The multidomain adaptor protein syntenin is known to mediate cancer cell metastasis and invasion through its tandem PDZ1 and PDZ2 domains, leading to the postulation that the PDZ tandem may serve as a potential drug target for cancer treatment. Here we report the development of high-affinity peptide blockers to target the syntenin tandem PDZ domain, and elucidate that blocking syntenin correlates with the inhibition of cell migration and spreading. Two strategies are employed to derive high-affinity blockers from the low-affinity natural binding peptides: first, dimerization of the C termini of natural syntenin-binding peptides confers dimer peptides with much higher affinity than the monomers; second, unnatural amino acid substitution at P-1 and P-2 positions of the PDZ-binding sequence increases the binding affinity. Through several rounds of optimization, we discovered a dimeric peptide that binds tightly to syntenin tandem PDZ domain, with a dissociation constant of 0.21 μM based on fluorescence polarization measurement. The peptide dimer inhibits the migration and invasion of syntenin high-expression human cancer cells through attenuating the ERK phosphorylation of the MAPK kinase pathway. This work showcases an effective strategy to derive high-affinity blocker of multidomain adaptor proteins, which resulted in a syntenin-targeted antagonist with potential pharmaceutical values for the treatment of syntenin over-expressing cancers.

已知多域衔接蛋白Syntenin通过其串联的PDZ1和PDZ2结构域介导癌细胞的转移和侵袭，导致人们推测PDZ串联可能充当癌症治疗的潜在药物靶标。在这里，我们报道了针对亲和素串联PDZ域的高亲和力肽阻滞剂的发展，并阐明了阻滞Syntenin与抑制细胞迁移和扩散相关。有两种策略可用于从低亲和力的天然结合肽中获得高亲和力的阻断剂：首先，天然突触素结合肽的C末端的二聚化赋予了二聚体肽比单体高得多的亲和力。第二，PDZ结合序列的P-1和P-2位置的非天然氨基酸取代增加了结合亲和力。通过几轮优化，我们发现了一种二聚体肽，与二联蛋白串联PDZ域紧密结合，基于荧光偏振测量，其解离常数为0.21μM。肽二聚体通过减弱MAPK激酶途径的ERK磷酸化来抑制合成素高表达人类癌细胞的迁移和侵袭。这项工作展示了一种获得多域衔接子蛋白高亲和力阻滞剂的有效策略，该蛋白可产生针对Syntenin的拮抗剂，具有治疗Syntenin过表达的癌症的潜在药物价值。肽二聚体通过减弱MAPK激酶途径的ERK磷酸化来抑制合成素高表达人类癌细胞的迁移和侵袭。这项工作展示了一种获得多域衔接子蛋白高亲和力阻滞剂的有效策略，该蛋白可产生针对Syntenin的拮抗剂，具有治疗Syntenin过表达的癌症的潜在药物价值。肽二聚体通过减弱MAPK激酶途径的ERK磷酸化来抑制合成素高表达人类癌细胞的迁移和侵袭。这项工作展示了一种获得多域衔接子蛋白高亲和力阻滞剂的有效策略，该蛋白可产生针对Syntenin的拮抗剂，具有治疗Syntenin过表达的癌症的潜在药物价值。