CO-CRYSTALLIZATION OF EUKARYOTIC PROTEIN KINASE INHIBITORS WITH MYCOBACTERIAL ST

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。 最近在结核分枝杆菌中发现了一个类真核丝氨酸/苏氨酸蛋白激酶(STPKs)家族。是细胞周期的候选调节者，越来越多的证据表明它们参与了人肺泡巨噬细胞内生长、潜伏期和持久性的关键阶段。PKnB是结核分枝杆菌中一种保守的跨膜受体STPK，对结核分枝杆菌的生长是必不可少的，并被认为介导了结核分枝杆菌在生长和分裂之间的转换。我们实验室已经解决了细胞内激酶域的结构问题。由于该结构在全球范围内类似于真核同源物，我们已经筛选了一个含有结核分枝杆菌中各种STPKs的已知真核细胞激酶抑制剂的小型文库。虽然我们没有发现PnuB的强匹配，但我们已经成功地使用PnuB的活性位点突变体来紧密结合结合结核分枝杆菌中STPK类似物的抑制剂。我们已经进行了PnuB替代物与激酶抑制剂如星形孢子素、BIS1和JNK抑制剂sp600125的共结晶研究。我们已经将PnuB与sp600125和其他真核抑制剂共结晶，并在2.0-2.9A范围内观察到了初步衍射。目前，我们正在类似结晶条件下，用几种抑制剂配体优化PnuB突变体的晶体，此外，我们的实验室还使用PnuB解决了其他几种晶体，包括PounB-ATPGammaS和PnuB M92A-1NMPP1的凹坑复合体。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能够帮助功能和药物发现工作。目前，为了利用化学遗传学的方法收集有关PKnB的功能信息，正在使用PKnB替代物在体内被真核细胞激酶抑制剂选择性地抑制。我们提出的结构研究将揭示如何提高对结核分枝杆菌中真核样STPKs的选择性抑制，并使化学遗传学方法能够验证STPKs。

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.