CRBN
CC-885 acts as a cereblon modulator that recruits the translation termination factor GSPT1 (eRF3a) to the CRL4CRBN E3 ubiquitin ligase, leading to its ubiquitination and degradation. It also promotes cereblon-dependent degradation of Ikaros (IKZF1). [1]
CC-885 acts as a CRBN modulator, binding to CRBN and altering the substrate specificity of the CRL4-CRBN E3 ubiquitin ligase to induce ubiquitination and degradation of multiple neo-substrates. Its primary degradation targets include: GSPT1 (translation termination factor): Induces CRBN-dependent GSPT1 ubiquitination and degradation, with a dissociation constant of approximately 350 nM for CRBN-DDB1 binding to GSPT1 as determined by SPR PLK1 (Polo-like kinase 1): Selectively promotes CRBN- and p97-dependent PLK1 ubiquitination and degradation CDK4 (cyclin-dependent kinase 4): Induces CDK4 ubiquitination and degradation in multiple myeloma cells in a CRBN-dependent manner GOLM1 (Golgi phosphoprotein 73): Mediates GOLM1 degradation via the CRL4-CRBN E3 ligase, inhibiting hepatocellular carcinoma progression IKZF1 (Ikaros): Promotes cereblon-dependent degradation of IKZF1

CC-885 is applied at different concentrations to human peripheral blood mononuclear cells (PBMC), human liver epithelial cell line (THLE-2) and acute myeloblatlic leukemia (AML) cell lines, with IC50s of 10×-6-1 μM. With IC50s>10 μM, CC-885 has a greater effect on cell proliferation in AML cell lines, THLE-2, and human PBMC than Lenalidomide and Pomalidomide.In order to determine whether the cytotoxic effects of CC-885 are caused by the cereblon-dependent degradation of GSPT1, a GSPT1 mutant that maintains its normal function but does not exhibit CC-885-dependent cereblon binding is employed to differentiate GSPT1's function from that of other substrates. In 293T HEK cells that consistently express CC-885-sensitive or -resistant GSPT1 variants, CC-885 is tested. While overexpressing a CC-885-sensitive variant, GSPT1Δ(1-138), only partially protects against the CC-885-induced anti-proliferation, overexpressing a resistant variant, GSPT1Δ(1–138)/(G575N), completely abrogates the effect. AML cell lines yield comparable outcomes[1].

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CC-885 exhibits broad-spectrum anti-proliferative activity against 132 human cancer cell lines, with sub-nanomolar potency against 4 out of 5 patient-derived acute myeloid leukemia (AML) samples.[1]
CC-885 induces cereblon-dependent ubiquitination and degradation of GSPT1, as shown by immunoblot and ubiquitination assays in 293FT HEK and AML cell lines.[1]
CC-885 does not decrease GSPT1 mRNA levels, indicating a post-transcriptional mechanism.[1]
CC-885 also promotes degradation of Ikaros in NB-4 leukemia cells.[1]

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CC-885 exhibits potent antiproliferative activity against various cancer cell lines in vitro. It demonstrates broad-spectrum antiproliferative activity against 132 human cancer cell lines, with subnanomolar potency in 4 out of 5 patient-derived acute myeloid leukemia samples. In hepatocellular carcinoma cells (HCCLM3, HepG2, Huh7), CC-885 dose- and time-dependently inhibits cell viability, colony formation, migration, and invasion. In non-small cell lung cancer cells (A549, NCI-H1299), CC-885 synergizes with volasertib to produce enhanced antiproliferative effects. The IC₅₀ range for acute myeloid leukemia cell lines, THLE-2 cells, and human peripheral blood mononuclear cells is 10⁻⁶-1 μM, with antiproliferative activity more potent than lenalidomide and pomalidomide. Mechanistically, CC-885 induces CRBN-dependent GSPT1 ubiquitination and degradation and promotes PLK1 degradation, amplifying volasertib-induced G2/M arrest and apoptosis.

The study assessed the therapeutic efficacy of this combination in vivo by using nude mice bearing tumors. While volasertib and CC-885 alone inhibited tumor growth, the combination of both small molecular drugs markedly inhibited tumor growth and reduced tumor weights (Figures 1I and IJ). Taken together, these data clearly show that CC-885 synergizes with volasertib against NSCLC cells both in vitro and in vivo.

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In a mouse model of hepatocellular carcinoma, CC-885 significantly inhibits tumor growth and angiogenesis and impedes the progression of orthotopic liver tumors. In an A549 xenograft model in BALB/c nude mice, CC-885 (20 mg/kg, intraperitoneal injection) alone inhibits tumor growth, with more pronounced inhibition when combined with volasertib. The bioorthogonal prodrug Pro-CC-885, based on CC-885, successfully induces in vivo degradation of the target protein GSPT1 in tumor-bearing mouse models.

Surface plasmon resonance (SPR) was used to measure binding between cereblon-DDB1 and GSPT1 in the presence of CC-885, showing a dissociation constant (K_D) of approximately 350 nM.[1]
In vitro ubiquitination assays were performed using recombinant CUL4A-RBX1-DDB1-cereblon complex, Ube1 E1, UbcH5a E2, ubiquitin, GSPT1, and CC-885. Reactions were carried out at 30°C for 2 hours in ubiquitination assay buffer with or without ATP and CC-885, followed by immunoblot analysis.[1]

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Surface plasmon resonance technology is used to determine the binding affinity between cereblon-DDB1 and GSPT1 in the presence of CC-885, with a dissociation constant of approximately 350 nM. The specific protocol involves immobilizing recombinant cereblon-DDB1 protein on a chip, flowing GSPT1 solutions containing varying concentrations of CC-885, and measuring binding responses to calculate affinity constants. In vitro ubiquitination assay: Recombinant CUL4A-RBX1-DDB1-cereblon complex, Ube1 E1, UbcH5a E2, ubiquitin, GSPT1, and CC-885 are incubated in ubiquitination assay buffer at 30°C for 2 hours, followed by immunoblot analysis.

The growth medium-cultured human cancer cell lines are seeded into black 384-well plates with DMSO or test compounds like CC-885 (10×-6-1 μM). Every cell line's seeding density is carefully calibrated to facilitate cell growth within the linear range over the course of a three-day culture period. Black 96-well plates containing DMSO or test compounds like CC-885 are seeded with 5,000–10,000 cells per well in 200 μl complete culture media to test the compound effect on cell proliferation in acute myeloid leukemia (AML) cell lines. Cell proliferation is measured using the CellTiter-Glo (CTG) Luminescent Cell Viability Assay after 48 or 72 hours[1].

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Cell proliferation was assessed using CellTiter-Glo assay after treating cells with CC-885 for 3 days.[1]
Immunoprecipitation of Flag-HA-cereblon from 293T HEK cells was performed in the presence of CC-885 to identify GSPT1 binding via mass spectrometry and immunoblot.[1]
Co-immunoprecipitation of HA-tagged GSPT1 with cereblon was conducted in the presence of CC-885 to confirm enhanced binding.[1]
Protein half-life analysis was performed by treating 293FT HEK cells with cycloheximide and CC-885, followed by immunoblotting at various time points.[1]
Lentiviral shRNA was used to silence GSPT1 expression in 293T HEK, OCI-AML2, and MOLM-13 cells to assess the effect on cell proliferation.[1]

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Cell Viability Assay: Human cancer cell lines are seeded in 384-well or 96-well plates and treated with CC-885 at concentrations ranging from 10⁻⁶-1 μM. After 48-72 hours, cell proliferation is assessed using the CellTiter-Glo luminescent cell viability assay. Hepatocellular Carcinoma Cell Functional Assays: HCCLM3, HepG2, and Huh7 cells are treated with varying concentrations of CC-885 for 24-72 hours. Cell viability is measured by MTT assay; cell migration and invasion are evaluated using Transwell assays. Immunoblot Analysis: A549 and NCI-H1299 cells are treated with 1 μM CC-885 for 24 hours and then lysed. Target proteins including PLK1 and GSPT1 are detected by immunoblot analysis.

Hepatocellular Carcinoma Orthotopic Model: HCC cells are inoculated into mouse livers to establish orthotopic tumor models. Following CC-885 administration, tumor growth, angiogenesis, and tumor progression are evaluated. Non-Small Cell Lung Cancer Xenograft Model: BALB/c nude mice are subcutaneously inoculated with A549 cells. After tumor establishment, CC-885 is administered intraperitoneally at 20 mg/kg, alone or in combination with volasertib. Tumor volumes and weights are measured periodically. Bioorthogonal Prodrug Study: Tumor-bearing mice are co-administered with the GSH-responsive probe XZ2223 and Pro-CC-885. Tumor imaging is monitored by in vivo imaging systems, and GSPT1 degradation is detected by western blotting.

CC-885 has a molecular weight of 440.88 and molecular formula C₂₂H₂₁ClN₄O₄. Solubility: High solubility in DMSO; working solutions should be prepared fresh before use. In vivo formulation: 10% DMSO + 90% corn oil at a concentration of 2.5 mg/mL (5.67 mM), with dissolution aided by heating, ultrasonication, or vortexing. Route of administration: Intraperitoneal injection; typical dosage is 20 mg/kg.

Prodrug design strategies based on CC-885 can significantly reduce its systemic toxicity. Studies show that through bioorthogonal prodrug modification, Pro-CC-885 remains biologically inert in normal cells and is activated only within tumor cells to exert its degradation effects, thereby broadening the therapeutic window and reducing systemic toxicity.

[1]. A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature. 2016 Jul 14;535(7611):252-7.

CC-885 contains a glutarimide ring that binds to cereblon, similar to other immunomodulatory drugs, but with extended chemical groups that enable it to interact uniquely with GSPT1. [1]
The crystal structure of the cereblon-DDB1-CC-885-GSPT1 complex shows that GSPT1 binds to cereblon via surface turns, with key glycine residues forming hydrogen bonds with N351, H357, and W400 residues of cereblon. [1]
CC-885 exhibits reduced activity against normal lymphocytes compared to AML tumor cells. [1]
The E377V polymorphism in rodent cereblon confers resistance to CC-885. [1]

C22H21CLN4O4

440.88

440.13

C, 59.93; H, 4.80; Cl, 8.04; N, 12.71; O, 14.52

1010100-07-8

24788636

Off white to light purple solid powder

1.7

3

4

4

31

747

0

ClC1=C(C)C=CC(=C1)NC(NCC1C=CC2C(N(CC=2C=1)C1C(NC(CC1)=O)=O)=O)=O

DOEVCIHTTTYVCC-UHFFFAOYSA-N

InChI=1S/C22H21ClN4O4/c1-12-2-4-15(9-17(12)23)25-22(31)24-10-13-3-5-16-14(8-13)11-27(21(16)30)18-6-7-19(28)26-20(18)29/h2-5,8-9,18H,6-7,10-11H2,1H3,(H2,24,25,31)(H,26,28,29)

N-(3-chloro-4-methylphenyl)-N'-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1-oxo-1H-isoindol-5-yl]methyl]-urea

CC-885; CC 885; CC885.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 40~67.5 mg/mL ( 90.72~153.10 mM )

Solubility in Formulation 1: 2.25 mg/mL (5.10 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 22.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.25 mg/mL (5.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 22.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)