Publications
211 results found
Ritzefeld M, Zhang L, Xiao Z, et al., 2024, Design, synthesis and evaluation of inhibitors of hedgehog acyltransferase, Journal of Medicinal Chemistry, Vol: 67, Pages: 1061-1078, ISSN: 0022-2623
Hedgehog signaling is involved in embryonic development and cancer growth. Functional activity of secreted Hedgehog signaling proteins is dependent on N-terminal palmitoylation, making the palmitoyl transferase Hedgehog acyltransferase (HHAT), a potential drug target and a series of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines have been identified as HHAT inhibitors. Based on structural data, we designed and synthesized 37 new analogues which we profiled alongside 13 previously reported analogues in enzymatic and cellular assays. Our results show that a central amide linkage, a secondary amine, and (R)-configuration at the 4-position of the core are three key factors for inhibitory potency. Several potent analogues with low- or sub-μM IC50 against purified HHAT also inhibit Sonic Hedgehog (SHH) palmitoylation in cells and suppress the SHH signaling pathway. This work identifies IMP-1575 as the most potent cell-active chemical probe for HHAT function, alongside an inactive control enantiomer, providing tool compounds for validation of HHAT as a target in cellular assays.
Coupland CE, Andrei SA, Ansell TB, et al., 2021, Structure, mechanism, and inhibition of Hedgehog acyltransferase, Molecular Cell, Vol: 81, Pages: 5025-+, ISSN: 1097-2765
The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
Coupland CE, Andrei SA, Ansell TB, et al., 2021, Structure and Mechanism of Hedgehog Acyl Transferase, Molecular Cell, ISSN: 1097-2765
<jats:title>SUMMARY</jats:title><jats:p>The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin <jats:italic>O</jats:italic>-acyltransferase GOAT, and a template for future drug discovery.</jats:p>
Lanyon-Hogg T, Ritzefeld M, Zhang L, et al., 2021, Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT)., Angew Chem Weinheim Bergstr Ger, Vol: 133, Pages: 13654-13659, ISSN: 0044-8249
The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (K i=38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.
Lanyon-Hogg T, Ritzefeld M, Zhang L, et al., 2021, Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase, Angewandte Chemie International Edition, Vol: 60, Pages: 13542-13547, ISSN: 1433-7851
The mammalian membrane‐bound O ‐acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small‐molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small‐molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure‐activity relationship investigation identified single enantiomer IMP‐1575 , the most potent HHAT inhibitor reported to‐date, and guided design of photocrosslinking probes that maintained HHAT‐inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small‐molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP‐1575 ( K i = 38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.
Lanyon-Hogg T, Ritzefeld M, Zhang L, et al., 2020, Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound <i>O</i>-acyltransferase
<jats:title>Abstract</jats:title><jats:p>The mammalian membrane-bound <jats:italic>O</jats:italic>-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports development of a photochemical probe to interrogate the small-molecule binding site in the human MBOAT Hedgehog acyltransferase (HHAT) based on HHAT inhibitor RUSKI-201. Structure-activity relationship investigation identified the improved enantiomeric inhibitor <jats:bold>IMP-1575</jats:bold>, which is the most potent HHAT inhibitor reported to-date, and guided rational design of a photocrosslinking probe that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT inhibitor <jats:bold>IMP-1575</jats:bold> (<jats:italic>K</jats:italic><jats:sub>i</jats:sub> = 38 nM) and a strategy for mapping of interaction sites in MBOATs.</jats:p>
Lanyon-Hogg T, Ritzefeld M, Sefer L, et al., 2019, Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes, Chemical Science, Vol: 10, Pages: 8995-9000, 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.
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.
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) [1]. 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.
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.
Broncel M, Serwa RA, Ciepla P, et al., 2015, Myristoylation profiling in human cells and zebrafish., Data in Brief, Vol: 4, Pages: 379-383, ISSN: 2352-3409
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.
Broncel M, Serwa RA, Ciepla P, et al., 2015, 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 Edition, Vol: 54, Pages: 5948-5951, ISSN: 1521-3773
Novel multifunctional reagents were applied incombination with a lipid probe for affinity enrichment ofmyristoylated proteins and direct detection of lipid-modifiedtryptic peptides by mass spectrometry. This method enableshigh-confidence identification of the myristoylated proteomeon an unprecedented scale in cell culture, and allowed the firstquantitative analysis of dynamic changes in protein lipidationduring vertebrate embryonic development.
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.
Ciepla P, Magee AI, Tate EW, 2015, Cholesterylation: a tail of hedgehog, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 43, Pages: 262-267, ISSN: 0300-5127
- Author Web Link
- Cite
- Citations: 12
Magee T, Henderson P, Baker A, et al., 2015, A Tribute to Stephen Allan Baldwin, MOLECULAR MEMBRANE BIOLOGY, Vol: 32, Pages: 33-34, ISSN: 0968-7688
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
- Author Web Link
- Open Access Link
- Cite
- Citations: 46
Koleva MV, Rothery S, Spitaler M, et al., 2015, Sonic hedgehog multimerization: A self-organizing event driven by post-translational modifications?, MOLECULAR MEMBRANE BIOLOGY, Vol: 32, Pages: 65-74, ISSN: 0968-7688
- Author Web Link
- Cite
- Citations: 8
Ciepla P, Konitsiotis AD, Serwa RA, et al., 2014, New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish, Chemical Science, Vol: 5, Pages: 4249-4259, ISSN: 2041-6520
Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in theprogression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requiresattachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidatedShh has been challenging due to the limited array of tools available, and the roles of theseposttranslational modifications are poorly understood. Herein, we describe the development andvalidation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable itsvisualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown tobe an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shhfrom signalling cells, formation of multimeric transport complexes and signalling. We have used thisprobe to determine the size of transport complexes of lipidated Shh in culture medium and expressionlevels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitativechemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy anddetection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probesprovide a set of novel and well-validated tools that can be used to investigate the role of lipidation onactivity of Shh, and potentially other members of the Hedgehog protein family
Konitsiotis AD, Chang S-C, Jovanovic B, et al., 2014, Attenuation of Hedgehog Acyltransferase-Catalyzed Sonic Hedgehog Palmitoylation Causes Reduced Signaling, Proliferation and Invasiveness of Human Carcinoma Cells, PLOS ONE, Vol: 9, ISSN: 1932-6203
- Author Web Link
- Open Access Link
- Cite
- Citations: 31
McDonald G, Deepak S, Miguel L, et al., 2014, Normalizing glycosphingolipids restores function in CD4<SUP>+</SUP> T cells from lupus patients, JOURNAL OF CLINICAL INVESTIGATION, Vol: 124, Pages: 712-724, ISSN: 0021-9738
- Author Web Link
- Cite
- Citations: 106
McDonald G, Miguel L, Hall C, et al., 2013, TARGETING GLYCOSPHINGOLIPID BIOSYNTHESIS PATHWAYS RESTORES T-CELL FUNCTION IN PATIENTS WITH SLE, Annual Meeting of the British-Society-for-Rheumatology and British-Health-Professionals-in-Rheumatology, Publisher: OXFORD UNIV PRESS, Pages: 127-127, ISSN: 1462-0324
McDonald G, Miguel L, Hall C, et al., 2012, Targeting Glycosphingolipid Biosynthesis Normalises T Lymphocyte Function in Patients with Systemic Lupus Eyrthematosus, Annual Scientific Meeting of the American-College-of-Rheumatology (ACR) and Association-of-Rheumatology-Health-Professionals (ARHP), Publisher: WILEY-BLACKWELL, Pages: S370-S370, ISSN: 0004-3591
Konitsiotis A, Chang S, Masumoto N, et al., 2012, Hhat a Potential New Target in Treatment of Pancreatic Cancer, 22nd Biennial Congress of the European-Association-for-Cancer-Research, Publisher: ELSEVIER SCI LTD, Pages: S149-S149, ISSN: 0959-8049
Vincent J-P, Magee T, 2012, Tiki Casts a Spell on Wnt, CELL, Vol: 149, Pages: 1426-1427, ISSN: 0092-8674
- Author Web Link
- Cite
- Citations: 2
Plaza-Menacho I, Morandi A, Mologni L, et al., 2011, Focal Adhesion Kinase (FAK) Binds RET Kinase via Its FERM Domain, Priming a Direct and Reciprocal RET-FAK Transactivation Mechanism, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 286, Pages: 17292-17302
- Author Web Link
- Cite
- Citations: 43
Miguel L, Owen DM, Lim C, et al., 2011, Primary Human CD4<SUP>+</SUP> T Cells Have Diverse Levels of Membrane Lipid Order That Correlate with Their Function, JOURNAL OF IMMUNOLOGY, Vol: 186, Pages: 3505-3516, ISSN: 0022-1767
- Author Web Link
- Cite
- Citations: 58
Salmond RJ, Filby A, Pirinen N, et al., 2011, Mislocalization of Lck impairs thymocyte differentiation and can promote development of thymomas, BLOOD, Vol: 117, Pages: 108-117, ISSN: 0006-4971
- Author Web Link
- Cite
- Citations: 10
Heal WP, Jovanovic B, Bessin S, et al., 2011, Bioorthogonal chemical tagging of protein cholesterylation in living cells, CHEMICAL COMMUNICATIONS, Vol: 47, Pages: 4081-4083, ISSN: 1359-7345
- Author Web Link
- Open Access Link
- Cite
- Citations: 69
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.