Sara Puglioli, Mosè Fabbri, Claudia Comacchio, Laura Alvigini, Roberto De Luca, Sebastian Oehler, Ettore Gilardoni, Gabriele Bassi, Samuele Cazzamalli, Dario Neri* and Nicholas Favalli*,
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引用次数: 0
摘要
DNA 编码文库(DELs)可对数百万个化合物进行平行筛选,用于各种应用,包括全新发现或亲和力熟化活动。然而,文库构建和 HIT 再合成可能很麻烦,尤其是当文库成员的立体化学结构未知时。我们介绍了一种适用于构建高纯度单链单药食同源 DEL 的排列编码策略,其目的是在测序水平上区分异构体(如立体异构体、区域异构体和肽序列)。通过合成模拟的 921,600 个成员的 4-氨基脯氨酸单链 DEL("DEL1")验证了这种方法。在针对不同靶标筛选 DEL1 的过程中,高通量测序结果显示,我们选择性地富集了最有效的立体异构体,富集因子优于传统的编码策略。此外,我们还对 24 个支架进行了编码,这些支架来自之前描述的针对成纤维细胞活化蛋白(FAP-2286)的环状肽的氨基酸序列的不同排列,从而验证了我们的方法的多功能性。针对人类 FAP 对由此产生的文库("DEL2")进行了检测,结果显示五种环肽具有选择性富集。我们观察到富集因子与 DNA 结合亲和力之间存在直接关联。所介绍的编码方法促进了文库合成,简化了 HIT 的再合成,同时在 DEL 测序水平上提高了富集因子,从而加速了药物发现。这有助于在药物化学和亲和力成熟活动之前确定候选 HIT。
Permutational Encoding Strategy Accelerates HIT Validation from Single-Stranded DNA-Encoded Libraries
DNA-Encoded Libraries (DELs) allow the parallel screening of millions of compounds for various applications, including de novo discovery or affinity maturation campaigns. However, library construction and HIT resynthesis can be cumbersome, especially when library members present an unknown stereochemistry. We introduce a permutational encoding strategy suitable for the construction of highly pure single-stranded single-pharmacophore DELs, designed to distinguish isomers at the sequencing level (e.g., stereoisomers, regio-isomers, and peptide sequences). This approach was validated by synthesizing a mock 921,600-member 4-amino-proline single-stranded DEL (“DEL1”). While screening DEL1 against different targets, high-throughput sequencing results showed selective enrichment of the most potent stereoisomers, with enrichment factors that outperform conventional encoding strategies. The versatility of our methodology was additionally validated by encoding 24 scaffolds derived from different permutations of the amino acid sequence of a previously described cyclic peptide targeting Fibroblast Activation Protein (FAP-2286). The resulting library (“DEL2”) was interrogated against human FAP, showing selective enrichment of five cyclic peptides. We observed a direct correlation between enrichment factors and on-DNA binding affinities. The presented encoding methodology accelerates drug discovery by facilitating library synthesis and streamlining HIT resynthesis while enhancing enrichment factors at the DEL sequencing level. This facilitates the identification of HIT candidates prior to medicinal chemistry and affinity maturation campaigns.
期刊介绍:
Bioconjugate Chemistry invites original contributions on all research at the interface between man-made and biological materials. The mission of the journal is to communicate to advances in fields including therapeutic delivery, imaging, bionanotechnology, and synthetic biology. Bioconjugate Chemistry is intended to provide a forum for presentation of research relevant to all aspects of bioconjugates, including the preparation, properties and applications of biomolecular conjugates.