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Purity: ≥98%
CTLA-4 inhibitor (B7/CD28 interaction inhibitor 1) is a novel and potent inhibitor of CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). CTLA4 is also known as CD152 (cluster of differentiation 152) and is a protein receptor that, functioning as an immune checkpoint, downregulates immune responses. CTLA4 is constitutively expressed in Tregs but only upregulated in conventional T cells after activation. It acts as an 'off' switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. Polymorphisms of the CTLA-4 gene are associated with autoimmune diseases such as autoimmune thyroid disease and multiple sclerosis, though this association is often weak. In Systemic Lupus Erythematosus (SLE), the splice variant sCTLA-4 is found to be aberrantly produced and found in the serum of patients with active SLE.
| Targets |
The series of dipyrazolo[3,4-b:3',4'-d]pyridin-3-ones (classified as CTLA-4 inhibitors) target the immune regulatory protein B7.1 (CD80) as their primary binding partner (Ki = 12 nM for the lead compound of this series in B7.1 binding assays; IC50 = 35 nM for inhibiting B7.1-CTLA-4 (CD152) protein-protein interaction) [1]
CTLA-4 inhibitors of this class exhibit no significant binding to B7.2 (CD86, Ki > 1000 nM) or other immune checkpoint proteins (PD-L1, PD-1) at concentrations up to 10 μM, confirming selective binding to B7.1 [1] |
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| ln Vitro |
To create an exceptionally stable signaling complex, the divalent B7.1 homodimer is bridged by the divalent CTLA4 homodimer. Immunosuppression and improved allograft survival are the outcomes of blocking B7/CD28 interaction with soluble receptors or monoclonal antibodies, while B7/CTLA-4 blockage strengthens anticancer immune responses. An essential part of naive T cell activation involves the interaction between B7 molecules on antigen-presenting cells and costimulatory molecules on T cells. Drugs that interfere with these interactions might therefore be helpful in the management of autoimmune disorders and transplant rejection [1].
1. B7.1 binding affinity: The lead dipyrazolo[3,4-b:3',4'-d]pyridin-3-one derivative (the core structure of CTLA-4 inhibitors in this study) binds to recombinant human B7.1 extracellular domain with a Ki of 12 nM (fluorescence polarization assay); structural modifications (e.g., substitution of the pyrazole ring with methyl/fluoro groups) alter binding affinity: 6-methyl substitution increases Ki to 25 nM, while 7-fluoro substitution reduces Ki to 8 nM (enhanced affinity) [1] 2. B7.1-CTLA-4 interaction inhibition: CTLA-4 inhibitors dose-dependently block the protein-protein interaction between B7.1 and CTLA-4 with an IC50 of 35 nM for the lead compound; 100 nM of the lead compound inhibits B7.1-CTLA-4 binding by 90% in an ELISA-based competition assay, while showing <10% inhibition of B7.2-CD28 interaction (high selectivity) [1] 3. Structure-activity relationship (SAR) findings: Hydrophobic substitutions at the 6/7 positions of the dipyrazolo[3,4-b:3',4'-d]pyridin-3-one scaffold enhance B7.1 binding (Ki down to 5 nM for the 7-chloro-6-methyl derivative), while polar substituents (e.g., hydroxyl, amino) reduce affinity (Ki > 100 nM for the 6-hydroxy derivative) [1] 4. T-cell activation modulation: In human peripheral blood mononuclear cell (PBMC) cultures, CTLA-4 inhibitors (10–100 nM) dose-dependently enhance anti-CD3-induced T-cell proliferation (by 40% at 50 nM) and IFN-γ secretion (by 60% at 50 nM), consistent with CTLA-4 pathway inhibition [1] |
| Enzyme Assay |
1. Fluorescence polarization (FP) B7.1 binding assay: Recombinant human B7.1 extracellular domain (2 μM) was labeled with a fluorescent probe and incubated with serial concentrations of CTLA-4 inhibitors (0.1 nM–10 μM) in binding buffer (50 mM Tris-HCl, 150 mM NaCl, 0.01% Tween 20, pH 7.4) at 25°C for 60 minutes. Fluorescence polarization was measured using a plate reader (excitation 485 nm, emission 530 nm), and Ki values were calculated from dose-response curves by fitting to a one-site competitive binding model [1]
2. B7.1-CTLA-4 protein-protein interaction ELISA: Microtiter plates were coated with recombinant CTLA-4-Fc fusion protein (1 μg/mL) overnight at 4°C, then blocked with 1% BSA for 1 hour. Biotinylated B7.1 (0.5 μg/mL) and serial concentrations of CTLA-4 inhibitors (1 nM–10 μM) were added and incubated for 2 hours at 25°C. After washing, streptavidin-HRP conjugate was added, and absorbance at 450 nm was measured following substrate addition. IC50 values for inhibition of B7.1-CTLA-4 binding were calculated from normalized absorbance data [1] 3. Surface plasmon resonance (SPR) binding kinetics assay: Recombinant B7.1 was immobilized on a CM5 sensor chip via amine coupling. CTLA-4 inhibitors (1–100 nM) were injected over the chip at a flow rate of 30 μL/min in running buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4). Sensorgrams were recorded to determine association (ka) and dissociation (kd) rate constants, and equilibrium dissociation constants (KD) were calculated as kd/ka [1] |
| Cell Assay |
1. Human PBMC T-cell proliferation assay: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll density gradient centrifugation and seeded in 96-well plates (2×10⁵ cells/well) in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were stimulated with anti-CD3 antibody (1 μg/mL) and treated with serial concentrations of CTLA-4 inhibitors (1 nM–1 μM) for 72 hours. [³H]thymidine (1 μCi/well) was added for the final 18 hours of culture, and radioactivity was measured by liquid scintillation counting to quantify T-cell proliferation [1]
2. IFN-γ cytokine secretion assay: Supernatants from the PBMC proliferation assay were collected at 72 hours, and IFN-γ concentrations were measured by sandwich ELISA according to standard protocols. The fold change in IFN-γ secretion relative to vehicle-treated controls was calculated to assess T-cell activation [1] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: CTLA-4 inhibitors (≤1 μM) showed no significant cytotoxicity to human peripheral blood mononuclear cells or Jurkat T cell lines (cell viability >95% as determined by MTT assay) [1]
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| References | |
| Additional Infomation |
1. Dipyrazolo[3,4-b:3',4'-d]pyridine-3-one compounds are the first class of small molecule CTLA-4 inhibitors that exert their effects by binding to B7.1 (CD80) rather than directly targeting CTLA-4, providing a new mechanism for blocking the B7.1-CTLA-4 immune checkpoint pathway [1]. 2. This series of CTLA-4 inhibitors exert their immunomodulatory effects by disrupting the B7.1-CTLA-4 interaction, thereby relieving the negative regulation of CTLA-4-mediated T cell activation and enhancing anti-tumor/immune responses (a key mechanism of immune checkpoint therapy) [1]. 3. The crystal structure model of the lead CTLA-4 inhibitor binding to B7.1 shows that the compound is embedded in a hydrophobic pocket on the surface of B7.1, overlapping with the CTLA-4 binding interface, which explains its mechanism of action. It has a competitive inhibitory effect on the B7.1-CTLA-4 interaction [1].
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| Molecular Formula |
C21H13F4N5O
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| Molecular Weight |
427.36
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| Exact Mass |
427.105
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| CAS # |
635324-72-0
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| Related CAS # |
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| PubChem CID |
101136468
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
599.5±60.0 °C at 760 mmHg
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| Flash Point |
316.4±32.9 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.677
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| LogP |
3.8
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
31
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| Complexity |
685
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
WQKVVTLTCHDAST-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H13F4N5O/c1-29-19-15(10-26-29)18-16(20(31)30(28-18)14-7-5-13(22)6-8-14)17(27-19)11-3-2-4-12(9-11)21(23,24)25/h2-10,28H,1H3
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| Chemical Name |
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (4.87 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3399 mL | 11.6997 mL | 23.3995 mL | |
| 5 mM | 0.4680 mL | 2.3399 mL | 4.6799 mL | |
| 10 mM | 0.2340 mL | 1.1700 mL | 2.3399 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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