Discovery of a New-Generation S‑Adenosylmethionine-Noncompetitive Covalent Inhibitor Targeting the Lysine Methyltransferase Enhancer of Zeste Homologue 2

The
first-generation enhancer of zeste homologue 2 (EZH2) inhibitors
suffer from several limitations, such as high dosage, cofactor S-adenosylmethionine
(SAM) competition, and acquired drug resistance. Development of covalent
EZH2 inhibitors that are noncompetitive with cofactor SAM offers an
opportunity to overcome these disadvantages. The structure-based design
of compound 16 (BBDDL2059) as a highly potent and selective
covalent inhibitor of EZH2 is presented in this context. 16 inhibits EZH2 enzymatic activity at sub-nanomolar concentrations
and achieves low nanomolar potencies in cell growth inhibition. The
kinetic assay revealed that 16 is noncompetitive with
the cofactor SAM, providing the basis for its superior activity over
noncovalent and positive controls by reducing competition with cofactor
SAM and offering a preliminary proof for its covalent inhibition nature.
Mass spectrometric analysis and washout experiments firmly establish
its covalent inhibition mechanism. This study demonstrates that covalent
inhibition of EZH2 can offer a new opportunity for the development
of promising new-generation drug candidates.

首代zeste增强子同源物2（enhancer of zeste homologue 2, EZH2）抑制剂存在诸多临床局限，包括用药剂量偏高、需与辅因子S-腺苷甲硫氨酸（S-adenosylmethionine, SAM）竞争结合位点，以及易诱导获得性耐药。开发不与辅因子SAM产生竞争的共价EZH2抑制剂，为克服上述缺陷提供了全新的解决思路。本文报道了基于结构设计的化合物16（BBDDL2059），其是一种强效且高选择性的EZH2共价抑制剂。该化合物在亚纳摩尔浓度下即可抑制EZH2的酶促活性，并在细胞增殖抑制实验中达到低纳摩尔级的药效效价。动力学实验结果显示，化合物16与辅因子SAM不存在竞争性结合，这一特性为其相较于非共价抑制剂及阳性对照的优异活性提供了理论依据——通过降低与辅因子SAM的竞争结合，同时也为其共价抑制的作用机制提供了初步佐证。质谱分析与洗脱实验进一步确证了该化合物的共价抑制机制。本研究证实，对EZH2实施共价靶向抑制，可为新一代极具开发潜力的候选药物研发提供全新方向。