Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Illustrative of these are the many screens that have been conducted to find inhibitors for the Hedgehog (Hh) signaling pathway, a major developmental pathway with many implications in health and disease, with many hits but very few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We developed a PROTAC based on the downstream Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using our Hedgehog Pathway PROTAC (HPP) we identified and validated BET bromodomains to be the cellular targets of HPI-1. Furthermore, we found that HPP-9 has a unique mechanism of action as a long-acting Hh pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that has answered the longstanding question of the cellular target of HPI-1 and yielded the first PROTAC that acts on the Hh pathway.
Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Many screens have been conducted to find inhibitors for the Hedgehog signaling pathway – a developmental pathway with many implications in health and disease – yielding many hits but only few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We develop a PROTAC based on Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using this Hedgehog Pathway PROTAC (HPP) we identify and validate BET bromodomains as the cellular targets of HPI-1. Furthermore, we find that HPP-9 is a long-acting Hedgehog pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that answers the longstanding question of the cellular target of HPI-1 and yields a PROTAC that acts on the Hedgehog pathway.
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