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

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.