Mechanisms of NMDA Receptor Inhibition by Sepimostat-Comparison with Nafamostat and Diarylamidine Compounds

N-methyl-D-aspartate (NMDA) receptors are inhibited by many amidine and guanidine compounds. In this work, we studied the mechanisms of their inhibition by sepimostat-an amidine-containing serine protease inhibitor with neuroprotective properties. Sepimostat inhibited native NMDA receptors in rat hippocampal CA1 pyramidal neurons with IC50 of 3.5 +/- 0.3 mu M at -80 mV holding voltage. It demonstrated complex voltage dependence with voltage-independent and voltage-dependent components, suggesting the presence of shallow and deep binding sites. At -80 mV holding voltage, the voltage-dependent component dominates, and we observed pronounced tail currents and overshoots evidencing a foot-in-the-door open channel block. At depolarized voltages, the voltage-independent inhibition by sepimostat was significantly attenuated by the increase of agonist concentration. However, the voltage-independent inhibition was non-competitive. We further compared the mechanisms of the action of sepimostat with those of structurally-related amidine and guanidine compounds-nafamostat, gabexate, furamidine, pentamidine, diminazene, and DAPI-investigated previously. The action of all these compounds can be described by the two-component mechanism. All compounds demonstrated similar affinity to the shallow site, which is responsible for the voltage-independent inhibition, with binding constants in the range of 3-30 mu M. In contrast, affinities to the deep site differed dramatically, with nafamostat, furamidine, and pentamidine being much more active.

Automated summary

This study investigated the inhibition mechanism of N-methyl-D-aspartate (NMDA) receptors by sepimostat, an amidine-containing serine protease inhibitor with neuroprotective properties. The results showed that sepimostat inhibits NMDA receptors through a complex voltage-dependent mechanism, with both shallow and deep binding sites. Compared to other structurally-related amidine and guanidine compounds, sepimostat demonstrated similar affinity to the shallow site but lower affinity to the deep site.