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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.

Original publication

DOI

10.1021/acschembio.6b00896

Type

Journal article

Journal

ACS Chem Biol

Publication Date

2016

Volume

11

Pages

3256 - 3262

Keywords

Acyltransferases/*antagonists & inhibitors/metabolism Animals Cell Line, Tumor Enzyme Inhibitors/*chemistry/*pharmacology HEK293 Cells Hedgehog Proteins/*metabolism Humans Lipoylation/drug effects Mice NIH 3T3 Cells Neoplasms/*drug therapy/metabolism Signal Transduction/*drug effects