GNE-6776 HCl   中文名称: GNE-6776盐酸

USP7 (IC50 = 1.4 μM)

在15μM时，GNE-6776显著抑制USP7。GNE-6776[1]是一种对内源性细胞去泛素酶和重组酶具有高度选择性的USP7抑制剂[1].
GNE-6776诱导肿瘤细胞死亡，并增强化疗药物和靶向化合物（包括PIM激酶抑制剂）的细胞毒性。结构研究表明，GNE-6640和GNE-6776非共价靶向距离催化半胱氨酸12Å的USP7。这些化合物减弱泛素结合，从而抑制USP7去泛素酶活性。GNE-6640和GNE-6776与酸性残基相互作用，酸性残基介导与泛素Lys48侧链的氢键相互作用，表明USP7优先与具有游离Lys48侧面链的泛素部分相互作用并切割泛素部分。 GNE-6776 促进人类异种移植物中的靶向途径调节。尽管仅短暂地达到有效暴露，但 GNE-6776 会导致适度但显着的 EOL-1 异种移植物生长延迟。
鉴于这些抑制剂的有利特性，我们在动物模型中研究了它们的疗效。药效学和药代动力学研究表明，GNE-6776具有口服生物可利用性，并促进人类异种移植物的靶向通路调节（扩展数据图4e-i）。尽管有效的暴露只是暂时的，但GNE-6776引起了EOL-1异种移植物生长的适度但显著的延迟（扩展数据图4j）。开发具有改善药物样性质的USP7抑制剂对于全面评估USP7在体内的抑制作用是必要的。

GNE-6776 促进人类异种移植物中的靶向途径调节。尽管仅短暂地达到有效暴露，但 GNE-6776 会导致适度但显着的 EOL-1 异种移植物生长延迟。
鉴于这些抑制剂的有利特性，我们在动物模型中研究了它们的疗效。药效学和药代动力学研究表明，GNE-6776具有口服生物可利用性，并促进人类异种移植物的靶向通路调节（扩展数据图4e-i）。尽管有效的暴露只是暂时的，但GNE-6776引起了EOL-1异种移植物生长的适度但显著的延迟（扩展数据图4j）。开发具有改善药物样性质的USP7抑制剂对于全面评估USP7在体内的抑制作用是必要的。

USP7 enzymatic analysis/USP7酶活分析[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

---

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）

---

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

---

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。

Immunodeficient C.B-17 SCID mice with an EOL1 AML xenograft, aged 12–16 weeks[1]
100 or 200 mg/kg

---

DMPK analysis. [1]
In vitro DMPK studies were performed using standard protocols. GNE-6776 was formulated as a suspension in 0.5% methylcellulose/0.2% Tween-80 and was administered at 200 mg kg−1 (body weight) by oral gavage to female C.B-17 SCID mice, aged 12–16 weeks (n = 3 per time point). No randomization was used for DMPK studies. At 0.5, 1, 2, 4, 8 and 24 h post-dose, blood samples were collected by terminal cardiac puncture into anticoagulant tubes (EDTA). Clarified plasma was then transferred to a fresh tube and snap frozen. GNE-6776 plasma concentrations were determined by LC–MS/MS.

---

In vivo pharmacodynamic response. [1]
For EOL-1 AML xenograft studies, immunodeficient C.B-17 SCID mice, aged 12–16 weeks, were inoculated subcutaneously on the right flank with five million cells in a 50:50 suspension of HBSS:Matrigel (100 μl). When tumour volumes reached between approximately 285 and 500 mm3, mice were distributed into volume-matched cohorts (n = 4). For MCF7 breast-cancer xenograft studies, immunodeficient nu/nu mice, aged 6–8 weeks, were implanted with 0.36 mg oestrogen pellets via trochar 1–3 days before tumour cell inoculation. Ten million MCF7-Ser cells, an in vivo-optimized MCF7 variant, were injected orthotopically into the 2/3 mammary fat pad of each mouse in a 50:50 suspension of HBSS:Matrigel in a total volume of 100 μl. When tumour volumes reached between approximately 285 and 450 mm3, mice were distributed into volume-matched cohorts (n = 4). GNE-6776 was formulated as a suspension in 0.5% methylcellulose/0.2% Tween-80 and administered at 200 mg kg−1 (body weight) by oral gavage at 0 and 4 h. 0.5% Methylcellulose/0.2% Tween-80 control or GNE-6776-treated samples were collected at 8 h after the first dose and excised tumours were flash-frozen on dry ice. Tumours were lysed in RIPA buffer containing protease inhibitors and 300 mM NaCl using a Qiagen TissueLyser. Samples were incubated on ice for 15 min and then centrifuged at 20,000g at 4 °C for 10 min. Protein levels in clarified lysates were quantified using a Pierce BCA assay kit and concentrations were normalized with sample buffer. Samples were run on gels, and proteins were transferred to membranes and western blotted as described above.

---

In vivo efficacy study. [1]
For EOL1 AML xenograft studies, immunodeficient C.B-17 SCID mice (Charles River Laboratories), aged 12–16 weeks, were inoculated subcutaneously on the right flank with five million cells in a 50:50 suspension of HBSS:Matrigel (100 μl). When tumours became established (150–300 mm3), mice were distributed into tumour-volume-matched cohorts (n = 7, mean tumour volume ~250 mm3). GNE-6776 was formulated as a suspension in 0.5% methylcellulose/0.2% Tween-80 and was administered at 100 or 200 mg kg−1 (body weight) by oral gavage on a once or twice daily schedule. Tumour volume measurements, body weight and body condition data were collected two or three times per week. The maximum tumour volume limit of 2,000 mm3 was not reached in any animal.

[1]. Usp7 inhibitor compounds and methods of use. US20160272588A1.

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] 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]

C21H21N3O2

347.41

348.16

C, 68.95; H, 5.79; N, 16.08; O, 9.18

2009273-60-1

122531799

Off-white to light yellow solid powder

3.5

88.2Ų

3

4

4

26

456

0

UCYSSYGGXOFJKK-UHFFFAOYSA-N

InChI=1S/C20H20N4O2/c1-3-15-16(13-6-9-17(23-10-13)20(26)22-2)11-24-19(21)18(15)12-4-7-14(25)8-5-12/h4-11,25H,3H2,1-2H3,(H2,21,24)(H,22,26)

6'-Amino-4'-ethyl-5'-(4-hydroxyphenyl)-N-methyl-[3,3'-bipyridine]-6-carboxamide

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.99 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 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。

---

配方 2 中的溶解度: ≥ 2.08 mg/mL (5.99 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 20.8 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。

---

View More

配方 3 中的溶解度: ≥ 2.08 mg/mL (5.99 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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

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。