GNE-6640

USP7 (IC50 = 0.75 μM); USP7 (IC50 = 0.43 μM); USP43 (IC50 = 20.3 μM); Ub-MDM2 (IC50 = 0.23 μM)

GNE-6640 encourages the natural ubiquitination of MDM2 through Lys48 (K48)-linked polyubiquitin chains, thereby guiding the process of proteasomal degradation13. GNE-6640 focuses on the p53, MDM2, and USP7 signaling pathways in cells. With an IC50 ≤ 10 μM, GNE-6640 reduces the viability of 108 cell lines. Combining GNE-6640 with DNA-damaging drugs like doxorubicin or cisplatin may trigger the p53 response and improve the effectiveness of USP7 inhibitors. GNE-6640 may cause the death of tumor cells. PIM kinase inhibitors and other targeted substances, such as chemotherapeutic agents, increase the cytotoxicity of GNE-6640[1].

GNE-6640是USP7的另一种新型选择性非共价抑制剂，其IC50值为0.75μM（全长USP7）、0.43μM（USP7催化结构域）和20.3μM（全长USP43）。泛素特异性蛋白酶-7（USP7）控制p53肿瘤抑制因子和对肿瘤细胞存活至关重要的各种其他蛋白质的稳定性。GNE-6640促进内源性MDM2泛素化，并靶向细胞USP7和p53信号通路，诱导肿瘤细胞死亡，增强细胞毒性，并降低约108个细胞系的存活率，IC50≤10μM，使用PIM激酶抑制剂等化疗药物[2]。

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USP7 enzymatic analysis[1]
使用1 nM USP7和一系列泛素-AMC底物滴定法对全长USP7进行Michaelis-Menten动力学测量。在Tecan Safire2平板读数器上使用Magellan软件确定底物水解的初始速率，并使用GraphPad Prism软件进行非线性回归分析建模动力学参数。标准误差由三个技术重复计算得出。对于使用USP7 D305/E308突变体的研究，样品在由50 mM HEPES（pH 7.5）、100 mM NaCl、2.5 mM二硫苏糖醇和0.1%（w/v）牛丙种球蛋白组成的缓冲液中反应。用于Michaelis-Menten分析的泛素-Rho110的起始底物浓度为100μM，连续稀释至781 nM。反应在室温下进行1小时，最终酶浓度为100 nM（三个独立的实验，见图表中的符号），在黑色100-μl体积96孔半面积板上进行。通过使用初始速度将数据与线性V0值拟合来计算酶活性，该线性V0值是通过使用485nm的激发和535nm的发射分析切割的Rho-110的荧光信号而测量的。

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Deubiquitinase selectivity analysis/去泛素酶选择性分析[1]
重组去泛素酶双泛素质谱裂解试验。如前所述，使用指定浓度的重组去泛素酶、二泛素底物和USP7抑制剂化合物进行MALDI-TOF DUB测定。在泛素的替代底物Ub-Ube2W（Ub-E2）上监测了GNE-6640和GNE-6776对UCH1家族成员的抑制效率。

Tumour cell-line panel viability/肿瘤细胞系存活率。[1]
如前所述，在441个细胞系中对GNE-6640和GNE-6641进行了3天的分析，并在185个细胞系的亚群中对GNE-6776、GNE-6640和GNE-641进行了5天的分析26。简而言之，使用三倍稀释法在九点剂量反应中筛选化合物。在加入化合物前24小时将细胞接种到384孔板中。然后将细胞与化合物一起孵育72小时或120小时，然后测定存活率。检测采用生物法，一式三份。在整个试验过程中，细胞在RPMI-1640、2.5%FBS（72小时试验）或5%FBS（120小时试验）和2 mM谷氨酰胺中孵育（37°C，5%CO2）。报告的IC50和平均存活率指标如下：IC50是相对于未处理孔的估计抑制率为50%的剂量（即绝对IC50）。

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Primary combination screen。[1]
在不存在或存在固定剂量的GNE-6776（0 nm、125 nm、250 nm、500 nm、1000 nm和2000 nm）或GNE-6640（400 nm）的情况下，筛选了一个包含589种按九点剂量反应排列的化合物库。简而言之，将5000个EOL-1细胞接种到384孔板中，24小时后加入化合物。在化合物加入后120小时测定细胞存活率（CellTiter Glo）。拟合曲线，计算IC50和平均存活率指标。IC50是相对于未处理的孔抑制50%的剂量。平均存活率是每个测试剂量下拟合存活率的平均值。平均存活率等于对数剂量/存活率曲线下的面积除以测试剂量的总数。平均存活率值用于扩展数据图6g中描述的分析。所有数据均使用Genedata Screener软件进行拟合。

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Primary combination screen analysis。[1]
在EOL-1细胞系中，在DMSO或浓度增加的GNE-6776（100 nM、250 nM、500 nM、1000 nM或2000 nM）或400 nM的GNE-6640存在下，测定了574种具有已知蛋白质或机制靶标的化合物的标准化平均存活率。对于每种化合物，我们评估了USP7抑制剂治疗和DMSO治疗之间的平均存活率差异。对于三种或多种化合物靶向的靶标，我们使用Wilcoxon秩和检验计算了每种浓度USP7抑制剂的高平均存活率差异的富集程度。为了可视化，我们通过取每个目标的−log10（转换后的P值）的平均值来组合所有浓度的结果。

[1]. USP7 small-molecule inhibitors interfere with ubiquitin binding. Nature. 2017 Oct 26;550(7677):534-538.

[2]. The role of deubiquitinating enzymes in cancer drug resistance. Cancer Chemother Pharmacol. 2020 Apr;85(4):627-639

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.[1]

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Herein we describe GNE-6640 and GNE-6776, selective USP7 inhibitors that possess a structurally defined mechanism of inhibition. Establishing stringent screening cascades was critical for selecting and optimizing on-target inhibitors. Combination studies revealed a previously undescribed intersection between USP7 deubiquitinase activity and PIM kinases in regulating cell viability. Co-crystal structures of GNE-6640 or GNE-6776 pointed to the importance of the complementary charged interactions between USP7-D305/E308 and ubiquitin-K48 side chains, which we confirmed using mutational analysis. Notably, D305G has been identified as a somatic loss-of-function mutant in patients with acute lymphoblastic leukaemia21. NMR analysis of USP7 binding to native mono-ubiquitin and differentially labelled di-ubiquitins revealed that USP7 preferentially interacts with ubiquitin moieties having free K48 side chains. It has been proposed that the inefficiency of some deubiquitinases to depolymerize longer substrate-conjugated K48-linked chains enables a threshold for proteasome-targeting polyubiquitination22; our studies substantiate this idea and provide a biophysical mechanism. Numerous proteins, including other deubiquitinases, ubiquitin ligases, DNA repair and endocytosis machinery, and epigenetic regulators are functionally dependent on ubiquitin binding23. Developing selective inhibitors that attenuate ubiquitin binding is an effective strategy for USP7 inhibition. Our studies demonstrate the feasibility of this approach, which may have broader applications for inhibiting other classes of ubiquitin-binding proteins. [1]

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Drug resistance is a well-known phenomenon leading to a reduction in the effectiveness of pharmaceutical treatments. Resistance to chemotherapeutic agents can involve various intrinsic cellular processes including drug efflux, increased resistance to apoptosis, increased DNA damage repair capabilities in response to platinum salts or other DNA-damaging drugs, drug inactivation, drug target alteration, epithelial-mesenchymal transition (EMT), inherent cell heterogeneity, epigenetic effects, or any combination of these mechanisms. Deubiquitinating enzymes (DUBs) reverse ubiquitination of target proteins, maintaining a balance between ubiquitination and deubiquitination of proteins to maintain cell homeostasis. Increasing evidence supports an association of altered DUB activity with development of several cancers. Thus, DUBs are promising candidates for targeted drug development. In this review, we outline the involvement of DUBs, particularly ubiquitin-specific proteases, and their roles in drug resistance in different types of cancer. We also review potential small molecule DUB inhibitors that can be used as drugs for cancer treatment.[2]

C20H18N4O

330.383123874664

330.15

C, 72.71; H, 5.49; N, 16.96; O, 4.84

2009273-67-8

122531786

White to off-white solid powder

3.8

87.8Ų

3

4

3

25

439

0

ZHYXJQQBKROZDX-UHFFFAOYSA-N

InChI=1S/C20H18N4O/c1-2-16-17(13-5-8-18-14(9-13)10-23-24-18)11-22-20(21)19(16)12-3-6-15(25)7-4-12/h3-11,25H,2H2,1H3,(H2,21,22)(H,23,24)

4-[2-amino-4-ethyl-5-(1H-indazol-5-yl)pyridin-3-yl]phenol

2934.99.03.00

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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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));
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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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