CP-10

Protein target name:

CDK6

Mechanism of action:

DegraderPROTAC

Recommended Concentration for use in cells:

100 nM

Probe Availability

MedChemExpress

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

CMGC

HUGO ID:

HGNC:1777

Entrez gene ID:

1021

UNIPROT ID:

Q00534

Target alias:

MCPH12; PLSTIRE

Probe Details

Chemical Probes Portal ID:

CP2140

PubChem CID:

138911323

SMILES string:

CC(C(C1=O)=C(C)C2=CN=C(NC3=CC=C(N4CCN(CC5=CN(CCOCCNC6=CC=CC(C(N7C8CCC(NC8=O)=O)=O)=C6C7=O)N=N5)CC4)C=N3)N=C2N1C9CCCC9)=O

InChi Key:

PACRWHUPEPCFHX-UHFFFAOYSA-N

Control compound:

CP-23

Orthogonal probe:

BSJ-03-123

Physico-chemical properties

NMR:

1H NMR (400 MHz, CDCl3) δ 10.34 (s, 1H), 8.79 (s, 1H), 8.22 (s, 1H), 8.12 (d, J = 9.1 Hz, 1H), 8.02 (d, J = 2.5 Hz, 1H), 7.75 (s, 1H), 7.51 (dd, J1 = 8.3 Hz, J2 = 7.3 Hz, 1H), 7.30 (dd, J1 = 9.1 Hz, J2 = 2.5 Hz, 1H), 7.14 (d, J = 7.3 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.63 (t, J = 5.3 Hz, 1H), 5.89–5.84 (m, 1H), 4.92–4.88 (m, 1H), 4.60–4.60 (m, 2H), 3.94–3.87 (m, 3H), 3.72–3.63 (m, 3H), 3.43–3.41 (m, 2H), 3.24–3.19 (m, 4H), 2.76–2.69 (m, 7H), 2.54 (s, 3H), 2.36–2.32 (m, 5H), 2.19–2.15 (m, 1H), 2.07–2.03 (m, 2H), 1.89–1.83 (m, 2H), 1.70–1.66 (m, 2H);

13C NMR (400 MHz, CDCl3) δ 202.87, 712.18, 169.99, 169.54, 167.62, 161.58, 158.25, 157.39, 155.68, 147.07, 141.99, 136.42, 132.69, 130.81, 126.09, 124.75, 117.17, 112.30, 110.93, 107.79, 77.39, 69.91, 69.61, 68.12, 54.23, 52.84, 52.49, 50.67, 49.18, 49.06, 42.40, 31.69, 31.55, 29.84, 28.20, 27.06, 25.87, 25.76, 23.20, 14.10.

Validation in Cells and Model Organisms

In cell validation

On target activity in cells

Potency in cells:

DC50, IC50, Dmax

2.1 nM (DC50), 10 nM MM.aS, 8 nM Mino, 72% @ 10 nM, 89% @ 100 nM,

Potency assay (cells):

western blot, proliferation assay in correlation with protein degradation

Phenotypic Control Experiment:

Control experiments clearly demonstrated that palbociclib, pomalidomide, or unconjugated PROTAC arms could not induce CDK6 degradation. Carfilzomib, a proteasome inhibitor, could completely disable the PROTAC effect.

Off target activity in cells

Probe Selectivity in Cell:

>50 fold selective over CDK4. Outside target family: Quantitative proteomic analysis confirmed CDK6 as the only target after 4 h incubation.

Primary Reference

Reference (doi):

10.1021/acs.jmedchem.9b00871

Reference (PMID):

31330105

Under Review

This probe is in the process of SAB review. Please continue to check back for new reviews and commentary.

SAB Rating

Rating for use in Cells

2 review(s)

Rating for use in Model Organisms

no rating

SAB Members

SAB Comments

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.

Feb 9 2021 - 8:58pm

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.

Feb 9 2021 - 9:00pm

CP-10

Probe Availability

Probe Information

Target Details

MCPH12; PLSTIRE

Probe Details

Physico-chemical properties

In cell validation

Primary Reference

Supporting Organizations

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CP-10

Probe Availability

Probe Information

Target Details

MCPH12; PLSTIRE

Probe Details

Physico-chemical properties

In cell validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?