CB-5083

p97 ( IC50 = 11 nM )
The primary target of CB-5083 is the p97 AAA ATPase, specifically the D2 ATPase domain. It is an ATP-competitive inhibitor with high selectivity for p97 over other ATPases, helicases, and kinases. The drug shows minimal interaction with DNA-PK (60-fold weaker binding than to p97) and no significant effects on other tested enzymes. The biochemical IC₅₀ for p97 inhibition is 11 nM [1,19]
- Additionally, CB-5083 has a reversible off-target effect on phosphodiesterase-6 (PDE6), with an inhibition constant of 80 nM, which is 7-fold smaller than that for sildenafil. This off-target activity may lead to potential visual side effects [26,37]

体外活性：在 A549 细胞中，CB-5083 引起显着的 K48 多聚泛素化蛋白和 CHOP 积累以及 p62 减少，并杀死肿瘤细胞，IC50 为 680 nM。激酶测定：将化合物在 DMSO 中稀释，从 10 μM 开始进行 3 倍 10 点连续稀释。该测定在 384 孔板中进行，每行作为单个稀释系列，每个化合物浓度点重复一次。在 5 μL 总体积中，添加 20 nM p97 六聚酶和 20 μM ATP 以启动反应。在轨道摇床中充分混合后，将板密封并在 37°C 下孵育 15 分钟。使用 Freedom Evo 通过自动液体处理进行化合物稀释、ATP 和酶添加。根据制造商的方案添加 ADP Glo 试剂 1 和 2。通过 Envision 读板器测量发光作为反应终点。每种化合物的 IC50 是通过将发光值拟合到四参数 S 形曲线得出的。细胞测定：A549 和其他肿瘤细胞系根据 ATCC 指南进行培养。细胞在黑色或白色、透明底部、经过组织培养处理的 384 孔板中培养。细胞用化合物的 10 点剂量滴定处理，一式两份。处理 72 小时后，将 CellTiter-Glo 添加到白板中以测量细胞活力。将发光值拟合到四参数 S 形曲线以确定 IC50 浓度。

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p97抑制作用：CB-5083强效抑制p97 ATPase活性，生化IC₅₀为11 nM。在10 μM浓度下，它选择性地阻断不可逆ATP探针与p97 D2区域的相互作用，而不影响p97 D1位点[1,19]
- 细胞活力：在A549人肺癌细胞中，CB-5083显示细胞毒性活性，72小时处理后的IC₅₀为0.68 μM。该化合物对多发性骨髓瘤细胞系和患者来源的多发性骨髓瘤细胞也显示相似效力，包括对蛋白酶体抑制剂耐药的细胞[31,32,39]
- 药效学标记物：CB-5083处理导致剂量依赖性的K48多泛素化蛋白(靶点参与标记物)、CHOP(通路抑制标记物)和p53(死亡诱导标记物)积累，同时p62蛋白水平(自噬标记物)降低。这些变化发生的浓度范围与导致细胞死亡的浓度一致[1,31]
- 作用机制：CB-5083通过抑制p97破坏蛋白质稳态，p97在泛素-蛋白酶体系统(UPS)和内质网相关降解(ERAD)中发挥关键作用。这导致错误折叠蛋白质积累，激活未折叠蛋白反应(UPR)，最终诱导细胞凋亡[1,8]

在携带人 HCT 116 结肠肿瘤异种移植物的小鼠中，CB-5083（75 mg/kg，口服）显着抑制肿瘤生长。在携带已建立的人 AMO-1 多发性骨髓瘤和 A549 肺癌肿瘤异种移植物的小鼠中，CB-5083（100 mg/kg，口服）也能产生显着的肿瘤生长抑制作用。

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抗肿瘤活性：在携带人HCT 116结肠癌异种移植物的小鼠中，口服给予CB-5083(75 mg/kg)显著抑制肿瘤生长[31]
- 药效学效应：在小鼠口服给药后，CB-5083导致肿瘤组织中多泛素化蛋白、CHOP和切割型多聚ADP-核糖聚合酶(cPARP)的快速和持续积累，证实了靶点参与和通路抑制[1]
- 与硼替佐米的比较：在临床前模型中，CB-5083在血液系统(AMO-1多发性骨髓瘤)和实体瘤(A549肺癌)模型中均显示抗肿瘤活性，口服剂量为100 mg/kg，每周给药4天、停药3天。相比之下，硼替佐米(一种蛋白酶体抑制剂)仅在血液系统模型中显示活性，在实体瘤模型中无活性[1]
- 脱靶效应：在小鼠中，15-30 mg/kg剂量的CB-5083显著降低视网膜敏感性，完全消除弱光刺激(0.0005 cd·s/m²)引起的ERG反应。这种效应是可逆的，与药物抑制PDE6有关[27]

从 10 μM 开始，使用 3 倍 10 点连续稀释方法在 DMSO 中稀释化合物。该测定在 384 孔板中进行，每行中的每个化合物浓度点重复代表一个稀释系列。为了启动反应，添加 20 μM ATP 和 20 nM p97 六聚酶，总体积为 5 μL。将板在轨道摇床中充分混合后，将其密封并在 37°C 下孵育 15 分钟。化合物稀释、ATP 添加和酶添加均通过 Freedom Evo 的自动液体处理进行。按照制造商的指示，添加 ADP Glo 试剂 1 和 2。使用 Envision 酶标仪测量发光作为反应终点。将每种化合物的发光值拟合成四参数 S 形曲线以确定其 IC50。

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p97 ATPase活性测定： 1. 使用ADP-Glo测定法(Promega)，以纯化的人p97酶为底物[1]
2. 将系列浓度的CB-5083(0.1 nM至10 μM)与纯化的p97在ATPase反应缓冲液(25 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM DTT)中孵育
3. 加入ATP(终浓度200 μM)启动反应，在30°C孵育30分钟
4. 加入ADP-Glo试剂将剩余ATP转化为ADP并终止反应
5. 然后加入激酶检测试剂将ADP转化为ATP，产生与ADP形成量成正比的发光
6. 使用酶标仪测量发光强度，从剂量-反应曲线计算IC₅₀值[1]
- PDE6抑制测定： 1. 将重组人PDE6酶与系列浓度的CB-5083(1 nM至1 μM)在测定缓冲液(50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT)中孵育
2. 加入cGMP(终浓度10 μM)启动反应，在30°C孵育20分钟
3. 加入EDTA至终浓度50 mM终止反应
4. 使用cGMP-Glo测定试剂盒(Promega)测量产生的GMP量，该试剂盒通过将GMP转化为ATP来检测PDE活性，产生发光
5. 测量发光强度，使用非线性回归分析计算抑制常数(Ki)[26,37]

ATCC 建议培养 A549 和其他肿瘤细胞系。干细胞在底部透明、黑色或白色的 384 孔板中生长。使用 10 点剂量滴定法将化合物一式两份地施用于细胞。通过在处理 72 小时后添加 CellTiter-Glo 来测量白板的细胞活力。寻找 IC50 浓度需要将光度值拟合到四参数 S 形曲线。

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细胞活力测定(CellTiter-Glo)： 1. 将A549人肺癌细胞以5×10³个/孔接种于96孔板，在37°C、5% CO₂条件下培养过夜[1,39]
2. 向细胞中加入系列浓度的CB-5083(0.1 nM至10 μM)，每个浓度设3个复孔
3. 孵育72小时后，向每孔加入CellTiter-Glo试剂并充分混合
4. 将板在室温下孵育10分钟以稳定发光
5. 使用酶标仪测量发光强度，计算细胞活力相对于溶剂对照组的百分比。从剂量-反应曲线确定IC₅₀值[1,39]
- 药效学标记物分析(免疫荧光)： 1. 将A549细胞以2×10⁴个/孔接种于24孔板的盖玻片上，培养过夜
2. 用CB-5083(0.1至10 μM)处理细胞6小时
3. 处理后，用4%多聚甲醛固定细胞15分钟
4. 用含0.2% Triton X-100的PBS通透10分钟，然后用含5% BSA的PBS封闭1小时
5. 加入抗K48多泛素化蛋白、CHOP、p62、p53或切割型caspase-3的一抗，4°C孵育过夜
6. 洗涤后，加入Alexa Fluor标记的二抗，室温孵育1小时
7. 用含DAPI的封片剂固定盖玻片，使用共聚焦显微镜观察
8. 使用图像分析软件量化荧光强度，确定标记物积累或减少的程度[1]

Suspended in 0.5% methylcellulose in water; 75 mg/kg, qd; oral administration Nu/Nu nude female mice bearing human HCT 116 colon tumor xenografts

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Tumor Xenograft Model: 1. Female nude mice (6-8 weeks old) were subcutaneously injected with 5×10⁶ HCT 116 human colon cancer cells in the right flank
2. When tumors reached approximately 100 mm³, mice were randomized into treatment groups (n=6 per group)
3. CB-5083 was formulated as a suspension in 0.5% methylcellulose aqueous solution and administered orally at 75 mg/kg once daily
4. Vehicle control groups received the same volume of 0.5% methylcellulose
5. Tumor volume (length × width² / 2) and body weight were measured twice weekly
6. After 4 weeks of treatment, mice were euthanized, tumors were excised and weighed, and tumor tissues were collected for immunohistochemical analysis [1,31]
- Retinal Function Assessment: 1. Male C57BL/6 mice (8-10 weeks old) were dark-adapted overnight
2. CB-5083 was dissolved in DMSO and diluted in saline to a final concentration of 10 mg/mL
3. Mice were treated with CB-5083 (15 or 30 mg/kg, i.p.) or vehicle control
4. After 1 hour, electroretinogram (ERG) recordings were performed under dim red light conditions
5. Mice were anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.) and pupils were dilated with tropicamide (1%)
6. ERG responses were recorded using a corneal electrode, with reference and ground electrodes placed subcutaneously
7. Flash stimuli of varying intensities (from 0.0005 to 10 cd·s/m²) were presented, and ERG waveforms were recorded and analyzed
8. The amplitude and latency of ERG responses were measured and compared between treatment groups [27]

Plasma Pharmacokinetics in Mice: - After intravenous administration (3.0 mg/kg) in female nude mice, CB-5083 shows a terminal elimination half-life (T₁/₂) of 2.83 hours, high plasma clearance (5.9 mL/min/kg), and large volume of distribution (418 mL/kg) [24,25,33]
- After oral administration (25 mg/kg), the drug reaches a maximum plasma concentration (Cmax) of 7.95 μM with moderate oral bioavailability (41%) in female nude mice [23,25]
- Metabolic Stability: - CB-5083 demonstrates good metabolic stability in preclinical species. In mouse liver microsomal stability studies, the drug has a T₁/₂ of 102 minutes [34,35,36]
- In hepatocyte stability studies, the T₁/₂ is 172 minutes, indicating relatively slow metabolism and potential for longer in vivo exposure [34,35,36]
- Absorption and Distribution: - The drug has moderate permeability as assessed by Caco-2 permeability assays, with an efflux ratio of less than 2, indicating minimal active efflux [1]
- Solubility studies show that CB-5083 has acceptable solubility across a range of pH conditions (pH 1-8), which is favorable for oral absorption [1]
- Plasma Protein Binding: - CB-5083 exhibits high plasma protein binding in preclinical species, with approximately 90% bound to plasma proteins in mouse, rat, dog, and monkey plasmas [1]

Off-Target Effects: - The most significant off-target effect of CB-5083 is its inhibition of PDE6, with an inhibition constant (Ki) of 80 nM. This is 7-fold more potent than sildenafil's inhibition of PDE6 [26,37]
- In mouse models, treatment with CB-5083 at 15-30 mg/kg significantly decreases retinal sensitivity, completely abolishing the ERG response to dim light stimuli. This effect is reversible and suggests potential visual side effects in humans [27]
- General Toxicity in Animals: - In tumor-bearing nude mice, administration of CB-5083 at therapeutic doses causes no significant changes in body weight, indicating low systemic toxicity [38]
- The maximum tolerated daily dose of CB-5083 in VCPR155H/+ mice is 25 mg/kg (defined as <20% weight loss) [30]
- Long-term treatment (5 months) with CB-5083 (15 mg/kg) in VCPR155H/R155H mice results in a steady increase in body weight and no significant changes in organ-to-body weight ratios [30]
- Hematological and Biochemical Parameters: - Blood toxicology analysis in VCPR155H/R155H mice treated with CB-5083 shows no increase in liver enzyme levels (AST and ALT) or creatine kinase levels. Interestingly, AST levels are significantly reduced in the treatment group, suggesting possible hepatoprotective effects in certain contexts [30]
- Immunological Effects: - Administration of CB-5083 during CD8 T cell expansion leads to strong toxicity in mice within hours, characterized by enhanced IL-6 levels in serum and accumulation of polyubiquitinated proteins. This suggests potential immune-related adverse effects when used in certain clinical settings [29]

[1]. J Med Chem . 2015 Dec 24;58(24):9480-97.

p97 Inhibitor CB-5083 is an orally bioavailable inhibitor of valosin-containing protein (VCP) p97, with potential antineoplastic activity. Upon oral administration, CB-5083 specifically binds to and inhibits the activity of p97. This prevents ubiquitin-dependent protein degradation and causes cellular accumulation of poly-ubiquitinated proteins. The inhibition of endoplasmic reticulum (ER)-associated protein degradation activates the ER-dependent stress response pathway, and leads to both an induction of apoptosis and inhibition of cell proliferation in susceptible tumor cells. p97, a type II AAA ATPase, plays a key role in cellular protein homeostasis. Its overexpression in many tumor cell types is associated with increased tumor cell proliferation and survival.

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Drug Development Status: - CB-5083 has been nominated as a drug candidate for the treatment of cancer and is currently being tested in phase 1 clinical trials for patients with relapsed/refractory multiple myeloma and advanced solid tumors [1,28,42]
- Structure-Activity Relationship: - The drug contains a 4-benzylamino-8-methoxyquinazoline core with a 2-aminobenzoimidazole moiety. Modifications to these groups significantly affect potency and selectivity [1]
- The 4-benzylamino group is essential for p97 binding, as substitutions or deletions result in loss of activity [1]
- Methylation of the amino group at the 2-position of the benzoimidazole increases biochemical potency approximately 4-fold, while larger alkyl groups such as ethyl are not tolerated [1]
- The indole moiety with a primary amide substituent at the 4-position is critical for high potency, likely due to its ability to form hydrogen bonds with amino acids S664 and K663 in the p97 D2 domain [1]
- Mechanism of Action Elucidation: - Docking studies show that CB-5083 binds to the D2 ATP binding site of p97, competing with ATP but in a slightly different orientation [1]
- Mutational analysis confirms that resistance to CB-5083 is associated with specific mutations in the p97 D2 ATPase region (N660 and T688), with a ~50-fold reduction in potency against mutant p97 [1]
- The drug's selective binding to the D2 domain of p97 is consistent with the D2 region being primarily responsible for the ATPase activity of p97 [1]
- Therapeutic Advantages: - CB-5083 represents a first-in-class approach to cancer treatment by targeting protein homeostasis through p97 inhibition, offering a novel mechanism distinct from traditional chemotherapies and other targeted agents [1,2]
- The drug's activity in both hematological and solid tumor models, unlike bortezomib which is only active in hematological malignancies, suggests a broader therapeutic window [1]
- The oral bioavailability of CB-5083 provides a significant advantage over many other anticancer agents that require intravenous administration [1]

C24H23N5O2

413.47

413.185

C, 69.72; H, 5.61; N, 16.94; O, 7.74

1542705-92-9

73051434

White to off-white solid powder

1.4±0.1 g/cm3

760.5±70.0 °C at 760 mmHg

413.8±35.7 °C

0.0±2.6 mmHg at 25°C

1.710

2.61

95.1

2

5

5

31

627

0

O1C([H])([H])C([H])([H])C2=C(C(N([H])C([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])=NC(=N2)N2C(C([H])([H])[H])=C([H])C3=C(C(N([H])[H])=O)C([H])=C([H])C([H])=C23)C1([H])[H]

RDALZZCKQFLGJP-UHFFFAOYSA-N

InChI=1S/C24H23N5O2/c1-15-12-18-17(22(25)30)8-5-9-21(18)29(15)24-27-20-10-11-31-14-19(20)23(28-24)26-13-16-6-3-2-4-7-16/h2-9,12H,10-11,13-14H2,1H3,(H2,25,30)(H,26,27,28)

1-[4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methylindole-4-carboxamide

CB5083; CB-5083; CB 5083

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 (6.05 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 (6.05 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 (6.05 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 0.5%CMC Na +5% Tween 80 : 30mg/mL

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配方 5 中的溶解度: 10 mg/mL (24.19 mM) in 50% PEG300 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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。