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NVP-DKY709 (NVP-DKY-709; DKY-709) is a first-in-class selective cereblon (CRBN) glue degrader of IKZF2 with anticancer and may be used for cancer immunotherapy. It is a molecular glue type of degrader that spares IKZF1 and IKZF3 from degradation. X-ray crystal structure of IKZF2 zinc fingers bound to DDB1:CRBN:NVP-DKY709 explained the target selectivity of NVP-DKY709. In vitro, NVP-DKY709 modulated Treg cell and Teff cell functions. In a mouse breast cancer xenograft model, daily oral dosing with NVP-DKY709 led to degradation of IKZF2 in tumour and peripheral blood Treg cells, reducing tumour growth similarly to that achieved with the anti-PD1 antibody PDR001. Oral NVP-DKY709 effectively degraded IKZF2 in monkeys and patients and is currently being evaluated in a first-in-human phase I trial in solid tumours.
| Targets |
IZKF2 (DC50 = 4 nM )
NVP-DKY709 is a selective molecular glue degrader that targets the IKZF2 (Helios) transcription factor by recruiting it to the CRBN E3 ubiquitin ligase. It selectively induces degradation of IKZF2 while sparing IKZF1 and IKZF3. It also degrades IKZF4 and SALL4 [2]. |
|---|---|
| ln Vitro |
NVP-DKY709 is a first-in-class and selective cereblon (CRBN) glue degrader of IKZF2, which spares IKZF1 and IKZF3 from degradation. Solving the X-ray crystal structure of IKZF2 zinc fingers bound to DDB1:CRBN:NVP-DKY709 explained the target selectivity of NVP-DKY709. In vitro, NVP-DKY709 modulated Treg cell and Teff cell functions [1].
- Cellular Degradation Selectivity: In Jurkat human T cells, NVP-DKY709 induced dose-dependent and selective degradation of endogenous IKZF2 (DC50 = 11 nM, Dmax = 69%) with no effect on IKZF1 up to 10 μM [2]. - Proteomics: Quantitative proteomics in Jurkat cells treated with 2.5 μM NVP-DKY709 for 16 h identified IKZF2 as the only significantly downregulated protein among 8,656 quantified proteins. IKZF1, IKZF3, and IKZF5 were not downregulated [2]. - Engineered Cell Assays: In 293T cells, NVP-DKY709 degraded engineered IKZF2 (Dmax = 53%, DC50 = 4 nM) and IKZF4 (DC50 = 13 nM, Dmax = 21%). It also degraded SALL4 (Dmax = 55%, DC50 = 2 nM for ProLabel; Dmax = 88%, DC50 = 13 nM for HiBit) [2]. - Functional Impact on T cells: In Jurkat cells stimulated with phytohemagglutinin (PHA), NVP-DKY709 increased IL-2 production in a dose-dependent manner. In primary human regulatory T cells (Treg), treatment resulted in IKZF2 degradation (DC50 = 11 nM, Dmax = 89%) and reduced their suppressive capacity. In an in vitro T effector cell exhaustion model, NVP-DKY709 reduced the percentage of IKZF2+ cells and increased IFN-γ expression in exhausted CD4 and CD8 T cells [2]. |
| ln Vivo |
In a mouse breast cancer xenograft model, daily oral dosing with NVP-DKY709 led to degradation of IKZF2 in tumour and peripheral blood Treg cells, reducing tumour growth similarly to that achieved with the anti-PD1 antibody PDR001. Oral NVP-DKY709 effectively degraded IKZF2 in monkeys and patients and is currently being evaluated in a first-in-human phase I trial in solid tumours [1].
- Humanized Mouse Xenograft Model: In NSG mice engrafted with human hematopoietic stem cells and implanted with MDA-MB-231 xenografts, oral administration of NVP-DKY709 (100 mg/kg, once daily) resulted in robust IKZF2 degradation in both tumor-infiltrating and peripheral blood Treg cells. Single-agent treatment led to reduced tumor growth, comparable to anti-PD1 antibody (PDR001) treatment [2]. - Cynomolgus Monkey Immunization Model: In cynomolgus monkeys, a single oral dose of NVP-DKY709 (0.01 to 1 mg/kg) led to maximum degradation (~90% at 1 mg/kg) of IKZF2 in circulating Treg cells 24 h post-dose. In a repeat-dose study (daily oral dosing of 0.1 or 3 mg/kg for 36 days) combined with KLH/squalene immunization, sustained IKZF2 degradation was observed [2]. |
| Enzyme Assay |
- SPR Binding Measurement: The binding affinity of NVP-DKY709 to CRBN was determined via surface plasmon resonance. DDB1:CRBN was immobilized on a sensor chip, and DKY709 was injected. The KD for the interaction of DKY709 to CRBN was 28 nM [2].
- Ternary Complex Formation: SPR was used to measure the binding of IKZF2 zinc finger constructs (ZF2, ZF2-3, ZF1-4) to the DDB1:CRBN:DKY709 complex. The binding of IKZF2 (ZF2-3) to the complex was determined, with an affinity greater than 20-fold higher than ZF2 alone [2]. - CRBN Binding Assay (Fluorescence Polarization): The ability of compounds to bind CRBN was determined by their ability to compete with a BodipyFL-conjugated lenalidomide probe. The assay was performed with CRBN/DDB1 protein and the probe. IC50 values were calculated [2]. |
| Cell Assay |
- ProLabel Degradation Assay: 293T cells stably expressing ProLabel-tagged proteins (IKZF1, IKZF2, etc.) were plated in 384-well plates and treated with compound for 24 h. ProLabel substrate was added, and luminescence was measured to determine protein degradation (Dmax and DC50) [2].
- NanoBiT Recruitment Assay: 293T cells were co-transfected with CRBN (SmBiT) and IKZF1/2 (LgBiT) vectors. After 24 h, cells were treated with the NAE1 inhibitor MLN4924 and then with test compounds for 10 min. NanoBiT substrate was added, and luminescence was measured to assess target protein recruitment to CRBN [2]. - HiBit Degradation Assay: 293GT cells stably expressing HiBit-gene of interest fusions were plated in 1536-well plates. After overnight incubation, compound was added using an Echo dispenser. After 20 h, HiBit Lysis Reagent was added, and luminescence was measured to determine protein degradation [2]. - Endogenous IKZF2 Degradation in T Cells: Jurkat cells or primary CD25+ enriched T cells were plated in 96-well plates and treated with compound for 18-24 h. Cells were stained for viability, fixed, and intracellularly stained for IKZF2 and Ikaros. Samples were analyzed by flow cytometry [2]. - Treg Suppression Assay: Expanded Treg cells were co-cultured with CFSE-labeled PBMCs at various ratios. T-cell activation was induced with beads or anti-CD3 antibody. After 3-4 days, proliferation of T effector cells was assessed by CFSE dilution using flow cytometry [2]. - T Cell Exhaustion Assay: T cells enriched from PBMCs were repeatedly stimulated with CD3/CD28 beads in the presence of compound. After 5 days, cells were restimulated with fresh beads. Cells were then stimulated with a cell stimulation cocktail and stained for viability, surface markers, and intracellular markers (IKZF2, IFN-γ) for flow cytometric analysis [2]. - IL-2 Production in Jurkat Cells: Jurkat cells were plated in 96-well plates and treated with compound for 24 h. Supernatants were collected, and IL-2 concentration was measured using an MSD assay kit [2]. |
| Animal Protocol |
- Humanized Mouse Xenograft Study: Immunodeficient NSG mice were irradiated or treated with busulfan and engrafted with human hematopoietic stem cells. MDA-MB-231 cells in matrigel were implanted subcutaneously. Seven days post-implantation, daily oral dosing of NVP-DKY709 (100 mg/kg) was initiated. Weekly intraperitoneal injections of anti-PD1 antibody (PDR001) were administered. After 21 days, blood and tumor samples were collected for analysis [2].
- Cynomolgus Monkey Study (PK/PD): Male cynomolgus monkeys were administered a single oral dose of NVP-DKY709 at various concentrations (0.01, 0.1, 0.5, 1 mg/kg). Blood was collected at multiple time points to assess IKZF2 degradation in circulating Treg cells [2]. - Cynomolgus Monkey Immunization Study: A non-GLP study was conducted in male cynomolgus monkeys. Animals were immunized intramuscularly with KLH/squalene. Daily oral treatment with NVP-DKY709 (0.1 or 3 mg/kg/day) or vehicle was initiated 5 days post-immunization and continued for 36 days. A recall immunization was administered on day 15. Blood was collected at various timepoints for immunophenotyping and assessment of IKZF2 degradation [2]. |
| ADME/Pharmacokinetics |
Oral effective concentration (F > 35%)
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| Toxicity/Toxicokinetics |
- NVP-DKY709 completely spares degradation of IKZF1 and IKZF3, potentially avoiding the on-target hematopoietic toxicities (such as neutropenia and myelosuppression) associated with IMiD drugs that degrade IKZF1/3 [2].
- It degrades SALL4, a factor linked to teratogenicity. However, SALL4 is not expressed in differentiated immune cells, suggesting a specific toxicity profile [2]. |
| References | |
| Additional Infomation |
Tumours can evade the immune system by recruiting immunosuppressive FOXP3+ regulatory T cells (Treg cells), which can limit the activation and proliferation of tumor-specific effector T cells (Teff cells). In recent years, IKZF2 (Helios), a member of the Ikaros transcription factor family—playing a crucial role in maintaining the function and stability of Treg cells—has become a highly attractive target for enhancing anti-tumor immune responses. However, targeting zinc finger transcription factors such as IKZF2 is extremely challenging because most of them have loose structures and lack ligand-binding sites.
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| Molecular Formula |
C25H27N3O3
|
|---|---|
| Molecular Weight |
417.50
|
| Exact Mass |
417.205
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| Elemental Analysis |
C, 71.92; H, 6.52; N, 10.06; O, 11.50
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| CAS # |
2291360-73-9
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| PubChem CID |
137519326
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| Appearance |
White to off-white solid powder
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| LogP |
2.3
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
4
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
31
|
| Complexity |
696
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
N1C(=O)CCC(N2CC3=C(C2=O)C=CC(C2CCN(CC4=CC=CC=C4)CC2)=C3)C1=O
|
| InChi Key |
OMISHRJQMYQPMG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H27N3O3/c29-23-9-8-22(24(30)26-23)28-16-20-14-19(6-7-21(20)25(28)31)18-10-12-27(13-11-18)15-17-4-2-1-3-5-17/h1-7,14,18,22H,8-13,15-16H2,(H,26,29,30)
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| Chemical Name |
3-[6-(1-benzylpiperidin-4-yl)-3-oxo-1H-isoindol-2-yl]piperidine-2,6-dione
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| Synonyms |
NVP-DKY709; DKY-709; NVP-DKY-709; 2291360-73-9; NVP-DKY 709; DKY709; 3-(5-(1-benzylpiperidin-4-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione; NVP-DKY709?; CHEMBL5077506; SCHEMBL20765247;
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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 |
| 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 : ~16.67 mg/mL (~39.93 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (2.40 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (2.40 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 10.0 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. View More
Solubility in Formulation 3: ≥ 1 mg/mL (2.40 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3952 mL | 11.9760 mL | 23.9521 mL | |
| 5 mM | 0.4790 mL | 2.3952 mL | 4.7904 mL | |
| 10 mM | 0.2395 mL | 1.1976 mL | 2.3952 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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