The Multi-Leu peptide inhibitor discriminates between PACE4 and furin and exhibits antiproliferative effects on prostate cancer cells

Abstract

The proprotein convertases (PCs) play an important role in protein precursor activation through processing at paired basic residues. However, significant substrate cleavage redundancy has been reported between PCs. The question remains whether specific PC inhibitors can be designed. This study describes the identification of the sequence LLLLRVKR, named Multi-Leu (ML)-peptide, that displayed a 20-fold selectivity on PACE4 over furin, two enzymes with similar structural characteristics. We have previously demonstrated that PACE4 plays an important role in prostate cancer and could be a druggable target. The present study demonstrates that the ML-peptide significantly reduced the proliferation of DU145 and LNCaP prostate cancer-derived cell lines and induced G0/G1 cell cycle arrest. However, the ML-peptide must enter the cell to inhibit proliferation. It is concluded that peptide-based inhibitors can yield specific PC inhibitors and that the ML-peptide is an important lead compound that could potentially have applications in prostate cancer.

Figure 1

PC prodomains as PACE4 inhibitors. (a) PCs prodomains were produced and purified to perform inhibition assays toward PACE4 and furin. The ratio between K_i for furin and PACE4, namely the specificity ratio, point out the selectivity of PC7 prodomain toward PACE4. This inhibitor is a 36-fold better inhibitor for PACE4 than furin. K_is in the table are means and standard deviations of three independent experiments. (b) PCs prodomain sequence alignment was performed for the region P7–P1 downstream primary cleavage site. Dark background indicates conserved residues, while light-gray background indicates residues of same type than consensus. Bold letters represent hydrophobic residues. UniProtKB accession numbers are the following: hfurin (P09958), mPC1/3 (P63239), hPC2 (P16519), mPC4 (P2921), hPC5/6 (Q92824), hPC7 (Q16549), and hPACE4 (P29122).

Figure 2

Use of SP-SPCL to profile PC-inhibitor recognition. To better understand the recognition patterns of PACE4 and furin, SP-SPCL was used toward both enzymes. For each sample in this table, the general recognition pattern RVKR is present in positions P1–P4 and P6 position are occupied by a unique amino acid. The other positions of those peptides are occupied by an equimolar mixture (X) of the 19 natural amino acids, cysteine excluded. K_i was calculated from IC₅₀ using the Cheng and Prussof equation for competitive inhibitors. K_is are means and standard deviations of at least two independent experiments.

Figure 3

Multi-Leucine peptides. To stabilize PC–inhibitor interaction, N-terminal leucine extensions were added to the core RVKR sequence. (a,b) Each peptide was assayed with PACE4 and furin in an inhibition assay. (c) K_is in this table are means and standard deviations of three independent experiments. The specificity ratio represents the relative inhibition preference toward PACE4. Peptides Ac-LLLRVKR-NH₂ and Ac-LLLLRVKR-NH₂ were the most potent and the most selective inhibitors of PACE4 of this library. The peptide Ac-LLLLRVKR-NH₂, named Multi-Leu peptide (ML), was selected as lead compound for further studies.

Figure 4

ML-peptide as an inhibitor of prostate cancer cell proliferation. In previous work, our research team proposed PACE4 as a therapeutic target against prostate cancer progression. (a) DU145, LNCaP, and PC3 prostate-cancer derived cell lines were first screened to compare their PCs expression levels using RT-qPCR. PACE4 was highly expressed in DU145 and LNCaP and almost absent from PC3. (b) To assess the efficiency of our new PACE4 inhibitor in such context, MTT assays were performed on those cell lines. ML is efficient to inhibit proliferation and metabolic activity in DU145 and LNCaP, two PACE4-expressing cell lines, indicating a possible role for ML as a prostate cancer therapeutic. (c) To prove that the inhibition observed is a PCs dependent mechanism, the peptide Ac-LLLLRVK[DArg]-NH₂ was used as negative control. Because the P1 position is a key residue of the recognition pattern, the replacement of P1 Arg by DArg significantly affected the K_i for this peptide. (d,e) As expected from inhibition constant values, the peptide Ac-LLLLRVK[DArg]-NH₂ is a poor proliferation inhibitor in a MTT assays with DU145 and LNCaP.

Figure 5

ML-peptide targets intracellular PACE4 to inhibit prostate cancer cell lines proliferation. (a) To determine whether the cell proliferation effects are mediated by cell surface or intracellular PACE4, ML N-terminal extensions were design to modify cell penetration properties of our inhibitor. Neither PEGylation (PEG8) nor alkylation (C8) modified the affinity of Multi-Leucine inhibitor toward PACE4 and furin, as determined in a kinetic assay. (b) The uptake of FITC-labeled peptides was tested on DU145 cells and analyzed by FACS. The FITC-β-Ala-ML-peptide has excellent cell penetration properties. Comparison of GeoMeans (numbers adjacent the peaks) obtained with these three peptides indicates that PEGylation prevents cell entry, whereas alkylation increases the uptake of the inhibitor. (c) Using a MTT metabolic assay, it was clearly demonstrated that alkylation increases inhibitory potency of ML peptide, whereas PEGylation leads to a poor proliferation inhibitor in DU145 cell line. (d) IC₅₀ in the table are means of five independent experiments. N.C. means the curve did not converged to 50% with doses up to 500 μM. This assay demonstrates that ML peptide must enter the cell in order to inhibit DU145 proliferation.

Figure 6

Cell viability and cell cycle analyses of ML-peptide treated LNCaP. The effect of PACE4 inhibition on cell cycle distribution was observed by flow cytometry. LNCaP cells were treated for 96 h with 100 or 200 μM of peptide. (a) Ac-LLLLRVKR-NH₂ or (b) Ac-[DLeu]LLLRVKR-NH₂. Cell cycle distribution was assessed from cellular DNA content analysis of cells treated with propidium iodine. The percentage of cells in each phases were calculated from total living cells. Hypodiploid DNA content (sub-G₁) represents cells undergoing apoptosis. The experiment was done three times in duplicates. The significance of the results were established from an unpaired two-tailed t test. (* p < 0.05; ** p < 0.01; ***p < 0.001). (c) Data in the table are mean and standard deviation of a representative experiment.

Figure 7

Homology model of PACE4. Stereoscopic views of (a) mouse furin crystal catalytic cleft (1P8J) and (b) PACE4 homology model. Asp and Glu negative charges are shown in red, whereas Arg and Lys positive charges are shown in blue. Green spheres represent dicationic calcium ions and decanoyl-RVKR-cmk inhibitor has been modified to Ac-RVKR-cmk for clarity. The homology model was built with Modeler 9v6 on a linux platform. (c) Alignment of hfurin and hPACE4 catalytic domain. Bold letters represent the catalytic triad D–H–S.