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.

Intracellular Activation of a Prostate Specific Antigen-Cleavable Doxorubicin Prodrug: A Key Feature Toward Prodrug-Nanomedicine Design

“…Despite the initially promising results, Dox-PSA did not advance beyond phase I clinical trials. The reasons behind this were likely toxicity concerns as it was shown that in four different species of laboratory animals there was approximately 30-40% non-PSA-mediated prodrug conversion into the active metabolite Dox 13,14 . Aiming at increasing PSA specificity, Aloysius et al modified the initial peptide sequence, Glutaryl-Hyp-Ala-Ser-Chg-Gln, by introducing modifications at position P5, which improved PSA hydrolysis rate and decreased non-PSA-specific cleavage 13 .…”

“…Several studies, have used a wide range of liposomes to efficiently deliver a variety of prodrugs, including a number of anticancer agents 36,37 , phospholipid prodrugs 38,39 , antiinflammatory prodrugs 40 and β-blockers prodrugs 41 . Interestingly, previous reports with PSA-cleavable prodrugs have relied on the enzymatically active extracellular PSA to activate the prodrug 13,15,20,42,43 . As a proof-of-concept, in this work we used pH-sensitive liposomes composed of DOPE:CHEMS to ensure a fast and efficient Dox-PSA release inside the acidified endosomes, which is essential for prodrug activation.…”

“…Aiming at improving the specificity of Glutaryl-Hyp-Ala-Ser-Chg-Gln as a PSAcleavable promoiety, Aloysius et al have recently designed the sequences Glutaryl-Ser-Ala-Ser-Chg-Gln and GABA-mGly-Ala-Ser-Chg-Gln. The authors have shown that while substitutions between P1 and P4 would adversely impact on PSA specificity, the given modifications at P5 improved PSA-mediated hydrolysis rate and decreased non-PSA-specific cleavage 13 . In the present work, we sought to explore a versatile approach where, rather than altering Dox-PSA prodrug design, we relied on its encapsulation inside a delivery system capable of improving Dox-PSA enzymatic stability.…”

“…A direct consequence of this is that our proposed approach would necessarily rely on Dox-PSA intracellular activation. However, to the best of our knowledge, previous studies with PSA-cleavable prodrugs have invariably grounded on the extracellular activation of the prodrug due to the secretory nature of PSA enzyme and the notion that high intracellular zinc concentrations inhibit PSA activity 12,13,20 . Yet, the dysregulation of several zinc transporters has been associated with low intracellular zinc levels in malignant prostate epithelial cells 21,22 .…”

“…Other PSA substrates were developed comprising non-natural amino acids in their peptide sequence in order to increase the chemical stability of the peptide–drug conjugate and mitigate hydrolysis by non-PSA proteases. One such example is the peptide–drug conjugate glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox (L-377,202- hereafter called Dox-PSA), which, despite having reached the clinical setting for mCRPC, did not advance beyond phase I trials due to toxicity concerns. , Although in preclinical studies in mice, rats, dogs, and monkeys, Dox-PSA selectively accumulated in the tumor, and the concentrations of Dox in the cardiac tissue (site of clinical dose-limiting toxicity) were much lower than those achieved when Dox was administered alone; there was 30–40% of non-PSA-specific metabolism of Dox-PSA . Moreover, while in the preclinical setting administration of the prodrug into nude mice bearing LNCaP xenografts greatly improved the therapeutic index, with 8- to 9-fold molar increase in the dose of Dox administered, the clinical scenario was largely dissimilar, where only 1.5-fold more molar equivalent of Dox could be administered.…”