Multivalent Inhibition of Integrin alphaVbeta3

这个项目是与Daniel Appella博士(LBC/NIDDK)的实验室合作的，他专门从事生物仿生的合成化学。这是他们工作的延伸，他们开发了DNA和肽核酸(PNA)分子的混合体，作为生物配体多价展示的可编程支架。PNA是具有肽样骨架和核酸类型碱基的低聚分子，即DNA和RNA的A、G、C和T/U，用于侧链。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的互补序列结合。通过在合成的PNA骨架上程序化地添加桥联分子，特定的配体被定位在PNA/DNA支架上的任何地方。为了证明原理，我们重点研究了细胞外基质蛋白中保守的RGD序列，它是整合素αVbeta3的优先配体。我们使用了该序列的5个残基的环肽类似物，类似于药物西伦吉肽(MerckSerono)，该药物目前正在进行针对胶质母细胞瘤和其他癌症的第二阶段研究。为了确定最佳构型，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交分子文库，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，一个由5个PNA片段组成的结构被认为是最佳的抑制剂，每个片段代表三个RGD配体。利用放射性标记的ecichatin置换实验，发现该优化构建体与αVbeta3结合的强度是单体RGD对照的400倍。最后，在小鼠模型中，这种最优的PNA/DNA抑制剂也被发现在阻断黑色素瘤细胞肺转移方面优于单体环状RGD。这部作品的手稿目前正在审阅中。

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.