Investigation of protein arginine methyltransferase bisubstrate inhibitors

Al-noori, Alaa (2020) Investigation of protein arginine methyltransferase bisubstrate inhibitors. PhD thesis, University of Nottingham.

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Protein arginine methylation is a significant post-translational modification which is catalysed by protein arginine methyltransferases (PRMTs). PRMTs catalyse the transfer of methyl group from the cofactor S-adenosyl-L-methionine (SAM) to arginine residues which are present in proteins. Dysregulation of these enzymes has been associated with cellular diseases such as prostate cancers, cardiovascular disease, and spinal muscular atrophy.

This thesis concerns the design and synthesis of SAM analogues, primarily as PRMT inhibitors, and evaluation of their activities against a selection of these enzymes. Bisubstrate inhibitors were designed to included SAM structure as well as those that mimic the peptidyl arginine substrate. The design of the inhibitors was based on potent and selective inhibitors of PRMTs, which had already been developed within the research group.2,3 The sulfonium centre of SAM was replaced with the more stable nitrogen atom to help produce robust SAM analogues which possess better chemical stability. In this thesis, we introduce a 2-aminopyridine as an isosteric replacement for arginine. Temporary protection of the pyridine nitrogen by complexation with borane was found to be essential for high target inhibitor yields. Additionally, a route for the development of SAM co-factor analogues featuring hydrophobic esters at the 2′,3′-hydroxyls of adenosine was also developed which may enable these compounds to pass through the cell membrane.

PRMT1 and CARM1 were produced which carried a His-tag at the N-terminus, and this His-tag was used to purify the proteins. Preliminary biological evaluation of the inhibitors showed that AzaSAM analogues, which included a 2-amino pyridine as a guanidine isostere were found to be potent inhibitors and were somewhat selective towards CARM1 over PRMT1.

Human CARM1 domain (residues 135-479) was cloned and produced as a maltose-binding protein (MBP) fusion protein with a C-terminus His-tag. Suitable conditions were determined for cleavage of the MBP tag and purification of CARM1 from the resulting mixture.

In order to improve the yield of the CARM1 domain (residues 135-479), it was cloned into a pRSF-13 vector as a 2GKG fusion protein which possessed a TEV cleavage site to allow subsequent removal of the 2GKG tag. CARM1 was successfully co-crystallised with two inhibitor compounds described in this thesis.

Human PRMT6 (residues 28-375 and 39-375-6×His) was cloned and produced with a soluble tag at the N-terminus and a His-tag at the C-terminus. PRMT6 (residue 28-375-6×His) was separated from contaminating chaperonin proteins and was shown to be activated via an in vitro methylation assay.

Moreover, full-length human PRMT2 (residues 1-431-6×His) and a slightly shorter PRMT2 construct (residues 95-431-6×His) were produced and purified using the His-tag present at the C-terminus.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Dowden, James
Dreveny, Ingrid
Keywords: proteins, co-factors, PRMTinhibitors
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Chemistry
Related URLs:
Item ID: 59769
Depositing User: Al-noori, Alaa
Date Deposited: 18 Oct 2023 14:34
Last Modified: 18 Oct 2023 14:34

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