donovani NMTs. Primary screening hits against either enzyme were tested for selectivity over both human NMT isoforms (Hs1 and Hs2) and for broad-spectrum anti-protozoan activity against the NMT from Trypanosoma brucei. Analysis of the screening results has shown that structure-activity relationships (SAR) for Leishmania NMT are divergent from all other NMTs tested, a finding not predicted by sequence similarity calculations, resulting in the identification of four novel series of Leishmania-selective NMT inhibitors. We found a strong overlap between the SARs for Plasmodium NMT and both human NMTs, suggesting that achieving an appropriate selectivity profile will be more challenging. However, we did discover two novel series with selectivity for Plasmodium NMT over the other NMT orthologues in this study, and an additional two structurally distinct series with selectivity over Leishmania NMT. We believe that release of results from this study into the public domain will accelerate the discovery of NMT inhibitors to treat malaria and leishmaniasis. Our screening initiative is another example of how a tripartite partnership involving pharmaceutical industries, academic institutions and governmental/non-governmental organisations such as Medical Research Council and Wellcome Trust can stimulate research for neglected diseases.

Rapid multilabel detection of geranylgeranylated proteins by using bioorthogonal ligation chemistry.

Post-translational prenylation of proteins is an essential process in eukaryotic cells, and plays a key role in signal transduction and vesicular trafficking. In the case of protein geranylgeranylation, geranylgeranyl pyrophosphate (GGpp) acts as the prenyl donor, and the process is catalyzed either by geranylgeranyl transferase-1 (GGT-1), or by Rab geranylgeranyl transferase (RGGT), the latter requiring a Rab escort protein (REP-1 or REP-2). GGT-1 targets Rho family proteins, whilst RGGT acts on the Rab small GTPases; both sets of proteins are prenylated at C-terminal prenylation motifs. Mis-prenylation of Rabs leads to several severe diseases, and a deeper understanding of this process is essential in order to design effective therapies. However, efficient detection and identification of prenylated proteins remains challenging, making use of quantities of HGGpp with detection times typically extending from days to weeks. Biotinylated or fluorophore-containing prenyl analogues offer enhanced detection, 6] but their flexibility is limited by the substrate specificity of the prenyl transferase and they are associated with a significant reduction in affinity for native RGGT. We and others have recently demonstrated that azide or alkyne tagging combined with bioorthogonal ligation chemistry can be used to detect post-translational protein lipidation (acylation or prenylation) in cell-free and live-cell systems. 7–10] These highly biocompatible and biomimetic tags are minimally disruptive and enable subsequent labeling with a wide range of potential reporters. We describe here a versatile azidetag/bioorthogonal ligation system (Figure 1) that enables the rapid detection and affinity purification of proteins geranylgeranylated by either RGGT or GGT-1 by using multiple labels, applicable both to recombinant proteins and proteins from cell lines or mammalian tissues. 16-Azido geranylgeranyl pyrophosphate (AzGGpp (1), Scheme 1) was selected as the azide-tagged GGpp analogue; similar reagents have been reported for tagging prenylated proteins through metabolic labeling, 12] and we predicted that 1 would be well tolerated by geranylgeranyl transferases. Attempts to synthesize the key intermediate 4 by using previously reported approaches 13] resulted in poor yields and purity. However, direct chlorination of geranylgeranyl acetate (2) with catalytic polymer-supported PhSeBr and N-chlorosuccinimide (NCS) enabled isolation of 3 in a much improved yield, and this was subsequently converted into 1 through a short reaction sequence. To establish 1 as a RGGT substrate, analytical gel filtration and competition assays were performed with RGGT/REP-1 and Rab1a as a representative Rab prenylation system. Under the conditions used (see the Supporting Information), 1 was transferred with an efficiency equal to that of GGpp (Figure S1). Compound 1 interconverts spontaneously Figure 1. Rapid multilabel detection of geranylgeranylated proteins across recombinant, cellular, and mammalian systems. Enzymatic transfer of a tagged geranylgeranyl pyrophosphate analogue to target proteins is followed by bioorthogonal ligation to a multilabel probe.