Dual protease activated peptides for specific targeting

项目摘要/摘要 在这里，我们开发了一个蛋白酶激活的多肽前药平台。我们最初将工作重点放在 抗菌肽(AMP)，具有广泛的细胞穿透、抗菌和细胞毒性作用 活动。然而，它们有多个局限性，包括靶外细胞毒性和蛋白降解不稳定性。至 为了避免这些有害的影响，线性蛋白酶激活的前药已经被使用。虽然有效 在加强主动有效载荷的活动方面，它没有充分解决上述副作用。至 减轻传统的酶激活前药方法的副作用，提出研究策略 在这里，试图通过将可切割的连接子偶联到侧链来设计支化的蛋白酶激活的前药 显著影响抗菌肽活性的氨基酸残基。作为概念验证，轰炸式 (一种对哺乳动物细胞具有细胞毒性的AMP)将被用作有效载荷，用caspase-3和成纤维细胞掩蔽 激活蛋白(癌症生物标记物)底物特异性连接物。通过修改Lys，Ser，Thr， 有了这些连接子，我们假设Bombolitin的物理化学性质将显著 改变，从而减少了肽与膜的相互作用，直到激活。在本研究中，我们提出了1)设计 以及合成在任一存在下裂解的单支化和双支化的蛋白酶激活的前药物 Caspase-3和/或成纤维细胞激活蛋白以产生活性多肽，2)确认caspase-3和成纤维细胞 用体外酶分析和生物物理方法从前药中切割接头激活蛋白 分析，以及3)执行基于细胞的分析以验证前药切割、靶标特异性、活性增强， 以及减少偏离目标的影响。

Project Summary/Abstract Here we develop a platform for protease-activated peptide prodrugs. We initially focus the effort on antimicrobial peptides (AMP), which have a broad spectrum of cell-penetrating, antimicrobial and cytotoxic activities. However, they have multiple limitations, including off-target cytotoxicity and proteolytic instability. To circumvent these unwanted effects, linear protease-activated prodrugs have been employed. Although effective at enhancing the activity of the active payload, it does not adequately address the side effects stated above. To mitigate the side effects of the traditional protease-activated prodrug approach, the proposed research strategy herein seeks to design a branched protease-activated prodrug by coupling a cleavable linker to the sidechains of amino acid residue that significantly impacts antimicrobial peptide activity. As a proof-of-concept, bombolitin (an AMP that is cytotoxic to mammalian cells) will be used as the payload, masked with caspase-3 and fibroblast activation protein (cancer biomarkers) substrate-specific linkers. By modifying the sidechains of Lys, Ser, Thr, and Tyr with these linkers, we hypothesize that the physicochemical properties of bombolitin will be significantly altered, thus reducing peptide-membrane interaction until activation. In this research, we propose to 1) design and synthesize single and double-branched protease-activated prodrugs cleaved in the presence of either caspase-3 and/or fibroblast activation protein to yield the active peptide, 2) confirm caspase-3 and fibroblast activation protein cleavage of linker from prodrugs by performing in vitro enzymatic assays and biophysical analysis, and 3) perform cell-based assays to validate prodrug cleavage, target specificity, activity enhancement, and reduction of off-target effects.