Multivalent Inhibition of Integrin alphaVbeta3

该项目是与丹尼尔Appella博士（LBC/NIDDK）的实验室合作，该实验室专门研究仿生合成化学。这是他们开发DNA和肽核酸（PNA）分子的杂交体作为生物配体的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的寡聚分子，即DNA和RNA的A、G、C & T/U作为侧链。因此，通过调节单体的序列，PNA可以被编程为以螺旋构象结合到DNA中的单个或串联重复的互补序列。通过向合成的PNA骨架编程添加桥接接头，特异性配体位于沿PNA/DNA支架沿着的任何位置。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，其是整合素α V β 3的优先配体。我们使用了该序列的5个残基的环状肽类似物，其类似于药物西仑吉肽（MerckSerono），西仑吉肽目前正处于胶质母细胞瘤和其他癌症的II期研究中。为了确定最佳配置，Appella实验室生成了具有系统变化的配体位置和密度的杂交分子库，然后在黑色素瘤细胞结合试验中对其进行筛选。平衡分子大小和效力，具有5个PNA片段的构建体（每个片段呈现三个RGD配体）被判定为最佳抑制剂。使用放射性标记的echistatin置换测定，发现该最佳构建体与α V β 3的结合比单体RGD对照强400倍。最后，还发现这种最佳PNA/DNA抑制剂在小鼠模型中阻断黑色素瘤细胞肺转移方面优于单体环状RGD。目前正在审查这项工作的手稿。

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. A manuscript of this work is currently in review.