Multivalent Inhibition of Membrane Receptors

膜受体的多价抑制

• 批准号: 9344179
• 负责人: Stewart Durell
• 金额: $ 5.75万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9344179
• 关键词: Affect; Agonist; Amines; Apoptosis; Area; Binding; Biological; Biological Assay; Biomedical Engineering; Biomimetics; Biotechnology; Cell Adhesion; Cell Surface Receptors; Cell membrane; Cells; Cilengitide; Clinical; Collaborations; Cyclic Peptides; DNA; Development; Drug Targeting; Equilibrium; Extracellular Matrix; Extracellular Matrix Proteins; Family; Glioblastoma; Goals; Hereditary Disease; Human; Hybrids; Infiltration; Integrin alphaVbeta3; Integrins; Investigation; Laboratories; Libraries; Ligands; Lung diseases; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Melanoma Cell; Membrane; Metastatic Neoplasm to the Lung; Molecular Conformation; Molecular Target; Nanotechnology; National Institute of Diabetes and Digestive and Kidney Diseases; Neoplasm Metastasis; Nerve Degeneration; Nucleic Acids; Parkinson Disease; Peptide Nucleic Acids; Peptides; Pharmaceutical Preparations; Phase; Positioning Attribute; Purinergic P1 Receptors; RGD (sequence); RNA; Research; Skin Cancer; Surface; Synthesis Chemistry; Tandem Repeat Sequences; Technology; Time; Vascular Endothelium; Vertebral column; Work; Xanthines; angiogenesis; base; cancer cell; cancer site; cancer therapy; chemotherapy; density; dimer; drug development; echistatin; inhibitor/antagonist; interest; melanoma; migration; molecular size; monomer; mouse model; overexpression; peptide analog; programs; receptor; scaffold; tumor

This project is in collaboration with the laboratory of Dr. Daniel Appella (LBC/NIDDK), which specializes in synthetic chemistry of biomimetics. This is an extension of their work developing hybrids of DNA and Peptide Nucleic Acid (PNA) molecules as programmable scaffolds for the multivalent display of biological ligands. PNAs are oligomeric molecules with peptide-like backbones and nucleic acid-type bases, i.e. the A, G, C & T/U of DNA and RNA, for sidechains. Thus, by adjusting the sequence of the monomers, PNAs can be programmed to bind in a helical conformation to a single or tandem repeated complementary sequence in DNA. Specific ligands are positioned anywhere along the PNA/DNA scaffold by the programmed addition of bridging linkers to the synthesized PNA backbone. For a proof of principle, we focused on the conserved RGD sequence in extracellular matrix proteins that is a preferential ligand of Integrin alphaVbeta3. We used a 5-residue cyclic-peptide analog of this sequence that is similar to the drug Cilengitide (MerckSerono), which is currently under Phase II investigation for Glioblastoma and other cancers. To determine the optimal configuration, the Appella lab generated a library of hybrid molecules with systematically-varied ligand positions and densities, and then screened it on a melanoma cell binding assay. Balancing molecular size and potency, a construct with 5 PNA segments, each presenting three RGD ligands, was judged the optimal inhibitor. Using a radiolabled echistatin displacement assay, this optimal construct was found to bind 400 times stronger to alphaVbeta3 than the monomeric RGD control. Finally, this optimal PNA/DNA inhibitor was also found to outperform monomeric cyclic-RGD in blocking lung metastasis of melanoma cells in a mouse model. Recently, we have expanded this project to target the human A2a adenosine receptor for multivalent binding of agonists and antagonists. Recently we expanded the application of this technology to the A2a adenosine receptor, a drug target for Parkinson's disease and other neurodegenerative conditions. Using a multivalent library of attached anatagonist ligands (Xanthine Amine Congener), we were able to identify the receptor in both dimer and higher order configurations in the cell membrane.

该项目是与 Daniel Appella 博士（LBC/NIDDK）实验室合作的，该实验室专门研究仿生学的合成化学。这是他们开发 DNA 和肽核酸 (PNA) 分子混合体作为生物配体多价展示的可编程支架的工作的延伸。 PNA 是具有肽样主链和核酸型碱基（即侧链 DNA 和 RNA 的 A、G、C 和 T/U）的寡聚分子。因此，通过调整单体的序列，PNA 可以被编程为以螺旋构象与 DNA 中的单个或串联重复互补序列结合。通过在合成的 PNA 主链上按程序添加桥接接头，将特定配体定位在 PNA/DNA 支架上的任何位置。为了进行原理证明，我们重点关注细胞外基质蛋白中的保守 RGD 序列，该序列是整合素 alphaVbeta3 的优先配体。我们使用了该序列的 5 残基环肽类似物，该类似物与药物 Cilengitide (MerckSerono) 相似，该药物目前正在进行胶质母细胞瘤和其他癌症的 II 期研究。为了确定最佳配置，Appella 实验室生成了一个具有系统变化的配体位置和密度的杂合分子库，然后通过黑色素瘤细胞结合测定对其进行筛选。平衡分子大小和效力，具有 5 个 PNA 片段（每个片段呈现三个 RGD 配体）的构建体被认为是最佳抑制剂。使用放射性标记的 echistatin 置换测定，发现这种最佳构建体与 alphaVbeta3 的结合比单体 RGD 对照强 400 倍。最后，在小鼠模型中，这种最佳的 PNA/DNA 抑制剂在阻止黑色素瘤细胞肺转移方面也优于单体环状 RGD。最近，我们扩展了该项目，以人类 A2a 腺苷受体为靶点，实现激动剂和拮抗剂的多价结合。最近，我们将该技术的应用扩展到 A2a 腺苷受体，这是帕金森病和其他神经退行性疾病的药物靶点。使用附着的拮抗剂配体（黄嘌呤胺同源体）的多价文库，我们能够识别细胞膜中二聚体和更高阶构型的受体。