Selective Peptidomimetic Inhibitors of NTMT1/2: Rational design, synthesis, characterization, and crystallographic studies

Abstract

Protein N-terminal methyltransferases (NTMTs) methylate the α -N-terminal amines of proteins starting with the canonical X-P-K/R motif. Genetic studies imply that NTMT1 regulates cell mitosis and DNA damage repair. Herein, we report the rational design and development of the first potent peptidomimetic inhibitors for NTMT1. Biochemical and co-crystallization studies manifest that BM30 (IC 50 of 0.89 ± 0.10 µM) is a competitive inhibitor to the peptide substrate and noncompetitive to the cofactor S-adenosylmethionine. BM30 exhibits over 100-fold selectivity to NTMT1/2 among a panel of 41 methyltransferases, indicating the potential to achieve high selectivity when targeting the peptide substrate binding site of NTMT1/2. Its cell-permeable analog DC432 (IC 50 of 54 ± 4 nM) decreases the N-terminal methylation level of SET protein in HCT116 cells. This proof-of principle study provides valuable probes for NTMT1/2 and highlights the opportunity to develop more cell-potent inhibitors to elucidate the function of NTMTs in future.

Structure–Activity Relationship Studies of Venglustat on NTMT1 Inhibition

“…The cocrystallization and structure determination of GD433 with NTMT1 were performed as previously described. 24 To 5 mL of purified NTMT1 (0.9 mg/mL in 1× TBS buffer, 0.036 mM) was added 270 μL (1.5 equiv) of SAM (1 mM in 1× TBS buffer), and the resulting mixture was incubated for 30 min under 4 °C. Then, 20 μL (11.3 equiv) of GD433 (100 mM in ddH 2 O) was diluted 10-folds with 1× TBS buffer, which was added to the previous mixture and incubated at 4 °C for 1 h. The mixture was concentrated to 25−30 mg/mL NTMT1 with Amicon Ultra centrifugal filters (10.0 KDa cutoff MW) at 1800 rpm under 4 °C.…”

“…The data was processed, and the structure was solved as previously described. 24 4.9. Molecular Docking and MM-GBSA Rescoring.…”