NU 2023-096
INVENTORS Ananya Angela Basu Xiaoyu Zhang*
SHORT DESCRIPTION A CRISPR-activation screen for discovering E3 ligases amenable to targeted protein degradation, and a novel PROTAC for FBXO22
BACKGROUND Traditional small molecule drugs function by directly interfering with the activities of proteins. However, many proteins lack suitable functional sites for rational drug design, presenting challenges in targeting them with small molecules. A promising alternative approach, termed targeted protein degradation (TPD), involves the modification of small molecules to guide target proteins to the endogenous cellular machinery responsible for proteolytic degradation, leading to the complete removal of the protein. While a growing number of proteins have demonstrated susceptibility to ligand-induced degradation, few E3 ligases have been identified to support TPD, and so there is great need to expand the repertoire of E3 ligases that can be utilized for this purpose.
ABSTRACT Targeted protein degradation (TPD) is a promising approach for the treatment of diseases related to traditionally “undruggable” protein targets. However, only a small subset of human E3 ligases have been identified to support TPD, and these often exhibit unique or restricted substrate preferences which are not easily predictable or controllable. Thus, there is a need to uncover additional E3 ligases with distinct properties to enable the design and optimization of novel therapeutics. A commonly employed technique for identifying E3 ligases suitable for TPD involves CRISPR-Cas9 knockout screens; however, results from knockout screens can suffer from high background noise in cases where the E3 ligase is lowly expressed, exhibits cell-type specific activity, or only partially degrades its target. To overcome this problem, Northwestern researchers have developed a CRISPR-activation platform, which boosts E3 ligase expression, to unbiasedly screen E3 ligases that can facilitate TPD. Importantly, this platform is adaptable to the confident assessment of candidate proteolysis targeted chimeras (PROTACs), heterobifunctional compounds for E3-target engagement that may be either covalent or non-covalent. The researchers validated this approach by identifying FBXO22 as a novel E3 ligase amenable to TPD of FKBP12, and developed a candidate PROTAC for FBXO22 (22-SLF) that induces FKBP12 degradation across multiple cancer cell lines. This FBXO22-based PROTAC can effectively degrade another endogenous protein, BRD4 (22-JQ1), demonstrating versatility of FBXO22 for targeting an array of protein targets. Overall, the platform enables the discovery of additional E3 ligases amenable to TPD and has the potential to identify novel compounds for therapeutic development across multiple cell and disease types.
APPLICATIONS
ADVANTAGES
PUBLICATIONS Basu, A. A., Zhang, C., Riha, I. A., Magassa, A., Ko, F., & Zhang, X. (2023). A CRISPR activation screen identifies FBXO22 as an E3 ligase supporting targeted protein degradation. bioRxiv: the preprint server for biology, 2023.09.15.557708
IP STATUS A provisional application has been filed
Left: Schematic representation of the steps in the CRISPR-Cas9 transcriptional activation screen. Right: Volcano plot showing the E3 ligase focused CRISPR-Cas9 transcriptional activation screen for FKBP12-EGFP degradation after treatment of 2 μM 22-SLF in HEK293T CRISPR-Cas9 transcriptional activation cells for 24 hours (n = 3 biological independent samples).