MACCHESS PROGRAM FOR AUTOMATION AND HIGH-THROUGHPUT

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 国际象棋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，RT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点。Arnold小组已经解决了野生型和耐药HIV-1 RT与各种抗病毒药物和HIV基因组模型片段复合的三维结构。这些研究，加上其他实验室的贡献， 对聚合酶结构-功能关系产生了许多见解，详细的耐药机制，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集团之间的合作导致了许多抑制剂的开发，这些抑制剂显示出作为艾滋病潜在治疗的巨大希望，其中两种目前正在美国和海外进行II期（TMC 278）和III期（TMC 125）临床试验[165-167]。HIV-1 RT与RNA酶H抑制剂复合的结构研究也在进行中[168]; RT的RNA酶H活性对于HIV复制也是必不可少的，但尚未开发出靶向RNA酶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，这些RT经过专门设计以产生新的晶体形式。已经获得了与杨森抑制剂TMC 278复合的工程化HIV-1 RT的衍射数据，其分辨率扩展到1.8“。工程化策略包括在共表达的p66/p51系统中对HIV-1 RT异二聚体的N-和C末端进行系统性变异，并在其中加入可去除的His-标签， 方便的纯化和选择的表面突变如Lys=>Ala。在高分辨率HIV-1 RT/TMC 278结构的值得注意的方面中（J. Bauman，K. Das等人，在制备中），尽管对这种复合物进行了数千次结晶尝试，但这种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意义是，进一步的基于结构的HIV-1 RT抑制剂药物设计可以更准确和快速地进行。他们已经能够收集到许多数据集，这些数据集在有或没有结合抑制剂的情况下扩展到2 ½分辨率或更高分辨率，现在正在寻找可以容纳核酸结合的更高分辨率的晶体形式。 在鉴定产生高分辨率晶体的几种工程RT构建体的过程中，使用来自CHESS F1和A1光束线的X射线评估了许多晶体形式。在这项工作之前，任何HIV-1 RT结构的最高出版分辨率为2.2 <$，大多数出版的RT结构在2.7-3.0 <$范围内。国际象棋高吞吐量能力的发展，以及 特别是，在CHESS F1中使用新的机器人自动计数器，使得有可能评估大量候选晶体，并将继续成为Arnold小组进行药物样片段与RT结合的系统筛选的极其宝贵的资源。这些研究预计将导致识别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.