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

使用原位点击化学设计 Akt1 亲和捕获剂

• 批准号: 8065747
• 负责人: Steven W Millward
• 金额: $ 5.22万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8065747
• 关键词: Affinity; Alkynes; Amino Acids; Azides; Binding; Biological; Breast; Cancer Patient; Cell Extracts; Cell Nucleus; Cell surface; Chemicals; Chemistry; Colon Carcinoma; Complex; Data; Development; Devices; Diagnostic; Early Diagnosis; Event; Generations; Genetic Transcription; Hormones; In Situ; Lead; Libraries; Ligands; Link; Lung; Malignant Neoplasms; Mammalian Cell; Maps; Measures; Methods; Molecular; Mono-S; Ovarian; Peptide Library; Peptides; Phase; Phosphorylation; Phosphotransferases; Preparation; Process; Prostate; Protein Isoforms; Protein Kinase; Sampling; Screening for cancer; Series; Signal Pathway; Signal Transduction; Solid; Specificity; Technology; Therapeutic; Thyroid Gland; Tissue Sample; Tissues; Triazoles; Tumor Cell Line; analog; base; cytokine; design; improved; inhibitor/antagonist; member; overexpression; response; small molecule; transmission process

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.

描述（由申请人提供）：激酶在信号事件从细胞表面（例如激素或细胞因子的结合）传递到细胞核中的细胞应答（例如转录）中起分子路由器的作用。一种激酶Akt 1在多种下游信号通路中起节点作用，并且已显示在多种癌症中过表达和/或过度活化。与这种激酶及其交替激活状态结合的配体将能够开发用于癌症研究和早期检测的诊断平台。 我们建议设计一系列基于肽的双配体，以高亲和力识别Akt的磷酸化状态。通过将化学单珠、单化合物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Akt 1及其亚型的每种常见磷酸化状态的双配体。这些配体将被整合到微阵列中，并用于定量复杂生物样品中Akt的浓度和活化状态。 开始，在炔修饰的ATP模拟物的第二文库存在下，针对完全活性的Akt 1筛选叠氮化物修饰的Akt 1抑制剂肽的小文库。Akt 1靶标将指导最佳肽和小分子的缀合，以形成高亲和力的三唑连接的双配体。在表征“命中”分子之后，通过用更大和更多样化的肽文库重新筛选，前导化合物的磷酸化状态特异性和亲和力将增加。配体成熟的最后一轮将采用原位点击化学结合携带炔的小分子的第二文库来选择具有改善的亲和力和生物稳定性的多官能化配体。将以类似的方式产生针对Akt 1、Akt 2和Akt 3的每种磷酸化状态的双配体。优化后，将亲和捕获剂固定在微阵列中，并用于测量哺乳动物细胞提取物中Akt 1、Akt 2和Akt 3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。在这项研究中产生的配体将被组合成一个廉价的，高通量的微阵列设备映射在正常，癌前和癌症组织中的Akt 1的激活状态的变化，能够快速确定组织样本中的Akt异构体的浓度和激活状态将是一个重要的一步，对许多癌症的早期诊断和癌症患者的最佳治疗方案的确定。

项目成果

Iterative in situ click chemistry assembles a branched capture agent and allosteric inhibitor for Akt1.

• DOI: 10.1021/ja2064389
• 发表时间: 2011-11-16
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Millward, Steven W.;Henning, Ryan K.;Kwong, Gabriel A.;Pitram, Suresh;Agnew, Heather D.;Deyle, Kaycie M.;Nag, Arundhati;Hein, Jason;Lee, Su Seong;Lim, Jaehong;Pfeilsticker, Jessica A.;Sharpless, K. Barry;Heath, James R.
• 通讯作者: Heath, James R.