Preparation Method

To determine the IC₅₀of 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 IC₅₀values were determined by non-linear fitting of the concentration-dependent curves with the four-parameter IC₅₀logistic equation.

Reaction Conditions

37℃ for 5 min

Applications

IC₅₀of 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 ℃ for 1 h. The absorbance at 450 nm (A450) was detected by a plate reader.

Reaction Conditions

48 h

Applications

IC₅₀of 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.

FK866 (APO866) is an inhibitor of nicotinamide phosphoribosyltransferase (NMPRTase). FK866 (APO866) protects against experimental colitis and colitis–associated tumorigenesis by suppression of activated leukocytes particularly macrophages, inflammatory monocytes and T cells. FK866(APO866) also reduced inflammatory responses of lamina propria mononuclear cells (LPMNC) from colonic biopsies of patients with IBD to a comparable extent as dexamethasone^[1].

The IC50 of FK866 (APO866) on NAMPT activity is 1.60±0.32 nmol/L^[2].. IC50 of FK866 (APO866) on HepG2 cells is 2.21±0.21 nmol/L. FK866 (APO866) treatment strongly reduced NF-κB phosphorylation consequent to LPS treatment. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway^[3].. FK866 (APO866) potently inhibited NAMPT activity as demonstrated by reduced mucosal NAD, resulting in reduced abundances and activities of NAD-dependent enzymes including PARP1, Sirt6 and CD38, reduced nuclear factor kappa B activation, and decreased cellular infiltration by inflammatory monocytes, macrophages and activated T cells^[1].

FK866 (APO866) significantly ameliorated all features of DSS-induced colitis in Rag1–/– mice and effectively suppresses inflammatory innate immune responses in the absence of adaptive immunity. FK866 (APO866) significantly reduced chemokine and cytokine release, many of those which are macrophage/monocyte derived. Remarkably, the observed suppression was in the range or even superior to well-established anti-inflammatory compounds such as dexamethasone and infliximab^[1].

References:
[1].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.
[2].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.
[3].Thakur BK, Dittrich T, Chandra P, Becker A, Lippka Y, Selvakumar D, Klusmann JH, Reinhardt D, Welte K. Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells. Biochem Biophys Res Commun. 2012 Aug 3;424(3):371-7.