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 ␣-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 IC 50 of 2 M. Inhibition of PAM resulted in a concomitant increase in the glycine-extended substance p (substance P-Gly) precursor peptide. In the presence of 3 M 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.The C-terminal amide is a prerequisite for full biological activity of many neuropeptides (1). These neuropeptides are typically synthesized as glycine-extended precursors and converted to the mature peptides by a family of enzymes involved in posttranslational modifications, e.g. O-glycosylation, phosphorylation, sulfation, and hydroxylation, as well as in proteolytic processing, e.g. endoproteolysis and exoproteolysis (2). The final step of posttranslational processing is catalyzed by an enzyme originally identified as peptidylglycine ␣-amidating monooxygenase (PAM) 1 (3-5). PAM is localized in secretory granules and requires copper, ascorbate, and molecular oxygen for activity (2, 6, 7). Recent studies have shown that PAM is actually a bifunctional enzyme that contains two distinct enzymatic activities and catalyzes the C-terminal amidation in a sequential manner. The first enzyme, peptidylglycine ␣-hydroxylating monooxygenase (PHM) (EC 1.14.17.3), requires the cofactors for activity and catalyzes the peptidylglycine ␣-hydroxylation reaction while the second enzyme, peptidyl ␣-hydroxyglycine ␣-amidating lyase (PAL) (EC 4.3.2.5), converts the intermediate into an ␣-amidated peptide and glyoxylate (8 -11). Among the glycine-extended neuropeptides examined, glycine-extended substance P (substance P-Gly) has been demonstrated to possess the highest affinity ...

New insights into copper monooxygenases and peptide amidation: structure, mechanism and function

“…Other inhibitors of PHM include benzylhydrazine [78] and N-acetylphenylalanyl acrylate [79]. N-substituted dipeptides with a C-terminal homocysteine are also potent inhibitors of PHM and effectively block production of amidated substance P in cultured dorsal root ganglion neurons [80,81]. Potent inhibitors of PAL include pyruvate-extended N-acetyl amino acids [82] and 2,4-dioxo-5-acetamido-6-phenylhexanoate [79].…”

Inactivation of peptidylglycineα-hydroxylating monooxygenase by cinnamic acid analogs

“…We find that inactivated PHM is indistinguishable from untreated enzyme and find no evidence for cinnamate oxidation during the inactivation reaction. The reversible Michael addition of an active site nucleophile to cinnamate could account for our data and may explain the inactivation of PHM by a variety of acrylates 20,30,35 , and the 2- and 3-alkenoates 32 as well as provide an explanation for the lack of PHM-labeling by 3 H-PBA 37 .…”

“…A large percentage of mammalian bioactive peptides, ~50%, possess an α-amidated C-terminus. As a consequence, a number of pathological conditions correlate to dysfunction in the expression of specific α-amidated peptides or one of the PAM/PHM/PAL proteins, including cancer 17,18 , arthritis 19 , inflammation 20,21 and Alzheimer’s disease 22 . Peptide amidation is important in insects as well because ≥80% of all insect bioactive peptides are α-amidated 23 .…”

Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid

“…These fluorogenic substrates have been intensively used to isolate and characterise novel endopeptidases (Quinto et al, 2000;Morty et al, 2005) and to screen for selective endopeptidase inhibitors (Jeng et al, 1997;Loffler, 2000). Their potential use in the specific measurement of individual enzyme activities in biological tissues or fluids is limited, however, as most of the fluorogenic substrates are cleaved by multiple closely related host enzymes.…”