Discovery of Novel Inhibitors and Fluorescent Probe Targeting NAMPT.

Xia Wang,Shu-Na Wang,Sai-Long Zhang,Ying-Ying Le,Xin-Zhu Liu,Guo-Qiang Dong,Chun-Quan Sheng,Yun-Feng Guan,Zhi-Yong Li,Tian-Ying Xu,Pei Wang,Chao-Yu Miao,Shu Zhuo

doi:10.1038/srep12657

Abstract

Nicotinamide phosphoribosyltransferase (NAMPT) is a promising antitumor target. Novel NAMPT inhibitors with diverse chemotypes are highly desirable for development of antitumor agents. Using high throughput screening system targeting NAMPT on a chemical library of 30000 small-molecules, we found a non-fluorescent compound F671-0003 and a fluorescent compound M049-0244 with excellent in vitro activity (IC50: 85 nM and 170 nM respectively) and anti-proliferative activity against HepG2 cells. These two compounds 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 π-π interactions in inhibitor binding. Imaging study provided the evidence that fluorescent compound M049-0244 (3 μM) significantly stained living HepG2 cells. Cellular fluorescence was further verified to be NAMPT dependent by using RNA interference and NAMPT over expression transgenic mice. Our findings provide novel antitumor lead compounds and a “first-in-class” fluorescent probe for imaging NAMPT.

Highlights

Inhibitors with novel chemotypes is still highly desirable to validate the druggability of Nicotinamide phosphoribosyltransferase (NAMPT) as an antitumor target
Our high throughput screening (HTS) targeting NAMPT was developed based on a fluorometric method for NAMPT activity assay by measuring the fluorescence of nicotinamide mononucleotide (NMN) derivative resulting from the NAMPT enzymatic product NMN through simple chemical reactions (Fig. 1A)
The 30000 compounds were dissolved with dimethyl sulfoxide (DMSO); most of compounds were soluble at 10 mM stock solution (94.1%), some compounds insoluble at 10 mM were soluble at 2.5 mM stock solution (4.4%), and the remaining compounds insoluble at 2.5 mM (1.5%) were diluted further during HTS experiments (Fig. 1B)

Summary

Introduction

Inhibitors with novel chemotypes is still highly desirable to validate the druggability of NAMPT as an antitumor target. The identification of novel chemical tools, fluorescent probes, is important to the better understanding of the biological function of NAMPT. Structurally diverse NAMPT inhibitors were identified by high throughput screening. We developed a first-in-class fluorescent probe to straight detection and snapshot of NAMPT at molecular and cellular level, which may provide a novel and convenient strategy to study NAMPT

Methods

Results

Conclusion