薬剤活性向上した大環状ペプチドを開発する新規戦略

ABC转运蛋白是一大类膜结合蛋白，参与真核生物和原核生物耐药性的形成，尤其是对抗癌药物和抗生素的耐药性。与R教授合作。Tampe（法兰克福大学），我们已经引发了从头天然产物样肽，与TmrAB相互作用-最接近的同源物与抗原加工相关的人类转运蛋白-使用RaPID选择平台。对TmrAB的外向和内向构象进行选择。深度测序显示，在6轮选择后，前10个序列占序列读数的>60%。最高序列富集至总群体的20-40%。所鉴定的肽是化学合成的并通过各种测定表征。所述肽显示出以低纳摩尔亲和力（KD值在2-30 nM的范围内）与蛋白质紧密结合。Hank表明，一些发现的肽通过抑制ATP水解来阻断TmrAB肽易位活性。有趣的是，针对面向外的构象异构体选择的肽在阻断活性方面总体上比针对面向内的构象异构体选择的肽更有效。如分析尺寸排阻色谱法所示，V-D-Tyr 3肽与TmrAB-ATP复合物结合最紧密，因此被选为结晶和结构解析的稳定配体。结晶化工作目前正在进行中。

ABC-transporters are a large class of membrane bound proteins involved in the formation of drug resistance in eukaryotes and prokaryotes, especially with regard to anti cancer drugs and antibiotics. In collaboration with Prof. R. Tampe (University of Frankfurt), we have elicited de novo natural product-like peptides that interact with TmrAB - the closest homolog to the human transporter associated with antigen processing - using the RaPID selection platform. Selections were performed against the outward and inward facing conformation of TmrAB.Deep sequencing showed that after 6 rounds of selection the top 10 sequences accounted for >60% of the sequence reads. The top sequences were enriched up to 20-40% of the total population. The identified peptides where chemically synthesized and characterized by various assays. The peptides were shown to bind tightly to the protein with low nanomolar affinities (KD values are in the range of 2-30 nM).Characterization by S. Hank showed that some of the discovered peptides block TmrAB peptide translocation activity through inhibition of ATP hydrolysis. Interestingly, the peptides selected against the outward facing conformer were overall more efficient in blocking activity than the peptides selected against the inward conformer. As revealed by analytical size exclusion chromatography the V-D-Tyr 3 peptide binds most tightly to the TmrAB-ATP complex and was therefore chosen as a stabilizing ligand for crystallization and structure elucidation. Crystallization efforts are currently ongoing.