Ohnishi Toshiyuki, Sakamoto Kotaro, Asami-Odaka Asano, Nakamura Kimie, Shimizu Ayako, Ito Takashi, Asami Taiji, Ohtaki Tetsuya, Inooka Hiroshi
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
Biochem Biophys Res Commun. 2017 Jan 29;483(1):101-106. doi: 10.1016/j.bbrc.2016.12.186. Epub 2016 Dec 31.
Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors.
原肌球蛋白受体激酶B（TrkB）是脑源性神经营养因子（BDNF）的已知受体。由于TrkB在神经元发育、成熟、存活等的调节中起关键作用，它是抗中枢神经系统疾病药物的良好靶点。在本研究中，我们旨在通过应用随机肽噬菌体展示技术来生成肽类TrkB激动剂。在用重组Fc融合的TrkB（TrkB-Fc）进行噬菌体淘选后，直接针对表达TrkB的HEK293细胞筛选激动性噬菌体。通过随后对首次筛选出的BM17肽衍生的聚焦文库进行筛选，我们成功获得了BM17d99肽，它与BDNF没有序列相似性，但具有TrkB结合能力。然后我们合成了一种二聚体BM17d99类似肽，它可以通过促进受体同源二聚化来磷酸化或激活TrkB。用二聚体BM17d99类似肽处理表达TrkB的HEK293细胞显著诱导了TrkB的磷酸化，表明二聚体肽增强了TrkB的同源二聚化。本报告表明我们的方法对于生成细胞表面受体的人工肽激动剂是有用的。
