Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog Pathway Inhibitor-1

Abstract

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.

Application of PROTACs in target identification and validation

“…Bagka et al designed a PROTAC molecule (HPP-9) based on the HPI-1 and quantified the protein treated with HPP-9 through label-free quantitative proteomics. The results showed that two members of the bromine domain and the end effector domain (BET) protein family (BRD3 and BRD4) were significantly downregulated [43], further confirming the involvement of the BET bromine domain in regulating the Hh signaling pathway [49]. Based on an evaluation of degradation activity and the underlying mechanism, BET was ultimately determined to be the target of HPI-1 (Figure 4).…”

“…It was the first time a systematic and explicit approach had been used to establish an experimental method and workflow for target identification using PROTAC technology. The concept of TGDO was proposed, which is widely recognized by the academic community [40][41][42][43][44] (Figure 4).…”

Recent advances in the understanding of cilia mechanisms and their applications as therapeutic targets

“…TCHP may be a candidate for this approach because the knockout mice are viable and show resistance to high-fat diet-induced obesity and increased regeneration following skeletal muscle injuries ( Yamakawa et al, 2021 ; Yamakawa et al, 2022 ). Chemicals can be developed that inhibit the interaction between AURKA and the binding partner or degrade ciliary proteins by proteolysis-targeting chimeras ( Janeček et al, 2016 ; Bagka et al, 2022 ). Tubastatin-A, an inhibitor of HDAC6, can also be used to treat diseases associated with ciliary defects by inducing ciliogenesis ( Gradilone et al, 2013 ; Yang et al, 2014 ).…”