Enzymatic stability, solution structure, and antiproliferative effect on prostate cancer cells of leuprolide and new gonadotropin-releasing hormone peptide analogs

Abstract

Analogs of GnRH, including [DLeu6, desGly1o]-GnRH-NHEt (leuprolide, commercial product), have been widely used in oncology to induce reversible chemical castration. Several studies have provided evidence that, besides their pituitary effects, GnRH analogs may exert direct antiproliferative effects on tumor cells. To study the effect of modifications in positions 4 and 6 of leuprolide on prostate cancer cell proliferation, we synthesized 12 new leuprolide analogs. All GnRH analogs lacked the carboxy-terminal Gly10-amide of GnRH, and an ethylamide residue was added to Pro9. Gly6 was substituted by DLys, Nepsilon-modified DLys, Glu, and DGlu. To improve the enzymatic stability, NMeSer was incorporated in position 4, and the rate of hydrolysis by alpha-chymotrypsin and subtilisin was investigated. Our results demonstrate that this incorporation increases enzymatic stability in all analogs of GnRH, whereas the antiproliferative effect on PC3 and LNCaP prostate cancer cells is similar to that of leuprolide. Conformational studies were performed to elucidate structural changes occurring on substitution of native residues and to study structure-activity relationship for these analogs. The solution models of [DLeu6, desGly10]-GnRH-NHEt (leuprolide), [NMeSer4, DGlu6, desGly10]-GnRH-NHEt, [Glu6, desGly10]-GnRH-NHEt, and [DGIu6, desGly10]-GnRH-NHEt peptides were determined through two-dimensional nuclear magnetic resonance spectroscopy in dimethylsulfoxide. Nuclear magnetic resonance data provide experimental evidence for the U-turn-like structure appeared in all four analogs, which could be characterized as beta-hairpin conformation. The most stable analog [NMeSer4, DGlu6, desGly10]-GnRH-NHEt against proteolytic cleavage forms a second extra backbone turn observed for residues 1-4.

Metabolism of peptide reporters in cell lysates and single cells

“…Since both N-methylated and D-amino acids have been shown to impart stability to inhibitor peptides, D-phenylalanine and (N-methyl)phenylalanine were also incorporated as replacements for phenylalanine. 27, 35, 36, 38 Peptides QW-IV-85B through QW-V-23E (Table 1) were synthesized, purified, and then incubated in the Baf/BCR-ABL cytosolic lysate for varying times. Aliquots were removed and electrophoresed to assess the formation of fluorescent fragments.…”

“…This strategy has been particularly successful in stabilizing inhibitor peptides. 30, 33–38 Extensive libraries of non-native amino acids provide numerous possibilities for the construction of selective inhibitors or substrates, as demonstrated by Lee et al . in 2004.…”

Inhibition of cell adhesion and immune responses in the mouse model of collagen‐induced arthritis with a peptidomimetic that blocks CD2‐CD58 interface interactions

“…Since in vivo studies were conducted in a mouse model, use of mouse serum is appropriate. The stability of compound 7 was assessed by comparing the intensity of the peak in the mass spectrum with that of the freshly prepared leuprolide that was used as internal standard. The recovery of compound 7 from mouse serum was detectable at all time points, and the half‐life for compound 7 in mouse serum was estimated to be around 2 h (Figure ).…”

“…MALDI‐TOF MS measurements were performed on Ultraflextreme (Bruker Daltonics, Billerica, MA). Leuprolide (Sigma‐Aldrich) was used as an internal standard (molecular weight 1209) . The relative intensity of the peak vs. time was plotted.…”

Evaluation of the Biological Properties and the Enzymatic Stability of Glycosylated Luteinizing Hormone-Releasing Hormone Analogs

“…Medium-to-high affinity binding sites were also reported for LHRH agonists in LHRH receptor-positive prostate cancer cells (23). The agonists exhibited their antiproliferative effect in a doseand time-dependent manner (24). LHRH agonists and antagonists were shown to reduce the enzymatic activity of the plasminogen activator system in prostate cancer cells leading to a decrease in the migration and invasiveness of cancer cells (25).…”