Active Compounds Screening Targeting Visfatin/Nampt

Nicotinamide adenine dinucleotide (NAD) plays a critical role in many cellular processes. It functions as a cofactor in more than 200 oxidation reduction reactions, which are linked to Ca2+ mobilization, genomic stability, apoptosis, metabolism, and other vital biological processes. Nampt is the rate-limiting enzyme in the NAD biosynthesis pathway in mammals, and it regulates the total cellular NAD level and mitochondrial NAD levels. In addition, Nampt maintains an energy supply by synthesizing NAD and plays a crucial role in cell survival.A great number of experimental evidences have indicated that Nampt performs a variety of beneficial physiological functions, such as promoting the cellular differentiation and maturation, participating in immune response and inflammation, antiapoptosis, and plays a potential role in cardiovascular disease, diabetes, and autoimmunity disease. Tumor cells are more sensitive to the NAD levels, making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Previous studies have indicated that Nampt might have proangiogenic activity and support the growth of some tumors. Therefore, inhibiting the activity of the enzyme Nampt could be a better way for antitumor therapy. These findings make Nampt to be an attractive target for pharmaceutical reasearch during recent years. Especially, visfatin, a new adipokine found in 2005, has the same enzyme activity as Nampt, making visfatin/Nampt to be a hot factor in biological research in the world. However, only four Nampt inhibitors have been reported, and no Nampt activator and no high-throughput screening (HTS) strategy for Nampt has been proposed to date, largely limiting the drug discovery targeting Nampt. To find the first Nampt activator and more Nampt inhibitors of different structure as potential antitumor drug candidates, we screened a compound library, using a screening system targeting Nampt which was established in our previous study.In the current study, more than 15,200 compounds were screened under the concentration of 20μM, which were purchased from the National Compound Resource Center (Shanghai). 747 compounds resulting in more than 25% relative activity and 23 compounds resulting in less than 40% relative activity were subjected to a secondary screening. By setting the design of control group in secondary screening, we ruled out the false positive results. Preliminary experiment of the half inhibitory concentration (IC₅₀) study was carried out on 102 compounds under the concentration of 20, 2, 0.2μM. Six novel compounds showing less than 50% relative activity at 2μM were selected out. Besides, these compounds were further confirmed as Nampt inhibitors by a concentration- dependent inhibition assay. All of them showed conventional inhibition curves. The IC₅₀ values were calculated by fitting the curves with the four-parameter IC₅₀ logistic equation, and they were 3.54μM, 9.87 nM, 0.149μM, 0.777μM, 0.855μM, 0.352μM for compound 9882, 735, 2161, 4162, 7391 and 6443, respectively. Quality control parameters from each assay plate were obtained and analyzed. Z' factors during our screening ranged from 0.52 to 0.88, CV values were between 0.85⁹.02%, and S/N ratios were greater than 8, in line with the requirements of high-throughput screening.Through library screening, secondary screening and IC₅₀ study in vitro, we found six low molecule weight compounds of potential Nampt inhibitors. Further, we investigated the influence of compound 733, 735, 2161, 4162 and 7391 on intracellular NAD content of HepG 2 human hepatocarcinoma cells. After 24 h incubation with compounds at concentration of 1, 10, 100μM, HepG 2 tumor cells were lysised by perchloric acid for determination of the NAD content by enzymatic cycling techniques. In comparison to vehicle treated HepG 2 cells, a significant reduction of the intracellular amount of NAD by 72.9% was observed as soon as after 24 h in 1μM compound 735-treated cells (10 and 100μM by 74.04% and 77.12%, separately); 2161 and 7391 in the 100μM concentration for 24 h, could make the NAD content down-regulate 60.2% and 49.7%, respectively; the inhibition of 733 and 4162 on NAD level was not significant.We selected 735 as a potential antitumor drug candidate. Further, the half inhibitory concentration of 735 on NAD level of HepG 2 tumor cells was measured: IC₅₀ = 93.7±3.4 nM. Meanwhile, the cytotoxicity of 735 was not observed on HepG 2 cells in the concentration range of 0¹0μM for 24 h incubation. But in this range, 735 could effectively reduce the amount of intracellular NAD in HepG 2 tumor cells. Therefore, we considered that 735 had no direct, immediate cytotoxicity, but might cause gradual NAD depletion through specific inhibition of Nampt.In addition, we observed and compared the proliferation inhibition in vitro of 735 on human hepatoma cell line HepG 2, human ovarian cancer cell line A2780 and human prostate cancer cell line DU145. A gradient of concentration of compounds were applied to the logarithmic phase of the cell lines 72 h, using SRB assay to detect the ability of cell proliferation inhibition of 735. The results revealed that 735 showed different degrees of inhibition on the proliferation of these cell lines. The IC₅₀ value of 735 on A2780 cells and DU145 cells was approximately 0.3μM, and on HepG 2 cells was about 19.3μM.In the subject, we first carried out a high-throughput screening targeting Nampt, and we have found six novel compounds that are completely different from the existing structures of Nampt inhibitors, of which 735 is an active compound confirmed in cell. The biological activity remains to be further validated in vivo environments by appropriate approaches, and the mechanism is also required indepth research. The validated lead compound may be further optimized and serve as tool in Nampt-related research, and it is expected to discover new drug candidate for antitumor therapy.