Department of Chemistry and Chemical Biology and the Howard Hughes Medical Institute, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
J Am Chem Soc. 2010 Aug 25;132(33):11779-91. doi: 10.1021/ja104903x.
DNA-templated organic synthesis enables the translation of DNA sequences into synthetic small-molecule libraries suitable for in vitro selection. Previously, we described the DNA-templated multistep synthesis of a 13,824-membered small-molecule macrocycle library. Here, we report the discovery of small molecules that modulate the activity of kinase enzymes through the in vitro selection of this DNA-templated small-molecule macrocycle library against 36 biomedically relevant protein targets. DNA encoding selection survivors was amplified by PCR and identified by ultra-high-throughput DNA sequencing. Macrocycles corresponding to DNA sequences enriched upon selection against several protein kinases were synthesized on a multimilligram scale. In vitro assays revealed that these macrocycles inhibit (or activate) the kinases against which they were selected with IC(50) values as low as 680 nM. We characterized in depth a family of macrocycles enriched upon selection against Src kinase, and showed that inhibition was highly dependent on the identity of macrocycle building blocks as well as on backbone conformation. Two macrocycles in this family exhibited unusually strong Src inhibition selectivity even among kinases closely related to Src. One macrocycle was found to activate, rather than inhibit, its target kinase, VEGFR2. Taken together, these results establish the use of DNA-templated synthesis and in vitro selection to discover small molecules that modulate enzyme activities, and also reveal a new scaffold for selective ATP-competitive kinase inhibition.
DNA 模板有机合成能够将 DNA 序列转化为适合体外选择的合成小分子文库。之前，我们描述了一种 13824 个成员的小分子大环化合物文库的 DNA 模板多步合成。在这里，我们报告了通过针对 36 种与生物医学相关的蛋白质靶标对该 DNA 模板小分子大环文库进行体外选择，发现了能够调节激酶酶活性的小分子。通过 PCR 扩增编码选择幸存者的 DNA，并通过超高通量 DNA 测序进行鉴定。在毫克级规模上合成了对应于选择多种蛋白激酶后富集的 DNA 序列的大环化合物。体外测定表明，这些大环化合物以低至 680 nM 的 IC50 值抑制（或激活）它们所针对的激酶。我们深入研究了一组在Src 激酶选择中富集的大环化合物，并表明抑制高度依赖于大环化合物构建块的身份以及骨架构象。在这个家族中，有两个大环化合物对 Src 激酶具有异常强的抑制选择性，即使是与 Src 密切相关的激酶也是如此。其中一个大环化合物被发现激活而不是抑制其靶激酶 VEGFR2。总之，这些结果确立了使用 DNA 模板合成和体外选择来发现调节酶活性的小分子的方法，并揭示了一种用于选择性 ATP 竞争性激酶抑制的新支架。
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