Desensitization of the human motilin receptor by motilides
Desensitization of the human motilin receptor by motilides
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DOI：
10.1124/jpet.104.081497
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发表时间：
2005-06-01
影响因子：
3.5
通讯作者：
Peeters, TL
中科院分区：
文献类型：
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作者：
Thielemans, L;Depoortere, I;Peeters, TL
快速耐受可能是导致吗替利特 ABT-229 [N-乙基，N-甲基 4" 脱氧红霉素 (EM)-B 烯醇醚] 在临床试验中失败的原因。我们比较了结构相关的吗替利特 [EM-A、EM-A 烯醇醚 (ME4)、N-乙基、N-甲基 EM-A(ME36)、EM-B 烯醇醚 (ME67)、 N-乙基、N-甲基 EM-A 烯醇醚 (EM523)、ABT-229 和 4" 脱氧 EM-A 烯醇醚 (KOS1326)] 在表达人胃动素受体 (MTLR) 的中国仓鼠卵巢 (CHO)-K1 细胞系和兔十二指肠节段中。在用胃动素刺激之前，将 CHO-MTLR 细胞与胃动素预孵育。计算将最大胃动素诱导的 Ca2+ 通量降低至 50% 的预孵育浓度的负对数 (pDC(50))。在含有增强型绿色荧光蛋白 (EGFP) 标记的 MTLR 的 CHO-K1 细胞中观察内化，并在结合实验中进行定量。在十二指肠节段中测量重复刺激的收缩反应。在 CHO-MTLR 细胞中，pDC(50) 为 ABT-229(8.78) > 胃动素 (7.77) > EM-A (4.78)，与其诱导 Ca2+ 释放的效力顺序 (pEC(50)) 不同：胃动素 (9.39) > ABT-229 (8.46) > EM-A (7.11)。在带有 EGFP 标记的 MTLR 的细胞中，ABT-229 使膜荧光降低了 25 +/- 2%，而胃动素为 16 +/- 2%，EM-A 为 8 +/- 2%。结合研究证实，EM-A 不会诱导 MTLR 内化（与胃动素相比，残余结合为 96 +/- 4%，为 31 +/- 3%，而 ABT-229 为 21 +/- 1%）。其他吗替利特 ME4 (5.90; 8.08)、ME67 (6.03; 8.12)、ME36 (3.32; 6.62)、EM-523 (6.02; 8.22) 和 KOS1326 (7.32; 8.14) 的 pDC(50) 和 pEC(50) 值的比较表明，强脱敏特性ABT-229 的 4"-OH 的去除主要与克拉定糖的 4"-OH 的去除有关。十二指肠节段收缩反应的下降与 pDC(50) 相关。MTLR 脱敏和内化的能力不仅由效力决定。这可能是开发临床上有用的化合物的重要标准。
Tachyphylaxis may have contributed to the failure of the motilide ABT-229 [N-ethyl, N-methyl 4" deoxy erythromycin (EM)-B enolether] in clinical trials. We compared the desensitizing potency of structurally related motilides [EM-A, EM-A enolether (ME4), N-ethyl, N-methyl EM-A(ME36), EM-B enolether (ME67), N-ethyl, N-methyl EM-A enolether (EM523), ABT-229 and 4" deoxy EM-A enolether (KOS1326)] in a Chinese hamster ovary (CHO)-K1 cell line expressing the human motilin receptor (MTLR) and in rabbit duodenal segments. CHO-MTLR cells were preincubated with motilides prior to stimulation with motilin. The negative logarithm of the preincubation concentration reducing the maximal motilin-induced Ca2+ flux to 50% was calculated (pDC(50)). Internalization was visualized in CHO-K1 cells containing an enhanced green fluorescent protein (EGFP)-tagged MTLR and quantified in binding experiments. The contractile response of repeated stimulations was measured in duodenal segments. In CHO-MTLR cells, the pDC(50) was ABT-229(8.78) > motilin (7.77) > EM-A (4.78), different from their order of potency to induce Ca2+ release (pEC(50)): motilin (9.39) > ABT-229 (8.46) > EM-A (7.11). In cells with the EGFP-tagged MTLR, ABT- 229 decreased membrane fluorescence by 25 +/- 2% compared with 16 +/- 2% for motilin and 8 +/- 2% for EM-A. Binding studies confirmed that EM-A did not induce MTLR internalization ( residual binding 96 +/- 4% compared with motilin, 31 +/- 3% and ABT-229, 21 +/- 1%). Comparison of the pDC(50) and pEC(50) values of the other motilides ME4 (5.90; 8.08), ME67 (6.03; 8.12), ME36 (3.32; 6.62), EM-523 (6.02; 8.22), and KOS1326 (7.32; 8.14) suggested that the strong desensitizing properties of ABT- 229 are mostly related to the removal of the 4"-OH of the cladinose sugar. The decline of the contractile response in duodenal segments correlated with the pDC(50). The ability to desensitize and internalize the MTLR is not only determined by potency. This may be an important criterion for the development of a clinically useful compound.