Study Of Active Compounds Targeting NAMPT

NAMPT is the short name for Nicotinamide phosphoribosyltransferase. It has three names, NAMPT/Visfatin/PBEF. The main form of human NAMPT consists of 491 amino acids with a molecular weight of approximately 55 k Da. The X-ray crystal structure of NAMPT has also been determined and established, which also has been proved to be a diametric class of type II phosphoribosyltransferases. NAMPT has many biological functions, such as regulating lipid metabolism, promoting vascular cells differentiation and maturation, promoting the pre B cells maturation, participating in the inflammatory reaction and inhibiting neutrophils apoptosis. Moreover the most important function of Nicotinamide phosphoribosyltransferase is becoming the rate-limiting enzyme in NAD biosynthesis pathways. Many recent studies have identified NAMPT as the antitumor therapeutic target as well as the antistroke target. Thus, developing small molecular compounds targeting NAMPT is important for the treatment of tumor and stroke. However, there are not enough compounds targeting NAMPT for drug discovery especially those for the activators targeting NAMPT. Therefore,we need more active compounds with different structures to develop drugs for tumor and stroke.The study of this dissertation aims to develop inhibitors and activators targeting NAMPT with new structures. We identified a series of active compounds by using the high throughput screening system targeting NAMPT. The investigation of structure-activity relationship, binding mode and mechanism would contribute to deeply understanding the action mode of NAMPT inhibitors which also could become solid foundation for further development of NAMPT compounds as anticancer and antistroke agents in the future.1. Discovering inhibitors based on the none-Chemdiv compounds library, its structure-activity relationship and mechanism study.Nicotinamide phosphoribosyltransferase(NAMPT) is a promising anticancer target. By using high throughput screening system targeting NAMPT, we have obtained a potent NAMPT inhibitor MSO(China Patent ZL201110447488.9) with excellent in vitro activity(IC50 = 9.87 ± 1.15 n M) and anti-proliferative activity against multiple human cancer cell lines including stem-like cancer cells. Currently several highly effective analogues have been yielded based on the study of structure-activity. These inhibitors could specially bound NAMPT rather than its downstream target NMNAT. We provided the first chemical case by using cellular thermal shift assay to explain the difference between in vitro and cellular activity in which MS7 showed best in vitro activity(IC50 = 0.93 ± 0.29 n M) but worst cellular activity due to poor target engagement in living cells. Through site-directed mutagenesis studies and crystal structure cultivation of NAMPT active enzyme, we have identified important residues and binding mode of inhibitors.2. Discovering inhibitors based on the Chemdiv compounds library, its structure-activity relationship and mechanism study.Novel NAMPT inhibitors with diverse chemotypes are highly desirable for the development of antitumor agents. By using high throughput screening system targeting NAMPT on a chemical library of 30000 small-molecules, we found a non-fluorescent compound F671-0003(IC50 = 85.05 ± 6.42 n M) and a fluorescent compound M049-0244(IC50 = 170 ± 12 n M) with excellent in vitro activity and anti-proliferative activity against Hep G2 cells. These two compounds could significantly depleted cellular NAD levels. And exogenous NMN rescued their anti-proliferative activity against Hep G2 cells. The binding mode of NAMPT inhibitor F671-0003 has been revealed by the study of structure-activity relationship, which also proved the importance of hydrogen bonding, hydrophobic, and ?-? interactions for inhibitor binding. Through imaging study we also found the evidence that shows fluorescent compound M049-0244(3 ?M) would significantly stain the NAMPT in living Hep G2 cells and its cellular fluorescence has been further verified to be NAMPT dependent through the study of NAMPT over expression transgenic mice and RNA interference technology.3. Discovering potential activators based on the compounds library, its structure-activity relationship and mechanism study.We identified 495 potential activators targeting NAMPT through our high throughput screening system based on two compounds library. After the primary and secondary screening, 4 kinds of compounds with different structures have been found performing better activity, which could become the candidates for future activators discovery.In summary, we screened the compounds library with nearly 55000 chemicals by using our high throughput screening system targeting NAMPT. On one hand, from the aspect of NAMPT inhibitors, we identified a highly effective compound called MSO with novel structure and a fluorescent compound M049-0244, which can significantly stain living cells. On the other hand, for potential NAMPT activators, we discovered 4 kinds of compounds with different structures, which could be the candidates for future activators discovery. The study of this dissertation provides novel antitumor lead compounds and a “first-in-class” fluorescent probe for imaging NAMPT which would contribute to deeply understanding the action mode of NAMPT inhibitors for further research of anti-cancer and antistroke agents in the future.