Dual protease activated peptides for specific targeting

用于特异性靶向的双蛋白酶激活肽

• 批准号: 10680341
• 负责人: Ethel Naa Odey Tackie-Yarboi
• 金额: $ 6.91万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680341
• 关键词: Address; Amino Acids; Bacterial Infections; Bee Venoms; Binding; Biological Assay; CASP3 gene; Calorimetry; Cell Line; Cell membrane; Cells; Cellular Assay; Cellular Membrane; Charge; Circular Dichroism; Coupling; Cytolysis; Cytotoxin; Derivation procedure; Diagnostic; Discrimination; Disease; Drug Delivery Systems; Erythrocytes; Extravasation; Fluorescence; Future; High Pressure Liquid Chromatography; In Situ; In Vitro; Incubated; Infection; Investments; Kinetics; Label; Link; Literature; Location; Malignant Neoplasms; Mammalian Cell; Masks; Membrane; Metabolic; Methods; Molecular Conformation; Monitor; Nerve Degeneration; Parents; Penetration; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Play; Prodrugs; Property; Proteins; Proteolysis; Research; Role; Serum; Side; Solid Neoplasm; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; System; Techniques; Testing; Therapeutic; Toxin; Training; Vesicle; WI 38 cell; Whole Blood; alpha helix; anti-cancer; anti-cancer therapeutic; antimicrobial; antimicrobial peptide; aqueous; biophysical analysis; biophysical techniques; cancer biomarkers; cytotoxic; cytotoxicity; design; drug market; expectation; fibroblast-activating factor; improved; microbial; overexpression; peptide drug; peptide hormone; physical property; prevent; prototype; response; side effect; skills; small molecule; tumor; unilamellar vesicle

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.

项目总结/摘要 在这里，我们开发了一个蛋白酶激活肽前药的平台。我们首先将精力集中在 抗菌肽（AMP）具有广谱的细胞穿透性、抗菌性和细胞毒性， 活动然而，它们具有多种局限性，包括脱靶细胞毒性和蛋白水解不稳定性。到 为了避免这些不需要的作用，已经使用了线性蛋白酶活化的前药。虽然有效 在增强有源有效载荷的活性方面，它没有充分解决上述副作用。到 减轻传统的蛋白酶激活前药方法的副作用，提出了研究策略， 本文试图通过将可裂解的接头偶联到侧链上来设计支化的蛋白酶活化的前药 显著影响抗菌肽活性的氨基酸残基。作为概念验证， (an对哺乳动物细胞具有细胞毒性的AMP）将用作有效载荷，用半胱天冬酶-3和成纤维细胞掩蔽 活化蛋白（癌症生物标志物）底物特异性接头。通过修饰Lys、Ser、Thr的侧链， 和Tyr与这些接头，我们假设，bombolitin的物理化学性质将显着 改变，从而减少肽-膜相互作用直到活化。在本研究中，我们建议1）设计 并合成单和双分支蛋白酶激活的前药， 半胱天冬酶-3和/或成纤维细胞活化蛋白，以产生活性肽，2）证实半胱天冬酶-3和成纤维细胞活化蛋白， 通过进行体外酶促测定和生物物理测定从前药上活化蛋白质裂解接头 分析，和3）进行基于细胞的测定以验证前药切割、靶特异性、活性增强， 和减少脱靶效应。