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Exploiting the DCAF16-SPIN4 interaction to identify DCAF16 ligands for PROTAC development

ABSTRACT: Traditional small molecule drugs often target protein activity directly, but challenges arise when proteins lack suitable functional sites. An alternative approach is targeted protein degradation (TPD), which directs proteins to cellular machinery for proteolytic degradation. Recent studies have identified new E3 ligases, expanding the potential of TPD. Among these, DCAF16 has shown promise in facilitating protein degradation through both PROTAC and molecular glue mechanisms. In this study, we developed a Homogeneous Time Resolved Fluorescence (HTRF) assay to identify additional DCAF16 ligands using an in-house electrophile library. This led to the identification of two diastereomeric compounds, with one engaging DCAF16 at cysteines C177-179 and another reducing its expression. We demonstrated that the compound engaging DCAF16 can be transformed into a PROTAC capable of degrading FKBP12.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Xiaoyu Zhang

LAB HEAD: Xiaoyu Zhang

PROVIDER: PXD055063 | Pride | 2024-12-06

REPOSITORIES: Pride

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			Action	DRS
	2G072F07_ABPP.raw	Raw
	2G07SLF_ABPP_DBIA_RTS_6plex_01.raw	Raw
	2G07SLF_ABPP_DBIA_RTS_6plex_02.raw	Raw
	2G07SLF_ABPP_DBIA_RTS_6plex_03.raw	Raw
	2G07SLF_ABPP_DBIA_RTS_6plex_04.raw	Raw

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Project description:Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. This expansion will broaden the scope of potential protein targets, accommodating those with varying subcellular localizations and expression patterns. In this study, we describe a CRISPR-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. This approach allows us to address the limitations associated with investigating candidate degrader molecules in specific cell lines that either lack or have low levels of the desired E3 ligases. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FKBP12 when the FBXO22 gene transcription is activated. 22-SLF induced the degradation of endogenous FKBP12 in a FBXO22-dependent manner across multiple cancer cell lines. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in FBXO22 to achieve the target degradation. Finally, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading another endogenous protein BRD4. This study uncovers FBXO22 as an E3 ligase capable of supporting ligand-induced protein degradation through electrophilic PROTACs. The platform we have developed can readily be applied to elucidate protein degradation pathways by identifying E3 ligases that facilitate either small molecule-induced or endogenous protein degradation.

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