20 results found
Tate E, Lanyon-Hogg T, Ritzefeld M, et al., 2019, Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes, Chemical Science, ISSN: 2041-6520
Posttranslational attachment of lipids to proteins is important for many cellular functions, and the enzymes responsible for these modifications are implicated in many diseases, from cancer to neurodegeneration. Lipid transferases and hydrolases are increasingly tractable therapeutic targets, but present unique challenges for high-throughput biochemical enzyme assays which hinder development of new inhibitors. We present Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) as the first universally applicable biochemical lipidation assay, exploiting the hydrophobic nature of lipidated peptides to drive a polarised fluorescence readout. Acyl-cLIP allows sensitive, accurate, real-time measurement of S- or N-palmitoylation, N-myristoylation, S-farnesylation or S-geranylgeranylation. Furthermore, it is applicable to transfer and hydrolysis reactions, and we demonstrate its extension to a high-throughput screening format. We anticipate that Acyl-cLIP will greatly expedite future drug discovery efforts against these challenging targets.
Lim C, Ha KP, Clarke R, et al., 2019, Identification of a potent small-molecule inhibitor of bacterial DNA repair that potentiates quinolone antibiotic activity in methicillin-resistant Staphylococcus aureus, Bioorganic and Medicinal Chemistry, Pages: 1-7, ISSN: 0968-0896
The global emergence of antibiotic resistance is one of the most serious challenges facing modern medicine. There is an urgent need for validation of new drug targets and the development of small molecules with novel mechanisms of action. We therefore sought to inhibit bacterial DNA repair mediated by the AddAB/RecBCD protein complexes as a means to sensitize bacteria to DNA damage caused by the host immune system or quinolone antibiotics. A rational, hypothesis-driven compound optimization identified IMP-1700 as a cell-active, nanomolar potency compound. IMP-1700 sensitized multidrug-resistant Staphylococcus aureus to the fluoroquinolone antibiotic ciprofloxacin, where resistance results from a point mutation in the fluoroquinolone target, DNA gyrase. Cellular reporter assays indicated IMP-1700 inhibited the bacterial SOS-response to DNA damage, and compound-functionalized Sepharose successfully pulled-down the AddAB repair complex. This work provides validation of bacterial DNA repair as a novel therapeutic target and delivers IMP-1700 as a tool molecule and starting point for therapeutic development to address the pressing challenge of antibiotic resistance.
Storck Saha E, Morales Sanfrutos J, Serwa R, et al., 2019, Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics, Nature Chemistry, ISSN: 1755-4330
Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.
Lanyon-Hogg T, Faronato M, Serwa RA, et al., 2017, Dynamic protein acylation: new substrates, mechanisms and drug targets, Trends in Biochemical Sciences, Vol: 42, Pages: 566-581, ISSN: 0968-0004
Post-translational attachment of lipids to proteins is found in all organisms, and is important for many biological processes. Acylation with myristic and palmitic acids are among the most common lipid modifications, and understanding reversible protein palmitoylation dynamics has become a particularly important goal. Linking acyltransferase enzymes to disease states can be challenging due to a paucity of robust models, compounded by functional redundancy between many palmitoyl transferases; however, in cases such as Wnt or Hedgehog signalling, small molecule inhibitors have been identified, with some progressing to clinical trials. In this review, we present recent developments in our understanding of protein acylation in human health and disease through use of chemical tools, global profiling of acylated proteomes, and functional studies of specific protein targets.
Clulow JA, Storck EM, Lanyon-Hogg T, et al., 2017, Competition-based, quantitative chemical proteomics in breast cancer cells identifies new target profiles for sulforaphane, Chemical Communications, Vol: 53, Pages: 5182-5185, ISSN: 1364-548X
Sulforaphane is a small molecule isothiocyanate which exhibits anticancer potential, yet its biological targets remain poorly understood. Here we employ a competition-based chemical proteomics strategy to profile sulforaphane's targets and identify over 500 targets along with their relative affinities. These targets provide a new set of mediators for sulforaphane's bioactivity, and aid understanding of its complex mode of action.
Lanyon-Hogg T, Patel NV, Ritzefeld M, et al., 2017, Microfluidic mobility shift assay for real-time analysis of peptide n-palmitoylation, SLAS Discovery, Vol: 22, Pages: 418-424, ISSN: 2472-5552
The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransferase (Hhat). Hhat is an attractive target for small-molecule inhibition to arrest Hedgehog signaling, and methods for assaying Hhat activity are central to understanding its function. However, all existing assays to quantify lipidation of peptides suffer limitations, such as safety hazards, high costs, extensive manual handling, restriction to stopped-assay measurements, or indirect assessment of lipidation. To address these limitations, we developed a microfluidic mobility shift assay (MSA) to analyze Shh palmitoylation. MSA allowed separation of fluorescently labeled Shh amine-substrate and palmitoylated Shh amide-product peptides based on differences in charge and hydrodynamic radius, coupled with online fluorescence intensity measurements for quantification. The MSA format was employed to study Hhat-catalyzed reactions, investigate Hhat kinetics, and determine small-molecule inhibitor IC50 values. Both real-time and stopped assays were performed, with the latter achieved via addition of excess unlabeled Shh peptide. The MSA format therefore allows direct and real-time fluorescence-based measurement of acylation and represents a powerful alternative technique in the study of N-lipidation.
Rodgers U, Lanyon-Hogg T, Masumoto N, et al., 2016, Characterization of hedgehog acyltransferase inhibitors identifies a small molecule probe for hedgehog signaling by cancer cells, ACS Chemical Biology, Vol: 11, Pages: 3256-3262, ISSN: 1554-8937
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.
Baker A, Lanyon-Hogg T, Warriner SL, 2016, Correction: Peroxisome protein import: a complex journey., Biochemical Society Transactions, Vol: 44, Pages: 1183-1183, ISSN: 1470-8752
Zhao W, Jamshidiha M, Lanyon-Hogg T, et al., 2016, Direct targeting of the Ras GTPase superfamily through structure-based design, Current Topics in Medicinal Chemistry, Vol: 16, Pages: 16-29, ISSN: 1873-4294
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.
Baker A, Hogg TL, Warriner SL, 2016, Peroxisome protein import: a complex journey, Biochemical Society Transactions, Vol: 44, Pages: 783-789, ISSN: 1470-8752
The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor-cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes.
Lanyon-Hogg T, Masumoto N, Bodakh G, et al., 2016, Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase, Data in Brief, Vol: 7, Pages: 257-281, ISSN: 2352-3409
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) . 1H 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.
Bhogal MS, Lanyon-Hogg T, Johnston KA, et al., 2016, Covalent Label Transfer between Peroxisomal Importomer Components Reveals Export-driven Import Interactions., The Journal of Biological Chemistry, Vol: 291, Pages: 2460-2468, ISSN: 1083-351X
Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal malate dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes because of high degrees of protease susceptibility or resistance, respectively. Here we present a means for analysis of in vitro import through a covalent biotin label transfer and employ this method to the import of PEX5C. Label transfer demonstrates that the PEX5C construct is monomeric under the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labeling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and, strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import.
Lanyon-Hogg T, Masumoto N, Bodakh G, et al., 2015, Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase, Analytical Biochemistry, Vol: 490, Pages: 66-72, ISSN: 1096-0309
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.
Masumoto N, Lanyon-Hogg T, Rodgers UR, et al., 2015, Membrane bound O-acyltransferases and their inhibitors, Biochemical Society Transactions, Vol: 43, Pages: 246-252, ISSN: 1470-8752
Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin Oacyltransferase(GOAT)] have been shown to acylate specific proteins or peptides. In this review, topologydetermination, development of assays to measure enzymatic activities and discovery of small moleculeinhibitors are compared and discussed for each of these enzymes.
Lanyon-Hogg T, Ritzefeld M, Masumoto N, et al., 2015, Modulation of Amide Bond Rotamers in 5-Acyl-6,7-dihydrothieno[3,2-c]pyridines, Journal of Organic Chemistry, Vol: 80, Pages: 4370-4377, ISSN: 1520-6904
2-Substituted N-acyl-piperidine is a widespread and important structuralmotif, found in approximately 500 currently available structures, and present in nearly30 pharmaceutically active compounds. Restricted rotation of the acyl substituent insuch molecules can give rise to two distinct chemical environments. Here wedemonstrate, using NMR studies and density functional theory modeling of the lowestenergy structures of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine derivatives, that the amideE:Z equilibrium is affected by non-covalent interactions between the amide oxygen andadjacent aromatic protons. Structural predictions were used to design molecules that promote either the E- or Z-amideconformation, enabling preparation of compounds with a tailored conformational ratio, as proven by NMR studies. Analysis ofthe available X-ray data of a variety of published N-acyl-piperidine-containing compounds further indicates that these moleculesare also clustered in the two observed conformations. This finding emphasizes that directed conformational isomerism hassignificant implications for the design of both small molecules and larger amide-containing molecular architectures.
Konitsiotis AD, Jovanovic B, Ciepla P, et al., 2015, Topological Analysis of Hedgehog Acyltransferase, a Multipalmitoylated Transmembrane Protein, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 290, Pages: 3293-3307
Tate EW, Kalesh KA, Lanyon-Hogg T, et al., 2015, Global profiling of protein lipidation using chemical proteomic technologies, CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 24, Pages: 48-57, ISSN: 1367-5931
Lanyon-Hogg T, Hooper J, Gunn S, et al., 2014, PEX14 binding to Arabidopsis PEX5 has differential effects on PTS1 and PTS2 cargo occupancy of the receptor, FEBS LETTERS, Vol: 588, Pages: 2223-2229, ISSN: 0014-5793
Lanyon-Hogg T, Warriner SL, Baker A, 2010, Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes, BIOLOGY OF THE CELL, Vol: 102, Pages: 245-263, ISSN: 0248-4900
O'Leary-Steele C, Pedersen PJ, James T, et al., 2010, Synthesis of Small Molecules with High Scaffold Diversity: Exploitation of Metathesis Cascades in Combination with Inter- and Intramolecular Diels-Alder Reactions, CHEMISTRY-A EUROPEAN JOURNAL, Vol: 16, Pages: 9563-9571, ISSN: 0947-6539
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