Discovery of novel inhibitors and fluorescent probe targeting NAMPT

Aim Nicotinamide phosphoribosyltransferase （NAMPT） is a promising therapeutic target for cardio-ce- rebrovascular diseases and tumor. Novel NAMPT inhibitors with diverse chemotypes are highly desirable for devel- opment of therapeutic agents. Methods We carried out a high throughput screening targeting NAMPT on a chemi- cal library of 30000 small-molecules in this study. Assays of NAD levels, anti-proliferative activity, imaging study, RNA interference were conducted in HepG2 cells or primary mouse hepatocytes. Results A non-fluorescent com- pound F671-0003 and a fluorescent compound M049-0244 were found with excellent in vitro activity （IC50：85 nmol · L^-1 and 170 nmol · L^-1 respectively） and anti-proliferative activity against HepG2 cells. These two com- pounds significantly depleted cellular NAD levels. Exogenous NMN rescued their anti-proliferative activity against HepG2 cells. Structure-activity relationship study proposed a binding mode for NAMPT inhibitor F671-0003 and highlighted the importance of hydrogen bonding, hydrophobic and -rr--rr interactions in inhibitor binding. Imaging study provided the evidence that fluorescent compound M049-0244 （3 μmol · L^-1） significantly stained living HepG2 cells. Cellular fluorescence was further verified to be NAMPT dependent by using RNA interference and NAMPT over expression transgenic mice. Conclusions This study provides novel lead compounds and a ＂first-in- class＂ fluorescent probe for imaging NAMPT.