Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance.
Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance.
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DOI：
10.1038/srep14538
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发表时间：
2015-09-30
影响因子：
4.6
通讯作者：
Azam M
中科院分区：
文献类型：
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作者：
Kesarwani M;Huber E;Kincaid Z;Evelyn CR;Biesiada J;Rance M;Thapa MB;Shah NP;Meller J;Zheng Y;Azam M
对激酶抑制剂的遗传耐药性的出现对持久的治疗反应提出了巨大挑战。在这里，我们报告了fedratinib（TG 101348）抑制JAK 2激酶的新机制，可防止遗传耐药性的出现。使用体外药物筛选，我们鉴定了211个氨基酸取代，这些氨基酸取代赋予对鲁索利替尼（INCB 018424）的耐药性以及对JAK 2抑制剂AZD 1480、CYT-387和来替尼的交叉耐药性。相比之下，这些耐药变体对fedratinib完全敏感。结构建模，加上诱变和生化研究，揭示了fedratinib的双重结合位点。使用纯化蛋白的体外结合试验显示，对底物结合位点的亲和力较强（Kd = 20 nM），而对ATP位点的亲和力较差（Kd = ~8 μM）。我们的研究表明，影响底物结合口袋的突变编码一个催化不合格的激酶，从而防止出现耐药变异。最重要的是，我们的数据表明，为了开发无耐药性的激酶抑制剂，下一代药物设计应该靶向底物结合位点。
Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib. In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.
DOI：
10.3791/51984
发表时间：
2014-12-07
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Kesarwani M;Huber E;Kincaid Z;Azam M
通讯作者：
Azam M