A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL

Abstract

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific protein targets in a proteasome-dependent manner.However, a major limitation to broader TPD applications is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent ligand for the E3 ligase RNF114. When linked to the BET family inhibitor JQ1, the resulting heterobifunctional PROTAC molecule was capable of selectively degrading BRD4 in cancer cells. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or in some cases inactivity. Further contrasting from cereblon or VHL-recruiting degradation, we show that BT1 treatment not only leads to BCR-ABL degradation, but also stabilizes the endogenous RNF114 substrate and tumor suppressor substrate p21. This leads to additional anti-proliferative effects in leukemia cancer cells beyond those observed with cereblon or VHL-recruiting BCR-ABL PROTACs. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs with unique additional benefits for oncology applications. We also further demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing and unpredictable selectivity for the degradation of neo-substrate proteins.

Chemoproteomics-Enabled Ligand Screening Yields Covalent RNF114-Based Degraders that Mimic Natural Product Function

“…4b-4c, Supplementary Fig. 5b) (Tong et al, 2020). Consistent with ML 2-23 engaging BCR-ABL in cells, we observed inhibition of CRKL phosphorylation, a downstream substrate of BCR-ABL signaling (Fig.…”

“…Indeed, while over 600 different E3 ligases have been annotated, only a small handful of these potential targets have succumbed to the PROTAC strategy. Discovering additional, more synthetically tractable E3 ligase recruiters is thus an important topic in expanding the scope of TPD and may help to address resistance mechanisms (Bond et al, 2020;Ottis et al, 2019;Zeng et al, 2020), promote differing selectivity or kinetic profiles of degradation (Bondeson et al, 2018;Huang et al, 2018;Tong et al, 2020), and lead to celltype or location-specific degradation.…”

PROTAC: A Novel Technology for Drug Development**

“…Crews and co‐workers have studied other E3 ligases, [95] most of which have better ability to degrade unnatural substrates, which fully proves that we still need to constantly develop new E3 ligases and find their suitable ligands. Other teams also had discovered and developed novel E3 ligases and ligands, for example, keap‐1, [50] RNF114, [96] RNF4, [97] DCAF15, [98] DCAF16 [99] ligase and ligands, which filled some gaps in this filed. Furthermore, recent studies reported some novel thalidomide analogs such as CC‐122, CC‐220 and CC‐885, which enriched E3 ligand library [100] .…”

A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes

“…Accordingly, the successful ubiquitination of neo-substrates often reflects their ability to form a de novo protein-protein interface with the recruited E3 ligase (Gadd et al, 2017;Nowak et al, 2018). For example, recent studies show that degradation by bifunctional multi-kinase degraders is dependent on stable ternary complex formation, and that degradation selectivity varies according to the recruited E3 ligase (Tong et al, 2020;Bondeson et al, 2018;Lai et al, 2016). We were therefore interested in using our maleimide-conjugation strategy to extend COFFEE to additional neo-substrate targets by appending VHL with dasatinib, a promiscuous kinase inhibitor that engages 38 kinases with an apparent Ki <100 nM, in addition to its primary target, BCR-ABL (Klaeger et al, 2017;Smith, et.…”

“…In order to extend our approach to additional neo-substrates, we synthesized a maleimidelinked dasatinib probe, Compound 2, using the reported exit vector for dasatinib-based bifunctional degraders (Tong et al, 2020). Compound 2 gave complete single labeling of VHL/EloB C89S /EloC, as monitored by intact mass spectrometry (Fig.…”