Bcr-Abl

Chromosomal translocation occurs in some cancer cells, which results in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate the BCR-ABL protein. We have devised a protein knockdown system by hybrid molecules named Specific and Non-genetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER), which is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins, and a couple of SNIPER(ABL) against BCR-ABL protein have been developed recently. In this study, we tested various combinations of ABL inhibitors and IAP ligands, and the linker was optimized for protein knockdown activity of SNIPER(ABL). The resulting SNIPER(ABL)-39, in which dasatinib is conjugated to an IAP ligand LCL161 derivative by polyethylene glycol (PEG) × 3 linker, shows a potent activity to degrade the BCR-ABL protein. Mechanistic analysis suggested that both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) play a role in the degradation of BCR-ABL protein. Consistent with the degradation of BCR-ABL protein, the SNIPER(ABL)-39 inhibited the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and Crk like proto-oncogene (CrkL), and suppressed the growth of BCR-ABL-positive CML cells. These results suggest that SNIPER(ABL)-39 could be a candidate for a degradation-based novel anti-cancer drug against BCR-ABL-positive CML[1].

Measurement of inhibitor activity of ABL1 inhibitor that bind to the ATP binding site[1]
Before addition to the assay plate, threefold concentrations of His‐ABL1 protein, Tb‐SA and biotinylated anti‐His antibody were mixed in the assay buffer and incubated for over 1 h at room temperature. Several concentrations of test inhibitors dissolved in the assay buffer were dispensed in the assay plate. Subsequently, the ABL/antibody/Tb‐SA premix was dispensed to each well and incubated for 120 min at room temperature. Reaction was initiated by addition of assay buffer containing 13.5 nM BODIPY‐dasatinib. The plate was incubated for 30 min at room temperature and the TR‐FRET signal was measured using an EnVision Multilabel Plate Reader. The final concentrations of Tb‐SA, biotinylated anti‐His, ABL1 protein and BODIPY‐dasatinib were 0.2, 0.4, 0.38 and 4.5 nM, respectively. The values of the 0 and 100% controls were the signals obtained in the absence and presence of 3 μM dasatinib, respectively.

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Measurement of inhibitory activity of IAP/peptide interaction[1]
His‐IAP proteins (XIAP, cIAP1 or cIAP2), FITC‐Smac, Tb‐SA and biotinylated anti‐His antibody were mixed in the assay buffer and incubated for over 1 h at room temperature before addition to the assay plate. Several concentrations of test inhibitors were dispensed in the assay plate and the protein‐probe premix was dispensed to each well. All assays were carried out using 0.6 nM of IAP proteins. The concentrations of FITC‐Smac were described as follows: 27 nM for XIAP, 12 nM for cIAP1 and 19 nM cIAP2. The final concentrations of Tb‐SA and biotinylated anti‐His antibody were 0.2 and 0.4 nM, respectively. After 1 h incubation at room temperature, the TR‐FRET signal was measured using an EnVision Multilabel Plate Reader. The values of the 0 and 100% controls were the signals obtained in the presence and absence of IAP proteins, respectively.

Cell viability assay[1]
Cell viability was determined using water‐soluble tetrazolium WST‐8 (4‐[3‐(2‐methoxy‐4‐nitrophenyl)‐2‐(4‐nitrophenyl)‐2H‐5‐tetrazolio]‐1,3‐benzene disulfonate) for the spectrophotometric assay according to the manufacturer's instructions. Cells were seeded at a concentration of 5 × 103 cells per well in a 96‐well culture plate. After 24 h, the cells were treated with the indicated compounds for 48 h. The WST‐8 reagent was added and the cells were incubated for 0.5 h at 37°C in a humidified atmosphere of 5% CO2[1].

[1]. Development of protein degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL kinase inhibitors and IAP ligands. Cancer Sci. 2017 Aug;108(8):1657-1666.

In this study, we tested various combinations of ABL inhibitors and IAP ligands to develop SNIPER(ABL) that induces the degradation of oncogenic kinase BCR‐ABL, and the linker length was optimized for the activity. We found that SNIPER(ABL)‐39, in which dasatinib is conjugated to an LCL161 derivative with PEG × 3 linker, shows the most potent activity to degrade the BCR‐ABL protein. SNIPER(ABL)‐39 showed an effective protein knockdown activity at 10 nM and the maximum activity was observed at 100 nM. Notably, the protein knockdown activity was rather attenuated at higher concentrations of SNIPER(ABL)‐39 (Fig. 2c). This is known as a high‐dose hook effect, where a certain pharmaceutical activity is interfered with higher concentration of drugs. We speculate that formation of ternary complex consisting of BCR‐ABL/SNIPER(ABL)‐39/IAP required for the protein knockdown activity would be suppressed by higher concentrations of SNIPER(ABL)‐39, and, therefore, the protein knockdown activity is attenuated.[1]

With respect to the small compounds that induce the degradation of BCR‐ABL protein, PROTAC against BCR‐ABL were reported by conjugation of dasatinib to a von Hippel‐Lindau (VHL) E3 ligase ligand or a thalidomide derivative, pomalidomide, that is a ligand for Cereblon (CRBN) E3 ligase.32 Interestingly, CRBN‐based PROTAC can reduce BCR‐ABL protein at 25 nM, whereas VHL‐based PROTAC cannot. Because IAP‐based SNIPER(ABL) can induce the degradation of BCR‐ABL protein, it is suggested that IAP and CRBN are appropriate E3 ligases to degrade BCR‐ABL protein when dasatinib is incorporated as a ligand for BCR‐ABL protein. Probably, SNIPER(ABL)‐39 and CRBN‐based PROTAC can recruit E3 ligases to an appropriate position so that the lysine residues on the surface of BCR‐ABL can be ubiquitylated. Thus, the combination of an E3 ligase ligand and a target ligand is critically important to develop the degradation inducers such as SNIPER and PROTAC.[1]

Consistent with the degradation of BCR‐ABL protein, SNIPER(ABL)‐39 inhibited the BCR‐ABL‐related signaling pathway and proliferation of BCR‐ABL positive CML, such as K562, KCL‐22 and KU812 cell, expressing native BCR‐ABL protein. However, in SK‐9 cells expressing T315I mutant BCR‐ABL protein, SNIPER(ABL)‐39 did not reduce the BCR‐ABL protein nor inhibit cell proliferation. This may be attributed that SNIPER(ABL)‐39 could not bind to the T315I mutant BCR‐ABL protein, because the T315I is a gatekeeper mutation that prevents the binding of dasatinib.39, 42 However, BCR‐ABL protein has multiple domains, such as pleckstrin homology, Src homology (SH) 2 and SH3 domains, to which novel ligands could be developed. Incorporation of such ligands into SNIPER would allow us to develop a novel SNIPER(ABL) that can induce the degradation of BCR‐ABL proteins resistant to kinase inhibitors, which could be a novel strategy to overcome drug resistance against kinase inhibitors.[1]

C21H22CLN7O2S

471.96

471.12

C, 53.44; H, 4.70; Cl, 7.51; N, 20.77; O, 6.78; S, 6.79

2112837-79-1

863127-77-9 (hydrate);302962-49-8 (free);2112837-79-1 (cabaldehyde);910297-52-8 (N-oxide);

138377562

White to off-white solid powder

3.7

2

8

5

32

654

0

C1C(C)=C(C(=CC=1)Cl)NC(C1SC(NC2=CC(=NC(C)=N2)N2CCN(CC2)C=O)=NC=1)=O

BKDGDNUWAYNWBA-UHFFFAOYSA-N

InChI=1S/C21H22ClN7O2S/c1-13-4-3-5-15(22)19(13)27-20(31)16-11-23-21(32-16)26-17-10-18(25-14(2)24-17)29-8-6-28(12-30)7-9-29/h3-5,10-12H,6-9H2,1-2H3,(H,27,31)(H,23,24,25,26)

N-(2-chloro-6-methylphenyl)-2-((6-(4-formylpiperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide

Dasatinib carbaldehyde; 2112837-79-1; 5-Thiazolecarboxamide, N-(2-chloro-6-methylphenyl)-2-[[6-(4-formyl-1-piperazinyl)-2-methyl-4-pyrimidinyl]amino]- (ACI); N-(2-Chloro-6-methylphenyl)-2-[[6-(4-formyl-1-piperazinyl)-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide (ACI); SCHEMBL21340693;

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 : ~33.33 mg/mL (~70.62 mM)

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.)