NAMPT活性化合物的研究

发布时间：2018-09-05 13:21

【摘要】：烟酰胺磷酸核糖转移酶(NAMPT,Nicotinamide phosphoribosyltransferase),主要由491个氨基酸组成,其分子量为55 k Da,晶体结构显示为二聚体形式存在的II型磷酸核糖转移酶。NAMPT具有广泛的生物学功能,除了调节脂质代谢、促进血管平滑肌细胞分化成熟、促进前B细胞增殖分化、参与机体炎症反应、抑制中性粒细胞凋亡等作用外,其最主要的功能就是作为生物体内NAD合成的限速酶。近年来大量研究表明,NAMPT是一种很有前景的抗肿瘤靶点,在多种肿瘤细胞中NAMPT为过表达的并与肿瘤的发生发展都成正相关性;与此同时,在脑卒中的保护中,NAMPT也是至关重要的,其直接产物NMN对于脑卒中有很好的保护作用。因此,开发基于NAMPT为靶点的小分子抑制剂和激活剂,对治疗肿瘤和脑卒中都起着至关重要的作用,而目前针对于NAMPT的小分子化合物的开发还处在起步阶段,对于研发临床药物而言,仍需要大量结构多样的NAMPT小分子化合物作为基础。本论文以开发新型结构NAMPT抑制剂和激活剂为主要研究方向,以基于NAMPT为靶点的高通量筛选检测技术为研究手段,着重进行全新结构的NAMPT抑制剂和激活剂的发现、优化及其机制研究。一、基于非Chemdiv化合物库进行抑制剂的筛选、化合物优化及其机制研究应用基于NAMPT特异性靶点的高通量筛选检测技术,我们获得了一个活性很好的NAMPT抑制剂MSO(中国专利号:ZL201110447488.9),其体外离体酶活性为(IC50=9.87±1.15 n M),并且能够抑制包括肿瘤干细胞样的多种肿瘤细胞的增殖。我们对MSO经过构效分析研究后,合成了一系列高活性的MSO结构类似物。这些抑制剂能够特异性地结合NAMPT,而不是其下游靶点NMNAT。我们首次提供了一个化学实例,应用新近报道的细胞热转移分析技术,分析了化合物在细胞内和细胞外造成活性差异的原因,例如由于MS7化合物在活体细胞中的靶点富集作用很差,使得其在体外离体酶活性实验中显示出了最好活性(IC50=0.93±0.29 n M),但是在细胞水平上其活性很差。通过对NAMPT活性酶的点突变研究以及晶体的培养,我们确认了抑制剂的结合模式及其重要的作用氨基酸残基。本研究为更深层次地了解NAMPT抑制剂分子作用模式和未来的抗肿瘤药物研发奠定了基础。二、基于Chemdiv化合物库进行抑制剂的筛选及其机制研究在研发抗肿瘤药物中,需要大量新型的结构多样的NAMPT抑制剂。在使用基于NAMPT靶点的高通量筛选检测平台时,针对于有3万个化合物组成的Chemdiv化合物库进行筛选,我们发现了两个活性较好的化合物,分别是非荧光性化合物F671-0003(IC50=85.05±6.42 n M)和荧光性化合物M049-0244(IC50=170±12 n M)。这两种化合物能够有效地抑制Hep G2细胞系的增殖,并且显著地降低细胞内NAD水平。外源性地加入NMN,能够补救对于HepG2的增殖抑制作用。构效分析的研究结果显示了化合物F671-0003的分子作用模式,并且发现氢键作用力,疏水性作用力和π-π作用力对于化合物的结合是非常重要的。另一方面,成像研究显示了荧光性化合物M049-0244在3μM的浓度下,能够显著地染色HepG2中的NAMPT,并且在经过4 h后,仍能够检测到荧光。我们通过RNA干扰技术和对NAMPT过表达小鼠的研究,确认了该化合物的荧光特异性。本部分研究发现了一个具有全新结构类型的抗肿瘤先导化合物,并且首次应用荧光探针进行NAMPT检测。三、基于化合物库进行NAMPT潜在激活剂的筛选及其机制研究应用基于NAMPT为靶点的高通量筛选检测平台对Chemdiv化合物和非Chemdiv化合物两个化合物库进行筛选,发现有潜在激活NAMPT作用的化合物共495个,通过初筛复筛最终选出4类结构类型的化合物,对NAMPT有潜在的激活作用,可以作为研发NAMPT激活剂的备选化合物。综上所述,本研究针对由54434个小分子化合物组成的化合物库,应用自主建立的基于NAMPT为靶点的高通量检测技术,对NAMPT小分子化合物进行了高通量筛选,在抑制剂方面选出了多个系列新型NAMPT抑制剂,特别是,具有全新结构类型的化合物MSO和具有NAMPT荧光探针作用的化合物M049-0244,并以MSO为基本结构,合成优化了46个结构类似物,深入探讨了他们的构效关系和分子机制;在潜在NAMPT激活剂筛选方面,选出了4类结构可作为研发NAMPT激活剂的备选化合物。本论文NAMPT小分子活性化合物的发现及进一步作用机制的研究,为未来的抗肿瘤药物和脑卒中保护药物的研发奠定了基础。
[Abstract]:Nicotinamide phosphoribosyltransferase (NAMPT) is composed of 491 amino acids with a molecular weight of 55 K Da. The crystal structure of NAMPT is a dimer type II phosphoribosyltransferase. NAMPT has a wide range of biological functions, in addition to regulating lipid metabolism and promoting vascular smooth muscle cell differentiation. In recent years, a large number of studies have shown that NAMPT is a promising anti-tumor target. In many tumor cells, NAMPT is overexpressed and associated with tumors. At the same time, NAMPT is also very important in the protection of stroke, and its direct product NMN has a very good protective effect on stroke. Therefore, the development of small molecule inhibitors and activators targeting NAMPT plays a vital role in the treatment of cancer and stroke, and currently targeted at The development of small molecule compounds of NAMPT is still in its infancy. For the development of clinical drugs, a large number of small molecule compounds of NAMPT with various structures are still needed as the basis. Meanwhile, we focused on the discovery, optimization and mechanism of novel NAMPT inhibitors and activators. First, based on the non-Chemdiv compound library, we screened inhibitors, optimized compounds and their mechanisms. Using high throughput screening and detection technology based on NAMPT specific targets, we obtained a NAMPT inhibitor M with good activity. SO (China Patent No. It is the downstream target NMNAT. We provide a chemical example for the first time. Using the recently reported technique of cell heat transfer analysis, we analyzed the reasons for the difference in activity between intracellular and extracellular compounds. The best activity was shown (IC50 = 0.93 + 0.29 n M), but its activity was poor at the cellular level. Through the study of point mutation of NAMPT enzyme and crystal culture, we confirmed the binding pattern of inhibitors and their important functional amino acid residues. This study is to understand the molecular action pattern and future of NAMPT inhibitors in a deeper level. Second, a large number of novel NAMPT inhibitors are needed in the development of anti-tumor drugs based on the Chemdiv compound library. Chemdiv chelation of 30,000 compounds is used in the high throughput screening platform based on NAMPT target. Two compounds with better activity, F671-0003 (IC50 = 85.05 (+6.42 n M)) and M049-0244 (IC50 = 170 (+12 n M)), were found to inhibit the proliferation of Hep G2 cell lines and significantly reduce the intracellular NAD level. The results of structure-activity analysis showed that the molecular interaction mode of compound F671-0003 and the hydrogen bonding force, hydrophobic force and pi-pi force were very important for the binding of the compounds. On the other hand, imaging studies showed that the fluorescent compound M049-0244 was at 3 mu M. NAMPT in HepG2 can be stained significantly at the concentration of NAMPT, and fluorescence can still be detected after 4 hours. The fluorescence specificity of the compound was confirmed by RNA interference technique and NAMPT overexpression in mice. NAMPT detection using fluorescent probes. 3. Screening of potential NAMPT activators based on compound libraries and their mechanisms; screening of hemdiv compounds and non-Chemdiv compounds libraries using a high throughput screening platform based on NAMPT as a target. A total of 495 compounds with potential NAMPT activation were found. In summary, this study aimed at the compound library composed of 54434 small molecule compounds, and applied the self-established high-throughput detection technology based on NAMPT as the target to small molecule NAMPT. A series of novel NAMPT inhibitors were selected, especially compounds MSO with novel structure and M049-0244 with NAMPT fluorescence probe. Forty-six structural analogues were synthesized and optimized with MSO as the basic structure. Their structure-activity relationship and composition were discussed in detail. In this paper, the discovery of NAMPT small molecule active compounds and the further study of the mechanism of action have laid the foundation for the future development of anti-tumor drugs and stroke protective drugs.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R91

【相似文献】