C188-9

The target of C188-9 is Signal Transducer and Activator of Transcription 3 (STAT3), and it also has potent activity against STAT1.
- For STAT3: In SPR assays, the IC₅₀ for inhibiting recombinant STAT3 binding to phosphododecapeptide ligand is not explicitly given in numerical value, but it binds to STAT3 with high affinity (K_{D} was calculated from sigmoidal binding curve with concentrations of C188-9 ranging from 0.305 to 10,000 nM) [3]
- For G-CSF-induced pSTAT3 in AML cell lines: IC₅₀ values are between 4 and 8 μM [2,4]
- For constitutive pSTAT3 in UM-SCC-17B (HNSCC cell line): IC₅₀ was calculated from dose-response curves with C188-9 concentrations ranging from 0 to 100 μM [3]
- For constitutive pSTAT1 in UM-SCC-17B (HNSCC cell line): IC₅₀ was calculated from dose-response curves with C188-9 concentrations ranging from 0 to 100 μM [3]

C188-9 (Kd = 4.7 nM) 是一种 Stat3 抑制剂 [1]。 C188-9 在 AML 细胞系和原发性 AML 样品中抑制 Stat3 激活的 IC50 值范围为 4-7 μM 和 8-18 μM。为了在细胞凋亡研究中测量膜联蛋白 V 标记细胞的凋亡细胞，将 C188-9 应用于 AML 细胞系和原代样品，持续 24 小时。从 6 μM 到超过 50 μM，诱导细胞凋亡的 EC50 表现出显着的变异性 [2]。

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1. 癌症恶病质相关细胞模型：C188-9可阻断C26结肠癌细胞条件培养基诱导的C2C12肌管中p-Stat3激活（处理15分钟）；减少C2C12肌管在C26条件培养基中孵育72小时后的平均尺寸损失；抑制C26条件培养基处理的C2C12肌管中caspase-3的表达与激活（包括pro-caspase-3和裂解型caspase-3），并抑制C/EBPδ和肌抑素（myostatin）的上调；此外，还可减少C26条件培养基诱导的C2C12肌管中肌球蛋白重链蛋白的丢失（处理72小时），降低C2C12肌管中MAFbx/Atrogin-1和MuRF-1的mRNA水平（处理24小时）[1]
2. 急性髓系白血病（AML）细胞系：C188-9以剂量依赖方式抑制G-CSF诱导的Stat3磷酸化（预处理1小时后用100 ng/mL G-CSF刺激15分钟），6种AML细胞系的IC₅₀值在4至8 μM之间，且不改变总Stat3蛋白水平；不影响酪氨酸激酶蛋白阵列中大多数酪氨酸激酶的酪氨酸磷酸化水平，也不抑制MAPK/ERK通路或Akt通路的活性（通过pERK1/2和pAkt免疫反应性评估）；抑制3种AML细胞系中G-CSF诱导的Stat3靶基因（SOCS3和PIM1 mRNA）表达（10 μM C188-9预处理1小时后用100 ng/mL G-CSF刺激1小时）；处理24小时后可诱导AML细胞系凋亡（通过膜联蛋白V–PE标记定量，EC₅₀值由剂量-反应曲线估算）[2,4]
3. 原发性儿童AML样本：C188-9以剂量依赖方式抑制G-CSF诱导的pStat3（通过流式细胞术检测）；孵育48小时后可诱导CD34⁺原发性AML细胞凋亡（通过膜联蛋白V染色定量）；减少含生长因子的甲基纤维素培养基中培养的原发性AML样本的集落形成（集落≥30个细胞，以未处理组为对照进行归一化）[2,4]
4. 头颈部鳞状细胞癌（HNSCC）细胞系：C188-9处理24小时后（浓度0至100 μM），以剂量依赖方式抑制UM-SCC-17B细胞中组成型pSTAT3和pSTAT1的表达；处理48小时后（浓度0至100 μM）抑制UM-SCC-17B细胞的贴壁依赖性生长（通过MTT法定量活细胞数，IC₅₀由剂量-反应曲线计算）；处理72小时后抑制多种HNSCC细胞系（SCC-35、SCC-61、UM-SCC-17B、HN30）的非贴壁依赖性生长（IC₅₀值由剂量-反应曲线计算）[3]

在大约 13,528 个可区分基因中，C188 修改了 37 个基因转录本的水平（17 个下调，20 个上调，fdr <0.01，倍数变化≥1.5），其中 7 个被确定为 STAT3 基因靶标。先前确定受 STAT3 调节的 76 个基因（38 个下调和 38 个上调）属于 C188-9 影响的大量肿瘤发生相关基因（总共 384 个，95 个下调和 289 个上调） ）。当然，C188-9 治疗会导致 38 个基因中的 24 个 (63%) 下调，而之前报道这些基因会被 STAT3 上调。此外，C188-9 下调了另外 10 个基因（fdr <0.01，倍数变化≥1.5），这些基因先前已被证明被 STAT1 上调。因此，STAT1 已被证明可以正向调节先前被 C188-9 下调的 48 个基因中的 40 个（83.3%），其中包括已被证明由 STAT3 和 STAT1 共同调节的 16 个基因。该分析表明，C188-9 可能同时影响 STAT3 和 STAT1，从而介导其对 HNSCC 肿瘤中基因转录水平的影响[3]。

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1. 癌症恶病质动物模型：给接种C26肿瘤5天的CD2F1小鼠每日两次施用C188-9或D5W，持续14天。C188-9可抑制小鼠体重和肌肉量（腓肠肌、胫骨前肌）的丢失，改善胫骨前肌肌纤维尺寸分布，提升肌肉握力，增强肌肉蛋白质合成，并降低比目鱼肌和趾长伸肌（EDL）的蛋白质降解；同时降低腓肠肌中C/EBPδ、肌抑素、MAFbx/Atrogin-1的表达及14 kDa肌动蛋白片段（caspase-3活性指标）的水平，抑制肌肉组织中的蛋白酶体活性（以荧光肽LLVY-AMC为底物检测）[1]
2. 头颈部鳞状细胞癌（HNSCC）异种移植模型：将1.5×10⁶个UM-SCC-17B细胞注射至8-10周龄雄性裸鼠舌部，待肿瘤形成（平均体积约15-20 mm³）后，每周5次给小鼠腹腔注射C188-9（100 mg/Kg）。C188-9可抑制肿瘤异种移植物的生长（每周两次测量肿瘤体积，以首次给药日体积为基准归一化，t检验P<0.05），并降低肿瘤裂解物中pSTAT3和pSTAT1的水平（通过Western blot检测，以β-肌动蛋白归一化）[3]

1. 检测STAT3-配体结合抑制的SPR实验：将重组STAT3（200 nM）与不同浓度的C188-9（0.1至1000 μM）孵育后，加入固定有磷酸十二肽配体（包含EGFR的pY1068及周边12个氨基酸）的Biacore传感器芯片；测量平衡结合水平并归一化（加药组共振值÷未加药组共振值×100），以Log [nM] C188-9为横坐标绘图，计算IC₅₀[3]
2. 检测STAT3结合亲和力的荧光实验：将恒定浓度（80 nM）的荧光标记STAT3（氨基酸残基127-722）与递增浓度的C188-9（0.305至10000 nM）孵育；持续测量红外激光照射前后的荧光强度，计算荧光变化值（F_{norm}，即加热前即刻与加热30秒后的荧光强度比值）；以C188-9浓度对数为横坐标绘图，从S型结合曲线计算解离常数K_{D}[3]
3. 定量pSTAT3/pSTAT1的Luminex实验：收集经C188-9处理的AML或HNSCC细胞裂解物，通过Luminex技术检测pSTAT3、pSTAT1和GAPDH的水平；将GAPDH归一化后的pSTAT3或pSTAT1值除以未处理组的比值并以百分比表示，以Log [M] C188-9为横坐标绘图，计算IC₅₀[3,4]

1. 癌症恶病质相关C2C12肌管实验：将C2C12肌管与C26结肠癌细胞条件培养基共孵育，同时加入或不加入C188-9，处理时长分别为15分钟、24小时、72小时；通过Western blot检测p-Stat3、Stat3、caspase-3、裂解型caspase-3、pro-caspase-3、C/EBPδ、肌抑素及肌球蛋白重链的表达；测量孵育72小时后C2C12肌管的平均尺寸；处理24小时后通过RT-PCR检测MAFbx/Atrogin-1和MuRF-1的mRNA水平[1]
2. AML细胞系实验：将AML细胞系（Kasumi-1、THP-1等）饥饿处理1小时，用C188-9（0至10 μM）预处理1小时后，加入100 ng/mL G-CSF刺激15分钟；通过流式细胞术分析pStat3水平（根据前向/侧向散射排除碎片，基于同型对照设定pStat3⁻/pStat3⁺门）；通过Western blot检测pStat3、总Stat3、pERK1/2和pAkt的表达；采用ΔΔC_{t}法通过实时定量RT-PCR检测SOCS3和PIM1的mRNA水平；用C188-9处理24小时后，通过膜联蛋白V–PE标记定量细胞凋亡[2,4]
3. 原发性AML细胞实验：用C188-9处理原发性儿童AML样本48小时，通过膜联蛋白V染色定量凋亡；集落形成实验中，将原发性AML样本接种于含生长因子的甲基纤维素培养基中，并加入递增剂量的C188-9，10-14天后计数集落（≥30个细胞），以未处理组为对照归一化[2,4]
4. HNSCC细胞实验：用C188-9（0至100 μM）处理UM-SCC-17B、SCC-35、SCC-61和HN30细胞24-72小时；通过Luminex技术检测细胞裂解物中pSTAT3/pSTAT1的表达；贴壁依赖性生长实验中，将细胞接种于96孔板培养48小时，通过MTT法定量活细胞数，计算相对存活率（处理组存活率÷未处理组存活率×100），以Log [M] C188-9为横坐标绘图并计算IC₅₀；非贴壁依赖性生长实验处理72小时后，以相同方式计算IC₅₀[3]

D5W (5g of dextrose in 100 ml of water); 12.5 mg/kg; i.p.
CD2F1 female mice received isogenic C26 tumor cell via s.c injection

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1. Cancer cachexia model (CD2F1 mice): C26 colon carcinoma cells were implanted into CD2F1 mice to induce cachexia. After 5 days of tumor implantation, mice were treated with C188-9 or D5W twice daily for 14 days (administration route not specified). At the end of treatment, body weight, muscle mass (gastrocnemius, tibialis anterior), myofiber size distribution, muscle grip strength, muscle protein synthesis/degradation rates, and protein expression (caspase-3, C/EBPδ, myostatin, MAFbx/Atrogin-1) in gastrocnemius muscles were measured. Proteasome activity in muscle tissues was assessed using fluorogenic peptide LLVY-AMC as substrate [1]
2. HNSCC xenograft model (nude mice): 1.5 × 10⁶ UM-SCC-17B cells were injected into the tongues of athymic, 8–10 week old, male nude mice. Once tumors reached an average volume of ~15-20 mm³, mice were randomized into groups (5 mice/group) and received intraperitoneal injections of DMSO, C188 (50 mg/Kg), or C188-9 (100 mg/Kg) 5 times a week. Tumor volumes were measured twice weekly (calculated as 0.5 × (long dimension) × (short dimension)²), normalized to the volume at the first day of treatment. After treatment, mice were euthanized, tumor lysates were prepared, and Western blot was performed to detect pSTAT3, total STAT3, β-actin, pSTAT1, and total STAT1 [3]

[1]. Inhibition of Stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system, leading to preservation of muscle mass in cancer cachexia. J Biol Chem. 2015 Apr 24;290(17):11177-87.

[2]. Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor. Blood. 2011 May 26;117(21):5701-9.

[3]. Small-molecule inhibition of STAT3 in radioresistant head and neck squamous cell carcinoma. Oncotarget. 2016 May 3;7(18):26307-30.

[4]. Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor. Blood. 2011;117(21):5701-5709.

STAT3 Inhibitor C-188-9 is an orally bioavailable, binaphthol-sulfonamide-based inhibitor of signal transducer and activator of transcription 3 (STAT3), with potential antineoplastic activity. Upon oral administration, the STAT3 inhibitor C-188-9 specifically targets and binds to the phosphotyrosyl peptide binding site within the Src homology 2 (SH2) domain of STAT3. This inhibits the Janus kinase (JAK)-mediated tyrosine phosphorylation and activation of STAT3. This impedes nuclear translocation of STAT3, prevents STAT3 binding to responsive gene promoters and blocks STAT3-mediated regulation of gene expression. STAT3 regulates the transcription of genes involved in several cellular functions. STAT3 is constitutively activated in a variety of human cancers and plays a key role in neoplastic transformation, uncontrolled tumor cell proliferation, resistance to apoptosis, metastasis, immune evasion, tumor angiogenesis, epithelial-mesenchymal transition (EMT) and the Warburg effect.

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1. C188-9 is a novel small-molecule Stat3 inhibitor derived from the scaffold of C188 (a previously identified STAT3 inhibitor) through a hit-to-lead program. It binds to STAT3 with high affinity and has substantial improvements over C188 in inhibiting STAT3 binding to its pY-peptide ligand, cytokine-stimulated pSTAT3, and constitutive pSTAT3/STAT1 activity in cancer cells [3]
2. Mechanism of action in cancer cachexia: C188-9 inhibits Stat3 activation, thereby suppressing caspase-3 (via reducing its transcription and activity) and the ubiquitin-proteasome system (via inhibiting C/EBPδ → myostatin → MAFbx/Atrogin-1/MuRF-1 pathway), leading to preservation of muscle mass by reducing protein loss in muscle [1]
3. Mechanism of action in cancer cells: C188-9 selectively inhibits Stat3 (and STAT1) phosphorylation and activity, blocks Stat3-dependent gene expression (SOCS3, PIM1), induces apoptosis in cancer cells, and inhibits cancer cell proliferation (anchorage-dependent and independent growth). In HNSCC, it modulates STAT3-regulated genes involved in oncogenesis and radioresistance, as well as radioresistance genes regulated by STAT1 [2,3,4]
4. Clinical potential: C188-9 has potential for use in treating cancer cachexia, acute myeloid leukemia (AML), and radioresistant head and neck squamous cell carcinoma (HNSCC), either alone or in combination with radiotherapy. Baylor College of Medicine (with DJT as primary inventor) filed patents on C188 and C188-9, and StemMed, Ltd. holds an exclusive license to these compounds [3]

C27H21NO5S

471.52

471.114

C, 68.78; H, 4.49; N, 2.97; O, 16.97; S, 6.80

432001-19-9

1324494

Pinky beige solid powder

1.4±0.1 g/cm3

680.9±65.0 °C at 760 mmHg

365.6±34.3 °C

0.0±2.2 mmHg at 25°C

1.732

5.24

104

3

6

5

34

776

0

S(C1C=CC(=CC=1)OC)(NC1=CC(=C(C2=CC=CC=C12)O)C1=C(C=CC2=CC=CC=C12)O)(=O)=O

QDCJDYWGYVPBDO-UHFFFAOYSA-N

InChI=1S/C27H21NO5S/c1-33-18-11-13-19(14-12-18)34(31,32)28-24-16-23(27(30)22-9-5-4-8-21(22)24)26-20-7-3-2-6-17(20)10-15-25(26)29/h2-16,28-30H,1H3

N-[4-hydroxy-3-(2-hydroxynaphthalen-1-yl)naphthalen-1-yl]-4-methoxybenzenesulfonamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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: ≥ 2.08 mg/mL (4.41 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.8 mg/mL澄清DMSO储备液加入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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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：
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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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