Methyl Arginine Processing Enzymes: Small Molecule Modulation, Biological Mechanisms and Therapeutic Applications

精氨酸残基的甲基化被越来越多地认为是一种重要的翻译后修饰（PTM）。该反应由蛋白质精氨酸甲基转移酶（PRMT）家族催化，其将精氨酸的胍氮甲基化。已经描述了PRMT的两个大家族：产生不对称二甲基精氨酸（ADMA）的I型和产生对称二甲基精氨酸（SDMA）的II型。这两种类型的PRMT也可以产生单甲基精氨酸（LNMMA），可能作为二甲基化的中间体。最近，由于三个原因，精氨酸甲基化引起了越来越多的兴趣：首先是因为含有甲基精氨酸的蛋白质的水解导致游离ADMA和L-NMMA的产生，这两者都可以抑制一氧化氮合酶（NOS）酶，从而影响细胞内信号传导。其次，因为已经发现精氨酸脱亚胺酶可以将蛋白质中的甲基精氨酸残基代谢为瓜氨酸，因此该修饰是可逆的，并且可能类似于蛋白质磷酸化。第三，加工甲基精氨酸的酶的某些同工型，即二甲基精氨酸二甲氨基水解酶（DDAH）、精氨酸脱亚胺酶（ADI）和肽基精氨酸脱亚胺酶，已经涉及病原菌的基本生化途径。因此，与精氨酸甲基化和去甲基化相关的途径的功能意义似乎相当大，并且可能涉及基本的细胞过程，例如细胞周期控制，以及涉及从癌症到冠心病到细菌感染的疾病过程。基于我们在这一领域的丰富经验，我们建议：申请人建议开展一项总体工作计划来研究这些酶。在这方面，我们将整合化学，生物化学，生物物理学，核磁共振，晶体学，药理学和实验医学的活动，以实现以下目标。(1)为了扩展我们最近的令人兴奋的发现，这些发现已经鉴定了这些酶/此类分子的新型小分子抑制剂/调节剂，其将在多种疾病状态中具有潜在的治疗价值，特别是细菌感染和癌症。迄今为止，我们的活动已经确定了新的小分子结构，选择性地抑制这些酶的人（已发表）或细菌形式（未发表）。我们还确定了细菌酶ADI的第一个小分子抑制剂（未发表）。(2)我们建议使用生物物理技术（Ladbury），晶体学（McDonald）和NMR仔细描述各种新的小分子实体与这些甲基精氨酸加工酶的相互作用的性质。DDAH的NMR研究（Drivel）和ITC（Ladbury）的DDAH研究已经帮助确定了天然和非天然小分子结合的相对位置。此外，两个团队（McDonald / Vallance）解决了DDAH细菌形式的第一个结构，目前拥有与DDAH结合的第一个小分子抑制剂的晶体。(3)我们建议在体内评估这些新的小分子-蛋白质相互作用的相关性。我们将评估它们作为抗菌剂的有效性，它们进入细胞的能力（使用适当标记的实体和各种显微镜技术）以及调节一氧化氮水平的能力，例如在内皮细胞中。

Methylation of arginine residues in proteins is increasingly recognised as an important post-translational modification (PTM). The reaction is catalysed by a family of protein arginine methyltransferases (PRMTs) that methylate the guanidine nitrogens of arginine. Two broad families of PRMTs have been described / type I that generates asymmetric dimethylarginine (ADMA) and type II that generates symmetric dimethylarginine (SDMA). Both types of PRMTs can also generate monomethyl arginine (LNMMA), probably as an intermediate en route to dimethylation. Recently there has been increased interest in arginine methylation for three reasons: first because hydrolysis of proteins containing methylarginines leads to the generation of free ADMA and L-NMMA, both of which can inhibit nitric oxide synthase (NOS) enzymes and thereby influence intracellular signalling. Secondly because it has been found that an arginine deiminase can metabolise methylarginine residues in proteins to citrulline and therefore the modification is reversible and may be analogous to protein phosphorylation. Thirdly certain isoforms of the enzymes which process methylarginines i.e. dimethylarginine dimethylaminohydrolase (DDAH); arginine deiminase (ADI) and peptidyl arginine deiminase have been implicated in basic biochemical pathways of pathogenic bacteria. Thus the functional significance of the pathways related to arginine methylation and demethylation appears considerable and may be implicated in fundamental cellular processes such as cell cycle control as well as being implicated in disease processes ranging from cancer to coronary heart disease to bacterial infection. Building on our extensive experience in this area we propose to:The applicants propose to carry out an overarching programme of work to study these enzymes. In this we will integrate activities in chemistry, biochemistry, biophysics, NMR, crystallography, pharmacology and experimental medicine to achieve the following.(1) To extend our recent exciting findings which have identified novel small molecule inhibitors / modulators of these enzymes / such molecules will have potential therapeutic value in a variety of disease states, in particular bacterial infection and cancer. To date our activities have identified novel small molecule structures which selectively inhibit either the human (published) or bacterial form (unpublished) of these enzymes. We have also identified the first small molecule inhibitors of the bacterial enzyme ADI (unpublished). (2) We propose to carefully delineate the nature of the interaction of a variety of new small molecule entities with these methylarginine processing enzymes using Biophysical techniques (Ladbury), Crystallography (McDonald) and NMR. NMR studies on DDAH (Driscoll) and ITC (Ladbury) studies on DDAH have already helped determine the relative positions of binding of natural and non-natural small molecules. Moreover two of the team (McDonald / Vallance) solved the first structure of the bacterial form of DDAH and currently have crystals of the first small-molecule inhibitors bound to DDAH.(3) We propose to evaluate the relevance of these novel small molecule-protein interactions in vivo. We will evaluate their effectiveness as anti-bacterial agents, their ability to enter cells (using appropriately labelled entities, and a variety of microscopy techniques) and the ability to modulate nitric oxide levels, for example in endothelial cells.