Structural and biochemical evaluation of bisubstrate inhibitors of protein arginine N-methyltransferases PRMT1 and CARM1 (PRMT4).

Emma A Gunnell,Ingrid Dreveny,Clare C Davies,Usama Muhsen,James Dowden,Alaa Al-Noori

doi:10.1042/bcj20190826

Abstract

Attenuating the function of protein arginine methyltransferases (PRMTs) is an objective for the investigation and treatment of several diseases including cardiovascular disease and cancer. Bisubstrate inhibitors that simultaneously target binding sites for arginine substrate and the cofactor (S-adenosylmethionine (SAM)) have potential utility, but structural information on their binding is required for their development. Evaluation of bisubstrate inhibitors featuring an isosteric guanidine replacement with two prominent enzymes PRMT1 and CARM1 (PRMT4) by isothermal titration calorimetry (ITC), activity assays and crystallography are reported. Key findings are that 2-aminopyridine is a viable replacement for guanidine, providing an inhibitor that binds more strongly to CARM1 than PRMT1. Moreover, a residue around the active site that differs between CARM1 (Asn-265) and PRMT1 (Tyr-160) is identified that affects the side chain conformation of the catalytically important neighbouring glutamate in the crystal structures. Mutagenesis data supports its contribution to the difference in binding observed for this inhibitor. Structures of CARM1 in complex with a range of seven inhibitors reveal the binding modes and show that inhibitors with an amino acid terminus adopt a single conformation whereas the electron density for equivalent amine-bearing inhibitors is consistent with preferential binding in two conformations. These findings inform the molecular basis of CARM1 ligand binding and identify differences between CARM1 and PRMT1 that can inform drug discovery efforts.

Highlights

Protein arginine methyltransferases (PRMTs, E.C. 2.1.1.125) catalyse the transfer of methyl groups from S-adenosylmethionine (SAM) to arginine residues of target proteins, giving a pattern of nitrogen methylations dictated by enzyme type (Figure 1A)
No such trend was observed for PRMT1, with inhibitors displaying similar inhibitor concentration giving half maximal inhibition (IC50) values for PRMT122–361 (7.2–11.8 mM) as co-activator associated arginine methyltransferase 1 (CARM1)
The resultant dissociation constant (Kd) values showed a similar pattern to the IC50 values, with all inhibitors binding with low micromolar affinity to both PRMT1 and CARM1 and again, CARM1 displayed a slight preference for a shorter alkyl linker length (Table 1)

Summary

Introduction

Protein arginine methyltransferases (PRMTs, E.C. 2.1.1.125) catalyse the transfer of methyl groups from S-adenosylmethionine (SAM) to arginine residues of target proteins, giving a pattern of nitrogen methylations dictated by enzyme type (Figure 1A). Promising CARM1 inhibitors include EPZ022302/EZM2302 (a 6 nM CARM1 inhibitor with anti-tumour activity in vivo) [23] and TP-064 (IC50 < 10 nM against CARM1 and >100-fold selective over other methyltransferases) [32] In both cases, ternary crystal structures with CARM1-SAH showed the inhibitor to occupy the substrate-binding pocket with kinetic data supporting noncompetitive inhibition with respect to both SAM and peptide substrate [23,32]. Extending the guanidinium group to a CARM1 substrate peptide achieved a greater degree of selectivity for CARM1 over PRMT1 (IC50 values of 0.09 and 25.5 mM, respectively for the most selective compound) [28] Whilst promising in their apparent selectivity, a key drawback of such peptide-fused mimics is their potential susceptibility to proteolysis, which limits their utility in cellular assays and in vivo applications. The influence of a specific active site residue on the orientation of the catalytic glutamate and inhibitor binding was evaluated with CARM1 N265Y mutant protein; crystal structures revealed that this mutation affects the conformation of key residues at the substrate-binding site

Experimental procedures

Results

Discussion