Kir4.1钾通道毛孔阻断剂VU0134992的发现，表征和对肾液和电解质排泄的影响。,Molecular Pharmacology

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Kir4.1钾通道毛孔阻断剂VU0134992的发现，表征和对肾液和电解质排泄的影响。
Molecular Pharmacology ( IF 3.2 ) Pub Date : 2018-06-12 , DOI: 10.1124/mol.118.112359
Sujay V Kharade ₁ , Haruto Kurata ₁ , Aaron M Bender ₁ , Anna L Blobaum ₁ , Eric E Figueroa ₁ , Amanda Duran ₁ , Meghan Kramer ₁ , Emily Days ₁ , Paige Vinson ₁ , Daniel Flores ₁ , Lisa M Satlin ₁ , Jens Meiler ₁ , C David Weaver ₁ , Craig W Lindsley ₁ , Corey R Hopkins ₁ , Jerod S Denton ₂

Affiliation

内向整流钾（Kir）通道Kir4.1（KCNJ10）在肾脏的上皮细胞，中枢神经系统的星形胶质细胞和内耳的血管纹中发挥重要的生理作用。KCNJ10中的功能丧失突变导致EAST / SeSAME综合征，其特征是癫痫，共济失调，肾盐消耗和感觉神经性耳聋。尽管遗传方法对于确定Kir4.1在这些组织的正常功能中的重要性是必不可少的，但仍缺乏可用于在遗传上正常的动物中紧急操纵Kir4.1活性的药理学工具。因此，我们从范德比尔特化学生物学研究所的文库中对76,575种化合物进行了高通量筛选，用于Kir4.1的小分子调节剂。鉴定出的最有效的抑制剂是2-（2-溴-4-异丙基苯氧基）-N-（2,2,6,6-四甲基哌啶-4-基）乙酰胺（VU0134992）。在全细胞膜片钳电生理实验中，VU0134992在-120时抑制Kir4.1的IC50值为0.97 µM，对Kir4.1 / 5.1串联通道（IC50 = 9 µM）的同构Kir4.1选择性为9倍毫伏在th（Tl +）通量测定中，对于Kir4.1，VU0134992的选择性是Kir1.1，Kir2.1和Kir2.2的30倍以上；对Kir2.3，Kir6.2 / SUR1和Kir7.1的激活较弱；对Kir3.1 / 3.2，Kir3.1 / 3.4和Kir4.2同样有效。这种效能和选择性特征优于Kir4.1抑制剂阿米替林，去甲替林和氟西汀。药物化学鉴定出VU0134992的成分对于抑制Kir4.1至关重要。膜片钳电生理学，分子建模，并且定点诱变将孔壁谷氨酸158和异亮氨酸159鉴定为阻断通道的关键残基。VU0134992在大鼠血浆中显示出较大的自由未结合分数（fu）（fu = 0.213）。与Kir4.1在肾功能中的已知作用一致，口服VU0134992可导致大鼠剂量依赖性利尿，利尿和利尿。因此，VU0134992代表了首个体内活性工具化合物，可用于探索Kir4.1作为治疗高血压的新型利尿靶标的治疗潜力。VU0134992的口服给药导致大鼠的剂量依赖性利尿，利尿和利尿。因此，VU0134992代表了首个体内活性工具化合物，可用于探索Kir4.1作为治疗高血压的新型利尿靶标的治疗潜力。VU0134992的口服给药导致大鼠的剂量依赖性利尿，利尿和利尿。因此，VU0134992代表了首个体内活性工具化合物，可用于探索Kir4.1作为治疗高血压的新型利尿靶标的治疗潜力。

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Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.

The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 value of 0.97 µM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50 = 9 µM) at -120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction (fu) in rat plasma (fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.

更新日期：2019-11-01

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