Cereblon; CDK

BSJ-03–123 (BSJ) induces G1 cell cycle arrest without appreciably boosting apoptosis, hence exerting strong antiproliferative effects in CDK6-dependent AML cell lines [1].

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BSJ-03–123 triggers homolog-selective degradation via differential ternary complex formation [1]
Next, we sought to understand the mechanism of CDK6 selectivity. In vitro kinase assays confirmed comparable affinity to both kinases (Figure S1A, Table S1). Similar cellular thermal stabilization of CDK4/6 further suggested that selectivity does not emerge from differential cellular target engagement (Figure 2A). We thus hypothesized that selectivity might stem from differential ternary complex formation. To monitor tripartite assembly in real time in intact cells, we designed a luciferase complementation assay based on NanoBiT® technology (Figure 2B). We expressed C-terminal CDK4/6 LgBit fusions along with an N-terminal SmBit-CRBN fusion in 293TCRBN−/− cells. BSJ induced rapid, dose-dependent ternary complex formation with CDK6 and CRBN, but not with CDK4 and CRBN (Figure 2C). Treatment with BSJ-bump failed to induce CDK6:CRBN interactions (Figure 2C). Ternary complex formation was prevented by blocking binding sites on CDK6 or CRBN via pretreatment with palbo or lenalidomide (Figure 2D). We thus concluded that BSJ exploits structural differences between CDK4 and CDK6 to achieve homolog-selective degradation of CDK6 via differential ternary complex formation. BSJ-03–123 can exploit homolog-selective dependency on CDK6 [1]
To identify a cellular system that is disproportionally dependent on CDK6 over CDK4, we turned to genome-scale CRISPR/Cas9 screens in 342 cancer cell lines (Meyers et al., 2017). We identified a pronounced enrichment of acute myeloid leukemia (AML) cell lines among the most CDK6-addicted models (Figure 3A). None of these cell lines showed a comparable dependency on CDK4 (Figure S2A) Intersection with gene expression data did not unveil a correlation between essentiality and mRNA transcript levels (Figure 3A, S2A) (Klijn et al., 2015). Accodingly, CRISPR/Cas9-mediated genetic depletion of CDK4 in the AML cell line MV4–11 revealed largely preserved growth kinetics and cell cycle distribution, supporting a dispensable role of CDK4 in the proliferation of AML cell lines (Figure S2B–D).

In vitro CRBN Binding Assay [1]
Compounds in Atto565-Lenalidomide displacement assay were dispensed in a 384-well microplate using D300e Digital Dispenser (HP) and normalized to 1% DMSO into 10 nM Atto565-Lenalidomide, 100 nM DDB1∆B-CRBN, 50 mM Tris pH 7.5, 200 mM NaCl, 0.1% Pluronic F-68 solution. The change in fluorescence polarization was monitored using a PHERAstar FS microplate reader for 30 cycles of 187s each. Data from three independent measurements (n = 3) was plotted and IC50 values estimated using variable slope equation in GraphPad Prism 7.

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Cellular Thermal Shift Assay [1]
4×106 MV4–11 CRBN−/− cells were treated for 3 h with 20 µM palbociclib, BSJ-03–123 or vehicle. 1×106 cells were spun down at 500 x g for 5 min, and the supernatant removed. Pellets were incubated at 46, 49, 52 or 55 °C for 3 min followed by 3 min incubat ion at room temperature. 30 µl of lysis buffer (20 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, protease inhibitors) were added and cells lysed by 3 rounds of snap freezing and thawing. Denatured proteins were removed by 20 min centrifugation at 14.000 x g at 4 °C and supernatants analyzed by Western Blotting.

Proliferation assays [1]
For growth over time experiments of suspension cell lines, 300.000 cells per well were seeded in 24-well plates in triplicates. Cells were counted and treatments renewed every 2 days. For growth over time experiments of adherent cell lines, 100.000 cells per well were seeded in 12-well plates in triplicates. Cells were trypsinized and counted and treatments renewed every 3 days. For colony formation assays, 1000 cells per well were seeded in 6-well plates in duplicates. Every 2 days, culture medium was exchanged and treatments were renewed. After 12 days, cells were fixed in 1% PFA in PBS for 15 min at room temperature, washed 3 times with PBS and stained in Crystal Violet solution (0.1% in 10% EtOH) for 15 min at room temperature. The cells were again washed 3 times with PBS and left to dry overnight.

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Cell cycle and apoptosis [1]
For cell cycle measurement, 106 cells per well were seeded in 24-well plates in triplicates. After 24 h of drug treatment, cells were harvested by centrifugation at 500g for 5 min and washed in 1 ml cold PBS. Cells were spun down again, resuspended in 200 µl PBS and fixed by addition of 800 µl 70% EtOH and incubation for 20 min at −20°C. Cells were washed w ith 1 ml PBS and stained with 500 µl propidium iodide solution (50 µg/ml propidium iodide, 200 µg/ml RNase A, in PBS). Cellular DNA content was measured by flow cytometry using FACSCalibur and analyzed using the FlowJo v10 software. Analysis of apoptotic cells was performed using Caspase-Glo 3/7 according to manufacturer’s instructions. 20 000 cells per well were seeded in triplicate in a white 96-well plate in a total volume of 50 µl. After 24 h incubation with the treatment, 45 µl Caspase-Glo substrate were added per well. The plate was incubated at room temperature in the dark for 1 h and luminescence measured on a Victor X3 microplate reader. Luminescent signal was normalized to vehicle-treated cells.

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Immunoblotting [1]
2×106 cells were lysed in RIPA buffer (150 nM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0), protease inhibitors, benzonase. Cell lysates containing 40 µg of protein were loaded on Bolt™ 4–12% Bis-Tris Plus Gels and ran for SDS-PAGE before transfer onto 0.45 µM nitrocellulose membrane. Following antibodies were used for detection:

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NanoBiT® assay [1]
The NanoBiT® assay was performed following manufacturer’s instructions. Full-length CDK4 and CDK6 were cloned into pBit1.1-C [TK/LgBiT] vector, full-length CRBN was cloned into pBit2.1-N [TK/SmBiT]. 400.000 CRBN-deficient HEK293T cells were seeded in a 6-well plate and transfected the day after with 750 µg each of CDK4/6-LgBit and CRBN-SmBit using Lipofectamine according to manufacturer’s instructions. After 24 h, cells were detached and 50.000 cells per well seeded in a 96-well plate and allowed to attach overnight. Before the assay, the medium was exchanged with 100 µl of ambient temperature fresh medium supplemented with 25 mM HEPES and the plate incubated for 10 min at room temperature to equilibrate. 25 µl Nano-Glo® Live Cell Reagent was added per well, incubated for 5 min and baseline luminescence measured on a Victor X3 with 2 s integration time. 10 µl of 13.5X drug stock were added per well and emission of luminescent signal monitored by continuous measurement every 2 min. Signals were normalized to baseline and vehicle treated cells.

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Expression proteomics BSJ-03–123 [1]
Sample preparation: [1]
15×106 MOLT4 cells were treated with 100 nM BSJ-03–123 for 2 h, in triplicate. Cells were washed three times with PBS, the supernatant aspirated and pellets snap-frozen in liquid N2. Each washed cell pellet was lysed separately in 40 µL of freshly prepared lysis buffer containing 50 mM HEPES (pH 8.0), 2% SDS, 0.1 M DTT, 1 mM PMSF, and protease inhibitor cocktail. Samples rested at RT for 20 minutes before heating to 99 °C for 5 min. After cooling down to RT, DNA was sheared by sonication using a Covaris S2 high performance ultrasonicator. Cell debris were removed by centrifugation at 20.000 × g for 15 min at 20 °C. Supernatant was transferre d to fresh eppendorf tubes and protein concentration determined using the BCA protein assay kit. FASP was performed using a 30 kDa molecular weight cutoff filter. Fifty microliters of each cleared protein extract were mixed with 200 µL of freshly prepared 8 M urea in 100 mM Tris-HCl (pH 8.5) (UA-solution) in the filter unit and centrifuged at 14.000 × g for 15 min at 20 °C to remove SDS. Any residual SDS was washed out by a second washing step with 200 µL of UA. The proteins were alkylated with 100 µL of 50 mM iodoacetamide in the dark for 30 min at RT. Afterward, three washing steps with 100 µL of UA solution were performed, followed by three washing steps with 100µL of 50 mM TEAB buffer. Proteins were digested with trypsin overnight at 37 °C. Peptides were recovered using 40 µL of 50 mM TEAB buffer followed by 50 µL of 0.5 M NaCl. Peptides were desalted using C18 solid phase extraction spin columns. After desalting, peptides were labeled with TMT 10plex™ reagents according to the manufacturer. After quenching of the labeling reaction, labeled peptides were pooled, organic solvent removed in a vacuum concentrator at 45°C and reconstituted in 5% acetonitrile contai ning 20mM ammonia formate buffer, pH 10 for offline fractionation using high pH reversed phase liquid chromatography (2D-RP/RP-HPLC).

[1]. Homolog-Selective Degradation as a Strategy to Probe the Function of CDK6 in AML. Cell Chem Biol. 2019 Feb 21;26(2):300-306.e9.

The development of highly selective small-molecules is a longstanding challenge in ligand discovery given the structural similarities of substrate- or cofactor binding sites. This is a particular concern with ATP-competitive kinase inhibitors where a lack of selectivity can limit the achievable therapeutic window. This hinders pharmacologic exploitation of genetically defined dependencies for therapeutic indications. Here, we present BSJ-03–123(BSJ), a homolog-selective CDK6 degrader. BSJ features proteome-wide selectivity for CDK6 via differential ternary complex formation. Selectivity of BSJ enables exploitation of genetic dependencies beyond a resolution achievable with dual CDK4/6 inhibitors. In particular, we show that BSJ is capable of exploiting a homolog-selective dependency of AML cells on CDK6, thus outlining the potential for selective CDK6 degraders for further translational investigation, conceivably at reduced overall toxicity. Degradation of CDK6 is fast and potent, allowing us to map global consequences of acute CDK6 disruption on downstream signaling networks and transcriptional programs. While we cannot rule out that, at saturating concentrations, BSJ also catalytically inhibits CDK4, no inhibition was measured at the assayed concentration. Comparative profiling of CDK6 degradation and CDK4/6 inhibition suggest that in AML, CDK6 integrates signaling and gene activity predominantly via its kinase activity. Our analysis uncovered several signaling nodes and transcriptional hubs previously not linked to CDK6, such as BCL11A and NCOR2, and future research will be necessary to explore the functional relevance of these pathways in AML and beyond. CDK6 features a prominent role in other malignancies as well as hematopoietic and leukemic stem cells. Selective degradation of CDK6 will facilitate differentiating relevant molecular mechanisms and further untangle kinase-dependent and -independent functions. Future medicinal chemistry efforts will be necessary to expand on the presented concept and to develop a toolbox of selective degraders to investigate the role of protein kinases at unprecedented precision and kinetic resolution. [1]

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The design of selective small molecules is often stymied by similar ligand binding pockets. Here, we report BSJ-03–123, a phthalimide-based degrader that exploits protein-interface determinants to achieve proteome-wide selectivity for the degradation of cyclin-dependent kinase 6 (CDK6). Pharmacologic CDK6 degradation targets a selective dependency of acute myeloid leukemia cells, and transcriptomics and phosphoproteomics profiling of acute degradation of CDK6 enabled dynamic mapping of its immediate role in coordinating signaling and transcription. [1]

C47H56N10O11

937

936.413

C, 60.25; H, 6.02; N, 14.95; O, 18.78

2361493-16-3

BSJ-03-123 HCl;2361493-16-3;

137628658

White to yellow solid powder

1.4±0.1 g/cm3

1.628

0.28

3

17

21

68

1870

0

O=C1C(C(C)=O)=C(C)C2=CN=C(NC3C=CC(=CN=3)N3CCN(CCOCCOCCOCCNC(COC4=CC=CC5=C4C(N(C5=O)C4C(NC(CC4)=O)=O)=O)=O)CC3)N=C2N1C1CCCC1

PHXAASMYZCBYHV-UHFFFAOYSA-N

InChI=1S/C48H57N9O11/c1-30-35-28-51-40(26-37(35)56(32-6-3-4-7-32)47(63)43(30)31(2)58)52-39-12-10-33(27-50-39)55-17-15-54(16-18-55)19-21-66-23-25-67-24-22-65-20-14-49-42(60)29-68-38-9-5-8-34-44(38)48(64)57(46(34)62)36-11-13-41(59)53-45(36)61/h5,8-10,12,26-28,32,36H,3-4,6-7,11,13-25,29H2,1-2H3,(H,49,60)(H,50,51,52)(H,53,59,61)

N-(2-(2-(2-(2-(4-(6-((3-acetyl-1-cyclopentyl-4-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)amino)pyridin-3-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide

BSJ-03123; BSJ03123; BSJ 03123; BSJ-03-123; 2361493-16-3; N-(2-(2-(2-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide; N-[2-[2-[2-[2-[4-[6-[(6-acetyl-8-cyclopentyl-5-methyl-7-oxopyrido[2,3-d]pyrimidin-2-yl)amino]pyridin-3-yl]piperazin-1-yl]ethoxy]ethoxy]ethoxy]ethyl]-2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxyacetamide; CHEMBL5204174; SCHEMBL22199840; BSJ-03-123; BSJ03-123; BSJ 03-123

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~106.72 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.67 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 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: ≥ 2.5 mg/mL (2.67 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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