title: Improving the Specificity of the Prostate-Specific Antigen Substrate Glutaryl-Hyp-Ala-Ser-Chg-Gln as a Promoiety.
abstract: To develop PSA peptide substrates with improved specificity and plasma stability from the known substrate sequence glutaryl-Hyp-Ala-Ser-Chg-Gln, systematic replacements of the N-terminal segment with D-retro-inverso-peptides were performed with the incorporation of 7-amino-4-methylcoumarin (7-AMC) after Gln for convenient fluorometric determination and ranking of the PSA substrate activity. The D-retro-inverso-peptide conjugates with P2-P5 D-amino acid substitutions were moderate but poorer PSA substrates as compared to the original peptide, suggesting that inversion of the amide bonds and/or incorporation of the additional atom as in the urea linker adversely affected PSA binding. However, P5 substitution of Hyp with Ser showed significant improvements in PSA cleavage rate; the resulting AMC conjugate, glutaryl-Ser-Ala-Ser-Chg-Gln-AMC (11), exhibited the fastest PSA cleavage rate of 351 pmol/min/100 nmol PSA. In addition, GABA←mGly-Ala-Ser-Chg-Gln-AMC (conjugate 6) was the second best PSA substrate and released 7-AMC at a rate of 225 pmol/min/100 nmol PSA as compared to 171 pmol/min/100 nmol PSA for the control conjugate glutaryl-Hyp-Ala-Ser-Chg-Gln-AMC. Incubations of selected AMC conjugates with mouse and human plasma revealed that GABA←D-Ser-ψ[NH-CO-NH]-Ala-Ser-Chg-Gln-AMC (5) and GABA←mGly-Ala-Ser-Chg-Gln-AMC (6) were most stable to non-PSA-mediated proteolysis. Our results suggest that the PSA specificity of glutaryl-Hyp-Ala-Ser-Chg-Gln is improved with Ser and mGly substitutions of Hyp at the P5.
authors.affiliation: Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.
authors.forename: Herve
authors.initials: H
authors.lastname: Aloysius
authors.affiliation: Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.|School of Pharmaceutical Sciences, Shanxi Medical University, Taiyuan, 030001, China.|The Cancer Institute of New Jersey, New Brunswick, NJ, 08901, USA.
authors.forename: Longqin
authors.initials: L
authors.lastname: Hu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057075:Enzyme Assays; D006801:Humans; D008297:Male; D051379:Mice; D010455:Peptides / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D017430:Prostate-Specific Antigen / Q000097:blood / Q000378:metabolism*; D059748:Proteolysis; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D017430:Prostate-Specific Antigen
keywords: 7-amino-4-methylcoumarin; D-retro-inverso-peptide; prostate-specific antigen