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

NAMPT:<25 nM

GMX1778 induces NAD+ depletion through inhibition of NAD+ biosynthesis, followed by ATP depletion and ultimately resulted in cell death. [1] GMX1778 induces programmed cell death with apoptotic features. [2] GMX1778 suppresses nuclear factor-kappa B activity in cancer cells through downregulation of IKK activity (IC50=8 nM). [3]

GMX1778 (250 mg/kg, p.o.) shows marked antitumoural activity against three different human neuroendocrine tumours, midgut carcinoid (GOT1), pancreatic carcinoid (BON), and medullary thyroid carcinoma (GOT2), transplanted in nude mice. [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)