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:8363513
- 负责人:
- 金额:$ 6.16万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2011
- 资助国家:美国
- 起止时间:2011-07-01 至 2012-06-30
- 项目状态:已结题
- 来源:
- 关键词:Acquired Immunodeficiency SyndromeAnti-HIV AgentsAntiviral AgentsAutomationBindingClinical TrialsCollaborationsComplexCrystallizationDBL OncoproteinDataData SetDevelopmentDrug Delivery SystemsDrug DesignDrug resistanceEngineeringFundingGenomeGrantHIVHIV-1LaboratoriesLeadModelingMutationNational Center for Research ResourcesNucleic Acid BindingPharmaceutical PreparationsPhasePolymerasePreparationPrincipal InvestigatorProcessPublishingRNA-Directed DNA PolymeraseResearchResearch InfrastructureResolutionResourcesReverse Transcriptase InhibitorsRibonuclease HRoboticsRoentgen RaysSiteSourceStructureStructure-Activity RelationshipSurfaceSystemUnited StatesUnited States National Institutes of HealthVariantWorkbasebeamlinecostdesigninhibitor/antagonistinsightnovelprogramssuccessthree 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. Primary support for the subproject
and the subproject's principal investigator may have been provided by other sources,
including other NIH sources. The Total Cost listed for the subproject likely
represents the estimated amount of Center infrastructure utilized by the subproject,
not direct funding provided by the NCRR grant to the subproject or subproject staff.
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.
这个子项目是许多利用资源的研究子项目之一
由NIH/NCRR资助的中心拨款提供。子项目的主要支持
而子项目的主要调查员可能是由其他来源提供的,
包括其他NIH来源。 列出的子项目总成本可能
代表子项目使用的中心基础设施的估计数量,
而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。
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,产生了许多见解聚合酶的结构-功能关系,详细的耐药机制,并提供了基于结构的RT抑制剂的设计基础。
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,并带有便于纯化的可去除的His-标签,以及选择的表面突变如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,特别是国际象棋F1比赛中新机器人自动上料机的使用,已经使得评估大量候选晶体成为可能,并且随着Arnold小组对药物结合进行系统筛选,这些研究预计将导致潜在的新靶位点的鉴定以及抑制剂开发的新线索。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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EDWARD ARNOLD其他文献
EDWARD ARNOLD的其他文献
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{{ truncateString('EDWARD ARNOLD', 18)}}的其他基金
STRUCTURAL STUDIES OF HIV-1 REVERSE TRANSCRIPTASE (RT)
HIV-1 逆转录酶 (RT) 的结构研究
- 批准号:
8170670 - 财政年份:2010
- 资助金额:
$ 6.16万 - 项目类别:
MACCHESS PROGRAM FOR AUTOMATION AND HIGH-THROUGHPUT
用于自动化和高吞吐量的 MACCHESS 程序
- 批准号:
8171485 - 财政年份:2010
- 资助金额:
$ 6.16万 - 项目类别:
HIV-1 reverse transcriptase structure: function, inhibition, and resistance
HIV-1逆转录酶结构:功能、抑制和抵抗
- 批准号:
7750025 - 财政年份:2009
- 资助金额:
$ 6.16万 - 项目类别:
MACCHESS PROGRAM FOR AUTOMATION AND HIGH-THROUGHPUT
用于自动化和高吞吐量的 MACCHESS 程序
- 批准号:
7955572 - 财政年份:2009
- 资助金额:
$ 6.16万 - 项目类别:
STRUCTURAL STUDIES OF HIV-1 REVERSE TRANSCRIPTASE (RT)
HIV-1 逆转录酶 (RT) 的结构研究
- 批准号:
7957252 - 财政年份:2009
- 资助金额:
$ 6.16万 - 项目类别:
HIV-1 reverse transcriptase structure: function, inhibition, and resistance
HIV-1逆转录酶结构:功能、抑制和抵抗
- 批准号:
8416384 - 财政年份:2009
- 资助金额:
$ 6.16万 - 项目类别:
HIGH RES STRUCT OF HUMAN VIRUSES & VIRAL PROTEINS: SYNCH RADIATION AT CHESS: HIV
人类病毒的高分辨率结构
- 批准号:
7955535 - 财政年份:2009
- 资助金额:
$ 6.16万 - 项目类别:
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