CDK Family PROTAC Profiling Reveals Distinct Kinetic Responses and Cell Cycle–Dependent Degradation of CDK2

Abstract

Targeted protein degradation using heterobifunctional proteolysis-targeting chimera (PROTAC) compounds, which recruit E3 ligase machinery to a target protein, is increasingly becoming an attractive pharmacologic strategy. PROTAC compounds are often developed from existing inhibitors, and assessing selectivity is critical for understanding on-target and off-target degradation. We present here an in-depth kinetic degradation study of the pan-kinase PROTAC, TL12-186, applied to 16 members of the cyclin-dependent kinase (CDK) family. Each CDK family member was endogenously tagged with the 11-amino-acid HiBiT peptide, allowing for live cell luminescent monitoring of degradation. Using this approach, we found striking differences and patterns in kinetic degradation rates, potencies, and Dmax values across the CDK family members. Analysis of the responses revealed that most of the CDKs showed rapid and near complete degradation, yet all cell cycle–associated CDKs (1, 2, 4, and 6) showed multimodal and partial degradation. Further mechanistic investigation of the key cell cycle protein CDK2 was performed and revealed CDK2 PROTAC-dependent degradation in unsynchronized or G1-arrested cells but minimal loss in S or G2/M arrest. The ability of CDK2 to form the PROTAC-mediated ternary complex with CRBN in only G1-arrested cells matched these trends, despite binding of CDK2 to TL12-186 in all phases. These data indicate that target subpopulation degradation can occur, dictated by the formation of the ternary complex. These studies additionally underscore the importance of profiling degradation compounds in cellular systems where complete pathways are intact and target proteins can be characterized in their relevant complexes.

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs

“…As ubiquitination has been shown to be correlated with degradation rate (4), we sought to Treatment with a serial dilution of 1 µM TL12-186 for 3 hours resulted in different ubiquitination potencies of CDK proteins, with EC50 ranking in strong agreement with the model predictions of productive ternary complexes (Table 1), as well as matching the previously published Dmax50 ranking (49). Ubiquitination was most potent on CDK12 and CDK13 (Figure 5E and Table 1), with these proteins having the highest percentage of productive ternary conformations (77% and 62%, respectively) as predicted by the model.…”

“…Expression plasmids preparation for ubiquitination assays CDK2 (NM_001798), CDK5 (NM_004935), and CDK9 (NM_001261) expression plasmids were obtained from Kazusa DNA Research Institute (Kisarazu, Japan) as pFN21A HaloTag CMV Flexi vectors (Promega). CDK2 and CDK5 were cloned into pFC32K vector (Promega) to generate a C-terminal NanoLuc fusion and CDK9 was cloned into pFN31K vector (Promega) to generate an N-terminal NanoLuc fusion, corresponding to the same termini tagging used previously for endogenous CDK proteins (49). Site-directed mutagenesis was performed on each vector to generate the lysine to arginine point mutants, CDK2 (K6R), CDK5 (K56R), and CDK9 (K88R), while maintaining the same NanoLuc tag placement as the corresponding wildtype CDK protein.…”

Unifying Catalysis Framework to Dissect Proteasomal Degradation Paradigms

“… 86 Of note, deconvolution of TL12-186, a pan-kinase PROTAC, illuminated the mechanistic profile with the required selectivity and temporal resolution to identify degradation of POI subpopulations. 88 Collectively, these reports showcase that a detailed mechanistic understanding of degradation profiles for bivalent degraders can be achieved in relevant cellular contexts. Given the breadth and depth afforded by HiBiT tagging, this approach is also well positioned to characterize and exploit kinetically favored degradation mechanisms across the induced cooperativity spectrum.…”

CDK9 inhibitors in multiple myeloma: a review of progress and perspectives

“…Ubiquitination is associated with the functionality of the CRBN gene, whose product is a receptor of E3 ubiquitin ligase; hence, CRBN expression may affect the therapeutic effectiveness of protein degraders [ 94 , 99 ]. This strategy seems promising, especially in the degradation of CDK 9–13, which are not associated with the cell cycle [ 100 ].…”