Design of Affinity Capture Agents for Akt1 Using in situ Click Chemistry

描述(由申请人提供)：在从细胞表面(例如激素或细胞因子的结合)到细胞核内的细胞反应(例如转录)的信号事件的传输中，激酶起到分子路由器的作用。一种名为Akt1的激酶在多种下游信号通路中起着节点的作用，已被证明在多种癌症中过度表达和/或过度激活。与该激酶及其交替激活状态结合的配体将使研究和早期发现癌症的诊断平台的开发成为可能。 我们建议设计一系列具有高亲和力的识别Akt磷酸化状态的多肽双配体。通过将化学单珠、单化合物多肽库和原位点击化学与ATP类似物相结合，我们可以快速生成一系列识别Akt1及其异构体的常见磷酸化状态的双配体。这些配体将被集成到微阵列中，并用于定量复杂生物样品中Akt的浓度和激活状态。 首先，叠氮化物修饰的Akt1抑制剂肽的小文库将在第二个炔修饰的ATP模拟物库存在的情况下与完全活性的Akt1进行筛选。Akt1靶标将引导最佳多肽与小分子的结合，形成高亲和力的三唑连接双配体。在表征“HIT”分子之后，通过用更大和更多样化的肽库重新筛选，将提高先导化合物的磷酸化状态特异性和亲和力。最后一轮的配体成熟将采用原位点击化学结合第二个含炔小分子文库，以选择亲和力和生物稳定性更好的多官能化配体。针对Akt1、Akt2和AKT3的每个磷酸化状态的双配体将以类似的方式产生。经过优化后，亲和捕捉剂将被固定在微阵列中，并用于测量哺乳动物细胞提取物中Akt1、Akt2和AKT3的每种磷酸化状态的浓度。 在卵巢癌、乳腺癌、甲状腺癌、前列腺癌、肺癌和结肠癌中观察到Akt表达和/或活性增加。本研究中产生的配体将被结合到一个廉价的高通量微阵列设备中，以绘制正常、癌前和癌症组织中Akt1激活状态的变化图，快速确定组织样本中Akt亚型的浓度和激活状态的能力将是朝着许多癌症的早期诊断和确定癌症患者的最佳治疗方案迈出的重要一步。

DESCRIPTION (provided by applicant): Kinases function as molecular routers in the transmission of signaling events from the cell surface (e.g. the binding of a hormone or cytokine) to the cellular response in the nucleus (e.g. transcription). One kinase, Akt1, serves as a node in multiple downstream signaling pathways and has been shown to be overexpressed and/or hyperactivated in a wide variety of cancers. Ligands which bind to this kinase and its alternate activation states would enable the development of diagnostic platforms for the study and early detection of cancer. We propose to design a series of peptide-based bi-ligands that recognize the phosphorylation states of Akt with high affinity. By combining chemical one-bead, one-compound peptide libraries and in situ click chemistry with ATP analogs, we can rapidly generate a series of bi-ligands that recognize each of the common phosphorylation states of Akt1 and its isoforms. These ligands will be integrated into microarrays and used to quantitate the concentration and activation states of Akt in complex biological samples. To begin, small libraries of azide-modified Akt1 inhibitor peptides will be screened against fully active Akt1 in the presence of a second library of alkyne-modified ATP-mimics. The Akt1 target will direct the conjugation of the optimal peptide and small molecule to form a high-affinity triazole-linked bi-ligand. Following characterization of the "hit" molecules, the phosphorylation state specificity and affinity of the lead compounds will be increased by re-screening with larger and more diverse peptide libraries. The final round of ligand maturation will employ in situ click chemistry in conjunction with a second library of alkyne-bearing small molecules to select multi-functionalized ligands with improved affinity and biostability. Bi-ligands against each of the phosphorylation states of Akt1, Akt2, and Akt3 will be generated in a similar manner. Following optimization, the affinity capture agents will be immobilized in a microarray and used to measure the concentrations of each phosphorylation state of Akt1, Akt2, and Akt3 in mammalian cell extracts. Increased Akt expression and/or activity has been observed in ovarian, breast, thyroid, prostate, lung, and colon cancers. The ligands generated in this study will be combined into an inexpensive, high-throughput microarray device to map the changes in the activation state of Akt1 in normal, pre-cancer, and cancer tissues, The ability to rapidly determine the concentration and activation state of Akt isoforms in tissue samples would be a major step toward the early diagnosis of many cancers and the determination of optimal therapeutic regimes for cancer patients.