In JoVE (1)

Other Publications (83)

Articles by Edward W. Tate in JoVE

 JoVE Biology

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

1Photonics Group, Department of Physics, Imperial College London, 2Institute for Chemical Biology, Department of Chemistry, Imperial College London, 3MRC Clinical Sciences Centre, Hammersmith Hospital, 4Chemical Biology Section, Department of Chemistry, Imperial College London, 5Retroscreen Virology Ltd, 6Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK, 7Centre for Histopathology, Imperial College London

JoVE 55119

Other articles by Edward W. Tate on PubMed

A Highly Enantioselective Total Synthesis of (+)-goniodiol

Organic & Biomolecular Chemistry. May, 2006  |  Pubmed ID: 16633562

A high-yielding enantioselective total synthesis of the bioactive styryllactone (+)-goniodiol has been realised, starting from readily available (S)-glycidol. A key step is an oxygen-to-carbon rearrangement of a silyl enol ether linked via an anomeric centre, facilitating the rapid and diastereoselective construction of this functionalised system.

Total Synthesis of the Macrocyclic Cysteine Knot Microprotein MCoTI-II

Chemical Communications (Cambridge, England). Jul, 2006  |  Pubmed ID: 17007393

The first total synthesis of MCoTI-II, a cysteine knot microprotein and potent trypsin inhibitor, is described; a synthetic strategy has been developed that combines efficient backbone construction via optimised solid phase peptide synthesis with one-pot 'thia-zip' native chemical ligation and refolding to yield the natural product.

Immobilized Protease-assisted Synthesis of Engineered Cysteine-knot Microproteins

Chembiochem : a European Journal of Chemical Biology. Jul, 2007  |  Pubmed ID: 17526063

Molecules Incorporating a Benzothiazole Core Scaffold Inhibit the N-myristoyltransferase of Plasmodium Falciparum

The Biochemical Journal. Dec, 2007  |  Pubmed ID: 17714074

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 microg x l(-1) to >400 microg x l(-1) by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale 'piggyback' inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 microM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 microM.

Site-specific N-terminal Labelling of Proteins in Vitro and in Vivo Using N-myristoyl Transferase and Bioorthogonal Ligation Chemistry

Chemical Communications (Cambridge, England). Jan, 2008  |  Pubmed ID: 18188474

N-Myristoyl transferase-mediated modification with azide-bearing substrates is introduced as a highly selective and practical method for in vitro and in vivo N-terminal labelling of a recombinant protein using bioorthogonal ligation chemistry.

N-myristoyltransferase: a Prospective Drug Target for Protozoan Parasites

ChemMedChem. Mar, 2008  |  Pubmed ID: 18324715

Chemical and Biomimetic Total Syntheses of Natural and Engineered MCoTI Cyclotides

Organic & Biomolecular Chemistry. Apr, 2008  |  Pubmed ID: 18385853

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.

N-Myristoyl Transferase-mediated Protein Labelling in Vivo

Organic & Biomolecular Chemistry. Jul, 2008  |  Pubmed ID: 18563263

N-Myristoyl transferase-mediated labelling using a substrate modified with an azide or alkyne tag is described as an efficient and site-selective method for the introduction of a bioorthogonal tag at the N-terminus of a recombinant protein. The procedure may be performed in vitro, or in a single over-expression/tagging step in vivo in bacteria; tagged proteins may then be captured using Staudinger-Bertozzi or 'click' chemistry protocols to introduce a secondary label for downstream analysis. The straightforward synthesis of the chemical and molecular biological tools described should enable their use in a wide range of N-terminal labelling applications.

Specific N-terminal Protein Labelling: Use of FMDV 3C Pro Protease and Native Chemical Ligation

Chemical Communications (Cambridge, England). Aug, 2008  |  Pubmed ID: 18633492

We report an effective strategy for generating N-terminal cysteinyl proteins by proteolytic cleavage using the enzyme 3C pro, suitable for a wide range of applications via native chemical ligation.

Recent Advances in Chemical Proteomics: Exploring the Post-translational Proteome

Journal of Chemical Biology. Nov, 2008  |  Pubmed ID: 19568795

Identification and quantification of multiple proteins from complex mixtures is a central theme in post-genomic biology. Despite recent progress in high-throughput proteomics, proteomic analysis of post-translationally modified (PTM) proteins remains particularly challenging. This mini-review introduces the emerging field of chemical proteomics and reviews recent advances in chemical proteomic technology that are offering striking new insights into the functional biology of post-translational modification.

Potent Inhibitors of Beta-tryptase and Human Leukocyte Elastase Based on the MCoTI-II Scaffold

Journal of Medicinal Chemistry. Oct, 2009  |  Pubmed ID: 19772295

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.

Protein Myristoylation in Health and Disease

Journal of Chemical Biology. Nov, 2009  |  Pubmed ID: 19898886

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.

N-myristoyltransferase from Leishmania Donovani: Structural and Functional Characterisation of a Potential Drug Target for Visceral Leishmaniasis

Journal of Molecular Biology. Mar, 2010  |  Pubmed ID: 20036251

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. donovani NMT 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 A resolution. The structure has as its defining feature a 14-stranded twisted beta-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.

Chemical Probes of Surface Layer Biogenesis in Clostridium Difficile

ACS Chemical Biology. Mar, 2010  |  Pubmed ID: 20067320

Clostridium difficile, a leading cause of hospital-acquired infection, possesses a dense surface layer (S-layer) that mediates host-pathogen interactions. The key structural components of the S-layer result from proteolytic cleavage of a precursor protein, SlpA, into high- and low-molecular-weight components. Here we report the discovery and optimization of the first inhibitors of this process in live bacteria and their application for probing S-layer processing. We also describe the design and in vivo application of activity-based probes that identify the protein Cwp84 as the cysteine protease that mediates SlpA cleavage. This work provides novel chemical tools for the analysis of S-layer biogenesis and for the potential identification of novel drug targets within clostridia and related bacterial pathogens.

Getting a Chemical Handle on Protein Post-translational Modification

Organic & Biomolecular Chemistry. Feb, 2010  |  Pubmed ID: 20135026

This article highlights the emerging field of chemical proteomics, a powerful technology for the study of post- and co-translational modification of proteins. Genome mapping and the study of protein post-translational modifications have revealed the astounding chemical complexity present in the proteome of even the simplest organisms. The identification and characterisation of the modifications present on specific proteins in such complex mixtures has become a central challenge for post-genomic functional studies in cell and systems biology. In the chemical proteomic approach to this problem, protein-modifying enzymes and bioorthogonal chemoselective elaboration are exploited to deliver chemical tags to specific modified residues, enabling new advances in our understanding of protein modification.

Interaction and Dynamics of the Plasmodium Falciparum MTIP-MyoA Complex, a Key Component of the Invasion Motor in the Malaria Parasite

Molecular BioSystems. Mar, 2010  |  Pubmed ID: 20174678

The myosin tail domain interacting protein-myosin A (MTIP-MyoA) protein complex is an essential element of the motor driving invasion of red blood cells by the Plasmodium species that cause malaria. Here we report the key determinants of binding at the MTIP/MyoA interface, and the first structural study on the complex in solution using protein NMR.

Rapid Multilabel Detection of Geranylgeranylated Proteins by Using Bioorthogonal Ligation Chemistry

Chembiochem : a European Journal of Chemical Biology. Apr, 2010  |  Pubmed ID: 20209562

Membrane Enhanced Peptide Synthesis

Chemical Communications (Cambridge, England). Apr, 2010  |  Pubmed ID: 20369190

This communication reports a new technology platform that advantageously combines organic solvent nanofiltration (a newly emerging technology capable of molecular separations in organic solvents) with solution phase peptide synthesis-Membrane Enhanced Peptide Synthesis (MEPS).

Comparing Experimental and Computational Alanine Scanning Techniques for Probing a Prototypical Protein-protein Interaction

Protein Engineering, Design & Selection : PEDS. Jan, 2011  |  Pubmed ID: 20656696

The central role of protein-protein interactions in a wide range of cellular processes makes them a target for research and drug discovery. A variety of methods, both experimental and theoretical, exist for probing protein interfaces for residues that affect activity and binding affinity. Using as an example a protein-protein complex between trypsin and a nine-residue synthetic peptide, we experimentally assay-binding affinities for a variety of mutants and determine their relative free energy of binding, ΔΔG, to rank the importance of interface residues to binding. We then compare how accurately, precisely and reliably computational methods for calculating ΔΔG can replicate these results. We find that a 'post-process alanine scanning' protocol of a single native complex trajectory gives results with better accuracy than running separate molecular dynamics (MD) trajectories for individual mutants. Compared across 10 independent simulations, we find that results from the post-process alanine scanning are also more precise and are obtained over five times faster than their equivalent with the 'full MD' protocol. These results suggest that, although not suitable in every case, post-process alanine scanning is a useful and reliable tool in predicting important residues at protein interfaces with potential for modulation.

Activity-based Probes: Discovering New Biology and New Drug Targets

Chemical Society Reviews. Jan, 2011  |  Pubmed ID: 20886146

The development and application of chemical technologies enabling direct analysis of enzyme activity in living systems has undergone explosive growth in recent years. Activity-based protein profiling (ABPP) is a key constituent of this broad field, and is among the most powerful and mature chemical proteomic technologies. This tutorial review introduces the essential features of ABPP and the design and application of activity-based probes (ABPs) from drug target elucidation and in vivo visualisation of enzyme activity to comprehensive profiling of the catalytic content of living systems, and the discovery of new biological pathways.

Bioorthogonal Chemical Tagging of Protein Cholesterylation in Living Cells

Chemical Communications (Cambridge, England). Apr, 2011  |  Pubmed ID: 21221452

We report the first chemical probe for bioorthogonal chemical tagging of post-translationally cholesterylated proteins with an azide in living cells. This enables rapid multiplexed fluorescence detection and affinity labelling of protein cholesterylation, as exemplified by Sonic hedgehog protein, opening up new approaches for the de novo identification of cholesterylated proteins.

Activity-based Profiling for Drug Discovery

Chemistry & Biology. Apr, 2011  |  Pubmed ID: 21513874

Activity-based protein profiling (ABPP) is emerging as a game-changing tool for drug discovery, target validation, and basic biology. In this issue, Chang et al. (2011) report the ABPP-facilitated discovery of JW480, a highly selective potent and orally bioavailable inhibitor of monoalkylglycerol ether hydrolase KIAA1363 that dramatically impairs in vivo growth of human prostate cancer cell lines.

Roles of Cysteine Proteases Cwp84 and Cwp13 in Biogenesis of the Cell Wall of Clostridium Difficile

Journal of Bacteriology. Jul, 2011  |  Pubmed ID: 21531808

Clostridium difficile expresses a number of cell wall proteins, including the abundant high-molecular-weight and low-molecular-weight S-layer proteins (SLPs). These proteins are generated by posttranslational cleavage of the precursor SlpA by the cysteine protease Cwp84. We compared the phenotypes of C. difficile strains containing insertional mutations in either cwp84 or its paralog cwp13 and complemented with plasmids expressing wild-type or mutant forms of their genes. We show that the presence of uncleaved SlpA in the cell wall of the cwp84 mutant results in aberrant retention of other cell wall proteins at the cell surface, as demonstrated by secretion of the proteins Cwp66 and Cwp2 into the growth medium. These phenotypes are restored by complementation with a plasmid expressing wild-type Cwp84 enzyme but not with one encoding a Cys116Ala substitution in the active site. The cwp13 mutant cleaved the SlpA precursor normally and had a wild-type-like colony phenotype. Both Cwp84 and Cwp13 are produced as proenzymes which are processed by cleavage to produce mature enzymes. In the case of Cwp84, this cleavage does not appear to be autocatalytic, whereas in Cwp13 autocatalysis was demonstrated as a Cys109Ala mutant did not undergo processing. Cwp13 appears to have a role in processing of Cwp84 but is not essential for Cwp84 activity. Cwp13 cleaves SlpA in the HMW SLP domain, which we suggest may reflect a role in cleavage and degradation of misfolded proteins at the cell surface.

Highlights from the 46th EUCHEM Conference on Stereochemistry, Bürgenstock, Switzerland, May 2011

Chemical Communications (Cambridge, England). Oct, 2011  |  Pubmed ID: 21874182

Novel Inhibitors of Surface Layer Processing in Clostridium Difficile

Bioorganic & Medicinal Chemistry. Jan, 2012  |  Pubmed ID: 21752656

Clostridium difficile, a leading cause of hospital-acquired bacterial infection, is coated in a dense surface layer (S-layer) that is thought to provide both physicochemical protection and a scaffold for host-pathogen interactions. The key structural components of the S-layer are two proteins derived from a polypeptide precursor, SlpA, via proteolytic cleavage by the protease Cwp84. Here, we report the design, synthesis and in vivo characterization of a panel of protease inhibitors and activity-based probes (ABPs) designed to target S-layer processing in live C. difficile cells. Inhibitors based on substrate-mimetic peptides bearing a C-terminal Michael acceptor warhead were found to be promising candidates for further development.

A Fluorescence-based Assay for N-myristoyltransferase Activity

Analytical Biochemistry. Feb, 2012  |  Pubmed ID: 22051857

N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.

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

Topics in Current Chemistry. 2012  |  Pubmed ID: 22102218

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.

Multifunctional Protein Labeling Via Enzymatic N-terminal Tagging and Elaboration by Click Chemistry

Nature Protocols. Jan, 2012  |  Pubmed ID: 22193303

A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.

A New Chemical Handle for Protein AMPylation at the Host-pathogen Interface

Chembiochem : a European Journal of Chemical Biology. Jan, 2012  |  Pubmed ID: 22213418

Discovery of Plasmodium Vivax N-myristoyltransferase Inhibitors: Screening, Synthesis, and Structural Characterization of Their Binding Mode

Journal of Medicinal Chemistry. Apr, 2012  |  Pubmed ID: 22439843

N-Myristoyltransferase (NMT) is a prospective drug target against parasitic protozoa. Herein we report the successful discovery of a series of Plasmodium vivax NMT inhibitors by high-throughput screening. A high-resolution crystal structure of the hit compound in complex with NMT was obtained, allowing understanding of its novel binding mode. A set of analogues was designed and tested to define the chemical groups relevant for activity and selectivity.

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

PLoS Neglected Tropical Diseases. 2012  |  Pubmed ID: 22545171

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.

A Role for the Vesicle-associated Tubulin Binding Protein ARL6 (BBS3) in Flagellum Extension in Trypanosoma Brucei

Biochimica Et Biophysica Acta. Jul, 2012  |  Pubmed ID: 22609302

The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet-Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.

Regulation of the Plasmodium Motor Complex: Phosphorylation of Myosin A Tail-interacting Protein (MTIP) Loosens Its Grip on MyoA

The Journal of Biological Chemistry. Oct, 2012  |  Pubmed ID: 22932904

The interaction between the C-terminal tail of myosin A (MyoA) and its light chain, myosin A tail domain interacting protein (MTIP), is an essential feature of the conserved molecular machinery required for gliding motility and cell invasion by apicomplexan parasites. Recent data indicate that MTIP Ser-107 and/or Ser-108 are targeted for intracellular phosphorylation. Using an optimized MyoA tail peptide to reconstitute the complex, we show that this region of MTIP is an interaction hotspot using x-ray crystallography and NMR, and S107E and S108E mutants were generated to mimic the effect of phosphorylation. NMR relaxation experiments and other biophysical measurements indicate that the S108E mutation serves to break the tight clamp around the MyoA tail, whereas S107E has a smaller but measurable impact. These data are consistent with physical interactions observed between recombinant MTIP and native MyoA from Plasmodium falciparum lysates. Taken together these data support the notion that the conserved interactions between MTIP and MyoA may be specifically modulated by this post-translational modification.

Design and Synthesis of Inhibitors of Plasmodium Falciparum N-myristoyltransferase, a Promising Target for Antimalarial Drug Discovery

Journal of Medicinal Chemistry. Oct, 2012  |  Pubmed ID: 23035716

Design of inhibitors for N-myristoyltransferase (NMT), an enzyme responsible for protein trafficking in Plasmodium falciparum , the most lethal species of parasites that cause malaria, is described. Chemistry-driven optimization of compound 1 from a focused NMT inhibitor library led to the identification of two early lead compounds 4 and 25, which showed good enzyme and cellular potency and excellent selectivity over human NMT. These molecules provide a valuable starting point for further development.

Discovery of Novel and Ligand-efficient Inhibitors of Plasmodium Falciparum and Plasmodium Vivax N-myristoyltransferase

Journal of Medicinal Chemistry. Jan, 2013  |  Pubmed ID: 23170970

N-Myristoyltransferase (NMT) is an attractive antiprotozoan drug target. A lead-hopping approach was utilized in the design and synthesis of novel benzo[b]thiophene-containing inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) NMT. These inhibitors are selective against Homo sapiens NMT1 (HsNMT), have excellent ligand efficiency (LE), and display antiparasitic activity in vitro. The binding mode of this series was determined by crystallography and shows a novel binding mode for the benzothiophene ring.

Automated Fluorescence Lifetime Imaging Plate Reader and Its Application to Förster Resonant Energy Transfer Readout of Gag Protein Aggregation

Journal of Biophotonics. May, 2013  |  Pubmed ID: 23184449

Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z' factors exceeding 0.6 are realised for this FLIM FRET assay.

Chemical Proteomics: a Powerful Tool for Exploring Protein Lipidation

Biochemical Society Transactions. Feb, 2013  |  Pubmed ID: 23356258

The study of post-translational modifications such as protein lipidation is a non-trivial challenge of the post-genomic era. In recent years the field of chemical proteomics has greatly advanced our ability to identify and quantify protein lipidation. In the present review, we give a brief overview of the tools available to study protein acylation, prenylation and cholesterylation, and their application in the identification and quantification of protein lipidation in health and disease.

Unique Apicomplexan IMC Sub-compartment Proteins Are Early Markers for Apical Polarity in the Malaria Parasite

Biology Open. 2013  |  Pubmed ID: 24244852

The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite.

N-Myristoyltransferase As a Potential Drug Target in Malaria and Leishmaniasis

Parasitology. Jan, 2014  |  Pubmed ID: 23611109

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.

Validation of N-myristoyltransferase As an Antimalarial Drug Target Using an Integrated Chemical Biology Approach

Nature Chemistry. Feb, 2014  |  Pubmed ID: 24451586

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.

Attenuation of Hedgehog Acyltransferase-catalyzed Sonic Hedgehog Palmitoylation Causes Reduced Signaling, Proliferation and Invasiveness of Human Carcinoma Cells

PloS One. 2014  |  Pubmed ID: 24608521

Overexpression of Hedgehog family proteins contributes to the aetiology of many cancers. To be highly active, Hedgehog proteins must be palmitoylated at their N-terminus by the MBOAT family multispanning membrane enzyme Hedgehog acyltransferase (Hhat). In a pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and transfected HEK293a cells Hhat localized to the endoplasmic reticulum. siRNA knockdown showed that Hhat is required for Sonic hedgehog (Shh) palmitoylation, for its assembly into high molecular weight extracellular complexes and for functional activity. Hhat knockdown inhibited Hh autocrine and juxtacrine signaling, and inhibited PDAC cell growth and invasiveness in vitro. In addition, Hhat knockdown in a HEK293a cell line constitutively expressing Shh and A549 human non-small cell lung cancer cells inhibited their ability to signal in a juxtacrine/paracrine fashion to the reporter cell lines C3H10T1/2 and Shh-Light2. Our data identify Hhat as a key player in Hh-dependent signaling and tumour cell transformed behaviour.

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. Mar, 2014  |  Pubmed ID: 24641010

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme and an attractive drug target in parasitic infections 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 and displays activity in vivo against a rodent malaria model. Here we describe the discovery of 34c through optimization of a previously described series. Development, guided by targeting a ligand efficiency dependent lipophilicity (LELP) score of less than 10, yielded a 100-fold increase in enzyme affinity and a 100-fold drop in lipophilicity with the addition of only two heavy atoms. 34c was found to be equipotent on chloroquine-sensitive and -resistant cell lines and on both blood and liver stage forms of the parasite. These data further validate NMT as an exciting drug target in malaria and support 34c as an attractive tool for further optimization.

A Succinyl Lysine-based Photo-cross-linking Peptide Probe for Sirtuin 5

Organic & Biomolecular Chemistry. Jul, 2014  |  Pubmed ID: 24848608

A succinylation-specific photo-cross-linking peptide probe has been developed for the NAD(+)-dependent hydrolase Sirtuin 5. The probe, not only displayed robust labelling performance with purified Sirt5, but also enabled sensitive detection of the hydrolase in the presence of large excess of cellular proteins. It is anticipated that this probe, and future generations of it, will provide useful chemical tools for the functional analysis of Sirt5 and for the recently discovered PTM of lysine succinylation.

Genome-wide Functional Analysis of Plasmodium Protein Phosphatases Reveals Key Regulators of Parasite Development and Differentiation

Cell Host & Microbe. Jul, 2014  |  Pubmed ID: 25011111

Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria.

Diverse Modes of Binding in Structures of Leishmania Major N-myristoyltransferase with Selective Inhibitors

IUCrJ. Jul, 2014  |  Pubmed ID: 25075346

The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT) has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed.

Crystal Structures of Stapled and Hydrogen Bond Surrogate Peptides Targeting a Fully Buried Protein-helix Interaction

ACS Chemical Biology. Oct, 2014  |  Pubmed ID: 25084543

Constrained α-helical peptides are an exciting class of molecule designed to disrupt protein-protein interactions (PPIs) at a surface-exposed helix binding site. Complexes that engage more than one helical face account for over a third of structurally characterized helix PPIs, including several examples where the helix is fully buried. However, no constrained peptides have been reported that have targeted this class of interaction. We report the design of stapled and hydrogen bond surrogate (HBS) peptides mimicking the helical tail of the malaria parasite invasion motor myosin (myoA), which presents polar and hydrophobic functionality on all three faces in binding its partner, myoA tail interacting protein (MTIP), with high affinity. The first structures of these different constrained peptides bound to the same target are reported, enabling a direct comparison between these constraints and between staples based on monosubstituted pentenyl glycine (pGly) and disubstituted pentenyl alanine (pAla). Importantly, installation of these constraints does not disrupt native interactions in the buried site, so the affinity of the wild-type peptide is maintained.

Peptidomimetic Inhibitors of N-myristoyltransferase from Human Malaria and Leishmaniasis Parasites

Organic & Biomolecular Chemistry. Nov, 2014  |  Pubmed ID: 25230674

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.

Structure-based Design of Potent and Selective Leishmania N-myristoyltransferase Inhibitors

Journal of Medicinal Chemistry. Oct, 2014  |  Pubmed ID: 25238611

Inhibitors of Leishmania N-myristoyltransferase (NMT), a potential target for the treatment of leishmaniasis, obtained from a high-throughput screen, were resynthesized to validate activity. Crystal structures bound to Leishmania major NMT were obtained, and the active diastereoisomer of one of the inhibitors was identified. On the basis of structural insights, enzyme inhibition was increased 40-fold through hybridization of two distinct binding modes, resulting in novel, highly potent Leishmania donovani NMT inhibitors with good selectivity over the human enzyme.

Global Profiling of Co- and Post-translationally N-myristoylated Proteomes in Human Cells

Nature Communications. Sep, 2014  |  Pubmed ID: 25255805

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.

Crystal Structure of the Human, FIC-domain Containing Protein HYPE and Implications for Its Functions

Structure (London, England : 1993). Dec, 2014  |  Pubmed ID: 25435325

Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein,HYPE, which has remained poorly characterized.Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of auto AMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition.

Using a Non-image-based Medium-throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

PLoS Neglected Tropical Diseases. Dec, 2014  |  Pubmed ID: 25522361

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.

New Chemical Probes Targeting Cholesterylation of Sonic Hedgehog in Human Cells and Zebrafish

Chemical Science. May, 2014  |  Pubmed ID: 25574372

Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in the progression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requires attachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidated Shh has been challenging due to the limited array of tools available, and the roles of these posttranslational modifications are poorly understood. Herein, we describe the development and validation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable its visualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown to be an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shh from signalling cells, formation of multimeric transport complexes and signalling. We have used this probe to determine the size of transport complexes of lipidated Shh in culture medium and expression levels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitative chemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy and detection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probes provide a set of novel and well-validated tools that can be used to investigate the role of lipidation on activity of Shh, and potentially other members of the Hedgehog protein family.

Targeting a Dynamic Protein-protein Interaction: Fragment Screening Against the Malaria Myosin A Motor Complex

ChemMedChem. Jan, 2015  |  Pubmed ID: 25367834

Motility is a vital feature of the complex life cycle of Plasmodium falciparum, the apicomplexan parasite that causes human malaria. Processes such as host cell invasion are thought to be powered by a conserved actomyosin motor (containing myosin A or myoA), correct localization of which is dependent on a tight interaction with myosin A tail domain interacting protein (MTIP) at the inner membrane of the parasite. Although disruption of this protein-protein interaction represents an attractive means to investigate the putative roles of myoA-based motility and to inhibit the parasitic life cycle, no small molecules have been identified that bind to MTIP. Furthermore, it has not been possible to obtain a crystal structure of the free protein, which is highly dynamic and unstable in the absence of its natural myoA tail partner. Herein we report the de novo identification of the first molecules that bind to and stabilize MTIP via a fragment-based, integrated biophysical approach and structural investigations to examine the binding modes of hit compounds. The challenges of targeting such a dynamic system with traditional fragment screening workflows are addressed throughout.

Global Profiling of Protein Lipidation Using Chemical Proteomic Technologies

Current Opinion in Chemical Biology. Feb, 2015  |  Pubmed ID: 25461723

Protein lipidation is unique amongst post-translational modifications (PTMs) in enabling direct interaction with cell membranes, and is found in every form of life. Lipidation is important in normal function and in disease, but its intricate interplay with disease context presents a challenging for drug development. Global whole-proteome profiling of protein lipidation lies beyond the range of standard methods, but is well-suited to metabolic tagging with small 'clickable' chemical reporters that do not disrupt metabolism and function; chemoselective reactions are then used to add multifunctional labels exclusively to tagged-lipidated proteins. This chemical proteomic technology has opened up the first quantitative whole-proteome studies of the known major classes of protein lipidation, and the first insights into their full scope in vivo.

Topological Analysis of Hedgehog Acyltransferase, a Multipalmitoylated Transmembrane Protein

The Journal of Biological Chemistry. Feb, 2015  |  Pubmed ID: 25505265

Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo.

Synthesis of Unsaturated Phosphatidylinositol 4-phosphates and the Effects of Substrate Unsaturation on SopB Phosphatase Activity

Organic & Biomolecular Chemistry. Feb, 2015  |  Pubmed ID: 25515724

In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo.

Target Profiling of Zerumbone Using a Novel Cell-permeable Clickable Probe and Quantitative Chemical Proteomics

Chemical Communications (Cambridge, England). Mar, 2015  |  Pubmed ID: 25598414

Zerumbone is a phytochemical with diverse biological activities ranging from anti-inflammatory to anti-cancer properties; however, to date the cellular targets of this important compound have remained elusive. Here we report the global protein target spectrum of zerumbone in living cancer cells using competitive activity-based protein profiling of a novel cell-permeable clickable probe, combined with quantitative mass spectrometry.

Modulation of Amide Bond Rotamers in 5-Acyl-6,7-dihydrothieno[3,2-c]pyridines

The Journal of Organic Chemistry. May, 2015  |  Pubmed ID: 25713927

2-Substituted N-acyl-piperidine is a widespread and important structural motif, found in approximately 500 currently available structures, and present in nearly 30 pharmaceutically active compounds. Restricted rotation of the acyl substituent in such molecules can give rise to two distinct chemical environments. Here we demonstrate, using NMR studies and density functional theory modeling of the lowest energy structures of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine derivatives, that the amide E:Z equilibrium is affected by non-covalent interactions between the amide oxygen and adjacent aromatic protons. Structural predictions were used to design molecules that promote either the E- or Z-amide conformation, enabling preparation of compounds with a tailored conformational ratio, as proven by NMR studies. Analysis of the available X-ray data of a variety of published N-acyl-piperidine-containing compounds further indicates that these molecules are also clustered in the two observed conformations. This finding emphasizes that directed conformational isomerism has significant implications for the design of both small molecules and larger amide-containing molecular architectures.

Global Analysis of Protein N-myristoylation and Exploration of N-myristoyltransferase As a Drug Target in the Neglected Human Pathogen Leishmania Donovani

Chemistry & Biology. Mar, 2015  |  Pubmed ID: 25728269

N-Myristoyltransferase (NMT) modulates protein function through the attachment of the lipid myristate to the N terminus of target proteins, and is a promising drug target in eukaryotic parasites such as Leishmania donovani. Only a small number of NMT substrates have been characterized in Leishmania, and a global picture of N-myristoylation is lacking. Here, we use metabolic tagging with an alkyne-functionalized myristic acid mimetic in live parasites followed by downstream click chemistry and analysis to identify lipidated proteins in both the promastigote (extracellular) and amastigote (intracellular) life stages. Quantitative chemical proteomics is used to profile target engagement by NMT inhibitors, and to define the complement of N-myristoylated proteins. Our results provide new insight into the multiple pathways modulated by NMT and the pleiotropic effects of NMT inhibition. This work constitutes the first global experimental analysis of protein lipidation in Leishmania, and reveals the extent of NMT-related biology yet to be explored for this neglected human pathogen.

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

The Journal of Biological Chemistry. May, 2015  |  Pubmed ID: 25802338

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, 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 a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.

Multifunctional Reagents for Quantitative Proteome-wide Analysis of Protein Modification in Human Cells and Dynamic Profiling of Protein Lipidation During Vertebrate Development

Angewandte Chemie (International Ed. in English). May, 2015  |  Pubmed ID: 25807930

Novel multifunctional reagents were applied in combination with a lipid probe for affinity enrichment of myristoylated proteins and direct detection of lipid-modified tryptic peptides by mass spectrometry. This method enables high-confidence identification of the myristoylated proteome on an unprecedented scale in cell culture, and allowed the first quantitative analysis of dynamic changes in protein lipidation during vertebrate embryonic development.

Membrane Bound O-acyltransferases and Their Inhibitors

Biochemical Society Transactions. Apr, 2015  |  Pubmed ID: 25849925

Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early 2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin O-acyltransferase (GOAT)] have been shown to acylate specific proteins or peptides. In this review, topology determination, development of assays to measure enzymatic activities and discovery of small molecule inhibitors are compared and discussed for each of these enzymes.

Cholesterylation: a Tail of Hedgehog

Biochemical Society Transactions. Apr, 2015  |  Pubmed ID: 25849927

Cholesterylation is a post-translational attachment of sterol to proteins. This modification has been a characteristic of a single family of hedgehog proteins (Hh). Hh is a well-established morphogenic molecule important in embryonic development. It was also found to be involved in the progression of many cancer types. Herein, we describe the mechanism of biosynthesis of cholesterylated Hh, the role of this unusual modification on protein functions and novel chemical probes, which could be used to specifically target this modification, both in vitro and in vivo.

Creating a Customized Intracellular Niche: Subversion of Host Cell Signaling by Legionella Type IV Secretion System Effectors

Canadian Journal of Microbiology. Sep, 2015  |  Pubmed ID: 26059316

The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.

Chemoproteomic Evaluation of the Polyacetylene Callyspongynic Acid

Chemistry (Weinheim an Der Bergstrasse, Germany). Jul, 2015  |  Pubmed ID: 26079733

Polyacetylenes are a class of alkyne-containing natural products. Although potent bioactivities and thus possible applications as chemical probes have already been reported for some polyacetylenes, insights into the biological activities or molecular mode of action are still rather limited in most cases. To overcome this limitation, we describe the application of the polyacetylene callyspongynic acid in the development of an experimental roadmap for characterizing potential protein targets of alkyne-containing natural products. To this end, we undertook the first chemical synthesis of callyspongynic acid. We then used in situ chemical proteomics methods to demonstrate extensive callyspongynic acid-mediated chemical tagging of endoplasmic reticulum-associated lipid-metabolizing and modifying enzymes. We anticipate that an elucidation of protein targets of natural products may serve as an effective guide to the development of subsequent biological assays that aim to identify chemical phenotypes and bioactivities.

Legionella Pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor

Infection and Immunity. Oct, 2015  |  Pubmed ID: 26216420

Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.

Myristoylation Profiling in Human Cells and Zebrafish

Data in Brief. Sep, 2015  |  Pubmed ID: 26217820

Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223-6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.

Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics

Chemistry & Biology. Aug, 2015  |  Pubmed ID: 26256475

Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a generalized reduction in myristoylation of host proteins, whereas palmitoylation of host proteins, including regulators of interferon and tetraspanin family proteins, was selectively repressed. Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions.

Click Chemistry Armed Enzyme-linked Immunosorbent Assay to Measure Palmitoylation by Hedgehog Acyltransferase

Analytical Biochemistry. Dec, 2015  |  Pubmed ID: 26334609

Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.

Quantitative Lipoproteomics in Clostridium Difficile Reveals a Role for Lipoproteins in Sporulation

Chemistry & Biology. Nov, 2015  |  Pubmed ID: 26584780

Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C. difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C. difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen.

Discovery of High Affinity Inhibitors of Leishmania Donovani N-myristoyltransferase

MedChemComm. Oct, 2015  |  Pubmed ID: 26962429

N-Myristoyltransferase (NMT) is a potential drug target in Leishmania parasites. Scaffold-hopping from published inhibitors yielded the serendipitous discovery of a chemotype selective for Leishmania donovani NMT; development led to high affinity inhibitors with excellent ligand efficiency. The binding mode was characterised by crystallography and provides a structural rationale for selectivity.

Discovery of Pyridyl-based Inhibitors of Plasmodium Falciparum N-myristoyltransferase

MedChemComm. Oct, 2015  |  Pubmed ID: 26962430

N-Myristoyltransferase (NMT) represents an attractive drug target in parasitic infections such as malaria due to its genetic essentiality and amenability to inhibition by drug-like small molecules. Scaffold simplification from previously reported inhibitors containing bicyclic cores identified phenyl derivative 3, providing a versatile platform to study the effects of substitution on the scaffold, which yielded pyridyl 19. This molecule exhibited improved enzyme and cellular potency, and reduced lipophilicity compared to inhibitor 3. Further structure-based inhibitor design led to the discovery of 30, the most potent inhibitor in this series, which showed single-digit nM enzyme affinity and sub-μM anti-plasmodial activity.

Time-resolved FRET Reports FGFR1 Dimerization and Formation of a Complex with Its Effector PLCγ1

Advances in Biological Regulation. Jan, 2016  |  Pubmed ID: 26482290

In vitro and in vivo imaging of protein tyrosine kinase activity requires minimally invasive, molecularly precise optical probes to provide spatiotemporal mechanistic information of dimerization and complex formation with downstream effectors. We present here a construct with genetically encoded, site-specifically incorporated, bioorthogonal reporter that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of fibroblast growth factor receptor (FGFR). GyrB.FGFR1KD.TC contains a coumermycin-induced artificial dimerizer (GyrB), FGFR1 kinase domain (KD) and a tetracysteine (TC) motif that enables fluorescent labelling with biarsenical dyes FlAsH-EDT2 and ReAsH-EDT2. We generated bimolecular system for time-resolved FRET (TR-FRET) studies, which pairs FlAsH-tagged GyrB.FGFR1KD.TC and N-terminal Src homology 2 (nSH2) domain of phospholipase Cγ (PLCγ), a downstream effector of FGFR1, fused to mTurquoise fluorescent protein (mTFP). We demonstrated phosphorylation-dependent TR-FRET readout of complex formation between mTFP.nSH2 and GyrB.FGFR1KD.TC. By further application of TR-FRET, we also demonstrated formation of the GyrB.FGFR1KD.TC homodimer by coumermycin-induced dimerization. Herein, we present a spectroscopic FRET approach to facilitate and propagate studies that would provide structural and functional insights for FGFR and other tyrosine kinases.

Global Profiling of Huntingtin-associated Protein E (HYPE)-Mediated AMPylation Through a Chemical Proteomic Approach

Molecular & Cellular Proteomics : MCP. Feb, 2016  |  Pubmed ID: 26604261

AMPylation of mammalian small GTPases by bacterial virulence factors can be a key step in bacterial infection of host cells, and constitutes a potential drug target. This posttranslational modification also exists in eukaryotes, and AMP transferase activity was recently assigned to HYPE Filamentation induced by cyclic AMP domain containing protein (FICD) protein, which is conserved from Caenorhabditis elegans to humans. In contrast to bacterial AMP transferases, only a small number of HYPE substrates have been identified by immunoprecipitation and mass spectrometry approaches, and the full range of targets is yet to be determined in mammalian cells. We describe here the first example of global chemoproteomic screening and substrate validation for HYPE-mediated AMPylation in mammalian cell lysate. Through quantitative mass-spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification, we identified a total of 25 AMPylated proteins, including the previously validated substrate endoplasmic reticulum (ER) chaperone BiP (HSPA5), and also novel substrates involved in pathways of gene expression, ATP biosynthesis, and maintenance of the cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.

The Rab-binding Profiles of Bacterial Virulence Factors During Infection

The Journal of Biological Chemistry. Mar, 2016  |  Pubmed ID: 26755725

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection.

Design and Development of Histone Deacetylase (HDAC) Chemical Probes for Cell-based Profiling

Molecular BioSystems. May, 2016  |  Pubmed ID: 27021930

Histone deacetylases (HDACs) contribute to regulation of gene expression by mediating higher-order chromatin structures. They assemble into large multiprotein complexes that regulate activity and specificity. We report the development of small molecule probes with class IIa and pan-HDAC activity that contain photoreactive crosslinking groups and either a biotin reporter, or a terminal alkyne handle for subsequent bioorthogonal ligation. The probes retained inhibitory activity against recombinant HDAC proteins and caused an accumulation of acetylated histone and tubulin following cell treatment. The versatility of the probes has been demonstrated by their ability to photoaffinity modify HDAC targets in vitro. An affinity enrichment probe was used in conjunction with mass spectrometry proteomics to isolate HDACs and their interacting proteins in a native proteome. The performance of the probes in recombinant versus cell-based systems highlights issues for the development of chemoproteomic technologies targeting class IIa HDACs in particular.

Synthesis and Characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine Inhibitors of Hedgehog Acyltransferase

Data in Brief. Jun, 2016  |  Pubmed ID: 27077078

In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed "RU-SKI") class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article "Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase" (Lanyon-Hogg et al., 2015) [1]. (1)H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors.

N-Myristoyltransferase Inhibition Induces ER-Stress, Cell Cycle Arrest, and Apoptosis in Cancer Cells

ACS Chemical Biology. Aug, 2016  |  Pubmed ID: 27267252

N-Myristoyltransferase (NMT) covalently attaches a C14 fatty acid to the N-terminal glycine of proteins and has been proposed as a therapeutic target in cancer. We have recently shown that selective NMT inhibition leads to dose-responsive loss of N-myristoylation on more than 100 protein targets in cells, and cytotoxicity in cancer cells. N-myristoylation lies upstream of multiple pro-proliferative and oncogenic pathways, but to date the complex substrate specificity of NMT has limited determination of which diseases are most likely to respond to a selective NMT inhibitor. We describe here the phenotype of NMT inhibition in HeLa cells and show that cells die through apoptosis following or concurrent with accumulation in the G1 phase. We used quantitative proteomics to map protein expression changes for more than 2700 proteins in response to treatment with an NMT inhibitor in HeLa cells and observed down-regulation of proteins involved in cell cycle regulation and up-regulation of proteins involved in the endoplasmic reticulum stress and unfolded protein response, with similar results in breast (MCF-7, MDA-MB-231) and colon (HCT116) cancer cell lines. This study describes the cellular response to NMT inhibition at the proteome level and provides a starting point for selective targeting of specific diseases with NMT inhibitors, potentially in combination with other targeted agents.

Global Profiling and Inhibition of Protein Lipidation in Vector and Host Stages of the Sleeping Sickness Parasite Trypanosoma Brucei

ACS Infectious Diseases. Jun, 2016  |  Pubmed ID: 27331140

The enzyme N-myristoyltransferase (NMT) catalyzes the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localization of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labeling with an alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogue to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins, phosphatases, and many uncharacterized proteins as substrates of NMT in the parasite, providing a global view of the scope of this important protein modification and further evidence for the crucial and pleiotropic role of NMT in the cell.

Quantitative Chemical Proteomic Profiling of Ubiquitin Specific Proteases in Intact Cancer Cells

ACS Chemical Biology. Dec, 2016  |  Pubmed ID: 27779380

Deubiquitinating enzymes play an important role in a plethora of therapeutically relevant processes and are emerging as pioneering drug targets. Herein, we present a novel probe, Ubiquitin Specific Protease (USP) inhibitor, alongside an alkyne-tagged activity-based probe analogue. Activity-based proteome profiling identified 12 USPs, including USP4, USP16, and USP33, as inhibitor targets using submicromolar probe concentrations. This represents the first intact cell activity-based profiling of deubiquitinating enzymes. Further analysis demonstrated functional inhibition of USP33 and identified a synergistic relationship in combination with ATR inhibition, consistent with USP4 inhibition.

Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells

ACS Chemical Biology. Dec, 2016  |  Pubmed ID: 27779865

The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function.

Direct Targeting of the Ras GTPase Superfamily Through Structure- Based Design

Current Topics in Medicinal Chemistry. 2017  |  Pubmed ID: 27530972

The Ras superfamily of small monomeric GTPases includes some of the most prominent cancer targets for which no selective therapeutic agent has yet been successfully developed. The turn of the millennium saw a resurgence of efforts to target these enzymes using new and improved biophysical techniques to overcome the perceived difficulties of insurmountably high affinity for guanosine nucleotides and flat, flexible topology lacking suitable pockets for small molecule inhibitors. Further, recent investigations have begun to probe the dynamic conformational status of GTP-bound Ras, opening up new mechanisms of inhibition. While much of the literature has focused on the oncogenic Ras proteins, particularly K-Ras, these represent only a small minority of therapeutically interesting targets within the superfamily; for example, the Rab GTPases are the largest subfamily of about 70 members, and present an as yet untapped class of potential targets. The present review documents the key methodologies employed to date in structure-guided attempts to drug the Ras GTPases, and forecasts their transferability to other similarly challenging proteins in the superfamily.

Conformational Transition of FGFR Kinase Activation Revealed by Site-specific Unnatural Amino Acid Reporter and Single Molecule FRET

Scientific Reports. Jan, 2017  |  Pubmed ID: 28045057

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

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