【作者】 朱景宇；

【导师】 毛新良；

【作者基本信息】 苏州大学 ， 药理学， 2014， 博士

【摘要】 多发性骨髓瘤是第二大恶性血液肿瘤疾病，大约占血液系统肿瘤的10%。由于缺乏有效药物，多发性骨髓瘤的死亡率一直高居不下，占年癌症总死亡率的2%，因此急需开发新型的分子靶标抗骨髓瘤药物。PI3K/AKT通路与多发性骨髓瘤的存活、分化、迁移、血管生成以及耐药性都密切相关，因此PI3K/AKT已经成为开发抗骨髓瘤药物的一个理想靶标。目前已经有一些抑制剂被报道，有些已经进入了临床试验。随着组合化学、肿瘤生物学、生物信息学以及生物工程的高速发展，使得大规模筛选PI3K抑制剂的方法被越来越广泛地采用。与传统的高通量筛选相比，计算机虚拟筛选具有高效、快速、低成本的优点，使它成为高通量筛选的一个重要辅助方法。在本课题中，我们结合虚拟筛选和生物学实验的方法来发现新的PI3K抑制剂，并阐明其抗多发性骨髓瘤的分子机制。研究方法：(1)使用逐层虚拟筛选的方法来发现新的高效的PI3K虚拟抑制剂。(2)使用MTT实验法检测虚拟化合物对细胞活性水平的初筛，发现在细胞水平具有活性的化合物C98。(3)酶活性实验分析C98对PI3K的抑制作用。(4) Western blotting方法检测经C98处理之后细胞蛋白水平的表达，包括对PI3K通路的抑制以及对骨髓瘤细胞的促凋亡作用。(5)流式细胞技术检测C98对多发性骨髓瘤细胞的促凋亡作用。(6)免疫荧光实验用来检测AKT的细胞表达水平及亚细胞定位。(7)建立多发性骨髓瘤异种移植小鼠模型，检测C98对骨髓瘤生长的影响。(8)使用分子对接和分子动力学模拟方法在分子水平阐明C98抑制PI3K的作用机理。研究结果：(1)通过逐层虚拟筛选的方法对Specs和Chembridge化合物文库的80万个化合物进行筛选，经成药性检测以及聚类分析之后，得到了148个潜在的PI3K抑制剂。(2)采用MTT方法将上述148个化合物处理多发性骨髓瘤OPM2细胞，最后得到了6个具有抑制OPM2细胞增殖的化合物，然后使用IGF-1刺激法初步验证了这6个化合物中C98具有较好的PI3K抑制活性。(3)酶活性实验发现C98可以抑制I型PI3Ks，其中对PI3K和γ抑制活性最高达到纳摩尔级，但是对AKT及mTOR没有明显抑制作用。(4) Western blotting方法检测到C98特异性地抑制PI3K/AKT/mTOR通路，而对其他通路的蛋白（IGF-1、MAPK、ERK、p38、c-Src、STAT3等）没有抑制作用。(5)免疫荧光实验发现C98可以阻止AKT细胞质膜转移，从而抑制它的磷酸化。(6)伴随着对PI3K的抑制，C98可以诱导骨髓瘤细胞的凋亡。流式细胞术分析发现C98对骨髓瘤细胞的促凋亡作用呈浓度依赖性关系。Western blotting检测发现伴随着磷酸化AKT的抑制，C98可以引起细胞内Caspase-3和PARP的裂解激活。(7)使用C98处理OPM2和JJN3肿瘤模型小鼠，发现灌胃给药C98在3周内可以显著抑制肿瘤的生长，并呈剂量效应关系。C98对肿瘤细胞的抑制与AKT的磷酸化关联。(8)分子对接和分子动力学研究发现在C98与PI3K的相互作用中氢键以及范德华相互作用起了重要的作用。例如C98的氨基（-NH2）与各亚型的对应残基都形成了相应的氢键相互作用：PI3K的Ser854、PI3Kβ的Val848、PI3K的Ser831、PI3Kγ的Ala885。而PI3Ks与C98也形成了数目不等的π-H相互作用：PI3K的Tyr836、Ile848、Val850、Ile932；PI3Kβ的Ile930；PI3K的Tyr813、Ile930；PI3Kγ的Tyr867。这些相互作用对于稳定C98的结合同样起到了积极的作用。结论本课题结合逐层虚拟筛选和生物学实验的方法发现了I型PI3K广谱抑制剂C98。它可以与PI3K的ATP口袋相结合从而抑制PI3K的活性，并特异性地抑制PI3K/AKT信号通路，从而诱导多发性骨髓瘤细胞的凋亡，延缓肿瘤细胞的生长。由于C98表现出了良好的口服活性和较低的毒性，C98可以发展成为一个高效的抗多发性骨髓瘤药物。更多还原

【Abstract】 Multiple myeloma is the second most hematological malignancy, accounting formore than10%of all blood cancers and2%of annual cancer-related deaths due tolack of curable drugs. Novel and molecularly targeted anti-MM drugs are in urgentneed. The phosphatidylinositol3-kinase (PI3K)/AKT signaling pathway plays acritical regulatory role in the pathophysiology of multiple myeloma, includingsurvival, proliferation, migration, angiogenesis, and chemoresistance. PI3K hastherefore emerged as a key therapeutic target. Many potent inhibitors targeting thispathway have been developed and some have been moved for clinical evaluations formultiple myeloma. With the advances of combinatorial chemistry, cancer biology,informatics technology and bioengineering, large-scale screening become more andmore common in PI3K-targeted drug discovery. Comparing to traditional highthroughput screening, virtual screening is reliable, cost-effective and time-saving, ithas become an important complementary approach to experimental large-scalescreening. In this project, we combined virtual screening and wet-lab experiments todiscover new PI3K inhibitors and found a potential new candidate for targeted therapyin multiple myeloma.Methods:(1) A hierarchical virtual screening was employed to find novel PI3K inhibitors.(2) MTT assay was used to further identify potential PI3K inhibitors that inhibitMM cell proliferation.(3) Cell-free assay was used to determine PI3K inhibitory effects of C98.(4) Western blotting was performed to examine the expression of proteins after the treatment of C98that involved in cell apoptosis and PI3K/AKT signalingpathway.(5) Flow cytometry was used to measure apoposis of myeloma cells induced byC98.(6) Immunofluorescence analysis was used to determine the cell expression andcellular localization of p-AKT in OPM2cells.(7) Human myeloma xenograft models in nude mice were used to evaluate thetherapeutic effects of C98in vivo.(8) Molecular docking and molecular dynamic stimulation were employed toinvestigate the mechnism of PI3K inhibition by C98.Results:(1) The hierarchical virtual screening combined with drug-like test and clusteranalysis led to148virtual “top-hits” after screening against800,000of smallmolecule compounds from the Specs and Chembridge Chemicals Libraries.(2) Six compounds showed potent anti-proliferation in the multiple myeloma cellline OPM2. These compounds were further subject to PI3K inhibition evaluated incells after IGF-1stimulation, from which C98was found to show potent inhibition ofAKT activation.(3) Enzymatic reaction assays in the cell free system showed that C98inhibitshuman PI3K, β, and γ at nanomolar or low micromolar concentrations but does notsuppress AKT or mTOR activity.(4) C98specifically inhibits the PI3K signaling pathway and has no inhibitoryeffects on other signal transduction pathways such as IGF-1, MAPK, ERK, p38, c-Srcand STAT3.(5) Immunofluorescence assay showed that C98blocks AKT translocation to thecytoplamic membrane.(6) C98induces apoptosis of MM cells in parallel with its PI3K inhibition. Cellapoptosis was found in C98-treated cells as measured by Annexin V-FITC and PIstaining. The results of Western blotting showed that C98activated Caspase-3andPARP in parallel with its p-AKT inhibition. (7) C98significantly delayed myeloma tumor growth in OPM2and JJN3xenograft models. C98delayed MM tumor growth at least partly in associated with itsinhibition of PI3K/AKT signaling pathway.(8) Molecular docking and MD stimulations confirm that the H-bonds and van deWaals contacts are important for the interference of C98with the ATP active pocket ofPI3Ks. These interactions established between specific groups of C98and conservedhydrophobic residues of the ATP-binding cleft may account for its potency againstPI3K and selectivity profile over protein kinases.Conclusion: A successful Virtual Screen-based identification of PI3K inhibitorswas established which leads to discovery of C98, a pan-Class I PI3K inhibitor,confirmed by a series of biochemical, cellular and animal studies. By binding to andinterfering with the ATP active cleft in PI3K enzymes, C98specifically blocks thePI3K/AKT signaling pathway. The impressive oral activity and well tolerance of C98in in-vivo models highlights its candidacy in MM treatment.更多还原