Small molecule-mediated knockdown of DOT1L as a new epigenetic therapeutic for leukemia

DOT 1 L是由1537个氨基酸组成的组蛋白赖氨酸甲基转移酶。它负责组蛋白3核心赖氨酸79的单甲基化、二甲基化和三甲基化，使用S-腺苷甲硫氨酸或SAM作为其辅因子和甲基化试剂。在生理条件下，DOT 1 L与转录活性、DNA损伤反应和细胞周期调控有关，在造血中起重要作用。然而，由DOT 1 L引起的异常甲基化已被证明会增强导致ML重排白血病（例如HOXA 9，MEISI）发展的基因的表达。这种类型的白血病发生在5-10%的成人急性白血病和超过70%的婴儿急性淋巴细胞白血病中，并且与不良预后相关。酶的功能抑制已被广泛探索，导致一系列抑制剂的开发，其中一些已经进入临床试验。这些可以细分为两类：SAM竞争性抑制剂，其来源于SAM的结构并在SAM结合位点结合，以及诱导口袋抑制剂，其结构不同于SAM并在不同位置结合。非SAM抑制剂的结合诱导酶三级结构的构象变化，其结果是消除SAM结合位点并防止组蛋白甲基化。在这个项目中将采取一种完全不同的方法。将研究DOT 1 L的蛋白水解靶向嵌合体或PROTAC试剂的开发。PROTAC是异双功能分子，其一端包含E3泛素连接酶结合配体，另一端包含结合目标蛋白的配体，通过接头连接（图1）。由于这种双功能性，目的蛋白与连接酶非常接近，并被泛素化。在几轮的泛素化后，多聚泛素化的蛋白质解结合，随后在蛋白酶体发生降解。这最终导致蛋白质从细胞中完全去除而不是功能抑制，并停止组蛋白甲基化。PROTAC试剂已被证明具有催化作用。一旦多聚泛素化的蛋白质解结合，新的蛋白质分子可以结合，整个循环可以重新开始。与功能性抑制剂相比，该技术可以被认为是有利的，因为需要施用较低的剂量。

DOT1L is a histone lysine methyltransferase enzyme made up of 1537 amino acids. It is responsible for the mono-, di-, and tri-methylation of lysine 79 at the core of histone 3, using S-adenosyl methionine, or SAM, as its cofactor and methylating reagent. Under physiological conditions, DOT1L is associated with active transcription, the DNA damage response and cell cycle regulation and plays an important role in haematopoiesis. Abnormal methylation brought about by DOT1L however has been shown to enhance the expression of genes that are responsible for the development of MLL-rearranged leukemia (e.g. HOXA9, MEISI). This type of leukemia occurs in 5-10% of adult acute leukemias and in more than 70% of infant acute lymphoblastic leukemias, and is associated with poor prognosis. Functional inhibition of the enzyme has been extensively explored, leading to the development of an array of inhibitors, some of which have already progressed into clinical trials. These can be subdivided into two categories: SAM-competitive inhibitors which are derived from the structure of SAM and bind at the SAM binding site, and induced pocket inhibitors which are structurally different from SAM and bind at a different location. The binding of the non-SAM inhibitors induces a conformational change in the enzyme's tertiary structure which as a result abolishes the SAM binding site and prevents histone methylation. A completely different approach will be undertaken in this project. The development of a proteolysis targeting chimera, or PROTAC reagent, for DOT1L will be investigated. PROTACs are heterobifunctional molecule comprising of an E3 ubiquitin ligase binding ligand on one end, and a ligand which binds the protein of interest at the other end, connected via a linker (Figure 1). As a result of this bifunctionality, the protein of interest comes in close proximity with the ligase and becomes ubiquitinated. After several rounds of ubiquitination, the polyubiquitinated protein unbinds and consequent degradation takes place at the proteasome. This ultimately leads to complete removal of the protein from cells instead of functional inhibition, and stops histone methylation.PROTAC reagents have been shown to act catalytically. Once the polyubiquitinated protein unbinds, a new protein molecule can bind and the whole cycle can start again. This technology could be considered advantageous compared to functional inhibitors, since a lower dose will need to be administered.