Suppression of Substance P Biosynthesis in Sensory Neurons of Dorsal Root Ganglion by Prodrug Esters of Potent Peptidylglycine α-Amidating Monooxygenase Inhibitors

Abstract

Substance P as well as many other neuropeptides are synthesized as glycine-extended precursors and converted to the biologically active C-terminal amides by posttranslational modification. The final step of posttranslational processing is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM). In a previous study, N-substituted homocysteine analogs were found to be potent inhibitors of PAM partially purified from conditioned medium of cultured rat medullary thyroid carcinoma CA-77 cells. These compounds, however, were only modest inhibitors of substance P production in cultured dorsal root ganglion cells, possibly because of poor cell penetration. Several ester derivatives of hydrocinnamoyl-phenylalanyl-homocysteine, one of the most potent PAM inhibitors, were prepared to increase the intracellular accessibility of these compounds. Hydrocinnamoyl-phenylalanyl-(S-benzoyl-homocysteine) benzyl ester was identified as the most potent compound, inhibiting substance P biosynthesis in dorsal root ganglion cells with an IC50 of 2 microM. Inhibition of PAM resulted in a concomitant increase in the glycine-extended substance p (substance P-Gly) precursor peptide. In the presence of 3 microM benzyl ester derivative, the intracellular substance P-Gly level was 2.4-fold higher while the substance P level was 2.1-fold lower than the corresponding peptides in control cells. These results suggest that PAM inhibition represents an effective method for suppression of substance P biosynthesis and, therefore, may have therapeutic utility in conditions associated with elevated substance P levels. Furthermore, PAM inhibition may also prove useful in decreasing other amidated peptides.