X-ray Crystallographic Fragment Screening Core

摘要 X射线晶体片断筛选(XCFS)是鉴定新的药效团的有力工具 用于探针和药物设计以及生物化学和结构动力学表征 感兴趣的蛋白质。已经建立了XCFS核心，以使这一强大的工具可供多个母公司访问 研究HIV-1和蜂巢中相关蛋白质/复合体的项目。XCFS核心(Arnold组)具有 完成了针对病毒目标的三次XCFS运动：艾滋病毒-1逆转录酶、甲型流感 核酸内切酶和HIV-1整合酶催化核心区。在每种情况下，成功的筛查活动 发现了新的病毒，在HIV-1逆转录酶和流感核酸内切酶的情况下 发现了新的构象/口袋，用于了解蛋白质动力学和生物化学。 XCFS核心将与其他蜂巢项目和核心合作，以确定符合以下条件的晶体形态 适用于XCFS并根据需要对其进行优化。初步项目包括：艾滋病毒-1衣壳病毒(项目3， 艾滋病毒-1整合酶(项目1和项目4，Kvaratskhelia和Engelman)和艾滋病毒-1逆转 核酸底物转录酶(项目3，阿诺德)。小规模试点筛查将验证 用甚高通量同步加速器进行大规模筛选前XCFS的晶型 X射线结晶学光束线(具体目标1)。钻石之光的一条专门的光束线，XChem 源将被用于高效和快速地筛选1000-2000个小分子片段。XChem工具 开发了几种碎片浸泡、晶体处理、数据收集和处理技术，以 允许非常快速的XCFS活动。除了XChem，还有新的高速光束线在 布鲁克黑文国家实验室和其他地方能够收集数据的速度是两年无法达到的 那是以前的事了。新的光束线技术允许单独筛选来自大型文库的片段， 而之前需要使用碎片的混合物。筛选单个化合物的能力 允许设计新的化合物文库以利用这一新技术(具体目标2)。新的 将为XCFS设计具有点击反应和含SuFEx部分的片段库(项目6， 奥尔森和夏普莱斯)。 将对来自XCFS的数据进行处理和提炼，以确保准确的命中识别和命中验证 通过对衍生物的筛选和适当的正交试验，确定了最佳工艺条件。通信和数据管理 项目和核心之间的关系将确保明确的信息传递和后续化学/研究(具体 目标3)。

Abstract X-ray crystallographic fragment screening (XCFS) is a powerful tool for identification of new pharmacophores for probes and drug design as well as well as for biochemical and structural dynamic characterization of the protein of interest. An XCFS Core has been established to make this powerful tool accessible to multiple HIVE Projects studying HIV-1 and related proteins/complexes in the HIVE. The XCFS Core (Arnold group) has completed three XCFS campaigns against the viral targets: HIV-1 reverse transcriptase, influenza A endonuclease, and HIV-1 integrase catalytic core domain. In each case the successful screening campaign has discovered novel hits and in the cases of HIV-1 reverse transcriptase and influenza endonuclease has found novel conformations/pockets for understanding protein dynamics and biochemistry. The XCFS Core will collaborate with the other HIVE Projects and Cores to identify crystal forms that are appropriate for XCFS and optimize them as needed. Initial projects include: HIV-1 capsid (Project 3, Sarafianos), HIV-1 integrase (Projects 1 and 4, Kvaratskhelia and Engelman), and HIV-1 reverse transcriptase with nucleic acid substrate (Project 3, Arnold). Small pilot screening will verify the usefulness of the crystal form for XCFS before proceeding to large scale screening using very high-throughput synchrotron X-ray crystallography beamlines (Specific Aim 1). A specialized beamline, XChem, at the Diamond Light Source will be used to efficiently and rapidly screen 1000-2000 small molecule fragments. The XChem facility has developed several technologies for fragment soaking, crystal handling, data collection and processing to allow for very rapid XCFS campaigns. In addition to XChem, there are new high-speed beamlines at Brookhaven National Laboratories and elsewhere capable of data-collection speeds unobtainable two years ago. The new beamline technologies allow for fragments from a large library to be screened individually, whereas previously mixtures of fragments needed to be used. The ability to screen individual compounds allows for design of novel compound libraries to take advantage of this new technology (Specific Aim 2). New fragment libraries with click-reactive and SuFEx-containing moieties will be designed for XCFS (Project 6, Olson and Sharpless). Data from XCFS will be processed and refined to ensure accurate hit identification and hits will be verified through screening of derivatives and appropriate orthogonal assays. Communication and data management between the Projects and Cores will ensure clear information transfer and follow up chemistry/studies (Specific Aim 3).