Human acetyl-CoA carboxylase (hACC1) (IC50 = 2.1 nM); hACC2 (IC50 = 6.1 nM)

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].

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.

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.

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.

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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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

[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]

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.)