Development of chemical probes for NSD2 and its PHD fingers

1)表观遗传学是指在不改变DNA序列的情况下改变基因活性的机制，它对细胞分化和生理功能至关重要。表观遗传修饰，如组蛋白翻译后修饰（PTM），在染色质浓缩和调节基因表达中起重要作用，并且表观遗传蛋白的异常表达常常与各种疾病的发展相关，NSD 2属于核受体结合SET结构域（NSD），组蛋白甲基转移酶家族，参与组蛋白H3和H4上赖氨酸残基侧链的甲基化，主要参与体内H3 K36的二甲基化，显示对染色质可及性和细胞凋亡中关键调节因子的基因表达的影响，DNA修复和细胞粘附。历史上，NSD 2因其与Wolf-Hirschhorn综合征（WHS）的联系而闻名，其中含有NSD 2基因的染色体区域的半合子缺失导致胚胎发育障碍和畸形综合征。NSD 2过表达也见于几种癌症（例如乳腺癌、胶质母细胞瘤、前列腺癌、肺癌等）中。它与肿瘤的侵袭性和不良预后有关，但它似乎在多发性骨髓瘤（MM）和急性淋巴细胞白血病（ALL）的发展中尤其重要。具体地，具有t（4;14）易位的MM细胞显示NSD 2的异常表达，导致H3 K36 me 2水平的增加（其是基因表达的活性标记）和抑制性标记H3 K27 me 3的减少。这涉及改变细胞生长和粘附特性的发展，这些特性在使用shRNA敲低NSD 2亚型后丢失。因此，抑制NSD 2可能是一种干预癌症、开发新的治疗方法和了解NSD 2的作用的可能策略。NSD 2的结构由一个催化SET结构域、一个HMG（高迁移率族）盒、2个PWWP（脯氨酸-色氨酸-色氨酸-脯氨酸）结构域和4个PHD（植物同源结构域）锌指组成。NSD 2的抑制可以通过靶向SET结构域或变构结合位点来实现。不幸的是，已经鉴定了很少的NSD 2的小分子抑制剂，例如N-烷基Sinefungin、LEM-06和LEM-14，其显示出低选择性和效力。基于结构的药物设计与NSD 2的主要问题是在结晶的催化SET域的困难。此外，针对SET结构域的选择性抑制剂的开发受到具有挑战性的测定开发的阻碍，迄今为止仅已知核小体底物。为了克服这些困难，尝试新的靶向NSD 2的方法可能是有益的，例如使用环肽或靶向其阅读器结构域。2）项目的目的本项目的目的是开发作为NSD 2的化学探针的环肽，并进一步了解其PHD指在NSD 2活性调节中的作用。使用环肽可能是一种有前途的方法，在开发化学探针/抑制剂的'undruggable'目标，如NSD 2，因为他们能够与他们的目标在更大的表面积相互作用。此外，靶向NSD 2的阅读器结构域，特别是其PHD指，可能是靶向SET结构域的令人感兴趣的替代方案，因为阅读器结构域可以影响NSD 2向其靶基因座的募集及其催化活性。

1) IntroductionEpigenetics refers to mechanisms responsible for altering gene activity without changing the DNA sequence, which are essential for cell differentiation and physiological functions. Epigenetic modifications, such as histone post translational modifications (PTMs), play an important role in chromatin condensation and regulating gene expression, and an aberrant expression of epigenetic proteins are often associated with thedevelopment of various diseases, e.g. cancers and autoimmune disorders.NSD2 belongs to the nuclear receptor-binding SET Domain (NSD) histone methyltransferase family and is involved in methylation of the side chain of lysine residues on histone H3 and H4, predominantly in dimethylation of H3K36 in vivo, showing effects on chromatin accessibility and gene expression of key regulators in apoptosis, DNA repair and cell adhesion. Historically, NSD2 is known for its link with Wolf-Hirschhorn Syndrome (WHS), where its hemizygous deletion of chromosomal region containing NSD2 gene leading to disorder of embryonic development and malformation syndrome. NSD2 overexpression is also found in several cancers (e.g. breast cancers, glioblastoma, prostate, lung, etc.) and it is associated with tumour aggressiveness and poor prognosis, but it especially seems to be crucial in the development of Multiple Myeloma (MM) and Acute Lymphoblastic Leukaemia (ALL). Specifically, MM cells with t(4;14) translocations show an aberrant expression of NSD2 leading to an increase of H3K36me2 levels, which is an active mark of gene expression, and a reduction of the repressive mark H3K27me3. This involves the development of altered cell growth and adhesion proprieties, which are lost after the knockdown of NSD2 isoforms using shRNA. Thus, inhibition of NSD2 may be considered a possible strategy for intervention in cancer, to develop new therapeutic approaches, and to understand the roles of NSD2.Structurally, NSD2 is composed of a catalytic SET domain, an HMG (high mobility group) box, 2 PWWP (proline - tryptophan - tryptophan - proline) domains and 4 PHD (plant homeodomain) zinc fingers. Inhibition of NSD2 may be achieved by targeting the SET domain, or allosteric binding sites. Unfortunately, few small molecule inhibitors of NSD2 have been identified, e.g. N-Alkyl Sinefungin, LEM-06 and LEM-14, which show low selectivity and potency. The main problem for structure-based drug design with NSD2 is the difficulty in crystallizing the catalytic SET domain. In addition, the development of selective inhibitors against the SET domain is hampered by challenging assay development, with only nucleosome substrates known so far. To overcome some of these difficulties it may be beneficial to attempt new methods of targeting NSD2, such as using cyclic peptides or targeting its reader domains.2) Aims of the projectThe aims of this project are to develop cyclic peptides as chemical probes for NSD2, and to further the understanding of its PHD fingers in the regulation of NSD2 activity. Using cyclic peptides may be a promising approach in the development of chemical probes/inhibitors for 'undruggable' targets like NSD2, since they are able to interact with their targets over larger surface areas. Furthermore, targeting the reader domains of NSD2, especially its PHD fingers, may be an interesting alternative to targeting of the SET domain, since the reader domains can affect the recruitment of NSD2 to its target loci and its catalytic activity.