Human acetyl-CoA carboxylase
Firsocostat (S enantiomer) targets acetyl‑CoA carboxylase 1 (ACC1) and acetyl‑CoA carboxylase 2 (ACC2). IC50 values: hACC1 = 2.1 ± 0.2 nM (n=7); hACC2 = 6.1 ± 0.8 nM (n=15) [1].

ND-630 (also known as GS-0976; NDI-010976; firsocostat) is a potent inhibitor of ACC (acetyl-CoA carboxylase). As a potent allosteric protein-protein interaction inhibitor, ND-630 interacts within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibits the enzymatic activity of both ACC isozymes, reduces fatty acid synthesis and stimulates fatty acid oxidation in cultured cells and in animals, and exhibits favorable drug-like properties. ND-630 inhibits hACC1 (IC50=2.1±0.2 nM) and hACC2 (IC50=6.1±0.8 nM). Inhibition is reversible and highly specific for ACC. ND-630 inhibits ACC activity by interacting within the phosphopeptide-acceptor and dimerization site of the enzyme to prevent dimerization. ND-630 inhibits fatty acid synthesis with an EC50 of 66 nM in HepG2 cells without altering the total cell number, cellular protein concentration, and incorporation of acetate into cholesterol.

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Kinase Assay: ND-630 inhibits human ACC1 and ACC2 with IC50 values of 2.1 and 6.1 nM, respectively.

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Cell Assay: ND-630 interacts 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.

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Firsocostat (S enantiomer) inhibited recombinant hACC1 and hACC2 in a reversible manner with high specificity. At 10 μM, it showed no effect on the activity of 101 enzymes, receptors, growth factors, transporters, and ion channels (Ricerca DrugMatrix Panel) [1].
Mechanism: Firsocostat (S enantiomer) binds within the ACC dimerization site, preventing dimerization. In non‑denaturing PAGE, hACC2 BC migrated as a dimer in the absence of the compound and as a monomer in its presence (1:2, 1:1, and 2:1 mol ratios). Co‑crystal structure of ND‑646 (primary amide of Firsocostat) with hACC2 BC (2.6 Å) showed interactions with residues Arg277, Val587, Tyr683, and others, filling a narrow deep pocket near Val587 and Tyr683 [1].
In HepG2 cells, Firsocostat (S enantiomer) inhibited [¹⁴C]acetate incorporation into fatty acids with EC50 values of 66 nM (cells with 10% serum) and 8.7 nM (serum‑free medium) without affecting cell number, protein concentration, or [¹⁴C]acetate incorporation into cholesterol [1].
In C2C12 cells, Firsocostat (S enantiomer) increased [¹⁴C]palmitate oxidation (both ¹⁴CO₂ release and ¹⁴C acid‑soluble material production) in a concentration‑dependent manner [1].

Chronical administration of ND-630 to rats with diet-induced obesity reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. Chronical administration of ND-630 Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). ND-630 exhibits an aqueous solubility of 594 μM and human and rat plasma protein binding of 98.5% and 98.6%, respectively. Pharmacokinetic evaluation of ND-630 in male Sprague–Dawley rats [i.v. 3 mg/kg; orally (p.o.) 10 mg/kg] yields a plasma t1/2 of 4.5 h, bioavailability of 37%, clearance of 33 mL/min/kg, volume of distribution of 1.9 L/kg, oral time of maximum plasma concentration of 0.25 h.
Acute in vivo: In chow‑fed male Sprague‑Dawley rats, oral Firsocostat (S enantiomer) dose‑dependently reduced hepatic malonyl‑CoA (ED50 0.8 mg/kg), skeletal muscle malonyl‑CoA (gastrocnemius, EDL, soleus; ED50 3‑10 mg/kg), and hepatic fatty acid synthesis (ED50 0.14 mg/kg) as measured by [¹⁴C]acetate incorporation. It also reduced respiratory quotient (RQ) in high‑carbohydrate‑fed rats, indicating increased whole‑body fatty acid oxidation [1].
Chronic in diet‑induced obese (DIO) rats on high‑sucrose diet (HSD): 4‑wk oral QD dosing reduced cumulative body weight gain by up to 20% without affecting food intake, normalized plasma leptin, reduced hepatic steatosis (triglycerides), lowered plasma triglycerides and free fatty acids, increased plasma ketones, reduced plasma cholesterol, and improved insulin sensitivity (reduced insulin excursion and AUC in oGTT) at doses tested [1].
Chronic in DIO rats on high‑fat diet (HFD): 2‑wk oral QD dosing reduced cumulative weight gain by up to 26% without affecting food intake, reduced hyperleptinemia, reduced hyperinsulinemia without altering plasma glucose, reduced hepatic triglycerides (normalized at highest dose) and hepatic cholesterol, and improved insulin sensitivity (reduced insulin excursion and AUC in ipGTT) [1].
Chronic in Zucker diabetic fatty (ZDF) rats (8 wk old, prediabetic to overt diabetes): 37‑d oral b.i.d. dosing (5, 15, 30 mg/kg b.i.d.) dose‑dependently reduced hepatic triglycerides (up to 64%), free fatty acids (up to 60%), and cholesterol (up to 32%), increased plasma ketones, significantly increased glucose‑stimulated insulin secretion (GSIS) (up to 80% at 15 min after glucose challenge on day 21), and reduced hemoglobin A1c by 0.9% (from 10.2 ± 0.3% to 9.3 ± 0.2% at 5 mg/kg b.i.d., P=0.029) [1].

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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ACC enzyme activity was measured using a luminescent ADP detection assay (ADP‑Glo Kinase Assay Kit). Recombinant full‑length hACC1 (C‑terminal His‑tag) or hACC2 (C‑terminal His‑tag) was incubated with compound in DMSO for 15 min at room temperature in 384‑well plates. Substrate mixture (ATP, acetyl‑CoA, NaHCO₃, Brij35, DTT) was added to initiate the reaction. Final assay concentrations: 5 nM enzyme, 20 μM ATP, 10 μM (ACC1) or 20 μM (ACC2) acetyl‑CoA, 30 mM (ACC1) or 12 mM (ACC2) NaHCO₃, 0.01% Brij35, 2 mM DTT, 5% DMSO. After 60 min, ADP‑Glo Reagent was added to terminate the reaction and deplete remaining ATP (40 min). Kinase Detection Reagent was added (40 min) and luminescence was read [1].
For hACC2 BC expression and crystallization: hACC2 BC (residues 217‑775) with N‑terminal His‑tag was expressed in E. coli, purified, and crystallized with ND‑646 (primary amide of Firsocostat) using sitting‑drop vapor diffusion. Crystals diffracted to 2.6 Å; structure solved by molecular replacement [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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Fatty acid synthesis (FASyn) in HepG2 cells: Cells were cultured in DMEM with 10% FBS or serum‑free medium. Firsocostat (S enantiomer) and [2‑¹⁴C]acetate were added for 4 h. Lipids were extracted and radioactivity counted. EC50 values were calculated [1].
Fatty acid oxidation (FAOxn) in C2C12 cells: Cells were treated with Firsocostat (S enantiomer) and [1‑¹⁴C]palmitate for 6 h. Released ¹⁴CO₂ was trapped in NaOH, and acid‑soluble materials were precipitated and counted [1].

Aqueous solubility: 594 μM. Plasma protein binding: human 98.5%, rat 98.6%. In male Sprague‑Dawley rats, following i.v. (3 mg/kg) and oral (10 mg/kg) administration: t₁/₂ = 4.5 h; oral bioavailability = 37%; Cl = 33 mL/min/kg; Vdss = 1.9 L/kg; Tmax (oral) = 0.25 h; AUC₀‑∞ (oral) = 1,932 ng·h/mL; Cmax plasma = 6.0 μM; Cmax liver = 81 μM; Cmax quadriceps = 0.6 μM; liver:plasma:quadriceps exposure ratio at Tmax = 135:10:1. Firsocostat (S enantiomer) does not cross the blood‑brain barrier [1].

Maximum tolerated dose (single oral): rats (n=6/group) given 100, 300, or 1,000 mg/kg showed no significant differences in body weight, hematology, coagulation, or clinical chemistry [1].
28‑day repeat oral dose (QD) in rats (10 M and 10 F, doses up to 60 mg·kg⁻¹·d⁻¹, 428× FASyn ED50): no compound‑related clinical signs, changes in body weight, food consumption, hematology, coagulation, or clinical chemistries; no target organ toxicity and no mortality [1].
Cardiovascular safety in Beagle dogs (4 M and 4 F): ECG recordings (leads I, II, III, aVR, aVL, aVF) at pretreatment, after 1 d and after 4 wk of oral dosing (100 mg·kg⁻¹·d⁻¹, at Tmax 1‑4 h). All dogs maintained sinus rhythm; no drug‑related effects on heart rate, RR, PR, QRS, QT/QTc intervals [1].
No adverse cardiac effects were observed at supratherapeutic doses (e.g., 428× ED50) [1].

Proc Natl Acad Sci U S A.2016 Mar 29;113(13):E1796-805.

Simultaneous inhibition of acetyl-CoA carboxylase (ACC) isoenzymes ACC1 and ACC2 can simultaneously inhibit fatty acid synthesis and promote fatty acid oxidation, potentially having a beneficial effect on the morbidity and mortality associated with obesity, diabetes, and fatty liver. 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 reduced glycated hemoglobin A1c (by 0.9%). These data collectively suggest that the inhibitory effect of this series of compounds on ACC may contribute to the treatment of various metabolic diseases, including metabolic syndrome, type 2 diabetes, and fatty liver. [1]

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Firsocostat (S enantiomer) (ND‑630) is a first‑in‑class allosteric ACC inhibitor that binds to the dimerization domain (BC domain) and mimics the physiological inhibition of ACC by AMPK. Unlike active‑site directed CT domain inhibitors, its inhibition is not affected by postprandial increases in citrate or by relief of feedback inhibition from fatty acyl‑CoAs. The hydrophilic nature of the binding site enabled identification of inhibitors with favorable physicochemical properties. Firsocostat (S enantiomer) does not cross the blood‑brain barrier, so it does not affect hypothalamic malonyl‑CoA or increase food intake. It has potential therapeutic utility in metabolic syndrome, type 2 diabetes, nonalcoholic fatty liver disease, and related disorders. The compound was prepared as described in patent US 2013/0123231 [1].

C28H31N3O8S

569.626046419144

569.18318

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

2128714-16-7

124672214

White to off-white solid powder

3.2

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-HXUWFJFHSA-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-/m1/s1

2-[1-[(2S)-2-(2-methoxyphenyl)-2-(oxan-4-yloxy)ethyl]-5-methyl-6-(1,3-oxazol-2-yl)-2,4-dioxothieno[2,3-d]pyrimidin-3-yl]-2-methylpropanoic acid

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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