Nanomolar, Noncovalent Antagonism of Hedgehog Cholesterolysis: Exception to the "irreversibility Rule" for Protein Autoprocessing Inhibition

Andrew G. Wagner, Robert T. Stagnitta, Zihan Xu, John L. Pezzullo, Nabin Kandel, José Luis Giner, Douglas F. Covey, Chunyu Wang, Brian P. Callahan

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Hedgehog (Hh) signaling ligands undergo carboxy terminal sterylation through specialized autoprocessing, called cholesterolysis. Sterylation is brought about intramolecularly in a single turnover by an adjacent enzymatic domain, called HhC, which is found in precursor Hh proteins only. Previous attempts to identify antagonists of the intramolecular activity of HhC have yielded inhibitors that bind HhC irreversibly through covalent mechanisms, as is common for protein autoprocessing inhibitors. Here, we report an exception to the "irreversibility rule" for autoprocessing inhibition. Using a fluorescence resonance energy transfer-based activity assay for HhC, we screened a focused library of sterol-like analogues for noncovalent inhibitors and identified and validated four structurally related molecules, which were then used for structure-activity relationship studies. The most effective derivative, tBT-HBT, inhibits HhC noncovalently with an IC50of 300 nM. An allosteric binding site for tBT-HBT, encompassing residues from the two subdomains of HhC, is suggested by kinetic analysis, mutagenesis studies, and photoaffinity labeling. The inhibitors described here resemble a family of noncovalent, allosteric inducers of HhC paracatalysis which we have described previously. The inhibition and the induction appear to be mediated by a shared allosteric site on HhC.

Original languageEnglish
Pages (from-to)1022-1028
Number of pages7
JournalBiochemistry
Volume61
Issue number11
DOIs
StatePublished - Jun 7 2022

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