CHS-828 (GMX1778), a pyridyl cyanoguanidine, is an effective inhibitor of NAD+ biosynthesis enzyme NAMPT (IC50 <25 nM).

NAMPT:<25 nM

GMX1778通过抑制NAD+的生物合成引起NAD+耗尽，进而导致ATP耗竭并最终导致细胞死亡。[1] GMX1778诱导具有凋亡特征的程序性细胞死亡。[2] GMX1778通过下调IKK活性（IC50=8 nM）抑制癌细胞中的核因子-κB活性。[3]

GMX1778（250 mg/kg，p.o.）对于移植至裸鼠的三种不同人类神经内分泌肿瘤——中肠类癌（GOT1）、胰腺类癌（BON）和甲状腺髓样癌（GOT2）——展现了显著的抗肿瘤活性。[4]

In vitro coupled-enzyme NAMPT assay: Recombinant NAMPT activity is assessed using a coupled-enzyme assay based on the quantitation of NAD+. Reactions are performed at room temperature for 180 min using mixtures consisting of 50 mM HEPES (pH 7.4), 50 mM KCl, 5 mM MgCl2, 0.5 mM β-mercaptoethanol, 0.005% bovine serum albumin, 1% DMSO, 2.0 U/ml lactate dehydrogenase, 4 mM sodium L-lactate, 0.4 U/ml diaphorase, 6 μΜ resazurin sodium salt, 0.4 mM PRPP, 3.0 nM NMNAT1, 125 μM ATP, 50 μM NM, and 2 to 5 μM recombinant NAMPT. Fluorescence s measured with a Tecan Safire plate reader (excitation wavelength, 560 nm; emission wavelength, 590 nm). Ki values are calculated using Graphpad Prism 4.0 software and the Cheng-Prusoff equation.

Serial dilutions of GMX1778 in DMSO are performed to achieve a final concentration of 0.2% DMSO. Relative ATP levels after 72 h are determined using a ViaLight HS high-sensitivity cytotoxicity and cell proliferation BioAssay kit per the manufacturer's instructions. For GMX1778 cytotoxicity rescue experiments, cells are treated with NA (10 μM) or NMN (100 μM) simultaneously with GMX1778. Sigmoidal dose-response curves are generated by nonlinear regression analysis of variable slope using GraphPad Prism version 4.00 (GraphPad Software) to calculate 50% inhibitory (IC50) values.(Only for Reference)