Dignify Therapeutics, Research Triangle Park, North Carolina, United States of America.
Drug Design and Discovery, Aptuit an Evotec Company, Verona, Italy.
PLoS One. 2018 Oct 25;13(10):e0205894. doi: 10.1371/journal.pone.0205894. eCollection 2018.
A series of peptide NK2 receptor agonists was evaluated for affinity, potency, efficacy, and selectivity at human recombinant NK2 and NK1 receptors expressed in CHO cells to identify compounds with the greatest separation between NK2 and NK1 receptor agonist activity. Binding studies were performed using displacement of [125I]-NKA binding to NK2 receptors and displacement of [3H]-Septide binding to NK1 receptors expressed in CHO cells. Functional studies examining the increase in intracellular calcium levels and cyclic AMP stimulation were performed using the same cell lines. A correlation was demonstrated between binding affinities (Ki) and potency to increase intracellular calcium (EC50) for NK2 and NK1 receptors. Ranking compounds by their relative affinity (Ki) or potency (EC50) at NK2 or NK1 receptors indicated that the most selective NK2 agonists tested were [Lys5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 674; NK1/NK2 EC50 ratio = 105) and [Arg5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 561; NK1/NK2 EC50 ratio = 70). The endogenous peptide, NKA, lacked selectivity with an NK1/NK2 Ki ratio = 20 and NK1/NK2 EC50 ratio = 1. Of the compounds selected for evaluation in cyclic AMP stimulation assays, [β-Ala8]-NKA(4-10) had the greatest selectivity for activation of NK2 over NK1 receptors (NK1/NK2 EC50 ratio = 244), followed by [Lys5,MeLeu9,Nle10]-NKA(4-10) (ratio = 74), and NKA exhibited marginal selectivity (ratio = 2.8).
一系列肽 NK2 受体激动剂在人重组 NK2 和 NK1 受体上进行了亲和力、效力、功效和选择性评估，以鉴定具有最大 NK2 和 NK1 受体激动活性分离的化合物。结合研究使用 [125I]-NKA 与 NK2 受体的结合置换和 CHO 细胞中表达的 NK1 受体的 [3H]-Septide 结合置换进行。使用相同的细胞系进行了检查细胞内钙水平增加和环 AMP 刺激的功能研究。证明了 NK2 和 NK1 受体的结合亲和力（Ki）与增加细胞内钙（EC50）的效力之间存在相关性。根据化合物对 NK2 或 NK1 受体的相对亲和力（Ki）或效力（EC50）进行排序，表明测试的最具选择性的 NK2 激动剂是 [Lys5，MeLeu9，Nle10]-NKA(4-10)（NK1/NK2 Ki 比 = 674；NK1/NK2 EC50 比 = 105）和 [Arg5，MeLeu9，Nle10]-NKA(4-10)（NK1/NK2 Ki 比 = 561；NK1/NK2 EC50 比 = 70）。内源性肽 NKA 缺乏选择性，NK1/NK2 Ki 比为 20，NK1/NK2 EC50 比为 1。在环 AMP 刺激测定中选择进行评估的化合物中，[β-Ala8]-NKA(4-10)对 NK2 受体的激活具有最大的选择性，而对 NK1 受体的激活（NK1/NK2 EC50 比 = 244），其次是 [Lys5，MeLeu9，Nle10]-NKA(4-10)（比 = 74），而 NKA 则表现出边缘选择性（比 = 2.8）。
