Probing the Plasticity in the Active Site of Protein N-terminal Methyltransferase 1 Using Bisubstrate Analogues

Abstract

The bisubstrate analogue strategy is a promising approach to develop potent and selective inhibitors for protein methyltransferases. Herein, the interactions of a series of bisubstrate analogues with protein N-terminal methyltransferase 1 (NTMT1) were examined to probe the molecular properties of the active site of NTMT1. Our results indicate that a 2-C to 4-C atom linker enables its respective bisubstrate analogue to occupy both substrate- and cofactor-binding sites of NTMT1, but the bisubstrate analogue with a 5-C atom linker only interacts with the substrate-binding site and functions as a substrate. Furthermore, the 4-C atom linker is the optimal and produces the most potent inhibitor (K i,app = 130 ± 40 pM) for NTMT1 to date, displaying more than 3000-fold selectivity for other methyltransferases and even for its homologue NTMT2. This study reveals the molecular basis for the plasticity of the active site of NTMT1. Additionally, our study outlines general guidance on the development of bisubstrate inhibitors for any methyltransferases.

Chemoproteomic study uncovers HemK2/KMT9 as a new target for NTMT1 bisubstrate inhibitors

“…27 Previous studies demonstrated that NAH-C4-GPKRIA and NAH-C4-GPKR bisubstrate analogs containing a 4-C carbon link showed slightly higher potency than those with a 3-C atom linker. 25 However, the NAH-C3-GPKK bisubstrate analogue 1 with the 3-C atom linker (Ki, app = 7 ± 1 nM) exhibited similar inhibition as its 4-C linker analogue 2 (Ki, app = 5.0 ± 0.3 nM) (Table 1). Likewise, the NAH-C3-GPK bisubstrate analogue 3 with a 3-C carbon linker showed comparable inhibitory activity to compound 4 with a 4-C carbon linker, although the Ki, app values of 3 and 4 were increased by 6-8 fold compared to 1 and 2 due to the shortened peptide moiety from a tetrapeptide to a tripeptide.…”

“…The proposed 1-4 were synthesized in a convergent manner by reacting the primary amines (7-8) with α-bromo peptide on resin and followed by acid treatment as described before (Schemes 1). 23,25 Inhibition Evaluation. The inhibitory activities of compounds 1-4 against NTMT1 were evaluated in the SAH hydrolase (SAHH)-coupled fluorescence assay under the condition that both SAM and the peptide substrate GPKRIA were at their respective Km values.…”

“…The co-crystallization and structure determination of compound 1 with NTMT1 were performed as previously described. 24,25 Purified NTMT1 (30 mg/mL) was mixed with 1 at a 1:2 molar ratio, and the mixture was incubated overnight at 4°C. Using the hanging-drop vapor diffusion method and incubation at 20°C, the crystals of 1 were grown in 1.…”

“…[22][23][24][25] Furthermore, NAH-C4-GPKRIA exhibits selective for NTMT1 over its closely related homolog NTMT2, suggesting the unique active site of NTMT1 and high selectivity of NTMT1 bisubstrate inhibitors. 25 While NTMT1 bisubstrate inhibitors exhibit decent selectivity among multiple MTs against isolated enzymes in biochemical assays, there is no information on any other targets that those inhibitors may interact within cellular contexts to understand any off-target engagement and delineate the mechanism of action. Chemoproteomic profiling is a powerful approach to provide a global insight into the cellular targets of an inhibitor in a complex biological environment by utilizing immobilized or photoreactive inhibitors.…”

“…Selectivity Assays. The selectivity studies of G9a, SETD7, PRMT1, PRMT3, TbPRMT7, and SAHH were performed as previously described 24,25,36. MALDI-MS Methylation InhibitionAssay.…”