Design of allele-specific protein methyltransferase inhibitors
Design of allele-specific protein methyltransferase inhibitors
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DOI：
10.1021/ja011423j
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发表时间：
2001-11-28
影响因子：
15
通讯作者：
Gray, NS
中科院分区：
文献类型：
--
作者：
Lin, Q;Jiang, FY;Gray, NS
蛋白质精氨酸甲基转移酶催化甲基从S-腺苷甲硫氨酸（SAM）转移到靶蛋白的精氨酸侧链，调节转录。RNA加工和受体介导的信号传导。为了具体解决这个家族的各个成员的功能作用，我们采取了“凹凸”的方法，并设计了一系列N-6-取代的S-腺苷高半胱氨酸（SAH）类似物，其靶向酵母蛋白甲基转移酶RMT 1。在Rmt 1中发现了一个点突变（E117 G），使该酶对SAH类似物的选择性抑制敏感。基于质谱的酶测定揭示了两种化合物，N-6-苄基-和N-6-萘基甲基-SAH，可以抑制突变体酶超过野生型，选择性大于20。当E117 G突变被引入酿酒酵母染色体中时，Np 13 p（一种已知的体内Rmt 1底物）的甲基化可以被N-6-naphthylmethyl-SAH在所得等位基因中适度降低。此外，发现N-6-苄基-SAM类似物作为正交SAM辅因子。相对于野生型酶，该类似物优先被突变甲基转移酶利用，选择性大于67。这种特定的酶/抑制剂和酶/底物设计应适用于该蛋白质家族的其他成员，并在结合RNA表达分析时促进体内蛋白质甲基转移酶功能的表征。
Protein arginine methyltransferases, which catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to arginine side chains in target proteins, regulate transcription. RNA processing, and receptor-mediated signaling. To specifically address the functional role of the individual members of this family, we took a "bump-and-hole" approach and designed a series of N-6-substituted S-adenosylhomocysteine (SAH) analogues that are targeted toward a yeast protein methyltransferase RMT1. A point mutation was identified (E117G) in Rmt1 that renders the enzyme susceptible to selective inhibition by the SAH analogues. A mass spectrometry based enzymatic assay revealed that two compounds, N-6-benzyl- and N-6-naphthylmethyl-SAH, can inhibit the mutant enzyme over the wild-type with the selectivity greater than 20. When the E117G mutation was introduced into the Saccharomyces cerevisiae chromosome, the methylation of Np13p, a known in vivo Rmt1 substrate, could be moderately reduced by N-6-naphthylmethyl-SAH in the resulting allele. In addition, an N-6-benzyl-SAM analogue was found to serve as an orthogonal SAM cofactor. This analogue is preferentially utilized by the mutant methyltransferase relative to the wild-type enzyme with a selectivity greater than 67. This specific enzyme/inhibitor and enzyme/substrate design should be applicable to other members of this protein family and facilitate the characterization of protein methyltransferase function in vivo when combined with RNA expression analysis.