Targeted chemotherapeutic drug discovery enabled by direct screening of peptide-drug conjugate libraries

100多年来，科学界和医学界一直致力于研究治疗癌症的不同方法。在20世纪中期，化疗药物被引入作为辅助治疗，以消除手术和放疗后残留的癌细胞。对许多癌症患者来说，这种疗法已经能够减缓肿瘤的生长，甚至彻底治愈癌症。然而，传统的化疗药物无法区分健康细胞和癌细胞，导致一些不希望的副作用。在这方面，靶向化疗的药物携带的配体选择性结合特定受体的癌细胞被开发来解决这个问题。将已知的受体靶向肽偶联到已上市药物上是一种可靠的方法，可以产生生物活性的PDCs作为靶向递送策略。由于已知的生物活性肽序列数量有限，细胞渗透性往往在货物附着后降低，并且基于肽的药物通常面临较短的血浆半衰期，因此需要一种简化的PDC开发方法。在本项目中，这种策略是通过在溶液中直接筛选非天然肽-药物偶联物文库来描述的，这为揭示具有蛋白酶抗性的PDC从头肽序列提供了一种直接的方法。通过SPPS辅助裂解混合合成生成肽库，然后依次进行Pt（IV）前药偶联和从固体载体上裂解PDC生成PDCs库。跨膜蛋白HER2在多种癌细胞系中过表达，是一种经过测试的癌症治疗生物学靶点。在完成PDC与HER2结合的下拉实验后，将混合物进行nLC-MS/MS进行高通量解码，最终获得潜在的命中PDC。这些PDCs将用AFPS制备，PDCs与her2的结合将通过生物层干涉法确认。此外，荧光显微镜实验可用于评价PDC内化，MTT试验可用于评价其抗癌活性。命中PDCs的验证为阐明受体介导的内化机制提供了机会。因此，我想展示一种直接筛选PDC文库的方法，以发现新的靶向癌症治疗方法，并开发用于阐明新生物学的分子工具。

The scientific and medical community has been devoted to investigating different approaches for the treatment of cancer for over 100 years. In the mid-20th century, chemotherapeutic agents were introduced as adjuvant therapy to eliminate remaining cancer cells after surgery and radiation. Such therapies, for many cancer patients, have been able to slow tumor growth or even cure the cancer outright. However, conventional chemotherapy drugs are not able to differentiate between healthy cells and cancer cells, leading to several undesired side effects. In this regard, targeted chemotherapies with drugs that carry ligands for selective binding to specific receptors on cancer cells were developed to address this problem. Conjugating known receptor-targeting peptides to marketed drugs is a reliable method to generate bioactive PDCs as a targeted-delivery strategy. Because the number of known bioactive peptide sequences is limited, cell permeability often decreases after cargo attachment, and peptide-based drugs usually face short plasma half-life time, a streamlined approach for PDC development is needed. In this project, such a strategy is described by direct screening of an unnatural peptide-drug conjugate library in solution, which provides a straight forward approach to revealing de novo peptide sequences of PDC with protease resistance. A peptide library will be generated by SPPS assisted split-and-mix synthesis, followed by sequential Pt(IV) prodrug conjugation and PDC cleavage from solid support to yield a library of PDCs. The transmembrane protein, HER2, is overexpressed on various cancer cell lines and is a tested biological target for cancer treatment. After pull-down experiments for PDC binding to HER2, the mixture will be subjected to nLC-MS/MS for high throughput decoding, finally yielding potential hit PDCs. These PDCs will be prepared with an AFPS and PDC-HER2 binding will be confirmed by BioLayer Interferometry. Additionally, fluorescence microscopy experiments can be used to evaluate PDC internalization and MTT assays can assess their anticancer activity. The validation of hit PDCs offers opportunities to elucidate receptor-mediated internalization mechanisms. Thus, I would like to demonstrate an approach for the direct screening of PDC libraries to discover new, targeted cancer treatments as well as to develop molecular tools for elucidating new biology.