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Purity: ≥98%
PD1-PDL1 inhibitor 2 (also known as BMS-202, BMS 202, BMS202), is a novel and potent inhibitor of the PD-1 (Programmed death- 1)/PD-Ll (Programmed death-ligand 1) protein/protein interaction with potential antineoplastic activity. This information can be found in compound example 202 of patent WO/2015034820 A1. BMS-202 has good in vitro activity, with IC50 values of less than 50 nM in the cell-free assay, according to homogenous time-resolved fluorescence (HTRF) assay results. However, there are no known in vivo data for this compound. Small molecule BMS-202, which binds to and causes the dimerization of PD-L1, has a structural basis for blocking the PD-1/PD-L1 interaction.
Targets |
PD-1/PD-L1 (IC50 = 18 nM); PD-1/PD-L1 (KD = 8 μM)
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ln Vitro |
BMS-202 (0-100 μM; 4 days; SCC-3 or Jurkat cells) treatment prevents the proliferation of strongly PD-L1 positive SCC-3 cells (IC50 of 15 μM) and anti-CD3 antibody-activated Jurkat cells (IC50 10 μM) in vitro[2]. BMS-202 specifically induces PD-L1 thermal stabilization. BMS-202 causes PD-L1 dimerization in solution. At the homodimer's core, BMS-202 fills a large hydrophobic pocket, facilitating numerous additional interactions between the monomers. PD-1/PD-L1 interaction is physiologically mediated by hydrophobic surfaces, and BMS-202 interacts with both PD-L1 molecules on these surfaces[1].
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ln Vivo |
BMS-202 (20 mg/kg; intraperitoneal injection; daily; for 9 days; NOG-dKO mice) treatment exhibits a distinct antitumor effect in comparison to the controls, in humanized MHC- dKO NOG mice[2].
Moreover, data from tumor-bearing nude-mice xenografts supported that BMS-202 significantly inhibited the growth of U251 cells in vivo. Above all, these in vivo and in vitro data demonstrated that BMS-202 significantly inhibited the growth of GBM cells without affecting normal glial cells, implicating a safe therapeutic window for its antitumor application in GBM. |
Enzyme Assay |
All binding studies are performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (with v) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-l-Ig/PD-Ll-His binding assay, inhibitors are pre-incubated with PD-Ll-His (10 nM final) for 15 m in 4 μL of assay buffer, followed by addition of PD-l-Ig (20 nM final) in 1 μL of assay buffer and further incubation for 15 m. PD-L1 from either human, cyno, or mouse are used. HTRF detection is achieved using europium crypate-labeled anti- Ig (1 nM final) and allophycocyanin (APC) labeled anti-His (20 nM final). Antibodies are diluted in HTRF detection buffer and 5 μL is dispensed on top of binding reaction. The reaction mixture is allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) is obtained using an En Vision fluorometer. Additional binding assays are established between PD-1-Ig/PD-L2-His (20, 5 nM, respectively), CD80-His/PD-Ll-Ig (100, 10 nM, respectively) and CD80-His/CTLA4-Ig (10, 5 nM, respectively).
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Cell Assay |
The programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) interaction plays a dominant role in the suppression of T cell responses, especially in a tumor microenvironment, protecting tumor cells from lysis. PD-1/PD-L1 inhibitor 2 is reported to prevent the interaction of PD-L1 with PD-1 with an IC50 value of 18 nM.
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Animal Protocol |
In an in vivo study using humanized MHC-double knockout (dKO) NOG mice, BMS-202 showed a clear antitumor effect compared with the controls; however, a direct cytotoxic effect was revealed to be involved in the antitumor mechanism, as there was no lymphocyte accumulation in the tumor site. These results suggest that the antitumor effect of BMS-202 might be partly mediated by a direct off-target cytotoxic effect in addition to the immune response-based mechanism. Also, the humanized dKO NOG mouse model used in this study was shown to be a useful tool for the screening of small molecule inhibitors of PD-1/PD-L1 binding that can inhibit tumor growth via an immune-response-mediated mechanism[2].
In vivo therapeutic tumor-bearing xenografts with BMS-202[3] According to our previous studies, 5 × 10~6 U251 cells were mixed with matrigel and subcutaneously injected into the male NOD/SCID nude mice, aged 4–6 weeks (n = 8). When the tumor volume (0.5 × length × width2) reached 100 mm3, the mice were randomly divided into the control group, treated with vehicle, and the BMS-202 group, intraperitoneally injected with 20 mg/kg BMS-202, twice per week. The therapeutic process was stopped until the tumor volumes in the control group reached the ethically approved maximum volume 2000 mm3. |
References |
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Additional Infomation |
Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has provided unprecedented results in cancer treatment in the recent years. Development of chemical inhibitors for this pathway lags the antibody development because of insufficient structural information. The first nonpeptidic chemical inhibitors that target the PD-1/PD-L1 interaction have only been recently disclosed by Bristol-Myers Squibb. Here, we show that these small-molecule compounds bind directly to PD-L1 and that they potently block PD-1 binding. Structural studies reveal a dimeric protein complex with a single small molecule which stabilizes the dimer thus occluding the PD-1 interaction surface of PD-L1s. The small-molecule interaction "hot spots" on PD-L1 surfaces suggest approaches for the PD-1/PD-L1 antagonist drug discovery.[1]
Recently, the first series of small molecule inhibitors of PD-1/PD-L1 were reported by Bristol-Myers Squibb (BMS), which were developed using a homogeneous time-resolved fluorescence (HTRF)-based screening investigation of the PD-1/PD-L1 interaction. Additional crystallographic and biophysical studies showed that these compounds inhibited the interaction of PD-1/PD-L1 by inducing the dimerization of PD-L1, in which each dimer binds one molecule of the stabilizer at its interface. However, the immunological mechanism of the antitumor effect of these compounds remains to be elucidated. In the present study, we focused on BMS-202 (a representative of the BMS compounds) and investigated its antitumor activity using in vitro and in vivo experiments. BMS-202 inhibited the proliferation of strongly PD-L1-positive SCC-3 cells (IC50 15 μM) and anti-CD3 antibody-activated Jurkat cells (IC50 10 μM) in vitro. Additionally, BMS-202 had no regulatory effect on the PD-1 or PD-L1 expression level on the cell surface of these cells. In an in vivo study using humanized MHC-double knockout (dKO) NOG mice, BMS-202 showed a clear antitumor effect compared with the controls; however, a direct cytotoxic effect was revealed to be involved in the antitumor mechanism, as there was no lymphocyte accumulation in the tumor site. These results suggest that the antitumor effect of BMS-202 might be partly mediated by a direct off-target cytotoxic effect in addition to the immune response-based mechanism. Also, the humanized dKO NOG mouse model used in this study was shown to be a useful tool for the screening of small molecule inhibitors of PD-1/PD-L1 binding that can inhibit tumor growth via an immune-response-mediated mechanism.[2] Recently, crystallographic studies have demonstrated that BMS-202, a small-molecule compound characterized by a methoxy-1-pyridine chemical structure, exhibits a high affinity to PD-L1 dimerization. However, its roles and mechanisms in glioblastoma (GBM) remain unclear. The objective of this study is to investigate the antitumor activity of BMS-202 and its underlying mechanisms in GBM using multi-omics and bioinformatics techniques, along with a majority of in vitro and in vivo experiments, including CCK-8 assays, flow cytometry, co-immunoprecipitation, siRNA transfection, PCR, western blotting, cell migration/invasion assays and xenografts therapeutic assays. Our findings indicate that BMS-202 apparently inhibits the proliferation of GBM cells both in vitro and in vivo. Besides, it functionally blocks cell migration and invasion in vitro. Mechanistically, it reduces the expression of PD-L1 on the surface of GBM cells and interrupts the PD-L1-AKT-BCAT1 axis independent of mTOR signaling. Taken together, we conclude that BMS-202 is a promising therapeutic candidate for patients with GBM by remodeling their cell metabolism regimen, thus leading to better survival.[3] |
Molecular Formula |
C25H29N3O3
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Molecular Weight |
419.52
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Exact Mass |
419.22
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Elemental Analysis |
C, 71.57; H, 6.97; N, 10.02; O, 11.44
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CAS # |
1675203-84-5
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Related CAS # |
N-deacetylated BMS-202;2310135-18-1
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PubChem CID |
117951478
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Appearance |
White to off-white solid powder
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Density |
1.1±0.1 g/cm3
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Boiling Point |
611.4±55.0 °C at 760 mmHg
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Flash Point |
323.6±31.5 °C
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Vapour Pressure |
0.0±1.8 mmHg at 25°C
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Index of Refraction |
1.575
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LogP |
3.99
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Hydrogen Bond Donor Count |
2
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Hydrogen Bond Acceptor Count |
5
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Rotatable Bond Count |
10
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Heavy Atom Count |
31
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Complexity |
526
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Defined Atom Stereocenter Count |
0
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SMILES |
O(C1C([H])=C([H])C(=C(N=1)OC([H])([H])[H])C([H])([H])N([H])C([H])([H])C([H])([H])N([H])C(C([H])([H])[H])=O)C([H])([H])C1C([H])=C([H])C([H])=C(C2C([H])=C([H])C([H])=C([H])C=2[H])C=1C([H])([H])[H]
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InChi Key |
JEDPSOYOYVELLZ-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C25H29N3O3/c1-18-22(10-7-11-23(18)20-8-5-4-6-9-20)17-31-24-13-12-21(25(28-24)30-3)16-26-14-15-27-19(2)29/h4-13,26H,14-17H2,1-3H3,(H,27,29)
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Chemical Name |
2-[2,6-dichloro-4-(3,5-dimethyl-1,2-oxazol-4-yl)anilino]-N-hydroxybenzamide
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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: ≥ 2.5 mg/mL (5.96 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 (5.96 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.96 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 4.05 mg/mL (9.65 mM) in 45% PEG300 5% Tween-80 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 2.3837 mL | 11.9184 mL | 23.8368 mL | |
5 mM | 0.4767 mL | 2.3837 mL | 4.7674 mL | |
10 mM | 0.2384 mL | 1.1918 mL | 2.3837 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.
J Med Chem.2017Jul 13;60(13):5857-5867. th> |
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J Med Chem.2017Jul 13;60(13):5857-5867. |
J Med Chem.2017Jul 13;60(13):5857-5867. |
Structural Biology of the Immune Checkpoint Receptor PD-1 and Its Ligands PD-L1/PD-L2.Structure.2017 Aug 1;25(8):1163-1174. th> |
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New Directions in Designing the Therapeutics Targeting the PD-1/PD-L1 Interaction.Structure.2017 Aug 1;25(8):1163-1174. td> |
Structural Basis of the PD-1/PD-L1 (PD-L2) Interaction.Structure.2017 Aug 1;25(8):1163-1174. td> |