Guitot Karine, Drujon Thierry, Burlina Fabienne, Sagan Sandrine, Beaupierre Sandra, Pamlard Olivier, Dodd Robert H, Guillou Catherine, Bolbach Gérard, Sachon Emmanuelle, Guianvarc'h Dominique
Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005, Paris, France.
Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), 75005, Paris, France.
Anal Bioanal Chem. 2017 Jun;409(15):3767-3777. doi: 10.1007/s00216-017-0319-5. Epub 2017 Apr 7.
Histone lysine methylation is associated with essential biological functions like transcription activation or repression, depending on the position and the degree of methylation. This post-translational modification is introduced by protein lysine methyltransferases (KMTs) which catalyze the transfer of one to three methyl groups from the methyl donor S-adenosyl-L-methionine (AdoMet) to the amino group on the side chain of lysines. The regulation of protein lysine methylation plays a primary role not only in the basic functioning of normal cells but also in various pathologies and KMT deregulation is associated with diseases including cancer. These enzymes are therefore attractive targets for the development of new antitumor agents, and there is still a need for direct methodology to screen, identify, and characterize KMT inhibitors. We report here a simple and robust in vitro assay to quantify the enzymatic methylation of KMT by MALDI-TOF mass spectrometry. Following this protocol, we can monitor the methylation events over time on a peptide substrate. We detect in the same spectrum the modified and unmodified substrates, and the ratios of both signals are used to quantify the amount of methylated substrate. We first demonstrated the validity of the assay by determining inhibition parameters of two known inhibitors of the KMT SET7/9 ((R)-PFI-2 and sinefungin). Next, based on structural comparison with these inhibitors, we selected 42 compounds from a chemical library. We applied the MALDI-TOF assay to screen their activity as inhibitors of the KMT SET7/9. This study allowed us to determine inhibition constants as well as kinetic parameters of a series of SET7/9 inhibitors and to initiate a structure activity discussion with this family of compounds. This assay is versatile and can be easily adapted to other KMT substrates and enzymes as well as automatized.
组蛋白赖氨酸甲基化与转录激活或抑制等重要生物学功能相关，这取决于甲基化的位置和程度。这种翻译后修饰由蛋白质赖氨酸甲基转移酶（KMTs）引入，该酶催化从甲基供体S-腺苷-L-甲硫氨酸（AdoMet）向赖氨酸侧链上的氨基转移一至三个甲基。蛋白质赖氨酸甲基化的调节不仅在正常细胞的基本功能中起主要作用，而且在各种病理过程中也起主要作用，KMT失调与包括癌症在内的疾病相关。因此，这些酶是开发新型抗肿瘤药物的有吸引力的靶点，并且仍然需要直接的方法来筛选、鉴定和表征KMT抑制剂。我们在此报告一种简单且稳健的体外测定法，用于通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF质谱）定量KMT的酶促甲基化。按照此方案，我们可以监测肽底物上随时间的甲基化事件。我们在同一光谱中检测修饰和未修饰的底物，并且这两种信号的比率用于定量甲基化底物的量。我们首先通过确定KMT SET7/9的两种已知抑制剂（(R)-PFI-2和杀稻瘟菌素）的抑制参数来证明该测定法的有效性。接下来，基于与这些抑制剂的结构比较，我们从化学文库中选择了42种化合物。我们应用MALDI-TOF测定法筛选它们作为KMT SET7/9抑制剂的活性。这项研究使我们能够确定一系列SET7/9抑制剂的抑制常数以及动力学参数，并启动与该类化合物的构效关系讨论。该测定法具有通用性，并且可以很容易地适用于其他KMT底物和酶，也可以实现自动化。
