| Nicotinamide adenine dinucleotide (NAD) plays a crucial role in a variety of cellular processes, and has profound impacts on gene expression, DNA repair, calcium mobilization, metabolism, proliferation and so on. Besides, NAD is closely related to the survival of cells. Therefore, the enzymes involving in NAD biosynthesis and metabolism have become extremely attractive targets of drug discovery. In mammals, Nicotinamide is catalyzed by Nampt to NMN, with NMN being subsequently converted to NAD by Nmnat. Most of the NAD in mammalian cells is synthesized by this pathway.We have witnessed that BL21-CondonPlus (DE3)-RIL was the suitable host bacterium for the expression of Nmnat and can significantly improve the expression efficiency of Nmnat in E. coli system. According to the results of the research, we determined the optimal conditions of Nmnat expression that it was induced for expression by 0.5mM IPTG for 8h in 2×YT medium containing 37ug/ml chloramphenicol and 75ug/ml kanamycin under the temperature of 28℃. We obtained the recombinant protein with high purity by means of Ni-NTA affinity chromatography. It was demonstrated by the enzyme-coupled fluorometric assay that the recombinant protein Nmnat had high enzymatic activity. Although the enzymatic activity of Nmnat performs a variety of beneficial functions on the chemotherapy of cancer, energy metabolism and signaling pathways, currently little has been reported about the regulation of its activity. Therefore, this section of our research laid a certain foundation for the screening of the compounds regulating its activity.In mammals, Nampt is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis and performs a variety of beneficial physiological functions, such as resistance to apoptosis, activation of adaptive immunity, tissue repair, promoting the cellular differentiation and maturation. In addition, it is the new target of the cardiovascular disease and cancer. In this section, we successfully expressed and purified recombinant protein Nampt from the prokaryotic E. coli system, as well as established the assay of Nampt enzymatic activity. After the reaction with acetophenone and formic acid, enzymatic product NMN can be converted into a fluorescent compound with the maximum fluorescence signal at the excitation wavelength of 382nm and emission wavelength of 460nm. We optimized the assay of Nampt activity, and furthermore, established a Nampt activity-based screening model according to the results analyzing the conditions of the detection reaction and the enzymology properties. The assay exhibited the Z'factor of 0.668 and the CV value of 3.0%, indicating the model met the requirements of the high-throughput screening. The preliminary screening was carried out on 84 compounds including Nampt inhibitor FK866. By virtue of the design of the control, we ruled out the positive result that the compound NCRC0553 up-regulated the Nampt activity. Besides, FK866, zl-1, zl-3 and gallotannin are active compounds effectively down-regulating the Nampt activity, with the IC50 at 0.9nM,17.8uM,6.72uM and 1.3uM respectively.High-throughput screening is an important way of the discovery of new drugs. We established a Nampt activity-based screening model for the first time, hoping to find the active molecules effectively regulating the Nampt activity. The lead compounds found in this way can be applied to the modulation of Nampt activity as well as the prevention and treatment of cancer and stroke, promoting the medical research and the studies of the Nampt physiological functions. |
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