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Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase

View ORCID ProfileThomas Lanyon-Hogg, View ORCID ProfileMarkus Ritzefeld, Leran Zhang, View ORCID ProfileBalazs Pogranyi, View ORCID ProfileMilon Mondal, Lea Sefer, View ORCID ProfileCallum D. Johnston, Claire E. Coupland, View ORCID ProfileSebastian A. Andrei, Joshua Newington, Anthony I. Magee, View ORCID ProfileChristian Siebold, View ORCID ProfileEdward W. Tate
doi: https://doi.org/10.1101/2020.10.16.342477
Thomas Lanyon-Hogg
aDepartment of Pharmacology, University of Oxford, Oxford, OX1 3QT, UK
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  • ORCID record for Thomas Lanyon-Hogg
Markus Ritzefeld
bDepartment of Chemistry, Imperial College London, London, W12 0BZ, UK
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Leran Zhang
bDepartment of Chemistry, Imperial College London, London, W12 0BZ, UK
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Balazs Pogranyi
bDepartment of Chemistry, Imperial College London, London, W12 0BZ, UK
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Milon Mondal
bDepartment of Chemistry, Imperial College London, London, W12 0BZ, UK
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Lea Sefer
cDivision of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
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Callum D. Johnston
bDepartment of Chemistry, Imperial College London, London, W12 0BZ, UK
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Claire E. Coupland
cDivision of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
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Sebastian A. Andrei
bDepartment of Chemistry, Imperial College London, London, W12 0BZ, UK
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Joshua Newington
bDepartment of Chemistry, Imperial College London, London, W12 0BZ, UK
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Anthony I. Magee
dCardioRespiratory Interface Section, National Heart & Lung Institute, Imperial College London, London, SW7 2AZ, UK
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Christian Siebold
cDivision of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
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Edward W. Tate
bDepartment of Chemistry, Imperial College London, London, W12 0BZ, UK
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  • For correspondence: e.tate@imperial.ac.uk
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Abstract

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 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 IMP-1575, 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 IMP-1575 (Ki = 38 nM) and a strategy for mapping of interaction sites in MBOATs.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵* E-mail: thomas.lanyon-hogg{at}pharm.ox.ac.uk, E-mail: e.tate{at}imperial.ac.uk

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted October 17, 2020.
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Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase
Thomas Lanyon-Hogg, Markus Ritzefeld, Leran Zhang, Balazs Pogranyi, Milon Mondal, Lea Sefer, Callum D. Johnston, Claire E. Coupland, Sebastian A. Andrei, Joshua Newington, Anthony I. Magee, Christian Siebold, Edward W. Tate
bioRxiv 2020.10.16.342477; doi: https://doi.org/10.1101/2020.10.16.342477
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Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase
Thomas Lanyon-Hogg, Markus Ritzefeld, Leran Zhang, Balazs Pogranyi, Milon Mondal, Lea Sefer, Callum D. Johnston, Claire E. Coupland, Sebastian A. Andrei, Joshua Newington, Anthony I. Magee, Christian Siebold, Edward W. Tate
bioRxiv 2020.10.16.342477; doi: https://doi.org/10.1101/2020.10.16.342477

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