Discovering Active Compounds Of Structural Diversity Targeting Nampt By High Throughput Screening

Nampt (nicotinamide phosphoribosyltransferase) is a protein with several suggested functions. It is potential involved with a wide range of disorders including cancer、diabetes and stroke. As the rate-limiting enzyme in the NAD biosynthesis pathway in mammals, it regulates the total cellular NAD level and mitochondrial NAD levels. NAD functions as a cofactor in oxidation reduction reactions, maintains an energy supply and plays a crucial role in cell survival. Therefore, Nampt, which is a key enzymes in NAD metabolic pathways, become an attractive target for drug discovery of various kinds of related diseases in recent years.Our previous study have identified that visfatin/Nampt is a endogenous nerve protector and suggested that visfatin/Nampt is a new therapeutic target for stroke prevention and treatment. Nampt activator will be of value for therapies of ischemic stroke. In the process of anti-tumor drug discovery, we find that tumor cells are more sensitive to the NAD levels. Nampt inhibitor can induce NAD depletion by inhibiting the enzyme activity, eventually leading to tumor cell death. However, Nampt inhibitors, which have been enrolled in the clinical trials, have not been observed expected effect because of low bioavailability and dose-dependent toxicities. In order to find the first Nampt activator and more Nampt inhibitors,we screened a large sample size of compound library, using a high throughput screen system targeting Nampt which was established in our previous work. Despite the fact that the primary amino acid sequence of visfatin/Nampt does not include a cytokine signal sequence, visfatin/Nampt is released extracellularly by a variety of cells and tissues sush as liver, cardiomyocytes, skeletal muscle and nerve cells. But how dose the extracellular form of visfatin/Nampt exhibit enzyme activity remains to be a matter of debate. To address this question, we use of Nampt enzyme activity detection method in screening system to detect the activity of Nampt in biological samples.In the first place, a total of39234compounds(492sample plates) were screened under the concentration of20μM, which were purchased from NCRC (Shanghai) and ChemDiv. There are347compounds resulting in less than40%relative enzyme activity and2042compounds resulting in more than125%relative enzyme activity, which of both were subjected to a secondary screen. By setting the design of control group in secondary screening, we ruled out the false positive results. There were695active compounds after confirmed by the second screen. Preliminary experiment of the half inhibitory concentration (IC50) study was carried out on286inhibiting compounds under the concentration of2μM、0.2μM、0.02μM. Compounds in dose-effect relationship showing less than50%relative enzyme activity at2μM were selected out. Besides, there were47compounds further confirmed as Nampt inhibitors by a concentration-dependent inhibition assay. The IC50values were calculated by fitting the curves with the four-parameter IC50logistic equation. There were8compounds which IC50values were under100nM. They were59.41、85.52、70.94、75.86、62.65、86.92、85.05、60.85nM for compound No.4、 No.22、No.23、No.24、No.36、No.39、No.44、No.60, accordingly. Quality control parameters-Z’factors、CV values/、S/N ratios from each assay plate were obtained and analyzed, in line with the requirements of high-throughput screening.In order to confirm the activity of8compounds mentioned above, we investigated the influence of these compounds on HepG2human hepatocarcinoma. After48h incubation with compounds at concentration of2μM, we used CCK8method to determine the cell vitality.The result shows that No.44compound has obvious inhibiting activity, the inhibiting rate is46%. Further, the half inhibitory concentration of No.44compound on HepG2tumor cells was measured:IC50=1.73±0.29μM. In order to investigate the structure activity relationship (SAR) as well as the molecular action mode, we find the47active compounds analogues, in a total of455, in our Chemdiv compound libraries. We confirmed the activities of these analogues through secondary screening. The result shows that the vast majority of structural analogues have no activity or poor activity(IC50>20μM).There were only12compounds structural analogues of enzyme activity in vitro of IC50values were between2μM-20μM, which is worse than previously found47active compounds. These repeated determinations further support the reliability of high-throughput screening.In addition, in order to confirm whether Nampt has extracellular activity, we measured the NMN levels in mouse plasma in our visfatin/Nampt activity-based Screening System. The concentrations of mouse plasma NMN were120±22.4nM. Next, we tested the influence of ATP concentrations on Nampt enzymatic activity. The enzymatic activity of extracellular visfatin maintained on a relatively low level when the ATP concentration was below105nM. In addition, we explored Middle Cerebral Artery Occlusion model in mice to detect extracellular ATP and visfatin/Nampt level under pathological condition.The plasma ATP and visfatin/Nampt concentration in MCAO model were significantly higher than that in normal condition. In conclusion, our results provide direct evidence that extracellular visfatin has Nampt enzymatic activity.In this program, we first adopt the method of high throughput screening of large compound libraries targeting Nampt. And we have found a great diversity of lead compounds which are different from the existing structures of Nampt inhibitors, of which No.44is an active compound confirmed on HepG2turner cell. Through the analysis of the analogues structure activity relationship, the validated lead compound may be further optimized and serve as tool in Nampt-related research. It is expected to discover new drug candidate for antitumor therapy and lay the foundation of Nampt molecular patterns research. Besides, we detect the enzyme activity of Nampt in biological samples through our HTS system, the result confirm that Nampt/visfatin has extracellular enzyme activity, which lay a foundation for clinical samples detection and further research of extracellular visfatin/Nampt.