Protoporphyrin IX enhancement by 5-aminolaevulinic acid peptide derivatives and the effect of RNA silencing on intracellular metabolism

Abstract

Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide derivatives of the haem precursor, 5-aminolaevulinic acid (ALA), was investigated in transformed PAM212 murine keratinocytes, together with studies of their intracellular metabolism. Porphyrin production was substantially increased compared with equimolar ALA using N-acetyl terminated phenylalanyl, leucinyl and methionyl ALA methyl ester derivatives in the following order: Ac-L-phenylalanyl-ALA-Me, Ac-L-methionyl-ALA-Me and Ac-L-leucinyl-ALA-Me. The enhanced porphyrin production was in good correlation with improved photocytotoxicity, with no intrinsic dark toxicity apparent. However, phenylalanyl derivatives without the acetyl/acyl group at the N terminus induced significantly less porphyrin, and the replacement of the acetyl group by a benzyloxycarbonyl group resulted in no porphyrin production. Porphyrin production was reduced in the presence of class-specific protease inhibitors, namely serine protease inhibitors. Using siRNA knockdown of acylpeptide hydrolase (ACPH) protein expression, we showed the involvement of ACPH, a member of the prolyl oligopeptidase family of serine peptidases, in the hydrolytic cleavage of ALA from the peptide derivatives. In conclusion, ALA peptide derivatives are capable of delivering ALA efficiently to cells and enhancing porphyrin synthesis and photocytotoxicity; however, the N-terminus state, whether free or substituted, plays an important role in determining the biological efficacy of ALA peptide derivatives.

The Use of Dipeptide Derivatives of 5-Aminolaevulinic Acid Promotes Their Entry to Tumor Cells and Improves Tumor Selectivity of Photodynamic Therapy

“…It is worth noting here that the amino acid residues that are usually found at the N-terminus of acetylated proteins include Ac-Met-, Ac-Ala-, and Ac-Ser- (61). This is in accordance with our previous results showing that one of our better performing ALA dipeptides was Ac-Met-ALA-Me (39).…”

“…Ac-Leu-and Ac-Phe-ALA-dipeptides are even better PpIX producers than Ac-Met-ALA-Me (39). The efficient release of ALA from Ac-Leu-and Ac-Phe-, however, appears consistent with the reported substrate and inhibitor preferences of APEH, which favors N-acetylated substrates with hydrophobic amino acid residues, and small-molecule inhibitors with aromatic substituents as side chain mimics.…”

“…N-acetyl termination appears to play an important role in metabolic processes leading to the production of PpIX from such compounds, whereas masking the C-terminus as a methyl ester does not exert a major effect (40). Using siRNA knockdown of acylaminoacyl peptide hydrolase (APEH) protein expression, we were able to show the involvement of APEH, a member of the prolyloligopeptidase family of serine peptidases, in the release of ALA from these N-acetylated peptide derivatives (39). APEH has been shown to be expressed in various normal tissues such as erythrocytes, liver, heart, testis, intestine, kidney, and brain, but its precise biologic activity is unknown (59)(60)(61)(62).…”

“…Indeed, prodrugs containing a D-amino acid component are incorporated but not processed, whereas in cell lines such as A549 epithelial carcinoma that have a low expression of acyl-peptide hydrolase (a protease shown to release ALA from these compounds; ref. 39), efficient prodrug uptake, but low ALA release and PpIX production were observed. These encouraging in vitro results and studies in skin explant models (40) highlight the possibility to design peptide prodrugs of this type that can be used to target disease-specific levels of a given enzymatic activity to provide selective ALA release.…”

Comparison of pharmacokinetics, intracellular localizations and sonodynamic efficacy of endogenous and exogenous protoporphyrin IX in sarcoma 180 cells

“…ALA itself is not a sensitizer and serves as the biological precursor in the haem biosynthesis pathway that is regulated by feedback inhibition in all nucleated cells [3]. Administration of exogenous ALA bypasses the negative feedback and induces preferential formation of endogenous PpIX (endo-PpIX) in tumor cells due to the lower ferrochelatase activity and the relatively higher activity of porphobilinogen deaminase in malignant tissues compared with that of normal cells [4,5].…”