MACCHESS PROGRAM FOR AUTOMATION AND HIGH-THROUGHPUT

用于自动化和高吞吐量的 MACCHESS 程序

基本信息

批准号：
8171485
负责人：
EDWARD ARNOLD
金额：
$ 8.52万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8171485
关键词：
Acquired Immunodeficiency Syndrome Anti-HIV Agents Antiviral Agents Automation Binding Clinical Trials Collaborations Complex Computer Retrieval of Information on Scientific Projects Database Crystallization DBL Oncoprotein Data Data Set Development Drug Delivery Systems Drug Design Drug resistance Engineering Funding Genome Grant HIV HIV-1 Reverse Transcriptase Institution Laboratories Lead Modeling Mutation Nucleic Acid Binding Pharmaceutical Preparations Phase Polymerase Preparation Process Publishing RNA-Directed DNA Polymerase Research Research Personnel Resolution Resources Reverse Transcriptase Inhibitors Ribonuclease H Robotics Roentgen Rays Site Source Structure Structure-Activity Relationship Surface System United States United States National Institutes of Health Variant Work base beamline design inhibitor/antagonist insight novel programs success three dimensional structure

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. CHESS has been an essential resource in the Arnold laboratory's effort to study the structure and function of reverse transcriptase (RT), a key component of the AIDS virus and the target of many of the most widely used anti-AIDS drugs. The Arnold group has solved three-dimensional structures of wild-type and drug-resistant HIV-1 RT in complexes with a variety of antiviral drugs and model segments of the HIV genome. These studies, together with contributions from other laboratories, have yielded numerous insights into polymerase structure-function relationships, detailed mechanisms of drug resistance, and provided the basis for structure-based design of RT inhibitors. A collaboration between the Arnold laboratory and the Janssen/Tibotec group led to the development of a number of inhibitors that show great promise as potential treatments for AIDS, two of which are currently in Phase II (TMC278) and Phase III (TMC125) clinical trials in the United States and overseas [165-167]. Structural studies of HIV-1 RT complexed with RNase H inhibitors [168] are also being pursued; the RNase H activity of RT is also essential for HIV replication, yet no drugs targeting RNase H have been developed. During the past year remarkable success has been achieved in terms of obtaining high-resolution diffraction from HIV-1 RT specifically engineered to yield novel crystal forms. Diffraction data extending to 1.8 ¿ resolution have been obtained for engineered HIV-1 RT in complex with the Janssen inhibitor TMC278. The engineering strategy has consisted of making systematic variations of the N- and Ctermini of the HIV-1 RT heterodimer in a coexpressed p66/p51 system, with a removable His-tag for convenient purification, and selected surface mutations such as Lys=>Ala. Among the noteworthy aspects of the high resolution HIV-1 RT/TMC278 structure (J. Bauman, K. Das et al., in preparation) is that this critical structure had been elusive despite literally thousands of crystallization attempts with this complex. An important implication of this new HIV-1 RT construct and crystal form is that further structurebased drug design for HIV-1 RT inhibitors can proceed both more accurately and rapidly. They have been able to collect numerous datasets extending to 2 ¿ resolution or better with and without bound inhibitors and are now searching for higher resolution crystal forms that can accommodate nucleic acid binding. Many crystal forms were evaluated using X-rays from the CHESS F1 and A1 beamlines in the process of identifying several engineered RT constructs that yielded high-resolution crystals. Prior to this work, the highest published resolution of any HIV-1 RT structure was 2.2 ¿, and most published RT structures are in the 2.7-3.0 ¿ range. The development of high-through put capability at CHESS, and in particular, the use of the new robotic automounter at CHESS F1, has made it possible to evaluate a large number of candidate crystals and will continue to be an extremely valuable resource as the Arnold group carries out a systematic screen for the binding of drug-like fragments to RT. These studies are expected to lead to the identification of potential new target sites as well as new leads for inhibitor development.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以出现在其他 CRISP 条目中列出的机构是。中心，不一定是研究者的机构。 CHESS 一直是阿诺德实验室研究逆转录酶 (RT) 结构和功能的重要资源，逆转录酶是艾滋病病毒的关键成分，也是许多最广泛使用的抗艾滋病药物的靶标。解析了野生型和耐药性 HIV-1 RT 与多种抗病毒药物的复合物和 HIV 基因组模型片段的三维结构。这些研究与其他实验室的贡献一起。对聚合酶结构-功能关系、耐药性的详细机制产生了许多见解，并为基于结构的 RT 抑制剂设计提供了基础。 Arnold 实验室和 Janssen/Tibotec 集团之间的合作开发了许多抑制剂，这些抑制剂显示出作为艾滋病潜在治疗方法的巨大前景，其中两种抑制剂目前处于 II 期 (TMC278) 和 III 期 (TMC125) 临床试验美国和海外也正在开展 HIV-1 RT 与 RNase H 抑制剂复合的结构研究 [168]。对于 HIV 复制也至关重要，但尚未开发出针对 RNase H 的药物。在过去的一年中，在从 HIV-1 RT 获得高分辨率衍射方面取得了显着的成功，该 RT 专门设计用于产生扩展至 1.8 ¿ 的新型晶体形式。已获得工程化 HIV-1 RT 与 Janssen 抑制剂 TMC278 复合物的分辨率。该工程策略包括在共表达的 p66/p51 系统中对 HIV-1 RT 异二聚体的 N 端和 C 端进行系统变异。可去除的 His 标签高分辨率 HIV-1 RT/TMC278 结构（J. Bauman、K. Das 等人，正在准备中）的一个值得注意的方面是，该关键结构具有方便的纯化和选择性表面突变，例如 Lys=>Ala。尽管对该复合物进行了数千次结晶尝试，但仍然难以捉摸。这种新的 HIV-1 RT 结构和晶体形式的一个重要意义是，进一步基于结构的 HIV-1 RT 抑制剂药物设计可以更准确、更快速地进行。他们已经能够收集扩展到 2 ¿ 的大量数据集。分辨率或更好，有或没有结合抑制剂，现在正在寻找可以适应核酸结合的更高分辨率的晶体形式。在鉴定几种产生高分辨率晶体的工程 RT 结构的过程中，使用来自 CHESS F1 和 A1 光束线的 X 射线评估了许多晶体形式，在此工作之前，任何 HIV-1 RT 结构的最高已发表分辨率为 2.2。 ¿ ，并且大多数已发表的 RT 结构都在 2.7-3.0 ¿ CHESS 的高吞吐量能力的发展，以及特别是，在 CHESS F1 上使用新型机器人自动安装机，使得评估大量候选晶体成为可能，并且随着阿诺德小组对药物结合进行系统筛选，这将继续成为极其宝贵的资源。这些研究预计将有助于识别潜在的新靶点以及抑制剂的新先导化合物。发展。