不同构建的SARS-Cov M pro 活性比较及其抑制剂评价 本文为读后感,原文见文后,读后感作者侯彭姣 由SARS-Cov-2感染引起的新冠疫情仍在全球蔓延。截止2020年5月31日,全世界累计确诊新冠肺炎患者已超过601万,累计死亡患者超过36万。疫情爆发至今已近6个月,有效的治疗选择仍然非常有限。除了隔离、治疗,加快特效药物和疫苗的研发将有助于更好更快地战胜病毒。 SARS-Cov-2 基因组由约30,000个核苷酸组成。可编码两个有重叠的多蛋白前体pp1a和pp1ab,通过广泛的水解酶的加工,生成病毒转录复制所需的蛋白。而参与这一水解过程的主要蛋白是其编码的Main protein ( M pro )。M pro 可识别不少于11个保守位点,并通过自切的方式,首先将其自身从pp1a和pp1ab中释放出来,继而水解并产生其他的蛋白成熟体。因此,M pro 作为一种关键的CoV酶,在介导病毒复制和转录中起关键作用,使其成为该病毒的有吸引力的药物靶标。 表达并纯化出产量高,活性好的蛋白酶,对药物开发和蛋白酶基础性质的研究都是一个瓶颈。因此,本文结合之前的研究结果,对SARS冠状病毒的M pro 的构建,表达进行优化,并对其酶活性进行评价。鉴于M pro 在冠状病毒家族中较高的保守性,因此,该研究结果对于新冠疫情相关药物的研发,筛选具有重要的意义。 首先,作者对SARS-Cov的M pro 的进行了不同构建和表达。 作者针对M pro 设计4种不同的构建,进行表达和纯化。具体描述如下: 1. GPLGS-WT :其N端残留5个氨基酸,这5个氨基酸为PGEX-6P-1载体上的氨基酸; 2. GS-WT : 在其N端仅残留2个氨基酸; 3. WT-GPH 6 :其C端残留8个氨基酸,分别是G,P和6个His; 4. WT: N 端和C端无残留的SARS-Cov M蛋白。 并对4种蛋白的酶活性进行了测定,评价和对比,如下图所示: 通过对比,可发现,WT的酶活性最高,Kcat/Km(M -1 s -1 )为26500;WT的酶活性是GPLGS-WT活性的150倍,GS-WT的20倍,仅是WT-GPH 6 的3倍。以上的研究结果提示,M pro N端和C端氨基酸的残留对其活性影响,其中C端氨基酸残留影响不大,而N端氨基酸的残留会严重影响其酶活性。 其次,作者通过晶体结构对酶活性之间的差异进行了进一步解释和阐明。 作者分别获得了GPLGS-WT和WT的晶体。GPLGS-WT的晶体属于P2 1 空间群,包括2个同源二聚体,分别为protomer A和B,每个protomer含3个结构域。但是只有protomer A是具有活性的,而protomer B由于其N端残留氨基酸使其S1部分破坏而不具酶活性。 而WT的晶体也含有两个同源二聚体,分别是protomer A*和B*,空间群属于C2,且protomer A*和B*均具有活性。这种结构上的变化,从一定程度解释为什么WT对比GPLGS-WT具有较高活性。 此外,作者发现GPLGS-WT活性低于GS-WT的活性,可能是由于GPLGS-WT的N端残留较多氨基酸,而N端残留氨基酸距离其酶活性中心较近所致。但由于C端残留氨基酸距离N端活性中心较远,所以WT-GPH 6 活性仅仅是WT的1/3,说明C端氨基酸的残留对酶活性的影响较小。 最后,作者对4种不同构建的SARS-Cov M pro 与抑制剂的结合进行了评价。使用的抑制剂为可与M pro 不可逆结合的抗病毒抑制剂N3,属于米氏受体的一种。评价结果如下所示: 由上可以看出,N3可将WT和WT-GPH 6 完全失活,却不能将GS-WT和GPLGS-WT完全失活。以上结果说明N端氨基酸残留对抑制剂的结合有很大影响,很大程度上影响酶的抑制剂评价。 以上研究结果提示,WT构建的SARS-Cov M pro 更适合蛋白的活性实验,以及抑制剂的筛选和评价。这对我们评价其他蛋白酶的活性具有重要意义和参考价值。 原文链接: 1. https://pubmed.ncbi.nlm.nih.gov/14585926/ 2. https://pubmed.ncbi.nlm.nih.gov/17189639/?from_single_result=10.1016%2Fj.jmb.2006.11.073expanded_search_query=10.1016%2Fj.jmb.2006.11.073 3. https://pubmed.ncbi.nlm.nih.gov/32272481/?from_term=Structure+of+Mpro+from+COVID-19+virus+and+discovery+of+its+inhibitorsfrom_pos=1
离子通道这里有vmd查看结构的例子: http://www.ks.uiuc.edu/Training/CaseStudies/pdfs/channels.pdf 还有基于NAMD的体系构建: \0 https://www.ks.uiuc.edu/Training/Tutorials/science/membrane/mem-tutorial.pdf \0 经典 MD 可以对实验上得到的构型进行稳定性验证。得到蛋白质的动态图像。 一般步骤如下: pdb2gmx error: Residue 5 named ARG of a molecule in the input file was mapped to an entry in the topology database, but the atom CG used in that entry is not found in the input file. Perhaps your atom and/or residue naming needs to be fixed. 经检查,发现 ARG64 太短了,没有侧链。 Some residues have missing atoms 。每个链中各有 5 个。怎么办? Charmm-gui 可以搞定。得到新的pdb。 再次pdb2gmx: error: Residue 'HSD' not found in residue topology database 改 charmm 力场可以解决。 再次pdb2gmx, error: Atom OXT in residue GLN 119 was not found in rtp entry GLN with 17 atoms while sorting atoms. 对比 rtp ,确实多了一个 O 去掉即可。 此外,water 改成 SOL 标准名称。 K+ 离子先去掉,生成后再加上。 Now there are 5882 atoms and 389 residues Total mass in system 40956.290 a.m.u. Total charge in system 4.000 e 即完成体系构建。 其他体系类似,可参考。
领导伦理的不确定性——建构焦点解决实践的本源(5) 高德明团队 译编 Griffin(2002)指出,许多讨论组织伦理的行为都会产生一个非此即彼的观点,要么是在不同情境中传统伦理定义的一般道德规范的应用,要么是后现代主义的立场强调与情境相对的道德选择和行为。传统定义假设组织领导者应该有绝对的道德标准。这也掩盖了许多一般道德准则在实际应用中的模糊性和不确定性。或者,后现代方法太过强调情境的模糊性和不确定性以至于认为一般道德规范无关紧要。 复杂反应过程的理论家提供了第三种关于领导伦理的对话基础。他们承认组织领导的许多不确定性,但他们也将一般道德规范作为组织伦理的潜在相关关系。他们通过把领导伦理作为自组织对话的一个方面,把这些独立的主题整合在了一起,也就是说,伦理参与关注的是社会互动和关系的细节。这些细节形成了真实情境中讨论道德准则的相关背景。Giffin(2002:213)解释了领导者和伦理同时在自组织对话中的显现;有效领导往往是那些能够发展更多的自主性和有能力处理那些正在进行的目的和互动任务的人。领导者是那些能够增强团队内成员负责任态度的人。他们能够加强团体内以及团体之间的沟通交流。 焦点解决实践者接受过对话知识和技能的训练,这些知识和技能有助于解决日常生活细节中的伦理问题。复杂反应过程理论家对焦点解决实践者造成的一个困难是,扩大他们对解决伦理方法的知识和技能,而这是在不确定和矛盾情境中的领导力的一个内容。再则,我们回顾组织是如何成为社交互动的点以及焦点解决顾问的责任是通过提升对话技巧和敏锐度来帮助组织成员变成更有效的领导者。 结 论 我已经指出,这篇文章与我在组织中的实践经验有关。这对焦点解决实践者的有用程度与他们的经历相关,也可能与如何看待顾问角色有关。对于隐含在本文中的假设——组织咨询的一个方面是为客户提供感受和处理组织生活中的矛盾、焦虑和困境提供资源,我称之为教学。焦点解决实践者可能会利用复杂反应过程的角度来提高他们教学活动中的两种相关方法。 首先,这是一种扩展实践者和客户词汇的资源。焦点解决实践者早就知道语言远超其所命名的事物,它们只是看到和想象的过去、现在和未来事实的基础。不幸的是,这些实践者有时在焦点解决对话过程中让极简主义的言论所蒙蔽,忽视了对话中关于改变的新词汇。第二种方式,对复杂反应过程的担忧可能增强了焦点解决实践者的教学,包括构建反思的社会背景。这个背景帮助焦点解决实践者和客户从经验中学习。这是Cheryl Mattingly和Maureen Hayes Fleming研究中的一个重要主题,职业治疗师如何能变得更熟练。虽然实践经验很重要,但是,不是所有有经验的治疗师都能产生相同的效果。 Mattingly和Fleming (1994: 30)认为,经验不是从事一些简单的事情,而是指做一些与反思有关或者使事情变得有意义的事。经验是有用的,但不是因为你经历过,而是因为它有意义。本研究与焦点解决实践者和客户的工作以及文章写作有关。如果这篇文章对你形成了一个语境反思,那么我就成功了。 ——高德明焦点解决高效教练督导团队译编 附:原文 Uncertainties of Ethical Leadership Griffin (2002) notes that many discussions about organizational ethics turn on an either/or choice between a traditional definition of ethics as the application of general moral codes to diverse situations and the postmodern stance that emphasizes the situational relativity of ethical choices and behavior. The traditional definition assumes that there are absolute moral standards to which organizational leaders should be held. It also glosses over the many ambiguities and uncertainties associated with applying general moral codes to actual situations. Alternatively, the postmodern approach assumes that the ambiguities and uncertainties of situations are so great that they render general moral codes irrelevant. Theorists of complex response processes offer a third basis for conversations about ethical leadership. The theorists acknowledge the many uncertainties of organizational leadership but they also treat general moral codes as potentially relevant to organizational ethics. They bring these separate themes together by treating ethical leadership as an aspect of self-organizing conversations, that is, an ethics of participation focused on the details of social interaction and relationships. The details form contexts for talking about the relevance of moral codes for actual situations. Griffin (2002: 213) explains that leaders and ethics emerge together in self-organizing conversations; Effective leaders tend to be those who have…developed more spontaneity and ability to deal with the on-going purpose and task of interaction. Leaders are individuals who have enhanced capacity for taking the attitudes of the other members of the group. They enhance communication within and between groups. Solution-focused practitioners are trained in conversational knowledge and skills that are potentially useful in addressing the ethical implications of the small details everyday life. A challenge that theorists of complex response processes pose for solution- focused practitioners is to extend their knowledge and skills to explicitly address ethics as an aspect of leadership in uncertain and paradoxical situations Once again, we come back to how organizations are sites of social interaction and to solution-focused consultants’ responsibility to help organization members become more effective leaders by enhancing their conversational skills and sensibilities. Conclusions I have already stated that this essay is connected to my practical experiences in organizations. It’s usefulness for solution-focused practitioners will probably vary based on their experiences. It is also likely to be related to how they think about their roles as consultants. Implicit in this essay is the assumption that one aspect of organizational consultation is providing clients with interpretive resources for making sense of and dealing with the paradoxes, anxieties and dilemmas of organizational life. I call this teaching. Solution-focused practitioners might use the complex response processes perspective to enhance their teaching activities in two interrelated ways. First, it is a resource for expanding practitioners’ and clients’ vocabularies. Solution- focused practitioners have long known that words are more than labels for naming things, they are standpoints for seeing and imagining past, present and future realities. It is unfortunate that these practitioners sometimes allow the rhetoric of minimalism in solution-focused discourse to blind them to the magic of new words in conversations about change. The second way that concern for complex response processes might enhance solution-focused practitioners’ teaching involves building social contexts of reflection. The contexts assist solution-focused practitioners and clients in learning from their experiences. This is a major theme in Cheryl Mattingly and Maureen Hayes Fleming’s6 study of how occupational therapists become more proficient. While practical experience is important, all experienced therapists are not equally effective. Mattingly and Fleming (1994: 30) explain that Experience is not simply doing something. It is doing something combined with reflecting on, or making meaning of, the event. Experience is useful, not because one has lived through it, but because one has made meaning of it. This research is also relevant to the work of solution-focused practitioners, their clients and to the writing of this essay. I have succeeded if this essay has formed a context of reflection for you. 学习、练习、实践、反思、督导,是一个SF取向工作者的快速成长之路。 敬请期待下期分享。