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: 7954327
• 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: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954327
• 关键词: Active Sites; Alveolar Macrophages; Binding; Cell Cycle; Complex; Computer Retrieval of Information on Scientific Projects Database; 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; structural biology; synchrotron radiation

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.

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。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 结核分枝杆菌（Mycobacterium tuberculosis，M.）结核病）是细胞周期的候选调节因子，越来越多的证据表明它们参与人肺泡巨噬细胞内生长、潜伏和持续的关键阶段。 PknB是M. tb，是生长所必需的3，并被认为介导M的生长和分裂之间的转换。TB. 本实验室已解决了胞内激酶结构域的结构。 由于其结构与真核同源物总体相似，我们筛选了一个已知真核激酶抑制剂的小库，其中含有M中的各种STPK。TB. 虽然我们没有发现PknB的强命中，但我们已经成功地使用PknB的活性位点突变体来紧密结合抑制剂，所述抑制剂结合M中的STPK旁系同源物。TB. 我们已经进行了PknB替代物与激酶抑制剂如星形孢菌素、Bis 1和JNK抑制剂sp 600125的共结晶研究。 我们已经将PknB与sp 600125和其他真核抑制剂共结晶，并且已经观察到2.0-2.9 A的初始衍射。 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突变体的晶体，添加到我们实验室使用PknB解决的几种其他晶体中，包括PknB-ATP γ S和PknB M92 A-1 NMPP 1的凹凸孔复合物。 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，以帮助功能以及药物发现的努力。 目前，正在使用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揭示如何提高选择性抑制真核生物样STPKs在M。结核病和使化学遗传学的方法来验证STPK。