William P. Heal

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.

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.

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.

Application of Activity-Based Protein Profiling to the Study of Microbial Pathogenesis

Activity-based protein profiling (ABPP) is a powerful technology for the dissection of dynamic and complex enzyme interactions. The mechanisms involved in microbial pathogenesis are an example of just such a system, with a plethora of highly regulated enzymatic interactions between the infecting organism and its host. In this review we will discuss some of the cutting-edge applications of ABPP to the study of bacterial and parasitic pathogenesis and virulence, with an emphasis on Clostridium difficile, methicillin-resistant Staphylococcus aureus, quorum sensing, and malaria.

Using a non-image-based medium-throughput assay for screening compounds targeting N-myristoylation in intracellular Leishmania amastigotes.

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.