CO-CRYSTALLIZATION OF EUKARYOTIC PROTEIN KINASE INHIBITORS WITH MYCOBACTERIAL ST

真核蛋白激酶抑制剂与分枝杆菌 ST 的共结晶

• 批准号: 7598234
• 负责人: CARL A MIECZKOWSKI
• 金额: $ 0.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598234
• 关键词: Active Sites; Alveolar Macrophages; Binding; Cell Cycle; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Crystallization; Family; Funding; Glean; Grant; Growth; Homologous Gene; Human; Institution; Libraries; Ligands; MAPK8 gene; Mediating; Mycobacterium tuberculosis; Phosphotransferases; Protein Kinase; Protein Kinase Inhibitors; Research; Research Personnel; Resources; SP600125; Source; Staging; Staurosporine; Structure; Subcellular structure; United States National Institutes of Health; adenosine 5' -O-(3-thiotriphosphate); chemical genetics; design; drug discovery; improved; in vivo; inhibitor/antagonist; insight; kinase inhibitor; mutant; mycobacterial; paralogous gene; protein kinase inhibitor; receptor

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. A recently discovered family of eukaryotic-like Ser/Thr protein kinases (STPKs) in Mycobacterium tuberculosis (M. tb.) are candidate regulators of the cell cycle, and there is growing evidence for their involvement in the critical stages of growth, latency, and persistence within human alveolar macrophages. PknB, a conserved transmembrane receptor STPK in M. tb, is essential for growth3 and is assumed to mediate a switch between growth and division in M. tb. The structure of the intracellular kinase domain has been solved by our lab. Because the structure globally resembles eukaryotic homologs, we have screened a small library of known eukaryotic kinase inhibitors with various STPKs in M. tb. Although we have found no strong hits for PknB, we have successfully used active-site mutants of PknB to tightly bind inhibitors that bind STPK paralogs in M. tb. We have performed co-crystallization studies of PknB surrogates with kinase inhibitors such as staurosporine, Bis1, and JNK inhibitor sp600125. We have co-crystallized PknB with sp600125 and other eukaryotic inhibitors and initial diffraction has been observed from 2.0-2.9 A. We are currently optimizing crystals of PknB mutants with several inhibitor ligands under similar crystallization conditions, adding to several other crystals our lab has solved using PknB, including PknB-ATPgammaS and a bump-hole complex of PknB M92A-1NMPP1. These structures will highlight the conservation of prokaryotic and eukaryotic protein kinases and enable the design of improved inhibitors to aid functional as well as drug discovery efforts. Currently, the use of PknB surrogates for their selective inhibition by eukaryotic kinase inhibitors in vivo is underway in order to glean functional information on PknB using a chemical genetics approach. Our proposed structural studies will shed insight on how to improve selective inhibition of eukaryotic-like STPKs in M. tb and enable a chemical genetics approach to validate the STPKs.

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。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 最近在结核分枝杆菌 (M. tb.) 中发现的真核样 Ser/Thr 蛋白激酶 (STPK) 家族是细胞周期的候选调节因子，并且越来越多的证据表明它们参与人类肺泡巨噬细胞的生长、潜伏期和持久性的关键阶段。 PknB 是结核分枝杆菌中的一种保守跨膜受体 STPK，对于生长至关重要，并且被认为介导结核分枝杆菌生长和分裂之间的转换。 我们实验室已经解析了细胞内激酶结构域的结构。 由于该结构在总体上类似于真核同源物，因此我们在结核分枝杆菌中筛选了一个已知真核激酶抑制剂的小型文库，其中包含各种 STPK。 尽管我们没有发现 PknB 的强烈命中，但我们已经成功地使用 PknB 的活性位点突变体来紧密结合结核分枝杆菌中与 STPK 旁系同源物结合的抑制剂。 我们对 PknB 替代物与激酶抑制剂（例如星孢菌素、Bis1 和 JNK 抑制剂 sp600125）进行了共结晶研究。 我们已经将 PknB 与 sp600125 和其他真核抑制剂共结晶，并在 2.0-2.9 A 范围内观察到初始衍射。我们目前正在类似的结晶条件下优化具有几种抑制剂配体的 PknB 突变体晶体，添加到我们实验室使用 PknB 解决的其他几种晶体，包括 PknB-ATPgammaS 和 PknB 的凹凸孔复合物 M92A-1NMPP1。 这些结构将突出原核和真核蛋白激酶的保守性，并使改进的抑制剂的设计成为可能，以帮助功能和药物发现工作。 目前，正在利用 PknB 替代物在体内通过真核激酶抑制剂进行选择性抑制，以便使用化学遗传学方法收集 PknB 的功能信息。 我们提出的结构研究将深入了解如何改善结核分枝杆菌中类真核 STPK 的选择性抑制，并启用化学遗传学方法来验证 STPK。