BMS-986397 targets casein kinase 1alpha (CK1alpha), a serine/threonine protein kinase that regulates multiple cellular processes including Wnt/beta-catenin signaling, DNA damage response, and cell cycle progression. The compound acts as a molecular glue degrader that simultaneously binds to cereblon (CRBN), a substrate receptor of the CUL4-RBX1-DDB1-CRBN E3 ubiquitin ligase complex, and CK1alpha. This ternary complex formation induces ubiquitination of CK1alpha, followed by proteasomal degradation. Degradation of CK1alpha leads to p53 activation (in TP53 wild-type cells), cell cycle arrest (G1 phase), and induction of apoptosis. The compound is highly selective for CK1alpha over other CRBN neosubstrates such as IKZF1, IKZF3 (Ikaros and Aiolos), and GSPT1. BMS-986397 is part of the CELMoD class of agents, which have improved properties compared to early immunomodulatory drugs (IMiDs) like lenalidomide and pomalidomide.

In vitro, BMS-986397 is a potent and selective molecular glue degrader of CK1alpha. It induces CK1alpha degradation in TP53 wild-type AML cell lines (e.g., MOLM-13, OCI-AML3, THP-1) with DC₅0 (degradation concentration 50%) in the low nanomolar to sub-nanomolar range (typically 0.1-10 nM). In MOLM-13 cells, treatment with 1 nM BMS-986397 for 4-8 hours leads to >90% reduction in CK1alpha protein levels as measured by Western blot. This degradation is CRBN-dependent; in CRBN-knockout cells, CK1alpha levels remain unchanged. Following CK1alpha degradation, p53 protein levels stabilize and accumulate (p53 is normally degraded by MDM2). p53 target genes, including p21 (CDKN1A) and PUMA, are upregulated. In TP53 wild-type AML cell lines, BMS-986397 (0.1-10 nM) induces G1 cell cycle arrest and rapid apoptosis (Annexin V positivity within 24-48 hours). The compound also induces differentiation of leukemic cells. In contrast, TP53 mutant AML cells (e.g., K562, HL60) are less sensitive, with IC₅0 values for cell viability inhibition 10-100 fold higher (100 nM to 1 uM). BMS-986397 is highly selective for CK1alpha over other CRBN neosubstrates: it does not degrade IKZF1, IKZF3, or GSPT1 at concentrations up to 100 nM, as measured by Western blot. It also shows minimal off-target kinase inhibition (IC₅0 > 1 uM for a panel of 100+ kinases). The compound is soluble in DMSO (96 mg/mL) and can be used at concentrations of 0.1-1000 nM in cell culture. It induces apoptosis in primary AML patient samples (blasts) with TP53 wild-type status, with EC₅0 values typically 0.5-10 nM. It also demonstrates activity against HR-MDS patient samples.

In vivo, BMS-986397 exhibits strong anti-leukemic activity in mouse xenograft models of AML and HR-MDS. In a MOLM-13 (TP53 wild-type AML) subcutaneous xenograft model in female athymic nude mice, BMS-986397 is administered orally at doses of 1, 3, 10, or 30 mg/kg once daily for 14-28 days. At 3 mg/kg/day, significant tumor growth inhibition (TGI) of 60-80% is observed compared to vehicle control. At 10 mg/kg/day, TGI of 80-95% is achieved, with some complete regressions. In a systemic (IV) MOLM-13-luciferase model (leukemia disseminated in bone marrow), oral BMS-986397 (10 mg/kg/day) reduces bioluminescence signal by >90%, prolongs survival (median survival 40-50 days vs. 20-25 days for vehicle). In an OCI-AML3 xenograft model, similar efficacy is observed. In a patient-derived xenograft (PDX) model of HR-MDS, oral BMS-986397 (10 mg/kg/day) reduces human CD45+ blast counts in the bone marrow and spleen, and restores normal hematopoiesis. The compound is well tolerated in mice, with no significant body weight loss (<10%) at doses up to 30 mg/kg/day. Pharmacodynamic markers: CK1alpha degradation in tumor tissue is observed 4-8 hours post-dose, with maximum degradation (>90%) at 8-16 hours, sustained for 24 hours. p21 upregulation and Ki-67 reduction (proliferation) are observed. No significant thrombocytopenia or neutropenia is reported, distinguishing BMS-986397 from standard chemotherapy. BMS-986397 has also been evaluated in combination with venetoclax (BCL-2 inhibitor) and azacitidine (hypomethylating agent), showing enhanced anti-leukemic activity and synergy.

For CK1alpha degradation assays (non-cell-based or protein-based), biochemical assays can be performed to measure ubiquitination. Recombinant human CK1alpha protein (1 ug) is incubated with CRBN-DDB1-CUL4-RBX1 E3 ligase complex (purified or from cell lysates of 293T cells expressing CRBN and components), ubiquitin (5 ug), E1 enzyme (0.1 ug), E2 enzyme (UBE2D2, 0.5 ug), and ATP (1 mM) in reaction buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 2 mM DTT, 100 mM NaCl) at 37degC for 60 minutes. BMS-986397 (0.1-1000 nM) is added to the reaction. CK1alpha ubiquitination is detected by Western blot using anti-CK1alpha antibody (smear pattern of high molecular weight bands indicates polyubiquitination). For compound binding to CRBN, a fluorescence polarization (FP) or TR-FRET assay can be used: recombinant human CRBN-DDB1 complex (biotinylated, 10 nM) is incubated with fluorescently labeled lenalidomide probe (e.g., FAM-lenalidomide, 5 nM) and BMS-986397 (0.1-1000 nM) in assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 0.01% Tween-20) for 60 minutes. Streptavidin-conjugated beads or streptavidin-labeled terbium donor are added, and FP or TR-FRET signal is measured. BMS-986397 binds to CRBN with an IC₅0 of 5-20 nM. For selectivity against other CRBN neosubstrates, similar assays using IKZF1 peptide substrates can be performed. However, BMS-986397 is selective for CK1alpha over IKZF1 in cellular assays.

For in vitro cellular assays, TP53 wild-type AML cell lines (MOLM-13, OCI-AML3, THP-1) are cultured in RPMI-1640 with 10% FBS and 1% penicillin-streptomycin. For degradation assays, cells are seeded in 6-well plates (5 × 10⁵ cells/mL) and treated with BMS-986397 (0.001-1000 nM) for 4-24 hours. Cells are harvested, lysed in RIPA buffer with protease inhibitors, and protein lysates (20-50 ug) are resolved by SDS-PAGE and Western blotted with anti-CK1alpha (1:1000, Cell Signaling), anti-p53 (1:1000), anti-p21 (1:1000), and anti-beta-actin (loading control). Densitometry is performed. DC₅0 is calculated as the concentration required for 50% CK1alpha degradation. For CRBN-dependent degradation, cells are pre-treated with CRBN siRNA or treated with lenalidomide (10 uM) as a competitor. For apoptosis assays, cells are seeded in 96-well plates (2 × 10⁴ cells/well) and treated with BMS-986397 (0.01-1000 nM) for 24-72 hours. Apoptosis is measured by Annexin V-FITC/PI staining and flow cytometry, or by caspase-3/7 activity using a fluorogenic substrate (DEVD-R110, Promega Caspase-Glo). IC₅0 for apoptosis induction is typically 0.1-1 nM in TP53 wild-type cells. For cell viability assays (MTT or CellTiter-Glo), cells are treated for 48-72 hours. IC₅0 for viability in TP53 wild-type cells is 0.1-5 nM; in TP53 mutant cells (K562, HL60), IC₅0 is 100-1000 nM. For cell cycle analysis, cells are treated with BMS-986397 (0.5-10 nM) for 24-48 hours, fixed in 70% ethanol, stained with propidium iodide (50 ug/mL) with RNase A (100 ug/mL), and analyzed by flow cytometry. G1 arrest is indicated by increased percentage of cells in G1 phase. For differentiation assays, cells are stained with CD11b or CD14 antibodies and analyzed by flow cytometry. For combination studies, cells are treated with BMS-986397 (0.1-10 nM) and venetoclax (1-100 nM) or azacitidine (0.1-10 uM), and synergy is assessed by calculating combination index (CI) using Chou-Talalay method (CI < 1 indicates synergy). For primary AML patient samples, mononuclear cells are isolated from peripheral blood or bone marrow of patients with TP53 wild-type AML, cultured in StemSpan with cytokines (SCF, FLT3L, TPO, IL-3), and treated with BMS-986397 (0.01-100 nM) for 48-72 hours. Viability is assessed by flow cytometry using CD34+ or CD33+ gating and Annexin V staining. Each condition should be tested in triplicate, and experiments repeated at least three times. Results are expressed as mean +/- SD. Statistical significance is determined by one-way ANOVA with post-hoc Tukey test or by Student's t-test. p < 0.05 is considered significant.

For in vivo xenograft studies, female athymic nude mice (nu/nu) or NSG mice (6-8 weeks old, 18-22 g, n=8-10 per group) are inoculated subcutaneously in the right flank with 5 × 10⁶ MOLM-13 (TP53 wild-type AML) cells in 100 uL PBS mixed with 100 uL Matrigel. When tumors reach approximately 150-200 mm3 (7-10 days post-inoculation), mice are randomized into treatment groups. BMS-986397 is formulated in a vehicle such as 5% DMSO, 40% PEG300, 5% Tween-80, 50% ddH2O (clear solution) or as a homogeneous suspension in 0.5% CMC-Na (for higher concentrations, ≥5 mg/mL). Mice receive BMS-986397 at doses of 1, 3, 10, or 30 mg/kg by oral gavage once daily for 14-28 days. Control groups receive vehicle alone (negative control) and, if applicable, lenalidomide (25 mg/kg oral, positive control) or cytarabine (50 mg/kg IP, positive control). For the systemic disseminated AML model, 1 × 10⁶ MOLM-13-luciferase cells are injected via tail vein. Tumor engraftment is confirmed by bioluminescence imaging (BLI, Xenogen IVIS) at day 7. BMS-986397 (10 mg/kg, oral, daily) is administered from day 7 to day 28. BLI is performed weekly; survival is monitored daily. For PDX models, primary AML or HR-MDS patient samples are engrafted into NSG-SGM3 mice (NSG mice expressing human SCF, GM-CSF, IL-3) by intravenous injection of 1-5 × 10⁶ blasts. After engraftment (4-8 weeks, confirmed by hCD45+ in peripheral blood >1%), mice are treated with BMS-986397 (10 mg/kg oral daily) for 28 days. Human CD45+ blast percentage in bone marrow (femur) and spleen is measured by flow cytometry using anti-human CD45 antibody. Tumor volumes are measured with calipers, and TGI is calculated. Body weight is monitored for toxicity. At study termination (day 21-28), tumors are harvested for Western blot (CK1alpha, p53, p21, cleaved PARP), histology (H&E, Ki-67, TUNEL), and flow cytometry (Annexin V). Blood is collected for hematology (CBC) and serum chemistry (ALT, AST, BUN, creatinine). Survival is analyzed by Kaplan-Meier with log-rank test. No significant toxicity (weight loss >15%, clinical signs, abnormal hematology) should be observed at therapeutic doses (≤30 mg/kg). All animal studies must be approved by the IACUC.

BMS-986397 is soluble in DMSO (96 mg/mL) and should be stored at -20degC. It is insoluble in water and ethanol. For oral administration, formulated in 5% DMSO, 40% PEG300, 5% Tween-80, 50% ddH2O (validated for clear solution at concentrations up to 3-5 mg/mL). For higher doses, a CMC-Na suspension (5 mg/mL) can be used. The compound has moderate oral bioavailability in mice (estimated 30-60%). After oral administration, Tmax is 1-2 hours. Elimination half-life in mice is approximately 2-4 hours. Plasma protein binding is high (>90%). The compound is metabolized by liver CYP enzymes, but specific isozymes are not identified. No human PK data are available.

Preclinical toxicity data: BMS-986397 is well tolerated in mice at doses up to 30 mg/kg/day for 28 days, with no mortality, no significant body weight loss (<10%), and no adverse effects on liver (ALT, AST) or kidney (BUN, creatinine) function. At higher doses (100 mg/kg), mild gastrointestinal distress and reduced food intake were observed. No genotoxicity or cardiovascular toxicity has been reported. Not a skin/eye irritant. Standard laboratory safety precautions apply. Not for human use.

BMS-986397 (CC-91633) is a first-in-class CELMoD (cereblon E3 ligase modulator) agent currently in preclinical research. Unlike thalidomide and lenalidomide, which primarily degrade IKZF1/IKZF3, BMS-986397 is selective for CK1alpha degradation. The compound is not FDA-approved; clinical trials have not been initiated. Potential clinical indications: TP53 wild-type AML, HR-MDS, and other hematologic malignancies. For research use only, not for therapeutic or diagnostic use.

C21H16CLF3N4O4

480.82

2564486-44-6

Typically exists as solids at room temperature

ClC1=CC=CN=C1[C@H](C(F)(F)F)NC(C1C=CC2C(N(CC=2C=1)[C@@H]1C(NC(CC1)=O)=O)=O)=O

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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