Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy.
Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy.
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DOI：
10.1085/jgp.200409059
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发表时间：
2004-08
期刊：
影响因子：
--
通讯作者：
Verkman AS
中科院分区：
文献类型：
--
作者：
Muanprasat C;Sonawane ND;Salinas D;Taddei A;Galietta LJ;Verkman AS
囊性纤维化跨膜电导调节蛋白 (CFTR) 是一种 cAMP 调节的上皮 Cl− 通道，当出现缺陷时，会导致囊性纤维化。对 100,000 种不同小分子的筛选揭示了四种新颖的 CFTR 抑制剂化学类别，其 Ki < 10 μM，其中一种（甘氨酸酰肼）具有许多活性结构类似物。对一系列合成的甘氨酸酰肼类似物的分析揭示了对 N-(2-萘基) 和 3,5-二溴-2,4-二羟基苯基取代基的最大抑制效力。化合物 N-(2-萘基)-[(3,5-二溴-2,4-二羟基苯基)亚甲基]甘氨酸酰肼 (GlyH-101) 可在 1 分钟内可逆地抑制 CFTR Cl− 电导。全细胞电流测量显示，GlyH-101 具有强烈的内向整流作用，具有电压依赖性 CFTR 阻断作用，使表观抑制常数 Ki 从 +60 mV 时的 1.4 μM 增加到 -60 mV 时的 5.6 μM。通过降低细胞外 Cl− 浓度来降低表观效力。膜片钳实验表明，在通道开放爆发期间，通道快速关闭，将平均通道开放时间从 264 毫秒减少到 13 毫秒（−60 mV 保持电位，5 μM GlyH-101）。 GlyH-101 抑制效力在 6.5-8.0 范围内与 pH 值无关，它主要以单价阴离子形式存在，在水中的溶解度约为 1 mM。小鼠局部外用 GlyH-101 (10 μM) 可快速、可逆地抑制毛喉素诱导的鼻电位差超极化。在霍乱闭环模型中，腔内 GlyH-101（2.5 μg）可减少约 80% 霍乱毒素诱导的肠液分泌。与噻唑烷酮 CFTR 抑制剂 CFTRinh-172 相比，GlyH-101 具有显着更高的水溶性和作用速度，以及涉及外部孔入口附近闭塞的新颖抑制机制。甘氨酸酰肼可用作 CFTR 孔结构的探针、创建 CF 动物模型以及作为肠毒素介导的分泌性腹泻的止泻药。
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Cl− channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with Ki < 10 μM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl− conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant Ki from 1.4 μM at +60 mV to 5.6 μM at −60 mV. Apparent potency was reduced by lowering extracellular Cl− concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (−60 mV holding potential, 5 μM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5–8.0, where it exists predominantly as a monovalent anion with solubility ∼1 mM in water. Topical GlyH-101 (10 μM) in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 μg) reduced by ∼80% cholera toxin–induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTRinh-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of CFTR pore structure, in creating animal models of CF, and as antidiarrheals in enterotoxic-mediated secretory diarrheas.
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4.8
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