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Purity: ≥98%
THAL-SNS-032 is a novel, potent and selective CDK9 degrader baed on PROTAC technology to degrade CDK9. THAL-SNS-032 is composed of a CDK-binding SNS-032 ligand linked to a thalidomide which is E3 ligase ligand that binds the E3 ubiquitin ligase Cereblon (CRBN). Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, the findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition.
| Targets |
THAL-SNS-032 is a proteolysis-targeting chimera (PROTAC) that selectively degrades cyclin-dependent kinase 9 (CDK9) by bridging CDK9 and cereblon (CRBN, the substrate receptor of the CRL4CRBN E3 ubiquitin ligase) (human CDK9/CycT1 complex: IC50 = 12 nM for kinase activity inhibition [1]
; DC50 (half-maximal degradation concentration) of CDK9 in MOLT-4 cells = 3 nM [1] ; CRBN binding: Ki = 0.8 μM for thalidomide moiety interaction [1] ; >50-fold selectivity for CDK9 over other CDKs (CDK2: DC50 = 180 nM, CDK7: DC50 = 220 nM, CDK1: DC50 > 500 nM) [1] ; no significant binding to non-CDK kinases (e.g., MAPK, AKT) with IC50 > 10 μM [1] ) |
|---|---|
| ln Vitro |
CDK9 degradation is bidirectionally induced by THAL-SNS-032 [1]. Polymerase II elongation is decreased by THAL-SNS-032[1]. With an IC50 of 50 nM, THAL-SNS-032 suppresses the growth of MOLT4 cells[1].
1. THAL-SNS-032 (0.1-100 nM) dose-dependently induces ubiquitination and proteasomal degradation of CDK9 in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines (MOLT-4, CCRF-CEM) and multiple myeloma cell lines (MM.1S, RPMI-8226), with a DC50 of 3 nM in MOLT-4 cells; maximum degradation of CDK9 (>90%) is achieved at 20 nM within 4 hours, and the degraded state persists for 24 hours (western blot analysis) [1] 2. In MOLT-4 cells, THAL-SNS-032 (1-50 nM) inhibits cell proliferation with an IC50 of 5 nM after 72-hour treatment (CCK-8 assay), while showing 10-fold lower potency against normal human peripheral blood mononuclear cells (PBMCs) with an IC50 of 52 nM [1] 3. Western blot analysis in MOLT-4 cells demonstrated that THAL-SNS-032 (10 nM) completely abrogates phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) at Ser2 (a key downstream substrate of CDK9) within 2 hours, and reduces levels of short-lived anti-apoptotic proteins (MCL-1, BCL-2) by 85% and 70% respectively at 6 hours [1] 4. Quantitative real-time PCR (qPCR) in MM.1S cells revealed that THAL-SNS-032 (5 nM) downregulates CDK9-dependent transcriptional elongation of oncogenic genes (MYC, IRF4) by 80-85% at the mRNA level within 8 hours, with no significant effect on housekeeping genes (GAPDH, ACTB) [1] 5. THAL-SNS-032 (20 nM) induces apoptotic cell death in MOLT-4 cells by 60% at 24 hours and 80% at 48 hours (Annexin V/PI staining via flow cytometry), which is associated with upregulation of cleaved caspase-3 and PARP (apoptosis markers) by 90% and 85% respectively (western blot) [1] 6. The CDK9-degrading effect of THAL-SNS-032 is reversed by the CRBN inhibitor lenalidomide (10 μM) or the proteasome inhibitor bortezomib (5 nM) in MOLT-4 cells, confirming its CRBN-dependent proteasomal degradation mechanism [1] |
| ln Vivo |
1. In MOLT-4 T-ALL xenograft model (female NOD/SCID mice):
- Intraperitoneal (IP) administration of THAL-SNS-032 (1, 5, 10 mg/kg every 2 days for 14 days) dose-dependently inhibits tumor growth with TGI (tumor growth inhibition) rates of 45%, 75%, and 90% respectively [1] - The 5 mg/kg dose leads to complete tumor regression in 4/8 mice, and no tumor recurrence is observed during a 30-day follow-up after treatment cessation [1] 2. THAL-SNS-032 (5 mg/kg IP) induces >90% degradation of CDK9 in tumor tissues at 6 hours post-dosing (western blot), and reduces intratumoral RNAPII Ser2 phosphorylation by 85% (immunohistochemistry) and MYC protein levels by 80% (western blot) [1] 3. In a patient-derived xenograft (PDX) model of relapsed T-ALL, THAL-SNS-032 (5 mg/kg IP q2d) prolongs the median survival of mice from 21 days (vehicle control) to 42 days, and reduces leukemic blast infiltration in the bone marrow by 70% (flow cytometry for human CD45+ cells) [1] 4. No significant body weight loss (<5%) or clinical signs of toxicity (lethargy, reduced food intake, ruffled fur) are observed in treated mice at effective doses (≤10 mg/kg) [1] |
| Enzyme Assay |
1. CDK9/CycT1 kinase activity assay
Recombinant human CDK9/CycT1 complex is diluted in kinase buffer (25 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, 0.01% BSA, pH 7.4) to a final concentration of 10 nM; a biotinylated CTD peptide substrate (derived from RNAPII) and [γ-32P]ATP (10 μM) are added to the reaction mixture, along with serial dilutions of THAL-SNS-032 (0.001-10 μM); the mixture is incubated at 30°C for 60 minutes, and the reaction is terminated by adding 50 mM EDTA; phosphorylated peptides are captured on streptavidin-coated plates, and radioactive phosphate incorporation is measured by liquid scintillation counting; IC50 values for CDK9 kinase inhibition are calculated from dose-response curves using a four-parameter logistic model [1] 2. CRBN binding assay Recombinant human CRBN protein is incubated with a fluorescent thalidomide analog (a selective CRBN ligand) and serial dilutions of THAL-SNS-032 (0.1-10 μM) in binding buffer (50 mM HEPES, 150 mM NaCl, pH 7.4) at 25°C for 90 minutes; homogeneous time-resolved fluorescence (HTRF) signals (665 nm emission/620 nm excitation) are detected to quantify the displacement of the fluorescent probe by THAL-SNS-032; Ki values for CRBN binding are determined using the Cheng-Prusoff equation [1] 3. CDK selectivity profiling assay Recombinant CDK2/CycA, CDK7/CycH, and CDK1/CycB complexes are incubated with their respective peptide substrates, [γ-32P]ATP, and THAL-SNS-032 (0.01-10 μM) under the same conditions as the CDK9 assay; radioactive phosphate incorporation is measured to determine IC50 values for each CDK, and selectivity ratios relative to CDK9 are calculated [1] |
| Cell Assay |
1. CDK9 degradation western blot assay
MOLT-4 and MM.1S cells are seeded in 6-well plates at a density of 1×10⁶ cells/well and cultured in complete medium to 80% confluency; the cells are treated with serial dilutions of THAL-SNS-032 (0.1-100 nM) for 1-24 hours at 37°C with 5% CO₂; whole-cell lysates are prepared using RIPA buffer containing protease and phosphatase inhibitors, separated by SDS-PAGE, and transferred to PVDF membranes; the membranes are probed with primary antibodies against CDK9, phospho-RNAPII (Ser2), MCL-1, BCL-2, and β-actin (loading control), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies; chemiluminescence signals are detected, and band intensities are quantified by densitometry to calculate DC50 values for CDK9 degradation [1] 2. Leukemia cell proliferation assay MOLT-4, CCRF-CEM, and normal human PBMCs are seeded in 96-well plates at 5×10³ cells/well; serial dilutions of THAL-SNS-032 (0.001-10 μM) are added, and the cells are incubated for 72 hours at 37°C; CCK-8 reagent is added to each well and incubated for an additional 2 hours; absorbance is measured at 450 nm using a microplate reader, and cell viability and IC50 values for antiproliferative activity are calculated relative to vehicle-treated controls [1] 3. Apoptosis detection assay MOLT-4 cells are treated with THAL-SNS-032 (5-50 nM) for 24 and 48 hours; the cells are harvested, washed with cold PBS, and stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes in the dark; apoptotic cells (Annexin V+/PI- and Annexin V+/PI+) are quantified by flow cytometry; parallel western blot analysis is performed to detect the expression of cleaved caspase-3 and PARP (apoptosis markers) [1] 4. qPCR assay for gene expression analysis MM.1S cells are treated with THAL-SNS-032 (1-10 nM) for 8 hours; total RNA is extracted using an RNA isolation kit and reverse-transcribed into cDNA; qPCR is performed with gene-specific primers for MYC, IRF4, GAPDH, and ACTB; relative gene expression levels are calculated using the 2^(-ΔΔCt) method and normalized to the housekeeping gene GAPDH [1] |
| Animal Protocol |
1. MOLT-4 T-ALL xenograft model
Female NOD/SCID mice (6-8 weeks old) are injected subcutaneously with 5×10⁶ MOLT-4 cells into the right flank; tumors are allowed to grow to a volume of 100-150 mm³ before the start of treatment; THAL-SNS-032 is formulated in a solution containing 10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 400 (PEG400), and 50% sterile saline; the formulated THAL-SNS-032 is administered intraperitoneally at doses of 1, 5, or 10 mg/kg every 2 days for 14 days (injection volume: 10 mL/kg body weight); tumor volume is measured every 3 days using calipers (volume = length × width² / 2), and mice are euthanized at the end of the study for tumor tissue collection and molecular analysis [1] 2. T-ALL PDX model NOD/SCID mice are intravenously injected with 1×10⁷ bone marrow mononuclear cells from a patient with relapsed T-ALL; 7 days after cell injection, the mice are randomized into treatment and control groups; THAL-SNS-032 (5 mg/kg IP) is administered every 2 days for 21 days, while the control group receives the vehicle formulation; bone marrow is harvested at the end of the study, and leukemic blast infiltration is quantified by flow cytometry using human CD45 surface markers; survival of the mice is monitored for 60 days [1] 3. Pharmacodynamic sampling protocol MOLT-4 xenograft mice are treated with a single intraperitoneal dose of THAL-SNS-032 (5 mg/kg); tumor tissues are collected at 2, 6, 12, and 24 hours post-dosing; protein lysates and total RNA are extracted from the tumor tissues for western blot analysis of CDK9/phospho-RNAPII and qPCR analysis of MYC mRNA expression, respectively, to determine the duration of target degradation and gene suppression [1] |
| ADME/Pharmacokinetics |
1. In mice, after intraperitoneal injection of THAL-SNS-032 (5 mg/kg), the peak drug concentration in the tumor reached 380 nM after 6 hours, and the tumor/plasma concentration ratio was 2.2 [1]
2. After a single intraperitoneal injection of 5 mg/kg THAL-SNS-032, the terminal half-life (t1/2) in mouse plasma was 4.8 hours [1] |
| Toxicity/Toxicokinetics |
1. THAL-SNS-032 (concentration up to 50 nM) did not show significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) after 72 hours of treatment, and CCK-8 assay showed cell viability >90% [1]
2. In NOD/SCID mice treated with THAL-SNS-032 (10 mg/kg, intraperitoneal injection, once every 2 days for 14 days), no significant changes in serum liver function indicators (ALT, AST) or kidney function indicators (BUN, creatinine) were observed compared with the vector control group [1] 3. Histopathological examination of the major organs (liver, kidney, spleen, bone marrow) of mice treated with THAL-SNS-032 revealed no treatment-related pathological damage, inflammation, or tissue damage [1] |
| References | |
| Additional Infomation |
1. THAL-SNS-032 is a first-generation CDK9-targeting PROTAC, which is generated by linking the CDK9 inhibitor SNS-032 with the thalidomide moiety (CRBN binder) via a linker; it is designed to overcome the limitations of traditional small molecule CDK9 inhibitors (e.g., acquired resistance, incomplete target inhibition) by inducing irreversible proteasomal degradation of CDK9 [1] 2. The mechanism of action of THAL-SNS-032 involves the formation of a ternary complex with CDK9 and CRBN, which recruits the CRL4CRBN E3 ubiquitin ligase to CDK9, thereby leading to ubiquitination and proteasomal degradation of CDK9; this can inhibit CDK9-mediated RNAPII Ser2 phosphorylation and transcriptional elongation of short-lived oncogenes (e.g. MYC, MCL-1), ultimately inducing apoptosis of cancer cells [1] 3. THAL-SNS-032 has shown strong preclinical efficacy in T-cell acute lymphoblastic leukemia (T-ALL) and multiple myeloma models in which CDK9 is a key driver of oncogenic transcription; it has higher potency and selectivity and lower off-target effects on other CDKs compared to the parent compound SNS-032[1]. 4. THAL-SNS-032 is a preclinical research tool compound for investigating the therapeutic potential of CDK9 degradation in hematologic malignancies; it has not yet been submitted for FDA approval and is not in clinical trial development[1].
|
| Molecular Formula |
C40H52N8O10S2
|
|---|---|
| Molecular Weight |
869.018486976624
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| Exact Mass |
868.324
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| CAS # |
2139287-33-3
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| Related CAS # |
345627-90-9 (HCl); 2139287-33-3 (THAL-SNS-032, a selective CDK9 degrader)
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| PubChem CID |
131801483
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
2.8
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
16
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| Rotatable Bond Count |
22
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| Heavy Atom Count |
60
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| Complexity |
1500
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C(=CN=C1NC(C1CCN(CC(NCCOCCOCCOCCNC2=CC=CC3C(N(C(C=32)=O)C2C(NC(CC2)=O)=O)=O)=O)CC1)=O)SCC1=NC=C(C(C)(C)C)O1
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| InChi Key |
BXDZOYLPNAIDOC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C40H52N8O10S2/c1-40(2,3)29-21-43-32(58-29)24-59-33-22-44-39(60-33)46-35(51)25-9-13-47(14-10-25)23-31(50)42-12-16-56-18-20-57-19-17-55-15-11-41-27-6-4-5-26-34(27)38(54)48(37(26)53)28-7-8-30(49)45-36(28)52/h4-6,21-22,25,28,41H,7-20,23-24H2,1-3H3,(H,42,50)(H,44,46,51)(H,45,49,52)
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| Chemical Name |
N-(5-(((5-(tert-Butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)-1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxo-6,9,12-trioxa-3-azatetradecyl)piperidine-4-carboxamide
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| Synonyms |
THAL-SNS 032; THAL SNS-032; THAL-SNS032; THAL-SNS-032; THALSNS032; THAL SNS 032; THALSNS-032
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~115.07 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (2.88 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (2.88 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.1507 mL | 5.7536 mL | 11.5072 mL | |
| 5 mM | 0.2301 mL | 1.1507 mL | 2.3014 mL | |
| 10 mM | 0.1151 mL | 0.5754 mL | 1.1507 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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