VER155008

HSP70 (IC50 = 0.5 μM); HSC70 (IC50 = 2.6 μM); Grp78 (IC50 = 2.6 μM)

VER-155008 对 Hsp90 无活性，IC50 > 200 μM，但它是 Hsc70 和 Hsp70 的抑制剂，对 Hsp70、Hsc70 和 Grp7 的 IC50 分别为 0.5 μM、2.6 μM 和 2.6 μM。 BT474、MB-468、HCT116 和 HT29 细胞属于 VER-155008 抑制增殖的人类结肠和乳腺肿瘤细胞系，其 GI50 分别为 10.4 μM、14.4 μM、5.3 μM 和 12.8 μM。 HCT116 和 BT474 癌细胞响应 VER-155008 (5–40 μM) 表现出客户蛋白降解。此外，VER-155008 会导致人类肿瘤细胞系发生凋亡[1]。在培养的神经元中，VER-155008 (0.05-5 μM) 可恢复 Aβ 诱导的轴突变性[2]。 VER-155008（10 μM 或 25 μM）抑制 Hsp70 并阻止 LNCaP95 细胞增殖。此外，VER-155008 还降低了雄激素受体剪接变异 7 (AR-V7) 和全长雄激素受体 (AR-FL) 蛋白的表达[3]。

在幼稚雌性 BALB/c 小鼠中，VER-155008（25 mg/kg，静脉注射）显示血浆清除率。在 HCT116 荷瘤裸 BALB/c 小鼠中，VER-155008（40 mg/kg，静脉注射）同样表现出快速血浆清除并降低肿瘤水平[1]。在 5XFAD 小鼠中，VER-155008（10 μmol/kg/天，腹腔注射）可减轻与淀粉样斑块相关的轴突肿胀，并改善记忆缺陷。 VER-155008（89.9 μmol/kg/天，腹膜内注射）还可减少 5XFAD 小鼠中的淀粉样斑块和淀粉样斑块相关蛋白 PHF-tau [2]。

Hsc70、Hsp70、Grp78和Hsp90荧光偏振（FP）测定[1]
如前所述，测定VER-155008对Hsp70和Hsp90的活性。Hsc70（aa 5-381）和Grp78（ATP酶结构域）的克隆和表达如下。制作了对应于氨基酸5-381的Hsc70的PCR片段。将纯化的PCR片段连接到pCR2.1-TOPO中。随后将其限制性消化并亚克隆到商业组氨酸标记载体pET101中用于表达分析。通过PCR扩增Grp78的ATP酶结构域，并将其克隆到pCR2.1-TOPO中。随后将其消化并连接到谷胱甘肽-s-转移酶标记的表达载体pGEX-4T-1中。按照Hsp70的描述，使用与FP探针相同的N 6-（6-氨基）己基-ATP-5-FAM进行Hsc70和Grp78的FP测定，但有以下修改。对于Hsc70，在22°C下孵育30分钟，蛋白质和探针浓度分别为0.3μM和20 nM，而对于Grp78，在22℃下孵育2小时，蛋白质和探头浓度分别为2μM和10 nM。FAM-ATP探针的K D对Hsc70为0.24μM，对Grp78为2μM。

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BiaCore测定化合物与Hsp70的结合[1]
所有实验均在Biacore T100上在25°C下以30μL/min的流速进行。缓冲系统与Hsp70荧光偏振分析中使用的缓冲系统相同，含有1%DMSO和0.05%吐温20。双His标记的Hsp70被固定在NTA传感器芯片的表面上；约2000RU的蛋白质被固定。注入化合物90秒，在80秒时根据平衡结合确定Kds。

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Hsp70 ATP酶测定[1]
使用ADP Hunter Plus检测试剂盒测量ATP周转率和随后产生的ADP。反应混合物在半体积、全黑色非结合板中制备，在标准试剂盒测定缓冲液中含有250 nM全长、GST标记的Hsp70、50μM ATP和浓度递增的VER-155008。反应在30°C下孵育90分钟，然后在FlexStation 3上读取，E x为530 nm，E m为590 nm，每孔读取6次。设置不含酶的反应来监测ATP的自发水解，并从含有Hsp70的反应中减去。

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热变性萤光素酶的复性[1]
该测定基本上如前所述[24,25]进行。简而言之，Hsc70/Hsp70抑制剂在pH 7.5的兔网织红细胞裂解物（RRL，核酸酶处理）中稀释，该裂解物含有10 mM Tris、100 mM KCl、3 mM EDTA、1 mM DTT和ATP再生系统。最终ATP浓度为0.5 mM。化合物在室温下在上述混合物中预孵育15分钟，然后加入25 ng热变性萤光素酶（在40°C下变性30分钟）以启动复性反应，并在30°C下孵育60分钟。使用Bright glo试剂测定萤光素酶活性。

细胞周期分析[1]
将HCT116细胞暴露于指定浓度的VER-155008和VER-82160中24小时。在用RNaseA/碘化丙啶染色之前，收集所有细胞并将其固定在冰上的70%乙醇中。在FACSArray细胞仪上收集数据，并用FACSDeva软件进行分析。

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雄激素剥夺疗法最初对晚期前列腺癌症患者有效；然而，前列腺癌症逐渐对雄激素剥夺疗法产生耐药性，称为去势耐药性前列腺癌症（CRPC）。雄激素受体剪接变异体7（AR-V7）是CRPC的原因之一，与对新一代AR拮抗剂（恩杂鲁胺）的耐药性和预后不良有关。已知热休克蛋白70（Hsp70）抑制剂可以降低全长AR（AR-FL）的水平，但对其对表达AR-V7的CRPC细胞的影响知之甚少。在本研究中，我们研究了Hsp70抑制剂槲皮素和VER155008在表达AR-V7的前列腺癌症细胞系LNCaP95中的作用，并探讨了Hsp7调节AR-FL和AR-V7表达的机制。槲皮素和VER155008降低了细胞增殖，增加了凋亡细胞的比例，并降低了AR-FL和AR-V7的蛋白水平。此外，VER155008降低了AR-FL和AR-V7 mRNA水平。用Hsp70抗体进行免疫沉淀和质谱鉴定，Y-box结合蛋白1（YB-1）是通过与Hsp70相互作用在转录水平调节AR-FL和AR-V7的分子之一。VER155008降低了YB-1的磷酸化及其在细胞核中的定位，表明Hsp70参与AR调节可能是通过YB-1的激活和核转位介导的。总的来说，这些结果表明，Hsp70抑制剂通过YB-1抑制降低AR-FL和AR-V7的表达，对CRPC具有潜在的抗肿瘤活性[3]。

All in vivo studies were carried out according to UK Home Office guidelines and the “Guidance on the Operation of the Animals (Scientific Procedures) Act 1986”. Female BALB/c mice were dosed intravenously with 25 mg/kg VER-155008 into the lateral tail vein as a solution in 10% DMSO/5% Tween 80/85% saline (v/v/v). Animals were sacrificed at 5, 15 and 30 min, 1, 2, 4 and 6 h post dose.[1]
Female BALB/c nu/nu mice were inoculated with 5 × 106 HCT116 cells into the right flank. Tumors were monitored for growth, and animals were dosed IV with 40 mg/kg VER-155008 on day 31 (tumor volume approximately 200–250 mm3). Animals were sacrificed at 15 min, 1 and 4 h post dose.[1]
Each timepoint contained three mice and blood samples were collected by cardiac puncture. Plasma was obtained by centrifugation and frozen at −20°C prior to analysis. Tumors were harvested, snap frozen and stored at −80°C.[1]

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Alzheimer's disease (AD) is a progressive neurodegenerative disorder resulting in structural brain changes and memory impairment. We hypothesized that reconstructing neural networks is essential for memory recovery in AD. Heat shock cognate 70 (HSC70), a member of the heat shock protein family of molecular chaperones, is upregulated in AD patient brains, and recent studies have demonstrated that HSC70 facilitates axonal degeneration and pathological progression in AD. However, the direct effects of HSC70 inhibition on axonal development and memory function have never been investigated. In this study, we examined the effects of a small-molecule HSC70 inhibitor, VER-155008, on axonal morphology and memory function in a mouse model of AD (5XFAD mice). We found that VER-155008 significantly promoted axonal regrowth in amyloid β-treated neurons in vitro and improved object recognition, location, and episodic-like memory in 5XFAD mice. Furthermore, VER-155008 penetrated into the brain after intraperitoneal administration, suggesting that VER-155008 acts in the brain in situ. Immunohistochemistry revealed that VER-155008 reduced bulb-like axonal swelling in the amyloid plaques in the perirhinal cortex and CA1 in 5XFAD mice, indicating that VER-155008 also reverses axonal degeneration in vivo. Moreover, the two main pathological features of AD, amyloid plaques and paired helical filament tau accumulation, were reduced by VER-155008 administration in 5XFAD mice. This is the first report to show that the inhibition of HSC70 function may be critical for axonal regeneration and AD-like symptom reversal. Our study provides evidence that HSC70 can be used as a new therapeutic target for AD treatment[2].

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Formulation method 1: VER-155008 was dissolved in 10% dimethyl sulfoxide (DMSO)-containing saline and administered intraperitoneally (10 mmol/kg/day) to 5XFAD mice for 18 days. [1] Formulation method 2: Female BALB/c mice were dosed intravenously with 25 mg/kg VER-155008 into the lateral tail vein as a solution in 10% DMSO/5% Tween 80/85% saline (v/v/v). [2]

BALB/c mice

[1]. A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells. Cancer Chemother Pharmacol. 2010 Aug;66(3):535-45.

[2]. Heat Shock Cognate 70 Inhibitor, VER-155008, Reduces Memory Deficits and Axonal Degeneration in a Mouse Model of Alzheimer's Disease. Front Pharmacol. 2018 Jan 30;9:48.

[3]. Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells. Cancer Sci. 2017 Sep;108(9):1820-1827.

4-[[(2R,3S,4R,5R)-5-[6-amino-8-[(3,4-dichlorophenyl)methylamino]-9-purinyl]-3,4-dihydroxy-2-oxolanyl]methoxymethyl]benzonitrile is a purine nucleoside.

C25H23CL2N7O4

556.40

555.118

C, 53.97; H, 4.17; Cl, 12.74; N, 17.62; O, 11.50

1134156-31-2

25195348

Typically exists as White to pink solids at room temperature

1.6±0.1 g/cm3

856.3±75.0 °C at 760 mmHg

471.7±37.1 °C

0.0±0.3 mmHg at 25°C

1.748

2.71

164.36

4

10

8

38

831

4

ClC1=C(C([H])=C([H])C(=C1[H])C([H])([H])N([H])C1=NC2=C(N([H])[H])N=C([H])N=C2N1[C@@]1([H])[C@@]([H])([C@@]([H])([C@@]([H])(C([H])([H])OC([H])([H])C2C([H])=C([H])C(C#N)=C([H])C=2[H])O1)O[H])O[H])Cl

ZXGGCBQORXDVTE-UMCMBGNQSA-N

InChI=1S/C25H23Cl2N7O4/c26-16-6-5-15(7-17(16)27)9-30-25-33-19-22(29)31-12-32-23(19)34(25)24-21(36)20(35)18(38-24)11-37-10-14-3-1-13(8-28)2-4-14/h1-7,12,18,20-21,24,35-36H,9-11H2,(H,30,33)(H2,29,31,32)/t18-,20-,21-,24-/m1/s1

5'-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)methyl]amino]-adenosine

VER 155008; VER155008; VER-155008; VER-155008; VER 155008; 5'-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)methyl]amino]-adenosine; VER155008; C25H23Cl2N7O4; 4-((((2R,3S,4R,5R)-5-(6-Amino-8-((3,4-dichlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)benzonitrile; 5'-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)-methyl]amino]-adenosine;

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 (4.49 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 (4.49 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 (4.49 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: 1% CMC+0.5% Tween-80: 30 mg/mL

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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。