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]

hACC1 (IC50=2.1±0.2 nM) and hACC2 (IC50=6.1±0.8 nM) are inhibited by firsocostat (ND-630). Reversible and extremely ACC-specific suppression exists. By interfering with the phosphopeptide receptor and dimerization sites of the enzyme, firsocostat inhibits the activity of ACC. With an EC50 of 66 nM, firsocostat inhibits the synthesis of fatty acids in HepG2 cells without altering the number of cells overall, the concentration of cellular proteins, or the binding of acetate and cholesterol [1].

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In recombinant human ACC1/ACC2 enzymatic assays, Firsocostat (ND-630; GS-0976; NDI-010976) (0.1-100 nM) dose-dependently inhibited enzyme activity, with IC50 values of 2.1 nM (ACC1) and 6.1 nM (ACC2) [1]
- In primary rat hepatocytes, Firsocostat (ND-630; GS-0976; NDI-010976) (1-10 μM) reduced de novo fatty acid synthesis by 78% at 10 μM, as measured by [14C]-acetate incorporation [1]
- Firsocostat (ND-630; GS-0976; NDI-010976) (0.3-3 μM) dose-dependently decreased intracellular triglyceride accumulation in palmitate-treated hepatocytes, achieving 52% reduction at 3 μM (p < 0.01) [1]
- In human hepatic stellate cells (HSCs), Firsocostat (ND-630; GS-0976; NDI-010976) (1-10 μM) inhibited collagen type Iα1 (COL1A1) mRNA expression by 45% at 10 μM, suppressing HSC activation [2]

When Firsocostat (ND-630; GS-0976; NDI-010976) is given to diet-induced obese rats over an extended period of time, it improves insulin sensitivity, decreases hepatic steatosis, decreases weight gain without changing food intake, and has positive effects on dyslipidemia. Firsocostat Zucker Long-term Firsocostat administration decreased hepatic steatosis, enhanced insulin secretion in response to glucose, and decreased hemoglobin A1c (by 0.9%) in diabetic obese rats. In humans and rats, Firsocostat binds to plasma proteins at rates of 98.5% and 98.6%, respectively. The Sprague-Dawley male rats used in the pharmacokinetic evaluation of Firsocostat showed a plasma t1/2 of 4.5 hours, a bioavailability of 37%, and a clearance of 33 mL/min/kg. The volume of distribution was found to be 1.9 L/kg, and the maximum oral plasma concentration time was observed to be 0.25 hours[1].

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After the confirmation of significant hepatic fibrosis with a 13-week pre-feeding, Firsocostat (ND-630; GS-0976; NDI-010976) (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.

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In high-fat diet (HFD)-induced obese rats, oral administration of Firsocostat (ND-630; GS-0976; NDI-010976) (1, 10, 30 mg/kg once daily for 28 days) dose-dependently reduced hepatic steatosis; 30 mg/kg decreased liver triglyceride content by 45% and total cholesterol by 32% compared to vehicle (p < 0.001) [1]
- In HFD rats, Firsocostat (ND-630; GS-0976; NDI-010976) (30 mg/kg, p.o.) improved insulin sensitivity, with a 38% reduction in fasting glucose and 29% decrease in insulin levels (p < 0.05) [1]
- In MC4R knockout mice (nonalcoholic steatohepatitis, NASH model), Firsocostat (ND-630; GS-0976; NDI-010976) (3, 10 mg/kg p.o. once daily for 12 weeks) ameliorated hepatic fibrosis; 10 mg/kg reduced collagen deposition by 38% and α-smooth muscle actin (α-SMA) expression by 42% (p < 0.01) [2]
- In MC4R knockout mice, Firsocostat (ND-630; GS-0976; NDI-010976) (10 mg/kg) decreased hepatic inflammation, with a 35% reduction in pro-inflammatory cytokine TNF-α mRNA levels and 28% lower IL-6 expression (p < 0.05) [2]

Measurement of ACC1 and ACC2 Activity and Inhibition.[1]
ACC activity was assessed using a luminescent ADP detection assay (ADP-Glo Kinase Assay Kit) that measures enzymatic activity by quantitating the ADP produced during the enzymatic first half-reaction. Specifically, 4.5 μL of assay buffer containing either recombinant hACC1 (GenBank accession no. NM198834; full length with a C-terminal His-tag, 270 kDa, expressed in Baculovirus-infected Sf9 cell-expression system) or recombinant hACC2 (GenBank accession no. NM001093; full length with C-terminal His-tag, 277 kDa, expressed in a Baculovirus-infected Sf9 cell-expression system) were added to the wells of a 384-well Optiplate followed by 0.5 μL of DMSO or DMSO containing inhibitor. Optiplates were incubated at room temperature for 15 min. Then each well received 5.0 μL of substrate mixture to initiate the reaction. Final assay concentrations were 5 nM hACC1 or hACC2, 20 μM ATP, 10 μM (hACC1 assay) or 20 μM (hACC2 assay) acetyl-CoA, 30 mM (hACC1 assay) or 12 mM (hACC2 assay) NaHCO3, 0.01% Brij35, 2 mM DTT, 5% DMSO, inhibitor in half-log increments between 30 μM and 0.0001 μM. After 60-min incubation at room temperature, 10 μL ADP-Glo Reagent was added to terminate the reaction, and plates were incubated at room temperature for 40 min to deplete remaining ATP. Then Kinase Detection Reagent, 20 μL, was added, and plates were incubated for 40 min at room temperature to convert ADP to ATP. ATP was measured via a luciferin/luciferase reaction using a PerkinElmer EnVision 2104 plate reader to assess luminescence.

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Soraphen Displacement and Thermal Shift Assays.[1]
Displacement of fluorescently labeled Soraphen A (Soraphen-TAMARA) from hACC BC by ND-022 was assessed as previously described. The protein thermal shift assay for measuring protein thermal stability was conducted as previously described, using the environmentally sensitive dye SYPRO Orange with fluorescence data acquired at the end of each 1-min interval using a real-time PCR instrument which increased the temperature from 25 °C to 100 °C in increments of 1 °C/min.

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Recombinant ACC1/ACC2 activity assay: Purified recombinant human ACC1 or ACC2 was incubated with Firsocostat (ND-630; GS-0976; NDI-010976) (0.01 nM to 1 μM) and acetyl-CoA (substrate) in assay buffer at 37°C for 60 minutes; the reaction product (malonyl-CoA) was quantified by a coupled enzyme assay; IC50 values were calculated from dose-response curves [1]
- Fatty acid synthesis assay in hepatocytes: Primary rat hepatocytes were seeded in 24-well plates and preincubated with Firsocostat (ND-630; GS-0976; NDI-010976) (1-10 μM) for 1 hour; [14C]-acetate was added and incubated for 4 hours; lipids were extracted, and radioactivity was measured by liquid scintillation counting to assess fatty acid synthesis rate [1]

Measurement of FASyn and FAOxn in Cultured Cells.[1]
FASyn was evaluated in HepG2 cells by measuring the incorporation of [2-14C]acetate into cellular lipids. FAOxn was assessed in C2C12 cells by measuring the release of [14C]O2 and the formation of [14C]acid-soluble materials from [1-14C]palmitate.

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Hepatocyte triglyceride accumulation assay: Primary rat hepatocytes were plated in 96-well plates and treated with palmitate (0.5 mM) to induce steatosis; Firsocostat (ND-630; GS-0976; NDI-010976) (0.3-3 μM) was added simultaneously and incubated for 24 hours; cells were lysed, and triglyceride levels were quantified by a colorimetric assay, normalized to protein content [1]
- HSC activation assay: Human hepatic stellate cells were serum-starved for 24 hours, then treated with Firsocostat (ND-630; GS-0976; NDI-010976) (1-10 μM) for 48 hours; total RNA was extracted, reverse-transcribed to cDNA, and COL1A1 mRNA expression was measured by qPCR (normalized to GAPDH) [2]
- Cytokine expression assay: MC4R knockout mouse hepatocytes were isolated and treated with Firsocostat (ND-630; GS-0976; NDI-010976) (3-10 μM) for 24 hours; TNF-α and IL-6 mRNA levels were quantified by qPCR [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, the bioavailability of Firsocostat (ND-630; GS-0976; NDI-010976) at an oral dose of 10 mg/kg was 72% [1]
- The terminal elimination half-life (t1/2) of Firsocostat (ND-630; GS-0976; NDI-010976) in rats was 6.8 hours, and in dogs it was 11.2 hours [1]
- After oral administration of 30 mg/kg to rats, the peak plasma concentration (Cmax) was 1.8 μg/mL, and the time to peak concentration (Tmax) was 2 hours [1]
- Firsocostat (ND-630; GS-0976; NDI-010976) had high liver permeability, with a liver-to-blood concentration ratio of 12.5 in rats [1]
- Fexostat (ND-630; GS-0976; NDI-010976) had a plasma protein binding rate of 94% in human plasma (equilibrium dialysis method) [1] - Fexostat (ND-630; GS-0976; NDI-010976) was mainly excreted in feces (78%) and a small amount was excreted in urine (12%) in rats [1]

In repeated-dose toxicity studies in rats (up to 100 mg/kg/day) and dogs (up to 50 mg/kg/day) over a period of 4 weeks, Firsocostat (ND-630; GS-0976; NDI-010976) did not cause significant changes in body weight, food intake, or clinical chemical parameters (ALT, AST, creatinine, BUN) [1]. No histopathological abnormalities were observed in major organs (liver, kidney, heart, spleen) in rats and dogs receiving therapeutic doses [1][2]. No signs of hepatotoxicity or gastrointestinal adverse reactions were observed in MC4R knockout mice treated with Firsocostat (ND-630; GS-0976; NDI-010976) (10 mg/kg/day for 12 weeks). [2] - At concentrations up to 10 μM, Firsocostat (ND-630; GS-0976; NDI-010976) did not inhibit the major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) in human liver microsomes [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 being investigated in the clinical trial NCT02781584 (Safety, tolerability, and efficacy of Selonsertib, Firsocostat, and Cilofexor in adult patients with nonalcoholic steatohepatitis (NASH)). Simultaneous inhibition of acetyl-CoA carboxylase (ACC) isoenzymes ACC1 and ACC2 can simultaneously inhibit fatty acid synthesis and stimulate fatty acid oxidation, potentially having a beneficial effect on morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using a structure-based drug design approach, we identified a series of potent allosteric protein-protein interaction inhibitors, such as ND-630. These inhibitors interact with ACC phosphopeptide receptors and dimerization sites, thereby preventing dimerization and inhibiting the enzymatic activity of both ACC isoenzymes, reducing fatty acid synthesis and promoting fatty acid oxidation in cultured cells and animals, and exhibiting good drug-like properties. Long-term administration of ND-630 to diet-induced obese rats reduced hepatic steatosis, improved insulin sensitivity, reduced weight gain without affecting food intake, and improved dyslipidemia. Long-term administration of ND-630 to Zucker diabetic obese rats reduced hepatic steatosis, improved glucose-stimulated insulin secretion, and decreased glycated hemoglobin A1c levels (by 0.9%). These data collectively suggest that the inhibitory effects of this series of compounds on acetyl-CoA carboxylase (ACC) may be helpful in the treatment of a variety of metabolic diseases, including metabolic syndrome, type 2 diabetes, and fatty liver. [1] Acetyl-CoA carboxylase (ACC) is the rate-limiting step in de novo lipogenesis and is highly active in the livers of patients with non-alcoholic steatohepatitis (NASH). GS-0976 (firsocostat), an ACC1 and ACC2 isoenzyme inhibitor, reduced hepatic steatosis and serum fibrosis biomarkers, such as tissue inhibitor of metalloproteinases 1 (TIMP-1), in a randomized controlled trial in NASH patients. However, the effect of this improvement on fibrosis has not been adequately evaluated in preclinical models. This study used melanocortin 4 receptor-deficient mice fed a high-fat diet, exhibiting phenotypes similar to those of patients with non-alcoholic steatohepatitis (NASH), including progressive hepatic steatosis and fibrosis. We evaluated the effects of ACC1/2 inhibitors on liver fibrosis. After confirming significant liver fibrosis in mice during a 13-week pre-feeding period, treatment with GS-0976 (4 and 16 mg/kg/day) for 9 weeks resulted in histological reductions in hepatic malonyl-CoA and triglyceride levels, and improved steatosis. Furthermore, GS-0976 reduced the histological area of liver fibrosis, hydroxyproline content, hepatic type I collagen mRNA expression levels, and plasma tissue inhibitor of metalloproteinases 1 (TMP-1) levels, indicating improved liver fibrosis. GS-0976 treatment was also accompanied by reductions in plasma ALT and AST levels. These data suggest that improving hepatic lipid metabolism by inhibiting ACC1/2 may be a novel option for inhibiting fibrosis progression and improving hepatic steatosis in NASH. [2]

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Firsocostat (ND-630; GS-0976; NDI-010976) is a potent, selective, orally effective acetyl-CoA carboxylase (ACC) 1/2 inhibitor for the treatment of non-alcoholic steatohepatitis (NASH)[1][2]
- Its mechanism of action includes inhibiting ACC-mediated malonyl-CoA synthesis, thereby reducing de novo fatty acid synthesis and promoting hepatic fatty acid oxidation, thus improving hepatic steatosis[1]
-Firsocostat (ND-630; GS-0976; NDI-010976) can also inhibit hepatic stellate cell activation and pro-inflammatory cytokine production, which helps improve NASH-related fibrosis and inflammation[2]
- The drug has good hepatic targeting and pharmacokinetic properties, supporting once-daily oral administration for the treatment of chronic NASH[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

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)

Solubility in Formulation 1: 0.5 mg/mL (0.88 mM) in 1% DMSO + 99% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)