CP-10

This molecule should have good selectivity for CDK6 based on the selectivity of the parent compound Palbociclib used to generate the PROTAC. However, the kinome or cellular selectivity have not actually been assessed. At the recommended concentration of 100 nM there was 89% degradation of CDK6 in U251 cells, thus there is predicted to be some CDK6 activity remaining.

CP-10 is a selective CDK6 degrader comprising Palbociclib linked to Pomalidomide. Used alongside the structurally matched negative controls CP-9 and CP-23, CP-10 can be a useful cell-based probe to explore the consequences of CDK6 removal in a concentration and time-dependent manner. Although 70 to 85-fold selectivity for CDK6 over CDK4 degradation is observed in U251 cells, selectivity appears to be cell line dependent and care should be taken to monitor CDK4 degradation in cell lines of interest.

Validation includes the following:

• In vitro, CP-10 inhibits CDK4 (IC₅₀ = 30nM) and CDK6 (IC₅₀ = 262nM) weaker than Palbociclib (CDK4 IC₅₀ = 4nM, CDK6 IC₅₀ = 11nM) under the same assay conditions
• In U251 cells, CP-10 preferentially degrades CDK6 (DC­₅₀ = 2.1nM) over CDK4 (DC­₅₀ = 150-180nM); CDKs 1, 2, 5 and 9 are not degraded by CP-10 at 500nM concentration.
• CP-10 elicits the degradation of CDK6 (72% degradation at 10 nM; 89% at 100 nM; DC₅₀ = 2.1nM in human glioblastoma U251 cells); degradation commences at 2h with complete degradation is observed after 6hr.
• CP-10-mediated CDK6 degradation is partially rescued by Palbociclib or Pomalidomide and is completely rescued by proteasome inhibitor Carfilzomib or neddylation inhibitor MLN4924 indicating that degradation is ubiquitin-proteasome mediated.
• Quantitative proteomic analysis (U251 cells) demonstrates preferential CDK6 degradation
• CP-10 inhibited the proliferation of CDK6-dependent hematopoietic cell lines more potently than Palbociclib indicative of a CDK6-driven effect; CP-10 also inhibited the proliferation of Palbociclib-resistant cell lines.
• CP-9 and CP-23 both N-ethyl imid derivatives serve as appropriate matched negative controls.
• Whilst CP-10 treatment (100nM) of U251 cells demonstrates a clear preference for CDK6 over CDK4 degradation, and similarly in HL-60, RPMI8226 and MM.1S cells, evidence for preferential degradation in THP-1 and Mino cells is less compelling, suggesting that the preferential degradation of CDK6 may be cell line dependent and should be carefully monitored.