Background
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].
参考文献:
[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.
FK866 (APO866) 是烟酰胺磷酸核糖转移酶 (NMPRTase) 的抑制剂。 FK866 (APO866) 通过抑制活化的白细胞特别是巨噬细胞、炎性单核细胞和 T 细胞来防止实验性结肠炎和结肠炎相关的肿瘤发生。 FK866(APO866) 还可降低 IBD 患者结肠活检组织固有层单核细胞 (LPMNC) 的炎症反应，其程度与地塞米松相当[1]。
FK866 (APO866) 对 NAMPT 活性的 IC50 为 1.60±0.32 nmol/L [2].. FK866 (APO866) 对 HepG2 细胞的 IC50 为 2.21±0.21 nmol/L。 FK866 (APO866) 处理强烈降低 LPS 处理后的 NF-κB 磷酸化。 FK866 对 NAMPT 的抑制或烟酰胺对 SIRT 的抑制减少了涉及 p53 乙酰化途径的 293T 细胞的增殖并触发了其死亡[3]..FK866 (APO866) 有效抑制了 NAMPT 活性，如粘膜减少所证明的NAD，导致包括 PARP1、Sirt6 和 CD38 在内的 NAD 依赖性酶的丰度和活性降低，核因子 kappa B 活化减少，炎症单核细胞、巨噬细胞和活化 T 细胞的细胞浸润减少[1] .
FK866 (APO866) 显着改善 Rag1-/- 小鼠中 DSS 诱导的结肠炎的所有特征，并在缺乏适应性免疫的情况下有效抑制炎症性先天免疫反应。 FK866 (APO866) 显着减少趋化因子和细胞因子的释放，其中许多是巨噬细胞/单核细胞衍生的。值得注意的是，观察到的抑制作用在范围内甚至优于地塞米松和英夫利昔单抗等公认的抗炎化合物 [1]。
Protocol
| Kinase experiment [1]: |
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Preparation Method
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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.
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Reaction Conditions
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37℃ for 5 min
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Applications
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IC50 of FK866 (APO866) on NAMPT activity is 1.60±0.32 nmol/L
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| Cell experiment [1]: |
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Cell lines
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HepG2
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Preparation Method
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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.
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Reaction Conditions
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48 h
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Applications
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IC50 of FK866 (APO866) on HepG2 cells is 2.21±0.21 nmol/L.
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| Cell experiment [2]: |
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Cell lines
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RAW 264.7 and MODE-K cells
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Preparation Method
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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.
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Reaction Conditions
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200nM FK866 (APO866) overnight
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Applications
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FK866 (APO866)treatment strongly reduced NF-κB phosphorylation consequent to LPS treatment.
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| Animal experiment [2]: |
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Animal models
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8-week-old female wild-type (WT) or Rag1tm1Mom/J (Rag1?/?) mice
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Preparation method
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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.
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Dosage form
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Intraperitoneally with 10mg/kg FK866 (APO866)
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Applications
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FK866 (APO866) significantly ameliorated all features of DSS-induced colitis in Rag1?/? mice.
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参考文献:
[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.
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