Denifanstat (TVB-2640)

Fatty Acid Synthase (FASN)(IC50 = 0.052 μM)

Denifanstat（化合物152）是FASN的强抑制剂[2]。脂肪酸合成酶（FASN）抑制严重急性呼吸系统综合征冠状病毒2型刺突蛋白的棕榈酰化[1]
脂肪酸合成酶（FASN）是非酒精性脂肪性肝炎（NASH）的一个有吸引力的治疗靶点，因为它驱动新的脂肪生成并介导促炎和纤维化信号传导。因此，我们在人类细胞培养和三种饮食诱导的NASH小鼠模型中测试了FASN的药理学抑制作用。使用了三种相关的FASN抑制剂；TVB-3664、TVB-3166和临床分期TVB-2640（denifanstat）。在人类原发性肝脏微问题中，FASN抑制剂（FASNi）降低甘油三酯（TG）含量，与直接抗脂肪变性活性一致。在人肝星状细胞中，FASNi降低了纤维化标志物，包括胶原1α（COL1α1）和α-平滑肌肌动蛋白（αSMA）。在暴露于NASH相关细胞因子的CD4+T细胞中，FASNi减少了Th17细胞的产生，并减少了LPS刺激的PBMC中IL-1β的释放[3]。

在饮食诱导的NASH 1小鼠中，FASNi可防止肝脂肪变性和纤维化的发展，并降低循环IL-1β。在已确定饮食诱导的NASH小鼠中，FASNi降低了NAFLD活性评分、纤维化评分、ALT和TG水平。在CCl4诱导的FAT-NASH小鼠模型中，FASN抑制使肝纤维化和纤维化标志物以及肝细胞癌（HCC）肿瘤的发展减少85%。这些结果表明，FASN抑制通过直接抑制免疫细胞和星状细胞来减弱NASH的炎症和纤维化驱动因素，而不仅仅是减少肝细胞中的脂肪积累。因此，FASN抑制为靶向NASH的三个关键特征提供了机会[3]。

生化和细胞效价测定[3]
如Chung等人37所述，使用基于荧光的硫醇定量测定法测定FASN酶活性。该测定监测辅酶A（CoA）的释放，辅酶A是FASN活性的副产物，与荧光染料CPM（7-二乙氨基-3-（4-马来酰亚胺苯基）-4-甲基香豆素）（Sigma C1484）反应并使其具有荧光性。对于FASN生化测定，从SKBr3细胞中提取人FASN蛋白。棕榈酸酯合成测定如前所述25，通过在含有13C-乙酸盐的培养基中培养细胞，并通过LC–MS分析测量转化为棕榈酸酯。显示了2至15个独立测定的平均值<小时> 活性测定[4]
通过使用CLARIOstar分光光度计（软件版本：5.20 R5）在334下监测NADPH氧化，测定有和没有Denifanstat的hFASN的KR结构域的活性 nm，在反式-1-十氢萘酮存在下。反应在37 96孔微孔板中的°C，最终体积为150 µl。测定混合物含有50 mM磷酸钠，pH 7.4，1 mM DTT，0.04%（v/v）吐温-20400 µM NADPH，5 mM反式-1-十氢萘酮和100 nM纯化的蛋白质。对于蛋白质抑制，10 使用µM Denifanstat。野生型L1097A和H878A-hFASN的DH结构域活性是通过在334处由NADPH氧化引起的吸光度降低用分光光度法测量的 nm。反应在37 96孔微孔板中的°C，最终体积为150 µl。反应混合物含有50 mM磷酸钠，pH 7.4，1 mM TCEP，1 mM EDTA，40 µM NADPH和800 µM DLβ-羟基丁酰基辅酶A和100 nM纯化的蛋白质。取5的校准曲线 分钟，然后再添加基质。除非另有说明，否则该图的误差是根据每个纯化的蛋白质制剂中具有技术重复条件的两次生物复制绘制的。更具体地，使用来自两个单独转染的两种蛋白质制剂（即，生物复制品）。对于每个生物复制品，在相同的96孔板（即技术复制品）上进行两次独立的测定。在添加衬底之后，将每条曲线相对于其第一个测量点进行归一化。每个测定重复在补充图10中单独绘制。所有测定数据都以excel文件的形式提供，其中包含原始和标准化的吸光度值以及每个时间点的计算NADPH浓度。

LMT[3]
LMT是使用冷冻保存的原代人类细胞创建的，并使用已建立的方案在96孔Akura平板中培养，如前所述27。总TG含量用TG-Glo测定法测量。

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LX-2[3]
按照前面所述的程序进行29。简言之，在6孔培养板中，每孔接种150000个LX-2细胞，并使血清饥饿过夜。两组培养皿平行放置。用载体（DMSO）或指示剂量的TVB-3664诱导两组LX-2细胞。在收获前TVB-3664处理48小时后收获一组LX-2细胞。在另一组细胞中，用含有10%FBS的正常DMEM培养基代替载体和TVB-3664，并在收获前再维持48小时。对于RNA表达，从收获的LX-2细胞中提取总RNA，合成cDNA，并通过qPCR定量纤维化基因的表达。未配对T检验用于比较每组的平均结果，因为这些是独立的组。

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HSC的免疫细胞化学[3]
原代人肝星状细胞（phHSC）是从未鉴定患者手术切除的人类肝脏的废弃残留物中分离、纯化和培养的。该方案由纽约西奈山伊坎医学院机构审查委员会（IRB）审查和批准，并获得知情同意。所有方法均按照赫尔辛基宣言进行。用免疫染色DAB技术测定TVB-3664小分子存在下LX-2或phHSC中的Col1α1和αSMA。将100000个LX-2细胞或80000个原代hHSC接种在玻璃盖玻片上。将细胞在补充有0.1%BSA（不含抗生素）的DMEM中饥饿过夜，以同步细胞的代谢活性。然后将细胞与载体或TVB-3664-FASNi以所指示的浓度和持续时间孵育。用1×PBS彻底洗涤细胞，并将其固定在4%多聚甲醛中，用在1×PBS中的0.5%吐温-20透化，并用Dako过氧化物酶阻断剂（0.03%H2O2，叠氮化钠）阻断。为了避免非特异性抗体结合，用Dako蛋白阻断无血清试剂重新阻断细胞。用兔抗胶原1或兔抗αSMA（Abcam，MA）一级抗体对细胞进行免疫染色过夜。平行运行一组无初级抗体对照细胞作为背景对照（数据未显示）。用于本研究的第二抗体是Dako标记的聚合物HRP抗兔，并孵育1小时。然后用Dako DAB发色团处理细胞。用苏木精进行核计数器染色。使用AxioImager Z2立式显微镜中的10×物镜，用Axiocam 503单摄像机捕获抗体信号。图像采集通过Zen2软件进行分析。

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PBMC和CD4+T细胞[3]
从加利福尼亚州帕洛阿尔托的斯坦福血液中心购买了用柠檬酸钠试管采集的健康人体供体血液。根据斯坦福血液中心的许可证，获得了适当的知情同意书和IRB批准(https://stanfordbloodcenter.org/research-updates/donating-for-research/). 所有样本均被取消鉴定。所有方法均按照赫尔辛基宣言进行。通过Ficoll-Paque-Plus密度梯度分离从新鲜收集的PBMC中分离。用Dynabeads™人CD4+T细胞分离试剂盒富集幼稚CD4+T淋巴细胞。 然后通过用1μg/mL抗-CD3和抗-CD28抗体包被的珠子将幼稚的人CD4+T细胞接种到48孔组织培养板（Costar）中来分化。将分化产生人Th17幼稚的人CD4+T细胞培养物置于含有以下添加剂的培养基中4天：来自R&D Systems的抗IL-2（30 ng/mL，抗IL-4抗体（20 ng/mL），抗IFNγ抗体（50 ng/mL）、重组人IL-6（10 ng/mL）和人TGFβ（1 ng/mL）（来自BD Biosciences的重组人IL-1β（10 ng/mL）和重组人IL-23（10 ng/mL。将产生的Th17细胞在补充有10%热灭活FCS和500单位青霉素-链霉素的IMDM GlutaMAX培养基中培养。根据制造商的说明，通过ELISA测量来自原代人PBMC上清液的IL-1β。

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流式细胞术[3]
使用对以下抗原特异性的单克隆抗体（并用所示的荧光标记物标记）：Foxp3-FITC（236A/E7；1:100）、IL-17A PE-Cy7（eBio64DEC17；1:50）、CD4-PerCP（SK3；1:200） 为了分析表面标记物，将细胞在含有0.25%BSA和0.02%叠氮化物的PBS中染色。通过LIVE/Dead Fixable Dead Cell Stain Kit排除死细胞。对于细胞内细胞因子染色，用Brefeldin A（5mg/mL）处理细胞2小时，并根据制造商的说明使用固定/渗透试剂盒进行染色。在FACSCalibur上获得荧光细胞的定量及其染色强度，并用CELLQuest软件分析数据。

Preventative NASH diet-induced model[3]
5-week old male C57BL/6J mice were used. CARE is a USDA certified (Certificate Number 84-R-0081) and OLAW accredited facility. The study design and animal usage were reviewed and approved by the CARE Research IACUC for compliance with regulations prior to study initiation (IACUC number 1628). Mice were fed a diet designed to imitate a WD, containing 40 kcal% fat (vegetable shortening), 20 kcal% fructose, and 2% cholesterol, or normal standard commercial rodent diet. Liver samples were processed and histology performed at IDEXX BioResearch. Masson’s Trichrome stain was used to assess the amount of fibrous connective tissue in the liver, and Oil Red O and adipophilin stained sections were used to characterize histologic features associated with steatosis. A qualified pathologist performed histologic evaluation using the following scoring system on H&E stained sections: 0 = normal; 1 = minimal changes; 2 = mild changes; 3 = moderate changes; 4 = marked changes and 5 = severe changes. Cytokine/chemokine assays were performed by Sagimet Biosciences using terminal bleed samples, by ELISA according to manufacturers instructions.

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Therapeutic NASH diet-induced model[3]
5-week old male C57BL/6J mice were used. All animal experiments were conducted in accordance with Gubra’s bioethical guidelines, fully compliant to internationally accepted principles for the care and use of laboratory animals. The experiments were covered by licenses for Jacob Jelsing (2013-15-2934-00784 and 2015-15-0201-00518) issued by the Danish committee for animal research. Mice were fed a diet designed to imitate a WD, containing 40% high trans-fat with high sugar (20% fructose, 9% sucrose) and 2% cholesterol (Research Diets, Inc., #D09100310), for 44 weeks prior to treatment with TVB-3664, and during treatment. Blood samples were collected in heparinized tubes and plasma separated and stored at -80 °C until analysis. TG, TC, ALT, AST were measured using commercial kits on the Cobas TM C-501 autoanalyzer according to the manufacturer’s instructions. Liver IHC used standardized procedures. NAS scoring used the criteria outlined in Kleiner et al.28. Fibrosis was scored on the basis of 0 (none), 1 (perisinusoidal or periportal), 2 (perisinusoidal and portal/periportal), or 3 (bridging fibrosis). Statistical analysis used one-way ANOVA with Dunnett’s multiple comparison test to compare treated to vehicle. For change in histology score per animal, Fisher’s exact test followed by adjustment for multiple correction using the Bonferroni method.

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Fibrosis and tumor (FAT)-NASH CCl4 model[3]
6-week old male C57BL/6J mice were used. This animal protocol was approved by the IACUC at the Icahn School of Medicine at Mount Sinai, NY (IACUC-2015-0112). The model has been previously described29. Mice were fed a diet designed to imitate a WD, containing 21.2% fat (42% Kcal), 41% sucrose and 1.25% cholesterol (Teklad Custom diet Cat# TD.120528, Envigo, WI), and access to sugar water solution (18.9 g/L d-(+)-Glucose and 23.1 g/L d-(−)-Fructose, and once weekly CCl4 IP injection (0.2 µ/g) for 24 weeks. Mice were treated with either 3 mg/kg TVB-3664 or vehicle (PEG400) for the last 12 weeks, after developing NASH and hepatic fibrosis. Blood serum analysis, fibrogenic gene and protein expression, collagen quantification by Bioquant and NAFLD activity score (NAS) were analyzed as previously described29. Data analysis was accomplished by using GraphPad Prism v7.4 statistical software. Standard error mean (± SEM) was calculated according to unpaired two tailed Mann–Whitney test where Gaussian distribution is non-parametric. Unless otherwise specified, p values < 0.05 were considered statistically significant.

[1]. Fatty Acid Synthase inhibition prevents palmitoylation of SARS-CoV2 Spike Protein and improves survival of mice infected with murine hepatitis virus. BioRxiv, December 21, 2020.

[2]. Heterocyclic modulators of lipid synthesis. WO2012122391A1.

Denifanstat is an orally bioavailable fatty acid synthase (FASN) inhibitor. Due to its antineoplastic activities, it is being investigated for various cancers.
Denifanstat is an orally bioavailable fatty acid synthase (FASN) inhibitor, with potential antineoplastic activity. Upon administration, denifanstat binds to and blocks FASN, which prevents the synthesis of palmitate needed for tumor cell growth and survival. This leads to a reduction in cell signaling, an induction of tumor cell apoptosis and the inhibition of cell proliferation in susceptible tumor cells. FASN, an enzyme responsible for the de novo synthesis of palmitic acid, is overexpressed in tumor cells and plays a key role in tumor metabolism, lipid signaling, tumor cell survival and drug resistance; tumor cells are dependent on increased fatty acid production for their enhanced metabolic needs and rapid growth.

C27H29N5O

439.5521

439.2372

C, 73.78; H, 6.65; N, 15.93; O, 3.64

1399177-37-7

1399177-37-7;

66548316

White to off-white solid powder

5.3

85.7Ų

1

4

4

33

728

0

O=C(C1=CC(C2=NNC(C)=N2)=C(C=C1C)C1CCC1)N1CCC(C2C=CC(C#N)=CC=2)CC1

BBGOSBDSLYHMRA-UHFFFAOYSA-N

InChI=1S/C27H29N5O/c1-17-14-24(22-4-3-5-22)25(26-29-18(2)30-31-26)15-23(17)27(33)32-12-10-21(11-13-32)20-8-6-19(16-28)7-9-20/h6-9,14-15,21-22H,3-5,10-13H2,1-2H3,(H,29,30,31)

4-(1-(4-Cyclobutyl-2-methyl-5-(5-methyl-4H-1,2,4-triazol-3- yl)benzoyl)piperidin-4-yl)benzonitrile

TVB 2640; Denifanstat; FASN-IN-2; ASC-40; TVB-2640; ASC40; TVB2640

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)

DMSO : ~100 mg/mL (~227.51 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.69 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 (5.69 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 (5.69 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 储备液加入到 900 μL 玉米油中并混合均匀。

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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;
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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。