Structural optimization, biological evaluation, and application of peptidomimetic prostate specific antigen inhibitors

Abstract

Prostate-specific antigen (PSA) is a serine protease produced at high levels by normal and malignant prostate epithelial cells that is used extensively as a biomarker in the clinical management of prostate cancer. To better understand PSA's role in prostate cancer progression, we prepared a library of peptidyl boronic acid-based inhibitors. To enhance selectivity for PSA vs other serine proteases, we modified the P1 site of the inhibitors to incorporate a bromopropylglycine group. This allowed the inhibitors to participate in halogen bond formation with the serine found at the bottom of the specificity pocket. The best of these Ahx-FSQn(boro)Bpg had PSA Ki of 72 nM and chymotrypsin Ki of 580 nM. In vivo studies using PSA-producing xenografts demonstrated that candidate inhibitors had minimal effect on growth but significantly altered serum levels of PSA. Biodistribution of (125)I labeled peptides showed low levels of uptake into tumors compared to other normal tissues.

Figure 1

Structure of peptide boronic acids with hydrophobic amino acid substituents in the P5 position.

Figure 2

PSA inhibitor blocks PSA binding to serum protease inhibitors and alters PSA blood levels. (A) Western blot analysis of inhibition of PSA complex formation with A2M and ACT. FP = control non-PSA containing female plasma; All mixtures were incubated for overnight at room temperature. (B) PSA measurements using Hybritech assay of 100 ng/mL PSA in RPMI + 1% bovine serum albumin + 1 µM 6 or 1 µM Z-SSKn-OH peptide + 6 or 1 µM 20. (C) Free and Total PSA levels in serum of mice bearing PSA-producing LNCaP prostate cancer xenografts either untreated or receiving 3 courses of 20 at dose of 10 mg/kg for 5 consecutive days/week. (D) Free and Total PSA levels per gram of tumor and ratio of Free/Total PSA in mice treated as in (C).

Figure 3

Evaluation of the activity of inhibitor 20 in presence of bovine serum in the media

Figure 4

In vivo evaluation of 20, Ahx-FSQn(boro)Bpg. Mice bearing subcutaneous PSA-producing LNCaP prostate cancer xenografts were either untreated (Tumor control) or received 3 courses of 20 at dose of 10 mg/kg for 5 consecutive days/week (treatment interval indicated by gray bars).

Figure 5

Evaluation of ¹²⁵I labeled PSA inhibitor in vivo. (A) Blood clearance of [¹²⁵I]SIB-Ahx-NaphSQn(boro)F from mice blood measured over time. Data shows counts per second normalized to starting counts obtained in plasma as 2 h. Four animals were sacrificed at each time point. (B) Biodistribution of [¹²⁵I]SIB-Ahx-NaphSQn(boro)F, shown as percent of injected dose per gram (%ID/gram). Two mice were sacrificed per time point. Tissues were harvested at 2, 4 and 6 h, weighted and counted in gamma counter. Mice had PSA expressing LNCaP xenografts in bilateral flanks. (C) Biodistribution of [¹²⁵I]SIB-Ahx-FSQn(boro)Bpg 26 and [¹²⁵I]SIB-Ahx-FSQn(boro-Pin)Bpg 27, shown as percent of injected dose per gram (%ID/gram). Each drug was injected into two mice and one animal was sacrificed per time point. Tissues were harvested at 2 and 4 h, weighted and counted in gamma counter. Mice had single PSA expressing LNCaP xenografts.

Scheme 1

Synthesis of pinanediol protected phenylalanine boronic acid⁴⁵, (boro-Pin)Phe.

Scheme 2

Schematic illustration of solid phase peptide synthesis and subsequent conjugation to boronic acids; (boro)Phe and (boro)Bpg are used as examples.

Scheme 3

PSA active site interactions with amino acids in P1 – P5 positions of peptide boronic acid 20. Substrate binding pockets on the protease are labeled as S.

Scheme 4

Peptide radiolabeling with [¹²⁵I]SIB and preparation of cold reference.