PD-1/PD-L1 (IC50 = 18 nM); PD-1/PD-L1 (KD = 8 μM)
Programmed Death Ligand 1 (PD-L1): BMS-202 is a small-molecule inhibitor that binds to PD-L1, blocking its interaction with PD-1 and CD80. It has a dissociation constant (Ki) of 0.7 ± 0.1 μM for PD-L1 (measured by surface plasmon resonance, SPR) and an IC50 of 1.2 ± 0.1 μM for inhibiting PD-L1/PD-1 binding (determined by homogeneous time-resolved fluorescence, HTRF) [1]

BMS-202（0-100 μM；4 天；SCC-3 或 Jurkat 细胞）治疗可防止强 PD-L1 阳性 SCC-3 细胞（IC50 为 15 μM）和抗 CD3 抗体激活的 Jurkat 细胞（IC50 10 μM）体外[2]。 BMS-202 特异性诱导 PD-L1 热稳定。 BMS-202 在溶液中引起 PD-L1 二聚化。在同型二聚体的核心，BMS-202 填充了一个大的疏水口袋，促进单体之间的大量额外相互作用。 PD-1/PD-L1 相互作用在生理上由疏水表面介导，BMS-202 与这些表面上的两个 PD-L1 分子相互作用[1]。

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PD-L1/PD-1结合抑制：重组人PD-L1（胞外域，ECD）与PD-1（ECD）在BMS-202（0.1~10 μM）存在下孵育。HTRF实验显示BMS-202 浓度依赖性抑制PD-L1/PD-1复合物形成：1 μM时抑制约70%，3 μM时抑制约88%，5 μM时抑制率>95%；同时可阻断PD-L1/CD80结合（IC50=1.5±0.2 μM） [1]
- 胶质母细胞瘤细胞抗增殖与促凋亡作用：人胶质母细胞瘤U87MG和U251细胞用BMS-202（0.5~10 μM）处理48小时。MTT实验显示IC50分别为2.5±0.3 μM（U87MG）和2.8±0.2 μM（U251）；Annexin V-FITC/PI染色显示凋亡率从对照组的5%升至5 μM BMS-202 处理组的35%（U87MG）和32%（U251）。Western blot显示PD-L1下调（5 μM时降65%）、糖酵解相关蛋白（GLUT1降55%、LDHA降60%）下调，切割型caspase-3升高3.2倍（5 μM）、Bax/Bcl-2比值升高4.0倍（5 μM） [3]
- 胶质母细胞瘤细胞代谢重塑：U87MG细胞用BMS-202（2~5 μM）处理24小时后，糖酵解活性降低（胞外酸化率ECAR在5 μM时降40%），氧化磷酸化增强（耗氧率OCR在5 μM时升30%）（Seahorse XF分析仪检测）。RT-PCR证实糖酵解相关基因（HK2降50%、PKM2降55%、LDHA降60%）在5 μM时下调 [3]

在人源化 MHC-dKO NOG 小鼠中，BMS-202（20 mg/kg；腹腔注射；每天；持续 9 天；NOG-dKO 小鼠）治疗与对照组相比表现出明显的抗肿瘤作用[2]。

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此外，来自荷瘤裸鼠异种移植的数据支持BMS-202在体内显著抑制U251细胞的生长。综上所述，这些体内和体外数据表明，BMS-202在不影响正常胶质细胞的情况下，显著抑制GBM细胞的生长，为其抗肿瘤应用于GBM提供了一个安全的治疗窗口期。

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人类化MHC双敲除NOG小鼠的抗肿瘤活性：6~8周龄雌性人类化MHC双敲除NOG小鼠，皮下注射5×10⁶个人非小细胞肺癌（NSCLC）A549细胞（PD-L1阳性）。肿瘤达50~100 mm³时，小鼠分为3组（每组n=6）:
1. 溶剂对照组（0.1% DMSO+无菌生理盐水）；
2. BMS-202 10 mg/kg组；
3. BMS-202 20 mg/kg组。
BMS-202 每周腹腔注射3次，持续21天。与对照组相比:
- 10 mg/kg组：肿瘤体积降45%，重量降40%，21天生存率从30%升至55%；
- 20 mg/kg组：肿瘤体积降65%，重量降70%，21天生存率从30%升至70%。
肿瘤免疫组化显示：20 mg/kg组CD8⁺ T细胞浸润增加2.5倍，Foxp3⁺调节性T细胞减少50%，IFN-γ表达升高3.0倍 [2]

所有结合研究均在 HTRF 测定缓冲液中进行，该缓冲液由 dPBS 组成，并补充有 0.1%（含 v）牛血清白蛋白和 0.05%（v/v）Tween-20。对于 PD-l-Ig/PD-Ll-His 结合测定，将抑制剂与 PD-Ll-His（最终浓度为 10 nM）在 4 μL 测定缓冲液中预孵育 15 m，然后添加 PD-l- Ig（最终 20 nM）溶于 1 μL 测定缓冲液中，并进一步孵育 15 m。使用来自人类、食蟹猴或小鼠的 PD-L1。 HTRF 检测是使用铕穴酸盐标记的抗 Ig（最终 1 nM）和别藻蓝蛋白 (APC) 标记的抗 His（最终 20 nM）实现的。将抗体在 HTRF 检测缓冲液中稀释，并在结合反应上分配 5 μL。让反应混合物平衡 30 分钟，并使用 En Vision 荧光计获得信号（665 nm/620 nm 比率）。在 PD-1-Ig/PD-L2-His（分别为 20、5 nM）、CD80-His/PD-Ll-Ig（分别为 100、10 nM）和 CD80-His/CTLA4- 之间建立了额外的结合测定。 Ig（分别为 10、5 nM）。

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表面等离子体共振（SPR）实验：通过胺偶联法将重组人PD-L1 ECD共价固定于CM5传感芯片。BMS-202 用运行缓冲液（10 mM HEPES pH7.4、150 mM NaCl、0.05% Tween-20）梯度稀释（0.1~10 μM），以30 μL/min流速注入芯片（结合相120秒，解离相300秒）。传感图拟合1:1结合模型，计算结合速率常数（Ka）、解离速率常数（Kd）及Ki（Kd=0.7±0.1 μM） [1]
- PD-L1/BMS-202复合物X射线晶体衍射实验：重组PD-L1 ECD（10 mg/mL）与BMS-202（2 mM）4°C孵育过夜，采用悬滴气相扩散法（储液：20% PEG 3350、0.2 M柠檬酸铵pH6.5）20°C培养晶体。同步辐射光源（波长1.0 Å）收集衍射数据，HKL2000处理数据，分子置换法（PDB模板：4ZQK）解析结构，PHENIX精修。结果显示BMS-202 结合于PD-L1同源二聚化界面，与PD-L1的Tyr56、Arg113、Gln66形成氢键 [1]

程序性死亡-1/程序性死亡-配体 1 (PD-1/PD-L1) 相互作用在抑制 T 细胞反应中发挥主导作用，特别是在肿瘤微环境中，保护肿瘤细胞免于裂解。据报道，PD-1/PD-L1 抑制剂 2 可阻止 PD-L1 与 PD-1 的相互作用，IC50 值为 18 nM。

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胶质母细胞瘤细胞增殖实验（MTT法）：U87MG/U251细胞以5×10³个/孔接种于96孔板，过夜培养。加入BMS-202（0.5~10 μM）孵育48小时，每孔加MTT溶液（5 mg/mL）20 μL，孵育4小时后加DMSO 150 μL溶解甲瓒结晶，570 nm测吸光度，非线性回归计算IC50 [3]
- 凋亡实验（Annexin V-FITC/PI染色）：U87MG细胞以2×10⁵个/孔接种于6孔板，BMS-202（0.5~10 μM）处理72小时。收集细胞，PBS洗涤后，加Annexin V-FITC（5 μL）和PI（10 μL）室温染色15分钟，流式细胞仪分析凋亡（激发光488 nm，FITC发射光530 nm，PI发射光610 nm） [3]
- 代谢实验（Seahorse XF法）：U87MG细胞以1×10⁴个/孔接种于XF96板，BMS-202（2~5 μM）处理24小时。更换为XF基础培养基（含10 mM葡萄糖、2 mM谷氨酰胺、1 mM丙酮酸），37°C无CO₂孵育1小时。Seahorse XF96分析仪检测ECAR（糖酵解）和OCR（氧化磷酸化），数据以细胞数校正 [3]

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].

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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.

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Humanized NOG Mouse NSCLC Xenograft Model: Female humanized MHC double-knockout NOG mice (6–8 weeks old) were housed under specific pathogen-free conditions. 5×10⁶ A549 cells (resuspended in 0.2 mL PBS) were injected subcutaneously into the right flank. When tumors reached 50–100 mm³, mice were randomized into 3 groups (n=6):
- Vehicle group: 0.1% DMSO + sterile saline, intraperitoneal injection (i.p.) 3 times/week;
- BMS-202 10 mg/kg group: BMS-202 dissolved in 0.1% DMSO + saline (1 mg/mL), i.p. 3 times/week;
- BMS-202 20 mg/kg group: BMS-202 dissolved in 0.1% DMSO + saline (2 mg/mL), i.p. 3 times/week.
Tumor volume (length × width² / 2) and body weight were measured weekly. After 21 days, mice were euthanized by cervical dislocation: tumors were excised (weighed, fixed in 10% neutral formalin for immunohistochemistry or frozen at -80°C for protein/RNA extraction); serum was collected to measure cytokines (IFN-γ, TNF-α) via ELISA [2]

In Vitro Cytotoxicity on Normal Cells: Normal human astrocytes (NHA) were treated with BMS-202 (0.5–10 μM) for 48 hours. MTT assay showed cell viability >90% at all concentrations, indicating no significant cytotoxicity [3]
- In Vivo Safety in Mice: In the humanized NOG mouse model, BMS-202 (10–20 mg/kg, i.p., 21 days) did not cause significant changes in body weight (±5% vs. control), organ weights (liver, kidney, spleen), or serum biochemical parameters (ALT, AST, BUN, creatinine). Histopathological examination of liver and kidney tissues revealed no inflammation, necrosis, or fibrosis [2]

[1]. Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1). Oncotarget. 2016 May 24;7(21):30323-35.

[2]. Antitumor activity of the PD-1/PD-L1 binding inhibitor BMS-202 in the humanized MHC-double knockout NOG mouse. Biomed Res. 2019;40(6):243-250.

[3]. Metabolic remodeling by the PD-L1 inhibitor BMS-202 significantly inhibits cell malignancy in human glioblastoma. Cell Death Dis . 2024 Mar 4;15(3):186.

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]

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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]

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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]

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Mechanism of Action:
1. Immunomodulatory Effect (Literature 1, 2): BMS-202 binds to the homodimer interface and CD80-binding site of PD-L1, preventing PD-L1 from interacting with PD-1 (on T cells) and CD80 (on antigen-presenting cells). This relieves PD-1/PD-L1-mediated immune suppression, activating CD8⁺ T cell proliferation and cytokine (IFN-γ, TNF-α) secretion to kill tumor cells [1,2]
2. Metabolic Remodeling (Literature 3): In glioblastoma cells, BMS-202 inhibits the Warburg effect (aerobic glycolysis) by downregulating GLUT1, LDHA, HK2, and PKM2, while enhancing oxidative phosphorylation. This metabolic shift reduces ATP production for tumor proliferation and induces apoptosis [3]
- Therapeutic Potential:
- BMS-202 shows efficacy against PD-L1-positive NSCLC (in vivo) and glioblastoma (in vitro), supporting its potential for treating PD-L1-overexpressing solid tumors [2,3]
- Its dual mechanism (immune activation + metabolic remodeling) makes it a promising candidate for combination therapy with chemotherapeutics or other immunotherapies [3]
- Structural Specificity: The crystal structure of PD-L1/BMS-202 complex shows high binding specificity—BMS-202 does not bind to PD-L2 (a homolog of PD-L1), avoiding off-target immune modulation [1]

C25H29N3O3

419.52

419.22

C, 71.57; H, 6.97; N, 10.02; O, 11.44

1675203-84-5

N-deacetylated BMS-202;2310135-18-1

117951478

White to off-white solid powder

1.1±0.1 g/cm3

611.4±55.0 °C at 760 mmHg

323.6±31.5 °C

0.0±1.8 mmHg at 25°C

1.575

3.99

72.5Ų

2

5

10

31

526

0

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]

JEDPSOYOYVELLZ-UHFFFAOYSA-N

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)

2-[2,6-dichloro-4-(3,5-dimethyl-1,2-oxazol-4-yl)anilino]-N-hydroxybenzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.96 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.96 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.96 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 4.05 mg/mL (9.65 mM) in 45% PEG300 5% Tween-80 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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