FK866 (APO866) (Synonyms: K 22.175)

Preparation Method

To determine the IC₅₀ of inhibitors, 5 μl FK866 (APO866) solutions (containing 10% DMSO) with various concentrations were added into 96-well plate. The plate was incubated at 37 ℃ for 5 min after addition of 16.5 μl reaction buffer containing NAMPT. The enzyme reactions were initiated by 4.5 μl NAM (1.11 μM) following NMN measurement as described above. The IC₅₀ values were determined by non-linear fitting of the concentration-dependent curves with the four-parameter IC₅₀ logistic equation.

Reaction Conditions

37℃ for 5 min

Applications

IC₅₀ of FK866 (APO866) on NAMPT activity is 1.60±0.32 nmol/L

Cell lines

HepG2

Preparation Method

Cells were seeded in 96-well plate and starved for over 12 h with serum-free DMEM at 60~70% confluency, then treated with FK866 (APO866) or vehicle for 24 h to 72 h according to experiment requirements.10 μl CCK-8 solution was added to the culture medium and incubated at 37 °C for 1 h. The absorbance at 450 nm (A450) was detected by a plate reader.

Reaction Conditions

48 h

Applications

IC₅₀ of FK866 (APO866) on HepG2 cells is 2.21±0.21 nmol/L.

Cell lines

RAW 264.7 and MODE-K cells

Preparation Method

Cells were stimulated with ultrapure 100ng/mL lipopolysaccharide (LPS) or 1µg/mL flagellin followed by incubation with or without 200nM FK866 (APO866) overnight. Supernatants were harvested and protein was extracted with NE-PER containing protease and phosphatase inhibitors and stored at -80℃ until further workup.

Reaction Conditions

200nM FK866 (APO866) overnight

Applications

FK866 (APO866)treatment strongly reduced NF-κB phosphorylation consequent to LPS treatment.

Animal models

8-week-old female wild-type (WT) or Rag1tm1Mom/J (Rag1−/−) mice

Preparation method

Acute colitis was induced in mice with 3.5% or 3% dextran sulfate sodium ad libitum for 5 consecutive days, followed by a tap water period until end of experiments. Control mice received tap water during the study period. Mice were injected intraperitoneally with 10mg/kg bodyweight FK866 (APO866) or vehicle control twice daily until termination of experiments.

Dosage form

Intraperitoneally with 10mg/kg FK866 (APO866)

Applications

FK866 (APO866) significantly ameliorated all features of DSS-induced colitis in Rag1−/− mice.

References:

[1]. Zhang SL, Xu TY, Yang ZL, Han S, Zhao Q, Miao CY. Crystal structure-based comparison of two NAMPT inhibitors. Acta Pharmacol Sin. 2018 Feb;39(2):294-301.

[2]. Gerner RR, Klepsch V, Macheiner S, Arnhard K, Adolph TE, Grander C, Wieser V, Pfister A, Moser P, Hermann-Kleiter N, Baier G, Oberacher H, Tilg H, Moschen AR. NAD metabolism fuels human and mouse intestinal inflammation. Gut. 2018 Oct;67(10):1813-1823.