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.
Highlights
§ Present address: Gensia Inc., San Diego, CA 92121. 1 The abbreviations used are: peptidylglycine ␣-amidating monooxygenase (PAM), peptidylglycine ␣-amidating mogranules and requires copper, ascorbate, and molecular oxygen for activity [2, 6, 7]
Since compounds that contain a charged group, such as a carboxylic acid, frequently exhibit poor cell penetration, we attributed the large difference in the potencies between the in vitro isolated enzyme and the cell assays to the inability of Compound 1 to enter dorsal root ganglion (DRG) cells
This compound only showed a slight improvement in the DRG cell assay; substance P biosynthesis was inhibited by 37% at 10 M (Table I)
Summary
Materials—N-[3-(dimethylamino)propyl]-NЈ-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT) were obtained from Aldrich. The organic layer was washed once with water, twice with 0.1 N HCl, and once with saturated NaHCO3 It was dried over sodium sulfate/magnesium sulfate and filtered, and the solvent was evaporated in vacuo. The resultant solid was dissolved in warm methylene chloride and purified by flash chromatography on silica gel (25% ethyl acetate/hexane) to give 1.1 g (51% overall, 3 steps) of hydrocinnamoyl-L-phenylalanyl-D,L-homocysteine thiolactone as a white solid. After stirring for 30 min at room temperature, the mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and evaporated to give a residue that was subsequently purified by filtration through silica gel (25% ethyl acetate/ether followed by 50% ethyl acetate/ether) to give 101 mg (100%) of product as a white solid (m.p. 118 –121 °C). The substance P-Gly levels in sensory neurons were calculated by subtracting the amount of substance P in samples without PAM treatment from the corresponding samples treated with the enzyme
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