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。20世纪中叶，化疗药物被引入作为辅助治疗，以消除手术和放射治疗后残留的癌细胞。对于许多癌症患者来说，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，传统的化疗药物不能区分健康细胞和癌细胞，导致了几个不受欢迎的副作用。在这方面，为了解决这一问题，开发了靶向化疗药物，这些药物携带有选择性地与癌细胞上的特定受体结合的配体。将已知的受体靶向多肽与上市药物偶联是产生生物活性PDCs作为靶向递送策略的可靠方法。由于已知的生物活性多肽序列数量有限，载药后细胞通透性往往下降，以及多肽类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，通过在溶液中直接筛选非天然多肽-药物共轭文库来描述这一策略，为揭示具有蛋白酶抗性的PDC从头开始的多肽序列提供了一种直接的方法。通过SPPS辅助裂解和混合合成，然后依次进行铂(IV)前药结合和PDC从固体载体中裂解，将生成多肽库，从而产生PDC库。跨膜蛋白HER2在各种癌细胞系上过度表达，是癌症治疗的测试生物靶点。在PDC与HER2结合的下拉实验后，混合物将经过NLC-MS/MS进行高通量解码，最终产生潜在的HIT PDC。这些PDCs将与AFPS一起制备，并且PDC-HER2的结合将通过BioLayer干涉术得到确认。此外，荧光显微镜实验可以用来评价PDC的内化作用，而四甲基偶氮唑盐比色法可以评价它们的抗癌活性。HIT 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，并开发分子工具来阐明新的生物学。

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.