MACCHESS PROGRAM FOR AUTOMATION AND HIGH-THROUGHPUT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在阿诺德实验室研究逆转录酶(RT)结构和功能的努力中，国际象棋一直是必不可少的资源。逆转录酶是艾滋病病毒的关键组成部分，也是许多最广泛使用的抗艾滋病药物的靶标。Arnold团队已经在与多种抗病毒药物和HIV基因组的模型片段的复合体中解决了野生型和抗药性HIV-1 RT的三维结构。这些研究，加上其他实验室的贡献，已经 对聚合酶结构与功能的关系，耐药的详细机制有了大量的见解，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小组之间的合作导致了许多有望成为艾滋病潜在治疗方法的抑制剂的开发，其中两种目前正在美国和海外进行第二阶段(TMC278)和第三阶段(TMC125)临床试验[165-167]。HIV-1RT与RNaseH抑制剂的结构研究也在进行中；RT的RNaseH活性对HIV复制也是必不可少的，但尚未开发出针对RNaseH的药物。 在过去的一年里，在从HIV-1 RT获得高分辨率衍射方面取得了显着的成功，这是专门为产生新的晶体形式而设计的。工程HIV-1 RT与Janssen抑制剂TMC278的络合物的衍射数据已扩展到1.8？分辨率。工程策略包括在共表达的p66/p51系统中对HIV-1 RT异源二聚体的N-和CTeri进行系统的变异，其中包括可移除的His-tag 方便的纯化，以及精选的表面突变，如Lys=&gt；Ala。高分辨率HIV-1 RT/TMC278结构(J.Bauman，K.Das等人，正在准备中)的值得注意的方面之一是，尽管对该复合体进行了数千次结晶尝试，但这一关键结构一直难以捉摸。 这种新的HIV-1 RT结构和晶体形式的一个重要含义是，进一步的基于结构的HIV-1 RT抑制剂的药物设计可以更准确和快速地进行。他们已经能够收集大量的数据集，在有或没有结合抑制剂的情况下，分辨率达到2？或更高，现在正在寻找能够适应核酸结合的更高分辨率的晶体形式。 在鉴定几个产生高分辨率晶体的工程RT结构的过程中，使用国际象棋F1和A1光束线的X射线评估了许多晶体形态。在这项工作之前，已发表的HIV-1 RT结构的最高分辨率为2.2？，大多数已发表的RT结构在2.7-3.0？范围内。国际象棋高通推力的发展，以及 特别是，在国际象棋F1上使用新的机器人自动挂载机，使评估大量候选晶体成为可能，并将继续成为一种极其宝贵的资源，因为Arnold小组对类药物片段与RT的结合进行了系统筛选。这些研究有望导致确定潜在的新靶点以及抑制剂的新线索 发展。

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.