In Vitro Evolution Reveals a Single Mutation as Sole Source of Src-Family Kinase C-Helix-out Inhibitor Resistance.
In Vitro Evolution Reveals a Single Mutation as Sole Source of Src-Family Kinase C-Helix-out Inhibitor Resistance.
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DOI：
10.1021/acschembio.0c00373
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发表时间：
2020-08-21
影响因子：
4
通讯作者：
Smithgall TE
中科院分区：
文献类型：
--
作者：
Patel RK;Patel YK;Smithgall TE
了解癌细胞对蛋白酪氨酸激酶抑制剂的耐药性（通常源于靶激酶的获得性突变）对于开发更持久的治疗方法至关重要。揭示给定抑制剂和激酶靶点的潜在耐药途径的实验系统在激酶抑制剂药物的临床前开发中具有重要作用。在这里，我们采用密码子诱变策略来定义HCK获得性耐药的突变景观，HCK是SRC酪氨酸激酶家族的成员，也是急性髓性白血病（AML）的治疗靶点。利用基于pcr的饱和诱变技术，我们创建了一个cDNA文库，旨在用所有可能的密码子替换HCK开放阅读框中的每个密码子。该HCK突变文库用于转化大鼠-2成纤维细胞，随后用a -419259筛选耐药菌落，a -419259是一种吡咯嘧啶HCK抑制剂和AML药物先导。x射线晶体学表明，a -419259结合诱导激酶结构域α c -螺旋向外旋转，这是一种与磷转移不相容的构象。值得注意的是，在a -419259选择过程中，只有一个抗性突变进化：在激酶结构域的守门人位置上，组氨酸取代了苏氨酸。深度测序证实了在整个HCK开放阅读框中几乎所有其他错义突变的存在。这一观察结果表明，在Hck是致癌驱动因素的AML病例中，a -419259和其他c-螺旋外src家族激酶抑制剂可能具有狭窄的获得性耐药途径。
Understanding cancer cell drug resistance to protein-tyrosine kinase inhibitors, which often arises from acquired mutations in the target kinase, is central to the development of more durable therapies. Experimental systems that reveal potential paths to resistance for a given inhibitor and kinase target have an important role in preclinical development of kinase inhibitor drugs. Here we employed a codon mutagenesis strategy to define the mutational landscape of acquired resistance in HCK, a member of the SRC tyrosine kinase family and therapeutic target in acute myeloid leukemia (AML). Using PCR-based saturation mutagenesis, we created a cDNA library designed to replace each codon in the HCK open reading frame with all possible codons. This HCK mutant library was used to transform Rat-2 fibroblasts, followed by selection for resistant colonies with A-419259, a pyrrolopyrimidine HCK inhibitor and drug lead for AML. X-ray crystallography has shown that A-419259 binding induces outward rotation of the kinase domain αC-helix, a conformation incompatible with phosphotransfer. Remarkably, only a single resistance mutation evolved during A-419259 selection: histidine substitution for threonine at the gatekeeper position in the kinase domain. Deep sequencing confirmed representation of nearly all other missense mutations across the entire HCK open reading frame. This observation suggests that A-419259 and other C-helix-out Src-family kinase inhibitors may have a narrow path to acquired resistance in the context of AML cases where Hck is an oncogenic driver.
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影响因子：
4.8
作者：
Pene-Dumitrescu, Teodora;Smithgall, Thomas E.
通讯作者：
Smithgall, Thomas E.
影响因子：
64.5
作者：
Azam, M;Latek, RR;Daley, GQ
通讯作者：
Daley, GQ
影响因子：
7.3
作者：
Shen, Kexin;Moroco, Jamie A.;Smithgall, Thomas E.
通讯作者：
Smithgall, Thomas E.
影响因子：
17.1
作者：
Saito, Yoriko;Yuki, Hitomi;Ishikawa, Fumihiko
通讯作者：
Ishikawa, Fumihiko
DOI：
10.1016/j.bbapap.2005.07.027
发表时间：
2005-12-30
影响因子：
3.2
作者：
Chong, YP;Ia, KK;Cheng, HC
通讯作者：
Cheng, HC