PDF(Pubmed)
Abstract：
Phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn2+ -dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to coordinate the catalytic zinc ion in HDACs. Using a previously evolved N𝜀 -acetyl-lysyl-tRNA synthetase in combination with tRNAPyl , we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon-obligate phagemid library in E. coli encoding Aoda, we generated an Aoda-containing phage-displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7-mer peptide GH8P01F1 with Aoda-flanking amino acid residues that matched existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+ -chelating ncAA S-2-amino-8-hydroxyamino-8-oxooctanoic acid (Asuha) led to an extremely potent compound GH8HA01, which has an HDAC8-inhibition Ki value of 0.67 nM. GH8HA01 and its 5-mer truncation analogue Ac-GH8HA01Δ1Δ7 that has an HDAC8-inhibition Ki value of 0.31 nM are two of the most potent HDAC8 inhibitors that have been developed. Furthermore, both are highly selective against HDAC8 compared with other HDACs tested, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets with conserved active sites among homologues.
摘要：
噬菌体辅助,活性位点定向配体进化（PADLE）是一种最近开发的技术，它使用琥珀密码子编码的非规范氨基酸（ncAA）作为锚将噬菌体展示的肽定向到靶标，以增强配体鉴定过程。2-氨基-8-氧代癸酸（Aoda）是大环肽天然产物Apicidin中的含酮ncAA残基，它是Zn2依赖性组蛋白脱乙酰酶（HDAC）的泛抑制剂。它的酮用作锚定点以协调HDAC中的催化锌离子。使用先前进化的Nβ-乙酰基-赖氨酰-tRNA合成酶与tRNAPyl的组合，我们表明，通过琥珀色抑制，Aoda被有效地掺入大肠杆菌的蛋白质中。通过在编码Aoda的大肠杆菌中繁殖琥珀密码子专性噬菌粒文库，我们产生了含有Aoda的噬菌体展示肽库。使用该文库针对HDAC8进行PADLE揭示了7聚体肽GH8P01F1,其具有与鉴定的HDAC8底物中的现有肽序列匹配的Aoda侧翼氨基酸残基。将GH8P01F1中的Aoda转换为更能螯合Zn2的ncAAS-2-氨基-8-羟基氨基-8-氧辛酸（Asuha）导致了一种非常有效的化合物GH8HA01，其HDAC8抑制Ki值为0.67nM。GH8HA01及其5-mer截短类似物Ac-GH8HA01Δ1Δ1Δ7具有0.31nM的HDAC8抑制Ki值，是已开发的两种最有效的HDAC8抑制剂。此外，与其他测试的HDAC相比，两者都对HDAC8具有高度选择性，证明了使用PADLE鉴定具有高度有效和选择性的配体的靶标的巨大潜力，这些配体在同源物中具有保守的活性位点。
