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.

表型筛选中小分子命中的目标反卷积提出了重大挑战。为了寻找 Hedgehog 信号通路（一种对健康和疾病具有多种影响的发育通路）的抑制剂，已经进行了许多筛选，取得了许多成果，但只有很少的确定的细胞靶标。我们在这里提出了一种基于蛋白水解靶向嵌合体（PROTAC）并结合无标记定量蛋白质组学的靶点识别策略。我们开发了一种基于 Hedgehog Pathway Inhibitor-1 (HPI-1) 的 PROTAC，HPI-1 是一种针对未知细胞靶点的表型筛选。使用此 Hedgehog 通路 PROTAC (HPP)，我们识别并验证 BET 溴结构域作为 HPI-1 的细胞靶标。此外，我们发现 HPP-9 通过延长 BET 溴结构域降解而成为一种长效 Hedgehog 通路抑制剂。总的来说，我们提供了一种强大的基于 PROTAC 的目标解卷积方法，它回答了 HPI-1 细胞目标的长期问题，并产生了作用于 Hedgehog 通路的 PROTAC。