Open AccessA High-Throughput Fluorescent Turn-On Assay for Inhibitors of DHHC Family Proteins
Author(s) -
Tian Qiu,
Saara-Anne Azizi,
Noah Brookes,
Tong Lan,
Bryan C. Dickinson
Publication year - 2022
Publication title -
acs chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.899
H-Index - 111
eISSN - 1554-8937
pISSN - 1554-8929
DOI - 10.1021/acschembio.2c00193
Subject(s) - high throughput screening , drug discovery , computational biology , chemical biology , biology , microbiology and biotechnology , chemistry , biochemistry
As the "writer" enzymes of protein S- acylation, a dynamic and functionally significant post-translational modification (PTM), DHHC family proteins have emerged in the past decade as both key modulators of cellular homeostasis and as drivers of neoplastic, autoimmune, metabolic, and neurological pathologies. Currently, biological and clinical discovery is hampered by the limitations of existing DHHC family inhibitors, which possess poor physicochemical properties and off-target profiles. However, progress in identifying new inhibitory scaffolds has been meager, in part due to a lack of robust in vitro assays suitable for high-throughput screening (HTS). Here, we report the development of palmitoyl transferase probes (PTPs), a novel family of turn-on pro-fluorescent molecules that mimic the palmitoyl-CoA substrate of DHHC proteins. We use the PTPs to develop and validate an assay with an excellent Z'-factor for HTS. We then perform a pilot screen of 1687 acrylamide-based molecules against zDHHC20, establishing the PTP-based HTS assay as a platform for the discovery of improved DHHC family inhibitors.