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.

该项目与Daniel Appella博士（LBC/NIDDK）的实验室合作，该项目专门研究生物元素的合成化学。这是他们开发DNA和肽核酸（PNA）分子的杂种的工作的扩展，作为生物配体多价显示的可编程支架。 PNA是具有肽样骨架和核酸型碱基的低聚分子，即DNA和RNA的A，G，C＆T/U用于侧链。因此，通过调整单体的序列，可以对PNA进行编程以在螺旋构象中与DNA中的单个或串联重复互补序列结合。特定的配体通过在合成的PNA主链中编程的桥接连接器进行编程的添加，将沿PNA/DNA支架的任何地方放置。为了证明原理，我们专注于细胞外基质蛋白中的保守RGD序列，该蛋白是整合素alphavbeta3的优先配体。我们使用了该序列的5个循环肽类似物，该序列类似于药物的纤毛（Merckserono），该药物目前正在对胶质母细胞瘤和其他癌症的II期研究中进行研究。为了确定最佳构型，Appella Lab生成了具有系统变化的配体位置和密度的混合分子库，然后将其筛选在黑色素瘤细胞结合测定中。平衡分子大小和效能，一种具有5个PNA段的构建体，每个构建体呈现三种RGD配体，被认为是最佳抑制剂。使用放射式Echistatin位移测定法，发现该最佳构建体与单体RGD控制相比，与Alphavbeta3结合了400倍。最后，在小鼠模型中，该最佳PNA/DNA抑制剂在阻断黑色素瘤细胞的肺转移方面的表现均优于单体环状RGD。最近，我们扩展了该项目，以针对人A2a腺苷受体来靶向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，这是帕金森氏病和其他神经退行性疾病的药物靶标。使用连接的Anatogonist配体（黄嘌呤胺同源物）的多价库，我们能够在细胞膜中识别二聚体和高阶构型的受体。