Venglustat Inhibits Protein N-Terminal Methyltransferase 1 in a Substrate-Competitive Manner

Abstract

Venglustat is a known allosteric inhibitor for ceramide glycosyltransferase, investigated in diseases caused by lysosomal dysfunction. Here, we identified venglustat as a potent inhibitor (IC50 = 0.42 μM) of protein N-terminal methyltransferase 1 (NTMT1) by screening 58,130 compounds. Furthermore, venglustat exhibited selectivity for NTMT1 over 36 other methyltransferases. The crystal structure of NTMT1-venglustat and inhibition mechanism revealed that venglustat competitively binds at the peptide substrate site. Meanwhile, venglustat potently inhibited protein N-terminal methylation levels in cells (IC50 = 0.5 μM). Preliminary structure–activity relationships indicated that the quinuclidine and fluorophenyl parts of venglustat are important for NTMT1 inhibition. In summary, we confirmed that venglustat is a bona fide NTMT1 inhibitor, which would advance the study on the biological roles of NTMT1. Additionally, this is the first disclosure of NTMT1 as a new molecular target of venglustat, which would cast light on its mechanism of action to guide the clinical investigations.

Structure–Activity Relationship Studies of Venglustat on NTMT1 Inhibition

“…Venglustat competitively binds at the NTMT1 substrate binding site, exhibiting a comparable IC 50 value of 0.5 μM in both biochemical and cellular inhibition assays. 27 As venglustat is also an allosteric inhibitor for ceramide glycosyltransferase (GCS), a comprehensive SAR study is needed to provide a foundation to tune the selectivity for NTMT1 over GCS. As shown before, the N atom of the quinuclidine moiety in the left region is important to the NTMT1 inhibition of venglustat through two hydrogen bonds with Asp177 and Asp180 (Figure 2).…”

“…As shown before, the N atom of the quinuclidine moiety in the left region is important to the NTMT1 inhibition of venglustat through two hydrogen bonds with Asp177 and Asp180 (Figure 2). 27 The middle region does not display any direct interaction with NTMT1 but orients venglustat in a U shape to fit the peptide binding pocket well (Figure 2B). The thiazole moiety connecting the middle and the right regions forms a hydrogen bond with Asp168 and a π−π interaction with Trp136.…”

“…Compounds 1−11 were synthesized following the reported methods (Scheme 1). 27 Starting with 4-fluorobenzothioamide, 1 was efficiently synthesized via a substitution−cyclization reaction. The dimethylation reaction followed by ester hydrolysis produced the intermediate 2, which was then subjected to the Curtius rearrangement to prepare 3− 9.…”

“…Thus, removal of the amine abolished the inhibition. 27 As the quinuclidine portion is a key component for venglustat to inhibit GCS, we attempted to replace it with other amine-containing groups. 30 With a 3piperidine group replacement, 4 failed to inhibit 50% of NTMT1 activity at 100 μM (Table 1).…”

“…20−23 Compared to bisubstrate inhibitors, peptidomimetic inhibitors inhibit both NTMT1 and 2 (NTMT1/2), displaying modest cellular potency in the micromolar range. 24−26 Following up on our recent discovery of venglustat as the first cell-potent and smallmolecule inhibitor of NTMT1 (Figure 1), 27 we describe here our efforts to systematically examine the structure−activity relationship (SAR) of venglustat to boost its potency and selectivity, as well as demonstrating its applicability for inhibiting Nα methylation that is catalyzed by NTMT1/2.…”