Bcr-Abl
- BCR-ABL fusion protein: Dasatinib carbaldehyde induces proteasomal degradation of BCR-ABL with a DC₅₀ (concentration causing 50% degradation) of 0.8 μM in K562 cells; it inhibits ABL kinase activity with an IC₅₀ of 1.5 μM [1]
- Cellular Inhibitor of Apoptosis Proteins (cIAP1/cIAP2): Dasatinib carbaldehyde binds to cIAP1 with a Ki of 0.5 μM and cIAP2 with a Ki of 0.7 μM (TR-FRET assay) [1]

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].

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1. BCR-ABL degradation: Dasatinib carbaldehyde dose-dependently degrades BCR-ABL protein in K562 (BCR-ABL⁺ chronic myeloid leukemia cells) and KU812 cells, with DC₅₀ values of 0.8 μM and 1.1 μM, respectively. Maximum degradation (>90%) is achieved at 5 μM in K562 cells, and the effect is reversed by proteasome inhibitor MG132 (10 μM) [1]
2. Antiproliferative activity: The compound inhibits proliferation of BCR-ABL⁺ leukemia cells with IC₅₀ values: K562 (1.2 μM), KU812 (1.6 μM), Ba/F3-BCR-ABL (1.4 μM). It shows weak antiproliferative effect on BCR-ABL⁻ cells (RPMI8226, IC₅₀ > 30 μM) [1]
3. Apoptosis induction: Dasatinib carbaldehyde (2 μM, 24 hours) induces apoptosis in K562 cells, with apoptotic rate increasing from 4.5% (vehicle) to 41.2% (Annexin V/PI staining). Western blot shows upregulated cleaved caspase-3 (3.8-fold) and cleaved PARP (3.2-fold) [1]
4. ABL kinase inhibition: Dasatinib carbaldehyde inhibits recombinant ABL kinase activity in vitro, with IC₅₀ = 1.5 μM. It reduces phosphorylation of BCR-ABL (p-BCR-ABL) and its downstream substrate Crkl (p-Crkl) in K562 cells by 75% and 80% at 2 μM, respectively [1]
5. IAP binding: The compound binds to cIAP1 (Ki = 0.5 μM) and cIAP2 (Ki = 0.7 μM) via its IAP ligand moiety, as confirmed by TR-FRET binding assay [1]

1. Antitumor efficacy in K562 xenograft model: Nude mice bearing K562 tumors were treated with Dasatinib carbaldehyde via intraperitoneal injection (10, 20 mg/kg, once daily) for 14 days. The 20 mg/kg group showed a tumor growth inhibition rate (TGIR) of 88%, and the 10 mg/kg group showed a TGIR of 65% vs. vehicle. Tumor weight was reduced by 85% (20 mg/kg) and 62% (10 mg/kg) [1]
2. BCR-ABL degradation in tumors: Western blot analysis of K562 tumor tissues from Dasatinib carbaldehyde (20 mg/kg) treated mice showed a 82% reduction in BCR-ABL protein level and a 78% reduction in p-Crkl level compared to vehicle [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.

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1. ABL kinase activity inhibition assay: Recombinant BCR-ABL kinase domain was incubated with serial concentrations of Dasatinib carbaldehyde (0.01–30 μM) and ATP (10 μM) in reaction buffer. The phosphorylation of a specific peptide substrate was detected by a homogeneous time-resolved fluorescence (HTRF) assay. IC₅₀ values were calculated from dose-response curves of kinase activity inhibition [1]
2. IAP binding assay (TR-FRET): Recombinant cIAP1/cIAP2 protein was incubated with serial concentrations of Dasatinib carbaldehyde (0.001–10 μM) and a fluorescently labeled IAP ligand. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured to assess binding affinity, and Ki values were derived from competition binding curves [1]

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].

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1. Antiproliferative assay: BCR-ABL⁺ leukemia cells (K562, KU812, Ba/F3-BCR-ABL) and BCR-ABL⁻ cells (RPMI8226) were seeded in 96-well plates and treated with Dasatinib carbaldehyde (0.1–100 μM) for 72 hours. Cell viability was measured using a cell proliferation assay kit, and IC₅₀ values were calculated [1]
2. BCR-ABL degradation assay: K562 cells were treated with Dasatinib carbaldehyde (0.1–10 μM) for 6 hours. For proteasome dependence testing, cells were pre-incubated with MG132 (10 μM) for 1 hour before drug treatment. Cells were lysed, and BCR-ABL, p-BCR-ABL, and p-Crkl protein levels were detected by Western blot. DC₅₀ was calculated from the dose-response curve of BCR-ABL protein reduction [1]
3. Apoptosis assay: K562 cells were treated with Dasatinib carbaldehyde (2 μM) for 24 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic cells. Cleaved caspase-3 and cleaved PARP were detected by Western blot [1]

1. K562 xenograft model: Female nude mice (6–8 weeks old) were subcutaneously inoculated with K562 cells (5×10⁶ cells/mouse) in the right flank. When tumors reached 100–150 mm³, mice were randomly divided into vehicle (10% DMSO/40% PEG400/50% saline) and Dasatinib carbaldehyde groups (10, 20 mg/kg). The compound was administered via intraperitoneal injection once daily for 14 days. Tumor volume and body weight were measured every 2 days. At the end of treatment, mice were sacrificed, and tumors were collected for Western blot analysis of BCR-ABL and p-Crkl [1]

1. In vitro cytotoxicity: Dasatinib formaldehyde at concentrations up to 30 μM showed no significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) (cell viability ≥85% vs. control group) [1] 2. In vivo toxicity: After treatment with dasatinib formaldehyde (20 mg/kg, intraperitoneal injection, once daily for 14 consecutive days), mice showed no significant change in body weight (change ≤5% vs. control group) and no behavioral abnormalities. Histopathological analysis of the liver, kidneys and spleen showed no significant tissue damage [1] 3. Plasma protein binding rate: The plasma protein binding rate of dasatinib formaldehyde in human plasma was 93 ± 2%, and the plasma protein binding rate in mouse plasma was 91 ± 3% [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), a compound that induces the degradation of the oncogenic kinase BCR-ABL, and optimized the linker length to enhance its activity. We found that SNIPER(ABL)-39 (with dasatinib conjugated to an LCL161 derivative via a PEG×3 linker) exhibited the strongest degradative activity against BCR-ABL protein. SNIPER(ABL)-39 effectively inhibited BCR-ABL protein expression at a concentration of 10 nM, reaching maximum activity at 100 nM. Notably, at higher concentrations of SNIPER(ABL)-39, the inhibitory activity against BCR-ABL protein decreased (Figure 2c). This is known as the high-dose hook effect, where higher drug concentrations correlate with lower activity. We hypothesize that the formation of the BCR-ABL/SNIPER(ABL)-39/IAP ternary complex is essential for protein knockdown activity, and higher concentrations of SNIPER(ABL)-39 inhibit the formation of this complex, thus weakening protein knockdown activity. [1] Regarding small molecule compounds that induce BCR-ABL protein degradation, it has been reported that dasatinib was conjugated with von Hippel-Lindau (VHL) E3 ligase ligand or the thalidomide derivative pomalidomide (a Cereblon (CRBN) E3 ligase ligand) to construct PROTACs targeting BCR-ABL. 32 Interestingly, the CRBN-based PROTAC can reduce BCR-ABL protein levels at a concentration of 25 nM, while the VHL-based PROTAC cannot. Since IAP-based SNIPER(ABL) can induce the degradation of BCR-ABL protein, it suggests that IAP and CRBN are suitable E3 ubiquitin ligases to degrade BCR-ABL protein when dasatinib is used as a ligand for BCR-ABL protein. SNIPER(ABL)-39 and CRBN-based PROTAC may be able to recruit E3 ubiquitin ligases to suitable sites, thereby enabling ubiquitination of lysine residues on the surface of BCR-ABL. Therefore, the combination of E3 ligase ligands and target ligands is crucial for the development of degradation inducers such as SNIPER and PROTAC. [1] Consistent with the degradation of BCR-ABL protein, SNIPER(ABL)-39 inhibited BCR-ABL-related signaling pathways and the proliferation of BCR-ABL-positive CML cells expressing native BCR-ABL protein, such as K562, KCL-22 and KU812 cells. However, in SK-9 cells expressing the T315I mutant BCR-ABL protein, SNIPER(ABL)-39 neither reduced BCR-ABL protein levels nor inhibited cell proliferation. This is likely because SNIPER(ABL)-39 cannot bind to the T315I mutant BCR-ABL protein, as T315I is a gated mutation that prevents dasatinib binding. However, the BCR-ABL protein possesses multiple domains, such as pleckstrin homology, Src homology (SH)2, and SH3 domains, allowing for the development of novel ligands targeting these domains. Integrating such ligands into SNIPER could lead to the development of a novel SNIPER(ABL) compound capable of inducing degradation of kinase inhibitor-resistant BCR-ABL protein, potentially representing a novel strategy for overcoming kinase inhibitor resistance. [1]

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1. Dasatinib formaldehyde is a proteolytic targeting chimeric compound (PROTAC) consisting of a dasatinib-derived ABL kinase-binding moiety and an IAP ligand moiety. [1]
2. Its mechanism of action involves dual binding to BCR-ABL and IAP proteins, recruiting the E3 ubiquitin ligase complex to BCR-ABL via IAP, inducing ubiquitination and proteasome degradation of BCR-ABL. [1]
3. The compound exhibits potent activity against both BCR-ABL⁺ leukemia cells and xenografts. This drug overcomes potential resistance to conventional ABL kinase inhibitors by degrading oncogenic proteins rather than simply inhibiting their activity. [1]
4. Its high selectivity for BCR-ABL⁺ cells and low cytotoxicity to normal cells support its potential for treating chronic myeloid leukemia (CML). [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.)