Endogenous Metabolite; human cathepsin K

The inhibitory effect of the tested compounds varied with respect to lag phase, specific growth rate, and biomass yield compared to the control cultures grown under the same conditions without addition of inhibitors. However, P. taiwanensis was able to oxidize vanillin and furfural to vanillic acid and 2-Furoic acid, respectively. Vanillic acid was further metabolized, whereas 2-Furoic acid was secreted outside the cells and remained in the fermentation broth without further conversion. Acetic acid and formic acid were completely consumed from the fermentation broth, while concentration of levulinic acid remained constant throughout the fermentation process. Analysis of free intracellular metabolites revealed varying levels when P. taiwanensis VLB120 was exposed to inhibitory compounds. This resulted in increased levels of ATP to export inhibitors from the cell and NADPH/NADP ratio that provides reducing power to deal with the oxidative stress caused by the inhibitors. Thus, adequate supply of these metabolites is essential for the survival and reproduction of P. taiwanensis in the presence of biomass-derived inhibitors [1].

2-Furoic acid considerably lowers blood levels of triglycerides and cholesterol [2]. Moreover, ATP-dependent citrate lyase, acetyl-CoA synthetase, acyl-CoA cholesterol acyltransferase, sn-glycerol 3-phosphate acyltransferase, phosphatidyl phosphohydrolase, and heparin induction lipoprotein lipase activity are all decreased by 2-furoic acid in the liver and gut[2]. The LD50 of 2-furoic acid in mice was 250 mg/kg ip in an acute toxicity study [2].

Inhibitors threshold concentration test [1]
The inhibitor threshold concentration affecting growth was evaluated using the Growth Profiler 960. The inhibitory compounds were added into minimal medium supplemented with 4.5 g L−1 of glucose in different concentration levels. The media pH was adjusted to 7.0 ± 0.03 with 5 M of sodium hydroxide before inoculation. The same medium without inhibitory compounds was used as control. Aerobic cultivations were carried out in 24-well clear bottom microplate working volume 750 µL at 30 °C, 225 rpm. The Growth Profiler was set to generate a scan of the plate every 20 min. Based on this scan, the Growth Profiler software was used to calculate the density of the cultures in each single well of a plate (green value; G value). A calibration curve was generated to convert the G values into optical density (OD) values. The following equation was obtained from the calibration curve and used throughout the study:

Measurement of inhibitors and extracellular metabolites [1]
The concentration of inhibitors and extracellular metabolites was measured by high-performance liquid chromatography (HPLC). More specifically, quantification of furfural, 5-HMF, vanillin, and their corresponding acid in media was performed on a Dionex Ultimate 3000 HPLC equipped with a Supelco Discovery HS F5-3 HPLC column (150 × 2.1 mm × 3 µm) and a UV detector (260, 277, 304, and 210 nm). Samples (1 µL) were analyzed using a gradient method with mobile phase A: 10-mM ammonium formate, pH 3, and B: acetonitrile. A flow rate of 0.7 mL min−1 was used and the column was held at 30 °C. The program started with 5% of solvent B for 0.5 min and increased linearly to 60% over 5 min. The gradient was thereafter increased to 90% B over 0.5 min and kept at this condition for 2 min. Finally, returned to 5% B and equilibrated until 10 min. Concentrations of glucose, gluconate, acetic acid, formic acid, and levulinic acid were determined using a Dionex Ultimate 3000 HPLC with an Aminex® HPX-87X Ion Exclusion (300 × 7.8 mm) column and RI-150 refractive index detector. Gluconate was measured by UV monitoring at 210 nm. The mobile phase consisted of 5-mM H2SO4, the flow rate was 0.6 mL min−1 and the column was kept at 60 °C. Samples were held at 5 °C during the analysis and 20-µL sample volume injected.

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Measurement of intracellular metabolites [1]
Metabolite measurement was performed on AB SCIEX Qtrap1 5500 mass spectrometer ion-pairing techniques operated in negative mode as previously described [14]. A sample of 20 uL was injected on to an XSELECT HSS XP (150 × 2.1 mm × 2.5 μm) column, which was equilibrated for 10 min before injecting with 100% eluent A (10 mM tributylamine, 10 mM acetic acid (pH 6.86), 5% methanol, and 2% 2-propanol). Gradient elution was set to 0% of eluent B (2-propanol) for the first 5 min, and increased to: 2% (5–9 min), 6% (9–12 min), 11% (12–13.5 min), 28% (13.5–15.5 min), and 53% (15.5–22.5 min), and returned back to 0% (22.5–23 min) and equilibrated for 10 min (23–33 min) with 100% eluent A. The flow rate was 0.4 mL min−1 (0–15.5 min), 0.15 mL min−1 (16.5–23 min), and 0.4 mL min−1 (27–33 min); oven temperature was set to 40 °C. The mass spectrometer was operated in multiple-reaction-monitoring (MRM) mode. The optimized parameters for 0.4-mL min−1 flow rate were as follows: ion-spray voltage, − 4.5 kV; curtain gas and CAD gas, 40 and 12, respectively. The capillary temperature was 500 °C.

2-Furoic acid was shown to be effective in lowering both serum cholesterol and serum triglyceride levels significantly in rats with an elevation of HDL cholesterol level at 20 mg/kg/day orally. LDL receptor activity was reduced in hepatocytes, aorta foam cells, small intestinal epithelium cells and fibroblasts. HDL receptor activity was elevated in the rat hepatocytes and small intestinal cells. These activities were correlated with inhibition of acyl CoA cholesterol acyl transferase activity. Neutral cholesterol ester hydrolase activity was elevated in rat hepatocytes and human fibroblasts. Thus, 2-furoic acid appears to interfere directly with activity of intracellular enzymes rather than affecting high affinity-mediated lipoprotein membrane receptors. In vivo treatment with 2-furoic acid led to reduction in the liver and small intestine ATP dependent citrate lyase, acetyl CoA synthetase, acyl CoA cholesterol acyl transferase, sn-glycerol 3-phosphate acyl transferase, phosphatidylate phosphohydrolase and heparin induced lipoprotein lipase activities. 2-Furoic acid reduced biliary cholesterol levels but the agent increased bile salts which are lithogenic. Acute toxicity studies in mice suggest that the agent has some hepatic toxicity effects. The LD50 was relatively low at 250 mg/kg IP in mice.

mouse LD50 intraperitoneal 100 mg/kg Pharmaceutical Research., 2(233), 1985
mouse LD50 oral 1 gm/kg Biochemical Journal., 34(1196), 1940

[1]. Tolerance and metabolic response of Pseudomonas taiwanensis VLB120 towards biomass hydrolysate-derived inhibitors. Biotechnol Biofuels. 2018 Jul 19;11:199.

[2]. The hypolipidemic effects of 2-furoic acid in Sprague-Dawley rats. Arch Pharm (Weinheim). 1993 Jan;326(1):15-23.

2-furoic acid is a furoic acid having the carboxylic acid group located at position 2. It has a role as an inhibitor, a human xenobiotic metabolite, a Saccharomyces cerevisiae metabolite, a plant metabolite and a bacterial xenobiotic metabolite. It is a conjugate acid of a 2-furoate.
2-Furoic acid has been reported in Phomopsis velata, Aspergillus stellatus, and other organisms with data available.
See also: 2-Furoate (annotation moved to).

C5H4O3

112.08

112.016

C, 53.58; H, 3.60; O, 42.82

88-14-2

2-Furoic acid-d3;40073-83-4

6919

White to off-white solid powder

1.3±0.1 g/cm3

230-232 ºC

129-133 ºC

137 ºC

0.0±0.5 mmHg at 25°C

1.513

0.64

1

3

1

8

99.8

0

SMNDYUVBFMFKNZ-UHFFFAOYSA-N

InChI=1S/C5H4O3/c6-5(7)4-2-1-3-8-4/h1-3H,(H,6,7)

2-Furancarboxylic acid

alpha-Furoic acid; 2-Furoic acid; 2-FUROIC ACID; Furan-2-carboxylic acid; 88-14-2; 2-Furancarboxylic acid; Pyromucic acid; 2-Carboxyfuran; FUROIC ACID; Furancarboxylic acid; alpha-Furancarboxylic acid; Kyselina 2-furoova; 2 Furoic 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)

DMSO : ≥ 100 mg/mL (~892.22 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (22.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (22.31 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (22.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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