A rapid in vitro screening for delivery of peptide-derived peptidase inhibitors as potential drug candidates via epithelial peptide transporters
A rapid in vitro screening for delivery of peptide-derived peptidase inhibitors as potential drug candidates via epithelial peptide transporters
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DOI：
10.1124/jpet.104.066480
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发表时间：
2004-08-01
影响因子：
3.5
通讯作者：
Daniel, H
中科院分区：
文献类型：
--
作者：
Foltz, M;Meyer, A;Daniel, H
通过同时靶向细胞膜载体以有效转运的方式将药物或前体药物靶向特定的酶，应该在作用部位提供最高的生物利用度和特异性。多肽转运蛋白PEPT1和PEPT2在多种组织中表达，包括小肠和肾脏上皮细胞的刷状缘膜。转运体可以接受广泛的底物，因此是转运体介导的药物输送的良好靶点。在这里，我们报道了一种结合两个独立表达系统的候选多肽药物的筛选方法：1)在表达哺乳动物PEPT1或PEPT2的转基因毕赤酵母细胞中进行竞争试验，以确定底物与转运体结合部位的相互作用；2)非洲爪哇多肽转运体的卵表达，用于评估候选药物的电转移。基于已知的二肽基肽酶IV(DPIV)抑制剂异亮氨酸-噻唑烷及其类肽结构的口服利用度和体内疗效，我们首先测试了该化合物是否为上皮肽转运蛋白的底物。此外，还分析了一系列与结构相关的抑制剂的转运情况。我们鉴定了作为肠肽转运体PEPT1底物的各种化合物。相反，这些DPIV抑制剂都没有表现出PEPT2的电转运，尽管各种化合物在表达PEPT2的细胞中表现出很好的竞争肽摄取的亲和力，这表明它们可能是有效的抑制剂。总之，我们应用了一种体外筛选系统，该系统预测了体内通过PEPT1有效的肠道吸收多肽基多肽酶抑制剂。
Targeting drugs or prodrugs to a specific enzyme by simultaneously targeting cell membrane carriers for efficient transport should provide the highest bioavailability along with specificity at the site of action. The peptide transporters PEPT1 and PEPT2 are expressed in a variety of tissues, including the brush-border membranes of epithelial cells of the small intestine and kidney. The transporters accept a wide range of substrates and are therefore good targets for a transporter-mediated drug delivery. Here, we report a screening procedure for peptidomimetic drug candidates combining two independent expression systems: 1) a competition assay in transgenic Pichia pastoris yeast cells expressing either mammalian PEPT1 or PEPT2 for identifying substrate interaction with the transporter binding site; and 2) a Xenopus laevis-based oocyte expression of the peptide transporter for assessing electrogenic transport of drug candidates. Based on the known oral availability and in vivo efficacy of the dipeptidyl peptidase IV (DPIV) inhibitor isoleucine-thiazolidide and its peptide-like structure, we first tested whether this compound is a substrate of epithelial peptide transporters. Additionally, a series of structurally related inhibitors were analyzed for transport. We identified various compounds that serve as substrates of the intestinal peptide transporter PEPT1. In contrast, none of these DPIV inhibitors showed electrogenic transport by PEPT2, although a variety of the compounds displayed good affinities for competition in peptide uptake in PEPT2-expressing cells, suggesting that they may serve as efficient inhibitors. In conclusion, we have applied an in vitro screening system that predicts efficient intestinal absorption of peptide-derived peptidase inhibitors via PEPT1 in vivo.