Brown Brandon M, Shim Heesung, Zhang Miao, Yarov-Yarovoy Vladimir, Wulff Heike
Department of Pharmacology (B.M.B., H.S., H.W.), Department of Physiology and Membrane Biology (V.Y.-Y.), School of Medicine, and Department of Chemistry (H.S.), University of California, Davis, California; and Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, California (M.Z.).
Department of Pharmacology (B.M.B., H.S., H.W.), Department of Physiology and Membrane Biology (V.Y.-Y.), School of Medicine, and Department of Chemistry (H.S.), University of California, Davis, California; and Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, California (M.Z.)
Mol Pharmacol. 2017 Oct;92(4):469-480. doi: 10.1124/mol.117.109421. Epub 2017 Jul 31.
Intermediate-conductance (K3.1) and small-conductance (K2) calcium-activated K channels are gated by calcium binding to calmodulin (CaM) molecules associated with the calmodulin-binding domain (CaM-BD) of these channels. The existing K activators, such as naphtho[1,2-]thiazol-2-ylamine (SKA-31), 6,7-dichloro-1-indole-2,3-dione 3-oxime (NS309), and 1-ethylbenzimidazolin-2-one (EBIO), activate both channel types with similar potencies. In a previous chemistry effort, we optimized the benzothiazole pharmacophore of SKA-31 toward K3.1 selectivity and identified 5-methylnaphtho[2,1-]oxazol-2-amine (SKA-121), which exhibits 40-fold selectivity for K3.1 over K2.3. To understand why introduction of a single CH group in five-position of the benzothiazole/oxazole system could achieve such a gain in selectivity for K3.1 over K2.3, we first localized the binding site of the benzothiazoles/oxazoles to the CaM-BD/CaM interface and then used computational modeling software to generate models of the K3.1 and K2.3 CaM-BD/CaM complexes with SKA-121. Based on a combination of mutagenesis and structural modeling, we suggest that all benzothiazole/oxazole-type K activators bind relatively "deep" in the CaM-BD/CaM interface and hydrogen bond with E54 on CaM. In K3.1, SKA-121 forms an additional hydrogen bond network with R362. In contrast, NS309 sits more "forward" and directly hydrogen bonds with R362 in K3.1. Mutating R362 to serine, the corresponding residue in K2.3 reduces the potency of SKA-121 by 7-fold, suggesting that R362 is responsible for the generally greater potency of K activators on K3.1. The increase in SKA-121's K3.1 selectivity compared with its parent, SKA-31, seems to be due to better overall shape complementarity and hydrophobic interactions with S372 and M368 on K3.1 and M72 on CaM at the K3.1-CaM-BD/CaM interface.
中电导（K3.1）和小电导（K2）钙激活钾通道通过钙与这些通道钙调蛋白结合域（CaM-BD）相关的钙调蛋白（CaM）分子结合而门控。现有的钾通道激活剂，如萘并[1,2 -]噻唑 - 2 - 胺（SKA - 31）、6,7 - 二氯 - 1 - 吲哚 - 2,3 - 二酮3 - 肟（NS309）和1 - 乙基苯并咪唑啉 - 2 - 酮（EBIO），以相似的效力激活这两种通道类型。在之前的化学研究中，我们针对K3.1选择性优化了SKA - 31的苯并噻唑药效团，并鉴定出5 - 甲基萘并[2,1 -]恶唑 - 2 - 胺（SKA - 121），其对K3.1的选择性比对K2.3高40倍。为了理解为什么在苯并噻唑/恶唑系统的5位引入单个CH基团能实现对K3.1比对K2.3如此高的选择性，我们首先将苯并噻唑/恶唑的结合位点定位到CaM-BD/CaM界面，然后使用计算建模软件生成K3.1和K2.3与SKA - 121的CaM-BD/CaM复合物模型。基于诱变和结构建模的结合，我们认为所有苯并噻唑/恶唑型钾通道激活剂在CaM-BD/CaM界面中结合相对“深”，并与CaM上的E54形成氢键。在K3.中，SKA - 121与R362形成额外的氢键网络。相比之下，NS309在K3.1中位置更“靠前”，并直接与R362形成氢键。将R362突变为丝氨酸（K2.3中的相应残基）会使SKA - 121的效力降低7倍，表明R362是钾通道激活剂对K3.1普遍具有更高效力的原因。与母体SKA - 31相比，SKA - 121对K3.1选择性的增加似乎是由于在K3.1 - CaM-BD/CaM界面处与K3.1上的S372和M368以及CaM上的M72具有更好的整体形状互补性和疏水相互作用。
