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| Targets |
Cereblon; GBD-9 is a dual-mechanism degrader that targets both Bruton Tyrosine Kinase (BTK) via PROTAC mechanism and G1 to S phase transition 1 (GSPT1) via molecular glue mechanism. It recruits the E3 ligase cereblon (CRBN) to induce the degradation of both target proteins [1].
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| ln Vitro |
GBD-9 (50 nM, 24 h) induced strong degradation of BTK and GSPT1 in DOHH2 DLBCL cells, reducing both target proteins to less than 20% of baseline levels [1].
The degradation occurred rapidly, with treatment at 100 nM GBD-9 achieving degradation within 4 hours [1]. Competition experiments showed that ibrutinib (20 μM) abolished BTK degradation but did not affect GSPT1 degradation, while pomalidomide (20 μM) blocked degradation of both targets, confirming that BTK degradation requires both ends of the molecule (PROTAC mechanism), whereas GSPT1 degradation primarily depends on the pomalidomide end (molecular glue mechanism) [1]. MLN-4924 (500 nM), an inhibitor of E1 ubiquitin-activating enzyme, completely blocked degradation of both BTK and GSPT1, confirming that GBD-9 induces degradation through the ubiquitin-proteasome system [1]. GBD-9 significantly inhibited the proliferation of DOHH2 cells with an IC50 of 133 nM, demonstrating much greater inhibitory effect compared to ibrutinib and the single BTK degrader L18l [1]. In DOHH2 cells, GBD-9 induced G1 phase cell cycle arrest, increasing the proportion of cells in G1 phase by approximately 20%. It also induced apoptosis, as evidenced by downregulation of anti-apoptotic proteins BCL-2, MCL-1, and XIAP, and cleavage of caspase-3 [1]. Quantitative proteomics analysis showed that GBD-9 exhibited fair selectivity, with BTK and GSPT1 being the primary downregulated proteins. No degradation of IKZF1, IKZF3, or CK1α was detected [1]. GBD-9 stably and concurrently degraded BTK and GSPT1 in various DLBCL (WSU-NHL, HBL-1) and AML (THP-1, MV4-11, HL-60) cell lines [1]. |
| Enzyme Assay |
Molecular Docking and Molecular Dynamics Simulation: The binding of designed molecules with GSPT1 and CRBN was simulated based on the CRBN-DDB1-CC-885-GSPT1 cocrystal structure (PDB code: 5HXB). Docking and molecular dynamics simulation results were used to predict which PROTAC molecules might also function as suitable molecular glues [1].
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| Cell Assay |
Western Blot Analysis for Degradation: DOHH2 cells were treated with various concentrations of GBD-9 for 24 hours, or with 100 nM GBD-9 for different time points (0-24 h). Cells were lysed, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against BTK, GSPT1, and β-actin. Protein band intensities were quantified by grayscale analysis [1].
Cell Viability Assay (CCK-8): DOHH2 cells (5000 cells/well) were seeded in 96-well plates and incubated with varying concentrations of GBD-9, ibrutinib, L18l, pomalidomide, or negative control for 72 hours at 37°C. Cell viability was determined using the CCK-8 assay. The IC50 value for GBD-9 was calculated as 133 nM [1]. Cell Cycle Analysis: DOHH2 cells were treated with different concentrations of L18l and GBD-9 for 24 hours. Cells were then fixed, stained with propidium iodide, and analyzed by flow cytometry to assess cell cycle distribution [1]. Quantitative Proteomics (TMT Labeling): DOHH2 cells were treated with 300 nM GBD-9 for 8 hours. Proteins were extracted, digested, and labeled with TMT reagents. Labeled peptides were analyzed by mass spectrometry to identify and quantify protein expression changes. Proteomics data have been deposited to the ProteomeXchange Consortium with identifier PXD025609 [1]. |
| Toxicity/Toxicokinetics |
The literature does not describe toxicity or toxicokinetic data for GBD-9. However, selectivity profiling via quantitative proteomics showed that GBD-9 did not degrade IKZF1, IKZF3, or CK1α, which are known neo-substrates associated with toxicities of immunomodulatory drugs [1].
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| References | |
| Additional Infomation |
Background: Targeted protein degradation (TPD) is a promising therapeutic strategy. PROTACs (Proteolysis Targeting Chimeras) and Molecular Glues (MGs) are two major modes of TPD. PROTACs are suitable for rational design but often have larger molecular weights and potential druggability issues. MGs have smaller molecular weights and better druggability but are difficult to design rationally. GBD-9 represents a novel dual-mechanism degrader that merges the PROTAC and MG strategies [1].
Design Strategy: GBD-9 was designed by shortening the linker of a BTK PROTAC (L18l) to incorporate molecular glue characteristics. The optimized molecule has a linker with 7-9 carbon atoms and features meta-substituted pomalidomide to achieve a more stretched conformation. This design allows GBD-9 to function as a PROTAC for BTK degradation and as a molecular glue for GSPT1 degradation [1]. Mechanism of Action: GBD-9 induces BTK degradation through a PROTAC mechanism requiring simultaneous binding to BTK and CRBN via the ibrutinib and pomalidomide ends, respectively. It induces GSPT1 degradation through a molecular glue mechanism primarily dependent on the pomalidomide end, forming a compact ternary complex with CRBN and GSPT1 [1]. Therapeutic Potential: GBD-9 overcomes the limitations of single-target BTK inhibitors and degraders in DLBCL and AML by concurrently degrading GSPT1. It shows significantly enhanced anti-proliferative effects across multiple DLBCL and AML cell lines compared to ibrutinib and single BTK degrader L18l, suggesting potential for treating these difficult-to-treat hematologic malignancies [1]. |
| Molecular Formula |
C44H47N9O6
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| Molecular Weight |
797.90068936348
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| Exact Mass |
797.364
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| Elemental Analysis |
C, 66.23; H, 5.94; N, 15.80; O, 12.03
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| CAS # |
2864408-92-2
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| PubChem CID |
168324318
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| Appearance |
Light yellow to green yellow solid powder
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| LogP |
5.5
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
15
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| Heavy Atom Count |
59
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| Complexity |
1490
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1C[C@H](CN(C1)C(=O)CCCCCCCCNC2=CC3=C(C=C2)C(=O)N(C3=O)C4CCC(=O)NC4=O)N5C6=NC=NC(=C6C(=N5)C7=CC=C(C=C7)OC8=CC=CC=C8)N
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| InChi Key |
DCJYQURCBHSFEP-OSRRZMJMSA-N
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| InChi Code |
InChI=1S/C44H47N9O6/c45-40-38-39(28-15-18-32(19-16-28)59-31-12-6-5-7-13-31)50-53(41(38)48-27-47-40)30-11-10-24-51(26-30)37(55)14-8-3-1-2-4-9-23-46-29-17-20-33-34(25-29)44(58)52(43(33)57)35-21-22-36(54)49-42(35)56/h5-7,12-13,15-20,25,27,30,35,46H,1-4,8-11,14,21-24,26H2,(H2,45,47,48)(H,49,54,56)/t30-,35?/m1/s1
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| Chemical Name |
5-[[9-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]-9-oxononyl]amino]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione
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| Synonyms |
GBD-9; 2864408-92-2; GBD 9; GBD9; 5-[[9-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]-9-oxononyl]amino]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione;
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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 and light. |
| 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 (125.3 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2533 mL | 6.2664 mL | 12.5329 mL | |
| 5 mM | 0.2507 mL | 1.2533 mL | 2.5066 mL | |
| 10 mM | 0.1253 mL | 0.6266 mL | 1.2533 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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