Zhang Jianchun, Ji Tao
Department of Pharmacy, Linyi People's Hospital, Linyi, 276003, China.
Department of Gastroenterology, Linyi People's Hospital, Linyi, 276003, China.
J Mol Model. 2017 Jan;23(1):1. doi: 10.1007/s00894-016-3177-8. Epub 2016 Dec 5.
Oncogenic BRaf V600E mutation is involved in the development, invasion and metastasis of colon cancer. Selective inhibition of BRaf mutant has been recognized as a therapeutic strategy for the cancer. Here, we carried out atomistic molecular dynamics (MD) simulations to characterize the structural basis, energetic property, and dynamics behavior of conformational change in BRaf activation loop upon the mutation. It is found that V600E mutation destabilizes inactive DFG-out conformation of activation loop and promotes its conversion to the active DFG-in conformation, thus conferring constitutive activity for BRaf kinase. A further analysis revealed that the conformational change is induced by electrostatic effect of the negatively charged mutant residue Glu600, which can form a potent salt bridge with the positively charged residue Lys570; this is naturally consistent with phosphorylation of activation loop to activate the kinase. Both of them introduce a negative charge to activation loop and, consequently, the DFG-out is destabilized and conversed to DFG-in. Energetic analysis unraveled that small-molecule kinase inhibitor PLX4720 has a similar selectivity profile for mutant over wild-type kinases and for phosphorylated and dephosphorylated kinases. This can be substantiated in part by in vitro kinase assay that the inhibitor exhibits 12.6 and 10.4-fold higher potencies against mutant than wild type and against phosphorylated than dephosphorylated, respectively. It is suggested that the activation loop conformation, but neither V600E mutation nor phosphorylation, directly determines inhibitor affinity; the mutation and phosphorylation can only indirectly influence inhibitor binding via regulation of activation loop conformation. Graphical Abstract Chemotherapeutic drug selectivity between wild-type and mutant BRaf kinases in colon cancerᅟ.
致癌性BRAF V600E突变参与结肠癌的发生、侵袭和转移。对BRAF突变体的选择性抑制已被视为一种癌症治疗策略。在此，我们进行了原子分子动力学（MD）模拟，以表征BRAF激活环突变后构象变化的结构基础、能量性质和动力学行为。研究发现，V600E突变使激活环的无活性DFG-out构象不稳定，并促进其向活性DFG-in构象的转变，从而赋予BRAF激酶组成型活性。进一步分析表明，构象变化是由带负电荷的突变残基Glu600的静电效应诱导的，它可以与带正电荷的残基Lys570形成强盐桥；这与激活环的磷酸化以激活激酶自然一致。它们都给激活环引入了一个负电荷，因此，DFG-out不稳定并转变为DFG-in。能量分析表明，小分子激酶抑制剂PLX4720对突变体与野生型激酶以及磷酸化和去磷酸化激酶具有相似的选择性。这可以通过体外激酶测定部分证实，该抑制剂对突变体的效力比对野生型高12.6倍，对磷酸化激酶比对去磷酸化激酶高10.4倍。结果表明，激活环构象直接决定抑制剂亲和力，而V600E突变和磷酸化均不直接决定；突变和磷酸化只能通过调节激活环构象间接影响抑制剂结合。结肠癌中野生型和突变型BRAF激酶之间的化疗药物选择性ᅟ。
