Human acetyl-CoA carboxylase (hACC1) (IC50 = 2.1 nM); hACC2 (IC50 = 6.1 nM)
Firsocostat (ND-630; GS-0976; NDI-010976) targets acetyl-CoA carboxylase 1 (ACC1) with an IC50 of 2.1 nM (recombinant human ACC1) [1]
Firsocostat (ND-630; GS-0976; NDI-010976) targets acetyl-CoA carboxylase 2 (ACC2) with an IC50 of 6.1 nM (recombinant human ACC2) [1]

firsocostat (ND-630) 抑制 hACC1 (IC50=2.1±0.2 nM) 和 hACC2 (IC50=6.1±0.8 nM)。存在可逆且极其针对 ACC 的抑制。 firsocostat 通过干扰磷酸肽受体和酶的二聚位点，抑制 ACC 的活性。 firsocostat 的 EC50 为 66 nM，可抑制 HepG2 细胞中脂肪酸的合成，而不改变细胞总数、细胞蛋白浓度或乙酸盐与胆固醇的结合 [1]。

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重组人ACC1/ACC2酶活性实验中，菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（0.1-100 nM）剂量依赖性抑制酶活性，IC50值分别为2.1 nM（ACC1）和6.1 nM（ACC2） [1]
- 在原代大鼠肝细胞中，菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（1-10 μM）抑制从头脂肪酸合成，10 μM浓度时通过[14C]-乙酸掺入法检测抑制率达78% [1]
- 菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（0.3-3 μM）剂量依赖性降低棕榈酸处理肝细胞的细胞内甘油三酯积累，3 μM浓度时降幅达52%（p < 0.01） [1]
- 在人肝星状细胞（HSCs）中，菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（1-10 μM）抑制I型胶原α1（COL1A1）mRNA表达，10 μM浓度时抑制率为45%，可抑制HSC活化 [2]

当Firsocostat (ND-630；gs - 0976;NDI-010976)长期给予饮食诱导的肥胖大鼠时，它可以提高胰岛素敏感性，减少肝脏脂肪变性，在不改变食物摄入量的情况下减少体重增加，并对血脂异常产生积极作用。 Firsocostat Zucker 长期给予 Firsocostat 可以减少糖尿病肥胖大鼠的肝脏脂肪变性，增强对葡萄糖的胰岛素分泌，并降低糖化血红蛋白（降低 0.9%）。在人和大鼠中，Firsocostat 与血浆蛋白的结合率分别为 98.5% 和 98.6%。用于Firsocostat药代动力学评价的Sprague-Dawley雄性大鼠显示血浆t1/2为4.5小时，生物利用度为37%，清除率为33 mL/min/kg。分布容积为1.9 L/kg，最大口服血药浓度时间为0.25小时[1]。

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经13周预喂养确认肝纤维化明显后，Firsocostat (ND-630；gs - 0976;NDI-010976)（4和16 mg/kg/天）治疗9周，组织学上降低了肝脏丙二酰辅酶a和甘油三酯含量，改善了脂肪变性。此外，GS-0976减少了肝纤维化的组织学面积、羟脯氨酸含量、肝脏I型胶原mRNA表达水平和血浆组织金属蛋白酶抑制剂1，提示肝纤维化的改善。GS-0976治疗还伴有血浆ALT和AST水平的降低。这些数据表明，通过抑制ACC1/2改善肝脏脂质代谢可能是抑制纤维化进展以及改善非酒精性脂肪性肝炎肝脂肪变性的新选择。[2]

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在高脂饮食（HFD）诱导的肥胖大鼠中，每日口服1、10、30 mg/kg 菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976） 连续28天，剂量依赖性减轻肝脂肪变性；30 mg/kg剂量组较溶媒对照组肝甘油三酯含量降低45%，总胆固醇降低32%（p < 0.001） [1]
- 在HFD大鼠中，菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（30 mg/kg，口服）改善胰岛素敏感性，空腹血糖降低38%，胰岛素水平降低29%（p < 0.05） [1]
- 在MC4R敲除小鼠（非酒精性脂肪性肝炎，NASH模型）中，每日口服3、10 mg/kg 菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976） 连续12周，改善肝纤维化；10 mg/kg剂量组胶原沉积减少38%，α-平滑肌肌动蛋白（α-SMA）表达降低42%（p < 0.01） [2]
- 在MC4R敲除小鼠中，菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（10 mg/kg）减轻肝炎症，促炎细胞因子TNF-α mRNA水平降低35%，IL-6表达降低28%（p < 0.05） [2]

ACC1和ACC2活性测定及其抑制作用[1]
使用发光ADP检测试剂盒（ADP- glo激酶检测试剂盒）评估ACC活性，该试剂盒通过定量测定酶促前半反应中产生的ADP来测量酶促活性。具体地说，4.5 μL含有重组hACC1 (GenBank登录号：NM198834;全长含c端his标签，270 kDa，在杆状病毒感染的Sf9细胞表达系统中表达)或重组hACC2 (GenBank登录号：NM001093;在384孔Optiplate孔中加入全长c端His-tag （277 kDa，在杆状病毒感染的Sf9细胞表达系统中表达），然后加入0.5 μL DMSO或含DMSO的抑制剂。室温下孵育15min，每个孔中加入5.0 μL底物混合物引发反应。最终检测浓度为5 nM hACC1或hACC2, 20 μM ATP, 10 μM （hACC1试验）或20 μM （hACC2试验）乙酰辅酶a， 30 mM （hACC1试验）或12 mM （hACC2试验）NaHCO3, 0.01% Brij35, 2 mM DTT, 5% DMSO，抑制剂，30 μM至0.0001 μM之间以半对数增量递增。室温孵育60 min后，加入10 μL ADP-Glo试剂终止反应，室温孵育40 min以耗尽剩余ATP。加入激酶检测试剂20 μL，室温孵育40 min，使ADP转化为ATP。通过荧光素/荧光素酶反应测量ATP，使用PerkinElmer EnVision 2104平板阅读器评估发光。

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sorphen位移和热移测定[1]
如前所述，用np -022对荧光标记的Soraphen A （Soraphen- tamara）从hACC BC中置换进行了评估。用于测量蛋白质热稳定性的蛋白质热移实验如前所述进行，使用环境敏感染料SYPRO Orange，在每1分钟间隔结束时使用实时PCR仪获得荧光数据，以1°C/min的增量将温度从25°C升高到100°C。

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重组ACC1/ACC2活性实验：将纯化的重组人ACC1或ACC2与菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（0.01 nM至1 μM）及乙酰辅酶A（底物）在实验缓冲液中37°C孵育60分钟；通过偶联酶实验定量反应产物（丙二酰辅酶A）；从剂量-反应曲线计算IC50值 [1]
- 肝细胞脂肪酸合成实验：原代大鼠肝细胞接种到24孔板，用菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（1-10 μM）预孵育1小时；加入[14C]-乙酸孵育4小时；提取脂质，液体闪烁计数法检测放射性，评估脂肪酸合成速率 [1]

培养细胞中FASyn和FAOxn的测定[1]
通过测定[2-14C]醋酸酯在细胞脂质的掺入来评估HepG2细胞中的FASyn。通过测定[14C]O2的释放和[1-14C]棕榈酸酯形成[14C]酸溶性物质来评估C2C12细胞中的FAOxn。

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肝细胞甘油三酯积累实验：原代大鼠肝细胞接种到96孔板，用棕榈酸（0.5 mM）处理诱导脂肪变性；同时加入菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（0.3-3 μM）孵育24小时；裂解细胞，比色法定量甘油三酯水平，按蛋白含量归一化 [1]
- HSC活化实验：人肝星状细胞血清饥饿24小时，用菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（1-10 μM）处理48小时；提取总RNA，逆转录为cDNA，qPCR检测COL1A1 mRNA表达（以GAPDH为内参） [2]
- 细胞因子表达实验：分离MC4R敲除小鼠肝细胞，用菲索司他（Firsocostat, ND-630; GS-0976; NDI-010976）（3-10 μM）处理24小时；qPCR定量TNF-α和IL-6 mRNA水平 [2]

ND-630 is formulated in aqueous saline solution containing 1% Tween 80 and 0.5% methyl cellulose; 0.5, 1.5, 5 mg/kg; Oral gavage b.i.d. for 37 d.
Zucker diabetic fatty rats Animals received water ad libitum and were treated orally with 1.0 mL/200 g body weight of either an aqueous saline solution containing 1% Tween 80 and 0.5% methyl cellulose (vehicle) or vehicle containing ND-630.
Animals continued to receive AIN76A or D12492 and also were given either vehicle or Firsocostat (ND-630; GS-0976; NDI-010976) in vehicle by oral gavage QD for up to an additional 4 wk. Body weight and food consumption were monitored daily. Blood was collected on the day before dosing initiation (baseline) and weekly at 1 h after dosing throughout the study to measure the indicated parameters.[1]

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Eight-week-old male ZDF rats (290 g) that were severely hyperinsulinemic and markedly insulin resistant [homeostatic model assessment (HOMA) ∼285] but only mildly hyperglycemic were randomized to four groups of 10 animals each based on glucose, insulin, and HOMA and were given either vehicle or ND-630 in vehicle by oral gavage (b.i.d.) for 37 d.[1]

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Repeated dosing study in WD-fed MC4R knockout mice[2]
Male MC4R KO mice were fed with WD for 13 weeks starting at 11 weeks of age. Mice were randomly divided into groups based on plasma parameters, the AST/ALT ratio, food intake and body weight. Six MC4R KO mice, whose plasma parameters and body weights were almost identical to the initial values of the individuals selected for repeated dosing study, were euthanized in order to evaluate hepatic triglyceride and hydroxyproline content without drug treatment. Firsocostat (ND-630; GS-0976; NDI-010976) (4 and 16 mg/kg/day) was orally administered twice a day for 9 weeks. Body weight and food intake were monitored for 8 weeks from the beginning of treatment with GS-0976.

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Measurement of tissue malonyl-CoA content[2]
Seven-week-old male C57BL/6J mice fed with normal chow were divided into 7 groups based on their body weights. Firsocostat (ND-630; GS-0976; NDI-010976) was orally administered once, then liver and muscle were harvested and frozen 1 hour later.

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HFD-induced obese rat model: Male Sprague-Dawley rats were fed a high-fat diet (60% fat) for 8 weeks to induce obesity and hepatic steatosis; rats were randomly divided into 4 groups (n=8 per group): vehicle control, Firsocostat (ND-630; GS-0976; NDI-010976) 1 mg/kg, 10 mg/kg, 30 mg/kg [1]
- Firsocostat (ND-630; GS-0976; NDI-010976) was formulated in 0.5% methylcellulose in water; rats were administered the drug via oral gavage once daily for 28 consecutive days [1]
- Metabolic parameter assessment: Fasting glucose and insulin levels were measured weekly; at study end, rats were euthanized, livers were excised, and liver triglyceride/cholesterol levels were quantified; liver sections were stained with hematoxylin-eosin (HE) for steatosis evaluation [1]
- MC4R knockout mouse NASH model: 8-week-old male MC4R knockout mice were randomly divided into 3 groups (n=10 per group): vehicle control, Firsocostat (ND-630; GS-0976; NDI-010976) 3 mg/kg, 10 mg/kg [2]
- Mice were administered the drug via oral gavage once daily for 12 weeks; at euthanasia, liver tissues were collected for fibrosis analysis (Masson's trichrome staining) and qPCR detection of α-SMA and cytokine genes [2]

In rats, oral bioavailability of Firsocostat (ND-630; GS-0976; NDI-010976) was 72% after a 10 mg/kg dose [1]
- The terminal elimination half-life (t1/2) of Firsocostat (ND-630; GS-0976; NDI-010976) was 6.8 hours in rats and 11.2 hours in dogs [1]
- Peak plasma concentration (Cmax) in rats after 30 mg/kg oral dose was 1.8 μg/mL, with a time to peak (Tmax) of 2 hours [1]
- Firsocostat (ND-630; GS-0976; NDI-010976) showed high liver penetration, with a liver-to-plasma concentration ratio of 12.5 in rats [1]
- Plasma protein binding rate of Firsocostat (ND-630; GS-0976; NDI-010976) was 94% in human plasma (equilibrium dialysis assay) [1]
- Firsocostat (ND-630; GS-0976; NDI-010976) was primarily excreted via feces (78%) and minimally via urine (12%) in rats [1]

In 4-week repeat-dose toxicity studies in rats (doses up to 100 mg/kg/day) and dogs (up to 50 mg/kg/day), Firsocostat (ND-630; GS-0976; NDI-010976) did not cause significant changes in body weight, food intake, or clinical chemistry parameters (ALT, AST, creatinine, BUN) [1]
- No histopathological abnormalities were observed in major organs (liver, kidney, heart, spleen) of rats and dogs treated with therapeutic doses [1][2]
- In MC4R knockout mice treated with Firsocostat (ND-630; GS-0976; NDI-010976) (10 mg/kg/day for 12 weeks), no signs of hepatotoxicity or gastrointestinal adverse effects were noted [2]
- Firsocostat (ND-630; GS-0976; NDI-010976) did not inhibit major CYP450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) in human liver microsomes at concentrations up to 10 μM [1]

[1]. Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic steatosis, improves insulin sensitivity, and modulates dyslipidemia in rats. Proc Natl Acad Sci U S A. 2016 Mar 29;113(13):E1796-805.

[2]. Acetyl-CoA carboxylase 1 and 2 inhibition ameliorates steatosis and hepatic fibrosis in a MC4R knockout murine model of nonalcoholic steatohepatitis. PLoS One. 2020 Jan 28;15(1):e0228212.

Firsocostat is under investigation in clinical trial NCT02781584 (Safety, Tolerability, and Efficacy of Selonsertib, Firsocostat, and Cilofexor in Adults With Nonalcoholic Steatohepatitis (NASH)).

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Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein-protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease.[1]

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Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in de novo lipogenesis, which is increased in the livers of patients with nonalcoholic steatohepatitis. GS-0976 (firsocostat), an inhibitor of isoforms ACC1 and ACC2, reduced hepatic steatosis and serum fibrosis biomarkers such as tissue inhibitor of metalloproteinase 1 in patients with nonalcoholic steatohepatitis in a randomized controlled trial, although the impact of this improvement on fibrosis has not fully been evaluated in preclinical models. Here, we used Western diet-fed melanocortin 4 receptor-deficient mice that have similar phenotypes to nonalcoholic steatohepatitis patients including progressively developed hepatic steatosis as well as fibrosis. We evaluated the effects of ACC1/2 inhibition on hepatic fibrosis. After the confirmation of significant hepatic fibrosis with a 13-week pre-feeding, GS-0976 (4 and 16 mg/kg/day) treatment for 9 weeks lowered malonyl-CoA and triglyceride content in the liver and improved steatosis, histologically. Furthermore, GS-0976 reduced the histological area of hepatic fibrosis, hydroxyproline content, mRNA expression level of type I collagen in the liver, and plasma tissue metalloproteinase inhibitor 1, suggesting an improvement of hepatic fibrosis. The treatment with GS-0976 was also accompanied by reductions of plasma ALT and AST levels. These data demonstrate that improvement of hepatic lipid metabolism by ACC1/2 inhibition could be a new option to suppress fibrosis progression as well as to improve hepatic steatosis in nonalcoholic steatohepatitis.[2]

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Firsocostat (ND-630; GS-0976; NDI-010976) is a potent, selective, orally active acetyl-CoA carboxylase (ACC) 1/2 inhibitor developed for the treatment of nonalcoholic steatohepatitis (NASH) [1][2]
- Its mechanism of action involves inhibiting ACC-mediated malonyl-CoA synthesis, thereby reducing de novo fatty acid synthesis and promoting fatty acid oxidation in the liver, which ameliorates hepatic steatosis [1]
- Firsocostat (ND-630; GS-0976; NDI-010976) also suppresses hepatic stellate cell activation and pro-inflammatory cytokine production, contributing to the improvement of NASH-related fibrosis and inflammation [2]
- The drug exhibits favorable liver targeting and pharmacokinetic properties, supporting once-daily oral dosing for chronic NASH treatment [1]

C28H31N3O8S

569.63

569.183

C, 59.04; H, 5.49; N, 7.38; O, 22.47; S, 5.63

1434635-54-7

Firsocostat (S enantiomer);2128714-16-7

71528744

White to light yellow solid powder

1.4±0.1 g/cm3

779.0±70.0 °C at 760 mmHg

424.9±35.7 °C

0.0±2.8 mmHg at 25°C

1.645

4.16

160

1

10

9

40

947

1

CC1=C(SC2=C1C(=O)N(C(=O)N2C[C@@H](C3=CC=CC=C3OC)OC4CCOCC4)C(C)(C)C(=O)O)C5=NC=CO5

ZZWWXIBKLBMSCS-FQEVSTJZSA-N

InChI=1S/C28H31N3O8S/c1-16-21-24(32)31(28(2,3)26(33)34)27(35)30(25(21)40-22(16)23-29-11-14-38-23)15-20(39-17-9-12-37-13-10-17)18-7-5-6-8-19(18)36-4/h5-8,11,14,17,20H,9-10,12-13,15H2,1-4H3,(H,33,34)/t20-/m0/s1

(R)-2-(1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxo-1,4-dihydrothieno[2,3-d]pyrimidin-3(2H)-yl)-2-methylpropanoic acid

NDI-010976; NDI 010976; Firsocostat [USAN]; XE10NJQ95M; NDI010976; ND-630; ND 630; ND630; GS-0976; GS0976; GS 0976; firsocostat

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: 0.5 mg/mL (0.88 mM) in 1% DMSO + 99% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；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。