CRISPR/Cas9技术及其在药物研发中的应用

CRISPR/Cas9系统是在细菌和古细菌中发现的一种为抵御病毒和质粒的不断攻击而演化来的获得性免疫防御机制,由规律成簇的间隔短回文重复(clustered regularly interspaced short palindromic repeats,CRISPR)和Cas(CRISPR-associated)蛋白组成。通过改造最简单的II型CRISPR系统,将特殊小向导RNA(small guide RNA,sg RNA)和Cas9核酸内切酶导入细胞内,即可在双链DNA特定位置上进行切割并实现基因敲除或敲入。CRISPR/Cas9系统因其高效基因编辑功能,已被应用于多种生物和多项科研领域。本文综合论述了CRISPR/Cas9技术在药物研发中的应用,如功能基因的筛选和定点编辑、药物靶点筛选和验证、动物模型构建和遗传疾病治疗等,总结了CRISPR/Cas9技术目前所存在的缺陷与改善的方向。国家自然科学基金资助项目(81422045,81603131

福建省药理学学科发展报告

福建省药理学学科的发展对于提高福建人民群众合理用药水平,促进医药企业新药研发,以及推动海西生物医药产业的发展,具有非常重要的意义。福建省药理学科在学科建设、人才培养和平台建设方面已取得一定成效,在药物靶点与先导化合物研究、中药与天然药物研究、小分子药物研究、基因药物研究和海洋药物研究等领域取得了初步成果。福建省药理学科将继续依托福建省药理学会平台,促进药理学术交流,加强药理学分支学科与专业委员会建设,充分发挥学科优势,服务福建经济建设与社会发展

控制肝脏组织发育、再生重塑与大小的关键蛋白质机器

肝脏损伤、功能衰竭及肝癌等疾病是我国重大健康问题,它们的发生发展与肝脏再生重塑及大小调控异常密切相关。针对肝脏发育、再生及大小控制机理的基础研究对于解决上述重大肝脏疾病的临床需求具有重要意义。前期研究发现Hippo信号通路在调控细胞增殖及分化、干细胞命运等方面起着关键作用,而Hippo通路的失调可导致肝脏等器官发育和大小异常,严重影响受损组织再生与重塑,或导致肿瘤迅速发生。本项目拟以肝脏为研究对象,聚焦Hippo相关信号网络如何感知器官大小并适时调控细胞生长、分化与死亡以实现器官发育、再生重塑和器官大小控制这一关键科学问题,整合基础、临床、药学领域的先进研究技术,拟通过基因编辑、内胚层干细胞肝向分化、肝细胞移植和肝脏重建、肝脏损伤修复等模型、临床病理分析、蛋白结构解析和靶向药物设计等方法,深入探讨该通路失控导致肝脏发育异常、再生重塑障碍、癌症发生的致病机理,并提出靶向干预新策略。控制肝脏组织发育、再生重塑与大小的关键蛋白质机器(2017YFA0504500

Impeded Nedd4-1-Mediated Ras Degradation Underlies Ras-Driven Tumorigenesis

RAS genes are among the most frequently mutated proto-oncogenes in cancer. However, how Ras stability is regulated remains largely unknown. Here, we report a regulatory loop involving the E3 ligase Nedd4-1, Ras, and PTEN. We found that Ras signaling stimulates the expression of Nedd4-1, which in turn acts as an E3 ubiquitin ligase that regulates Ras levels. Importantly, Ras activation, either by oncogenic mutations or by epidermal growth factor (EGF) signaling, prevents Nedd4-1-mediated Ras ubiquitination. This leads to Ras-induced Nedd4-1 overexpression, and subsequent degradation of the tumor suppressor PTEN in both human cancer samples and cancer cells. Our study thus unravels the molecular mechanisms underlying the interplay of Ras, Nedd4-1, and PTEN and suggests a basis for the high prevalence of Ras-activating mutations and EGF hypersignaling in cancer. © 2014 The Authors

Pharmacological targeting of STK19 inhibits oncogenic NRAS driven melanomagenesis

黑色素瘤是由黑色素细胞恶性转化产生的恶性程度极高的皮肤癌，含有NRAS激活突变的黑色素瘤约占20-30%，但至今还未有靶向NRAS的有效黑色素瘤治疗方案。针对这一难题，波士顿大学医学中心崔儒涛教授、厦门大学生命科学学院邓贤明教授、复旦大学附属肿瘤医院王鹏教授组成的联合研究团队利用激酶组siRNA文库筛选发现新颖的丝/苏氨酸激酶STK19是NRAS的上游激活子，进一步分子机制研究揭示STK19通过磷酸化NRAS的89位丝氨酸（S89）促进了NRAS介导的黑色素细胞恶性转化。该研究揭示了一种经由新颖激酶STK19调控NRAS突变黑色素瘤的分子机制，验证了STK19有望作为NRAS介导的黑色素瘤的有效靶标，为发展新的黑色素瘤靶向药物提供了先导化合物，同时也为发展其它素有“癌基因之王---RAS”驱动的相关肿瘤靶向药物发展提供了新思路。该论文由波士顿大学医学中心、厦门大学生命科学学院、复旦大学附属肿瘤医院等单位合作完成，共同第一作者厦门大学生命科学学院博士生张婷负责了该系列化合物的设计、合成与优化，崔儒涛教授、邓贤明教授和王鹏教授为共同通讯作者。【Abstract】Activating mutations in NRAS account for 20-30% of melanoma, but despite decades of research and in contrast to BRAF, no effective anti-NRAS therapies have been forthcoming. Here we identify a previously uncharacterized serine/threonine kinase STK19 as a novel NRAS activator. STK19 phosphorylates NRAS to enhance its binding to its downstream effectors and promotes oncogenic NRAS-mediated melanocyte malignant transformation. A recurrent D89N substitution in STK19 whose alterations were identified in 25% of human melanomas represents a gain-of-function mutation that interacts better with NRAS to enhance melanocyte transformation. STK19 D89N knockin leads to skin hyperpigmentation and promotes NRAS Q61R -driven melanomagenesis in vivo. Finally, we developed ZT-12-037-01 (1a) as a specific STK19-targeted inhibitor and showed that it effectively blocks oncogenic NRAS-driven melanocyte malignant transformation and melanoma growth in vitro and in vivo. Together, our findings provide a new and viable therapeutic strategy for melanomas harboring NRAS mutations.We thank Drs. Norman Sharpless and David Fisher for kindly providing the loxP/STOP/loxP NRAS Q61R knockin (LSL-NRAS Q61R ) mice. We thank Dr. Anurag Singh for kindly sharing cell lines. We also thank Drs. X. Shirley Liu, Tao Wang, Wantao Chen, Dali Liu, Chunxiao Xu, Jianming Zhang and Junrong Zou for discussion and assistance. This work was supported by grants from Boston University (to R.C.), the National Key R&D Program and the National Natural Science Foundation of China (No. 2017YFA0504504, 2016YFA0502001, 81422045, U1405223 and 81661138005 to X.D.), the Fundamental Research Funds for the Central Universities of China (No. 20720160064 to X.D.), and the Program of Introducing Talents of Discipline to Universities (111 Project, B12001).该研究得到了科技部重点研发计划、国家自然科学基金委和中央高校基本科研业务费等的资助

Pyrazolopyridines as inhibitors of the kinase LRRK2: a patent evaluation (WO2011141756)

Scientific evaluation of a patent aiming for the development of pyrazolopyridine derivatives as LRRK2 kinase inhibitors, a potential therapeutic target for combating Parkinson's disease.NIH [P41 GM079575-03]; Michael J Fox foundation for Parkinson's disease research; Fundamental Research Funds for the Central Universities of China [2011121030

Biodiversity Analysis and Bioactivity Detection of Actinomycetes from Rhizosphere Soil in Xiamen and Nanjing Area,China

为探究不同地区植物根际土壤中可培养放线菌的多样性,筛选具有抗菌及抗肿瘤活性的药源菌株,采用改良聚乳酸-明胶和海藻糖-脯氨酸2种培养基,选择分离采; 自厦门市翔安区香山风景区、南京市中山植物园及玄武湖公园的17份植物根际土壤样品中的放线菌,并进行16SrRNA基因鉴定、系统进化分析及抗菌和抗肿; 瘤生物活性测定.共分离到178株放线菌,其中链霉菌151株,其余27株为稀有放线菌,占总数的15.2%.稀有放线菌分布在9个属中:微杆菌属(Mi; crobacterium)、拟无枝酸菌属(Amycolatopsis)、韩国生工菌属(Kribbella)、野野村氏菌属(Nonomuraea); 、小单孢菌属(Micromonospora)、链孢囊菌属(Streptosporangium)、拟孢囊菌属(Kibdelosporangium); 、纤维微菌属(Cellulosimicrobium)和栖白蚁菌属(Isoptericola),包含2株新种.对分离得到的所有放线菌进行液体小量发; 酵,并测定其发酵粗提物的抗菌和抗肿瘤活性.结果显示:所测定的178株放线菌中,有82株对一种或多种指示菌表现出抗菌活性,占供测菌株的46.1%;; 有60株对一种或多种肿瘤细胞具有抑制作用,占供测菌株的33.7%.研究结果表明植物根际土壤中放线菌资源丰富,其中抗菌和抗肿瘤活性较强的菌株可为后; 续微生物药物研发提供有利资源.To investigate the diversity of actinomycetes from rhizosphere soil in; different regions and to screen the strains with antibacterial and; antitumor activities,the 16SrRNA gene sequencing and phylogenetic; analysis were used to explore the diversity of actinomycetes, which were; isolated from 17rhizosphere soil samples with two selective media.And; then 6bacteria,2fungi and 4tumor cells were used as indicators to; determine the biological activities of actinomycetes.In this study,a; total of 178actinomycetes were isolated from the rhizosphere; soil,including 27strains as rare actinomycetes,which accounted for a; total of 15.2%.The rare actinomycetes can be assigned to 9genera,which; are; Microbacterium,Amycolatopsis,Kribbella,Nonomuraea,Micromonospora,Strepto; sporangium, Kibdelosporangium,Cellulosimicrobium,Isoptericola,and; 2strains of them were novel species.Among the isolated actinomycetes,; 82strains(46.1%)had antibacterial activities against one or more; indicators,60strains(33.7%)had inhibitory activities against one or more; tumor cells.In conclusion,the results show that the rhizosphere soil is; a highly diverse actinobacterial community, in which most of them appear; to be novel candidate species.Therefore,the actinomycetes with; biological activities could be a promising source for secondary; metabolites separation and microbial pharmaceutical developments.国家自然科学基金; 福建省教育厅科技项

Leucine-rich repeat kinase 2 inhibitors: a patent review (2006-2011)

NIH [P41 GM079575-03]; Michael J Fox foundation for Parkinson's disease research; Fundamental Research Funds for the Central Universities of China [2011121030]; 111 Project of Education of China [B06016]Introduction: Leucine-rich repeat kinase 2 (LRRK2) has received considerable attention since the discovery of LRRK2 mutations in families with dominantly inherited Parkinson's disease (PD) in 2004. The missense mutation G2019S is the most common LRRK2 mutation and has been identified in both familial and sporadic PD cases. The G2019S mutation enhances kinase activity suggesting that LRRK2 could be an attractive therapeutic target for PD and small-molecule ATP-competitive LRRK2 kinase inhibitors are one way to investigate this possibility. Areas covered: Currently, LRRK2 kinase inhibitors are being actively pursued by industry and academia. Herein, patents detailing the discovery of LRRK2 kinase inhibitors from 2006 through 2011 and the corresponding publications from 2006 through July of 2012 are summarized. Expert opinion: Wild-type and mutant forms of LRRK2 are currently being actively pursued as therapeutic targets for the potential treatment of PD. The increasing number of patent applications being filed for inhibitors of LRRK2 is a testament to this activity. Numerous distinct chemo-types have been reported as LRRK2 inhibitors with some demonstrating exceptional potency and selectivity for LRRK2 relative to other kinases. These compounds are being used as pharmacological 'tools' to elucidate the physiological and pathophysiological function of LRRK2 and it appears likely that some will be investigated for their potential therapeutic effects for the treatment of PD