Photochemical probe identification of a small-molecule inhibitor binding site in Hedgehog acyltransferase (HHAT)
Siebold, C, Greenfield, J, Johnston, C, Coupland, C, Andrei, S, Pogranyi, B, Mondal, M, Zhang, L, Sefer, L, Lanyon-Hogg, T, Joshua Newington, Matthew J. Fuchter, Anthony I. Magee, Tate, E, Ritzefeld, M
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 rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site\nin 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 (Ki = 38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.