Development of chemical probes for NSD2 and its PHD fingers

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，这是细胞分化和生理功能所必需的。表观遗传修饰，如组蛋白翻译后修饰(PTM)，在染色质浓缩和基因表达调控中起着重要作用，表观遗传蛋白的异常表达经常与多种疾病的发展有关，如癌症和自身免疫性疾病。NSD2属于核受体结合集合域(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H3K36的二甲基化，影响染色质的可及性和细胞凋亡、DNA修复和细胞黏附中关键调控因子的基因表达。历史上，NSD2因其与Wolf-Hirschhorn综合征(WHS)的联系而闻名，其含有NSD2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。NSD2在几种癌症(如乳腺癌、胶质母细胞瘤、前列腺癌、肺癌等)中也有过表达。它与肿瘤的侵袭性和预后不良有关，尤其在多发性骨髓瘤(MM)和急性淋巴细胞白血病(ALL)的发生发展中起着至关重要的作用。特别是，t(4；14)易位的MM细胞表现出NSD2的异常表达，导致基因表达的活跃标志H3K36me2水平增加，抑制标志H3K27me3降低。这涉及到细胞生长和黏附特性的改变，在使用shRNA敲除NSD2亚型后，这些特性就消失了。因此，抑制NSD2可能被认为是一种可能的癌症干预策略，以开发新的治疗方法，并了解NSD2的作用。从结构上讲，NSD2由一个催化集结构域、一个高迁移率基团(HMG)盒、2个PRWP(Pro-Tryptophan-Tryptophan-Proline)结构域和4个PHD(植物同源结构域)锌指组成。对NSD2的抑制可以通过靶向SET结构域或变构结合位点来实现。遗憾的是，NSD2的小分子抑制剂还很少，如N-烷基辛芬净、LEM-06和LEM-14，它们的选择性和效力都很低。利用NSD2进行基于结构的药物设计的主要问题是难以结晶催化集结构域。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分析开发的阻碍，到目前为止只有核小体底物已知。为了克服其中的一些困难，尝试新的靶向NSD2的方法可能是有益的，例如使用环肽或靶向NSD2的读取域。2)本项目的目的本项目的目的是开发环肽作为NSD2的化学探针，并进一步了解其PhD手指在调节NSD2活性方面的作用。使用环肽可能是一种很有前途的方法，可以为像NSD2这样的不能下药的靶标开发化学探针/抑制剂，因为它们能够在更大的表面积上与靶标相互作用。此外，靶向NSD2的阅读器结构域，特别是它的PhD指，可能是靶向SET结构域的一个有趣的替代方案，因为阅读器结构域可以影响NSD2向其靶点的招募及其催化活性。

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.