Design, synthesis, and kinetic analysis of potent protein N-terminal methyltransferase 1 inhibitors

Abstract

The protein N-terminal methyltransferase 1 (NTMT1) methylates the α-N-terminal amines of proteins. NTMT1 is upregulated in a variety of cancers and knockdown of NTMT1 results in cell mitotic defects. Therefore, NTMT1 inhibitors could be potential anticancer therapeutics. This study describes the design and synthesis of the first inhibitor targeting NTMT1. A novel bisubstrate analogue (NAM-TZ-SPKRIA) was shown to be a potent inhibitor (Ki = 0.20 μM) for NTMT1 and was selective versus protein lysine methyltransferase G9a and arginine methyltransferase 1. NAM-TZ-SPKRIA was found to exhibit a competitive inhibition pattern for both substrates, and mass spectrometry experiments revealed that the inhibitor substantially suppressed the methylation progression. Our results demonstrate the feasibility of using a triazole group to link an S-adenosyl-L-methionine analog with a peptide substrate to construct bisubstrate analogues as NTMT1 potent and selective inhibitors. This study lays a foundation to further discover small molecule NTMT1 inhibitors to interrogate its biological functions, and suggests a general strategy for the development of selective protein methyltransferase inhibitors.

The ribosome: A hot spot for the identification of new types of protein methyltransferases

“…METTL11B was suggested to be primarily a monomethyltransferase that may prime substrates for the action of NTMT1 (55). Crystal structures of human NTMT1 in complex with substrate peptides have been solved (56,57) that support the substrate specificity of this enzyme determined from kinetic (58,59) and inhibitor studies (60). A variety of functions have been proposed for XPK N-terminal methylation of eukaryotic proteins (61), including regulating the affinity of protein binding to DNA (62,63), DNA repair (64-66) and protection from aminopeptidase attack (49).…”

Facile synthesis of SAM–peptide conjugates through alkyl linkers targeting protein N-terminal methyltransferase 1

“…29,30 So far, there is only one specific inhibitor available for this enzyme, which was synthesized through a click chemistry and reported by our laboratory. 24 To explore a new scaffold, we initiated our efforts to design a series of SAM–peptide conjugates by linking a SAM analog with peptides that start with either Ala or Gly through an alkyl group as our model system. Our crystal structure suggested that the distance between the sulfonium ion and the α- N -terminal nitrogen atom is 4.7 Å 30 And for most protein methyltransferases, this distance varies from 2.2 Å to 4.7 Å 29–33 Hence, we chose an ethylene or a propylene group as a linker and designed a series of compounds 1a–f targeting NTMT1 with substrate peptides that start with GPK/R.…”

“…At 30 μM, it did not show any significant inhibition of either G9a or PRMT1. We also examined how compound 1c affects the progression of α- N -amine methylation at 5 μM by MALDI-MS. 9,24,41 Triplicate samples of RCC1-10 peptide (SPKRIAKRRS) along with compound 1c were subjected to NTMT1 methylation assays. Following these assays, samples were analyzed at 20 min to monitor the methylation progression.…”

Design of a fluorescent ligand targeting the S-adenosylmethionine binding site of the histone methyltransferase MLL1

“…Compound 1 was obtained with high yield by condensation between the commercially available 5-FAM and the 2-azido-ethanamine with the coupling reagents HOBt and EDCI in anhydrous DCM. The synthesis of compound 2 to 5 started from 2′,3′- O -isopropylideneadenosine and followed the modified procedures reported in the literature 24, 25 . The sequential azidation and reduction of 2′,3′- O -isopropylideneadenosine yielded compound 3 .…”

“…The spectral data of compound 1 - 5 in this manuscript are consistent with the literature reports. 24, 25, 27 …”