PACE4 Inhibitors Research Articles

Enhancing the Drug-Like Profile of a Potent Peptide PACE4 Inhibitor by the Formation of a Host-Guest Inclusion Complex with β-Cyclodextrin.

The enzyme PACE4 has been validated as a promising therapeutic target to expand the range of prostate cancer (PCa) treatments. In recent years, we have developed a potent peptidomimetic inhibitor, namely, compound C23 (Ac-(DLeu)LLLRVK-4-amidinobenzylamide). Like many peptides, C23 suffers from an unfavorable drug-like profile which, despite our efforts, has not yet benefited from the usual SAR studies. Hence, we turned our attention toward a novel formulation strategy, i.e., the use of cyclodextrins (CDs). CDs can benefit compounds through the formation of "host-guest" complexes, shielding the guest from degradation and enhancing biological survival. In this study, a series of βCD-C23 complexes have been generated and their properties evaluated, including potency toward the enzyme in vitro, a cell-based proliferation assay, and stability in plasma. As a result, a new βCD-formulated lead compound has been identified, which, in addition to being more soluble and more potent, also showed an improved stability profile.

Enhanced anti-tumor activity of the Multi-Leu peptide PACE4 inhibitor transformed into an albumin-bound tumor-targeting prodrug

The proprotein convertase PACE4 has been validated as a potential target to develop new therapeutic interventions in prostate cancer (PCa). So far, the most effective compound blocking the activity of this enzyme has been designed based on the structure of a small peptide Ac-LLLLRVKR-NH2 known as the Multi-Leu (ML) peptide. Optimization of this scaffold led to the synthesis of compound C23 (Ac-[DLeu]LLLRVK-amidinobenzylamide) with a potent in vivo inhibitory effect on the tumor growth. However, further developments of PACE4 inhibitors may require additional improvements to counter their rapid renal clearance and to increase their tumor targeting efficiency. Herein, we explored the transformation of the ML-peptide into an albumin-binding prodrug containing a tumor specific release mechanism based on the prostate-specific antigen. Our data confirms that intravenous treatment using the ML-peptide alone has little effect on tumor growth, whereas by using the ML-prodrug in LNCaP xenograft-bearing mice it was significantly reduced. Additionally, excellent in vivo stability and tumor-targeting efficiency was demonstrated using a radiolabelled version of this compound. Taken together, these results provide a solid foundation for further development of targeted PACE4 inhibition in PCa.

Development of Novel DNA-Encoded PCSK9 Monoclonal Antibodies as Lipid-Lowering Therapeutics.

Elevated low-density lipoprotein cholesterol (LDL-C) is one of the major contributors to cardiovascular heart disease (CHD), the leading cause of death worldwide. Due to severe side effects of statins, alternative treatment strategies are required for statin-intolerant patients. Monoclonal antibodies (mAbs) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) have shown great efficacy in LDL-C reduction. Limitations for this approach include the need for multiple injections as well as increased costs associated with patient management. Here, we engineered a DNA-encoded mAb (DMAb) targeting PCSK9 (daPCSK9), as an alternative approach to protein-based lipid-lowering therapeutics, and we characterized its expression and activity. A single intramuscular administration of mouse daPCSK9 generated expression in vivo for over 42 days that corresponded with a substantial decrease of 28.6% in non-high-density lipoprotein cholesterol (non-HDL-C) and 10.3% in total cholesterol by day 7 in wild-type mice. Repeated administrations of the DMAb plasmid led to increasing expression, with DMAb levels of 7.5 μg/mL at day 62. daPCSK9 therapeutics may provide a novel, simple, less frequent, cost-effective approach to reducing LDL-C, either as a stand-alone therapy or in combination with other LDL-lowering therapeutics for synergistic effect.

Increasing C-Terminal Hydrophobicity Improves the Cell Permeability and Antiproliferative Activity of PACE4 Inhibitors against Prostate Cancer Cell Lines.

The serine protease, PACE4, is a proprotein convertase that plays a substantial role in malignancy of prostate cancer. Our initial selective PACE4 inhibitor (Ac-LLLLRVKR-NH2) has evolved to the current lead compound C23 (Ac-dLeu-LLLRVK-Amba), which is active both in vitro and in vivo. By screening natural residues, except Cys, in C-terminal P1' position, it was established that increasing hydrophobicity was improving cell permeability, which was directly translated into PCa cells antiproliferative activity. This cell antiproliferation enhancement seems independent from effect of P1' residue on PACE4 affinity. Replacement of P1-Amba of C23 by Acpa (( S)-2-amino-3-(4-carbamimidoylphenyl)propanoic acid) followed by addition of tryptamine in P1' resulted in compound 32 exhibiting superior PCa cells antiproliferative activity over the reference compound C23 (3-fold). This study sheds light on key factors that improve cell penetrating property and antiproliferative activity of PACE4 inhibitors.

Rational Design of a Highly Potent and Selective Peptide Inhibitor of PACE4 by Salt Bridge Interaction with D160 at Position P3.

PACE4, a member of the proprotein convertases (PCs) family of serine proteases, is a validated target for prostate cancer. Our group has developed a potent and selective PACE4 inhibitor: Ac-LLLLRVKR-NH2 . In seeking for modifications to increase the selectivity of this ligand toward PACE4, we replaced one of its P3 Val methyl groups with a basic group capable of forming a salt bridge with D160 of PACE4. The resulting inhibitor is eight times more potent than the P3 Val parent inhibitor and two times more selective over furin, because the equivalent salt bridge with furin E257 is not optimal. Moreover, the β-branched nature of the new P3 residue favors the extended β-sheet conformation usually associated with substrates of proteases. This work provides new insight for better understanding of β-sheet backbone-backbone interactions between serine proteases and their peptidic ligands.

Macrocyclization of a potent PACE4 inhibitor: Benefits and limitations

PACE4, one of the seven members of the proprotein convertase family, plays an important role in the progression of prostate cancer. Therefore, its inhibition has become an attractive target to develop new therapies against this disease. Recently, we have developed a highly potent and selective PACE4 inhibitor, known as the multi-Leu peptide with the following sequence Ac-LLLLRVKR-NH2. Herein, with the aim of improving the stability profile of this inhibitor for potential in vivo application, we investigated the impact of different cyclization strategies. The inhibitory activity of new peptides was tested and compared to their linear counterparts. The potent analogues were further selected for stability evaluation. Our results showed that the cyclization involving a C-terminal carboxylic acid (head-to-tail or side chain-to-tail) led to compounds with significantly diminished inhibitory potency towards PACE4, indicating that an appropriate balance between rigidity and flexibility of the structure is necessary to allow the optimal binding with the enzyme. On the other hand, the modification within a multi-Leu core in combination with the incorporation of a C-terminal 4-amidinobenzylamide (Amba) residue yielded potent cyclic analogues. The best compound derived from this group, (&)[Mpa]LLLC(&)RVK[Amba] (where & indicates cyclization, Mpa – 3-mercaptopropionic acid), exhibited promising overall profile comprising of potent inhibitory effect against PACE4 and prostate cancer cell lines as well as improved stability. We believe that this cyclic framework could be further used to design even more potent and stable PACE4 inhibitors.

Positional Scanning Identifies the Molecular Determinants of a High Affinity Multi-Leucine Inhibitor for Furin and PACE4

The proprotein convertase family of enzymes includes seven endoproteases with significant redundancy in their cleavage activity. We previously described the peptide Ac-LLLLRVK-Amba that displays potent inhibitory effects on both PACE4 and prostate cancer cell lines proliferation. Herein, the molecular determinants for PACE4 and furin inhibition were investigated by positional scanning using peptide libraries that substituted its leucine core with each natural amino acid. We determined that the incorporation of basic amino acids led to analogues with improved inhibitory potency toward both enzymes, whereas negatively charged residues significantly reduced it. All the remaining amino acids were in general well tolerated, with the exemption of the P6 position. However, not all of the potent PACE4 inhibitors displayed antiproliferative activity. The best analogues were obtained by the incorporation of the Ile residue at the P5 and P6 positions. These substitutions led to inhibitors with increased PACE4 selectivity and potent antiproliferative effects.

PACE4 is an important driver of ZR-75-1 estrogen receptor-positive breast cancer proliferation and tumor progression

Breast cancer is the most frequent and deadly malignancy in women worldwide. Despite national screening programs combined with new treatments relapse rate remain high and new therapies are needed. From previous work, we identified PACE4, a member of the proprotein convertase (PCs) family of endoproteases, as a novel therapeutic target in prostate cancer. In the present study we asked the question if PACE4 could also be a potential target in breast cancer. In clinical samples of breast adenocarcinoma, we observed a specific overexpression of PACE4 in the estrogen-receptor (ER) positive subtype. We therefore looked for a breast cancer cell line model which would be representative and thus focused on the ZR-75-1 since it both expresses PACE4 and is estrogen-receptor positive. We compared stable knockdowns of furin, PACE4 and PC7 in the estrogen-receptor-positive cell line ZR-75-1 to evaluate their respective contribution to cell growth and tumor progression. PACE4 was the only PC displaying an impact on cell growth. A PACE4 peptide-based inhibitor (C23) was tested and shown to decrease proliferation of ZR-75-1 cells in cell based assays. C23 also had potent effects of tumor progression in vivo on xenografts of the ZR-75-1 cell line in athymic nude mice. Thus, PACE4-silencing and systemic administration of a PACE4 inhibitor resulted in hindered tumor progression with reduction in proliferative indices and increased cell quiescence assessed with biomarkers. Our results suggest that PACE4 is a promising target for estrogen-receptor-positive breast cancer.

Novel Insights into Structure-Activity Relationships of N-Terminally Modified PACE4 Inhibitors.

PACE4 plays important roles in prostate cancer cell proliferation. The inhibition of this enzyme has been shown to slow prostate cancer progression and is emerging as a promising therapeutic strategy. In previous work, we developed a highly potent and selective PACE4 inhibitor, the multi-Leu (ML) peptide, an octapeptide with the sequence Ac-LLLLRVKR-NH2 . Here, with the objective of developing a useful compound for in vivo administration, we investigate the effect of N-terminal modifications. The inhibitory activity, toxicity, stability, and cell penetration properties of the resulting analogues were studied and compared to the unmodified inhibitor. Our results show that the incorporation of a polyethylene glycol (PEG) moiety leads to a loss of antiproliferative activity, whereas the attachment of a lipid chain preserves or improves it. However, the lipidated peptides are significantly more toxic when compared with their unmodified counterparts. Therefore, the best results were achieved not by the N-terminal extension but by the protection of both ends with the d-Leu residue and 4-amidinobenzylamide, which yielded the most stable inhibitor, with an excellent activity and toxicity profile.

Abstract 1707: PACE4-dependent cellular uptake and retention of the Multi-Leucine peptide inhibitor into cancer cells

Abstract The proprotein convertase PACE4 is strongly overexpressed in prostate cancer and plays an important role in tumor progression by promoting cell proliferation and tumor angiogenesis. Because the high expression levels and the necessity of this enzyme for cancer progression, we have developed a peptide-based inhibitor known as the Multi-Leucine (ML) peptide, which is a PACE4-specific inhibitor. Just like PACE4-downregulation through stable shRNA transfections, this compound displays anti-proliferative properties when applied on cancer cells. We showed its target-specific uptake into prostate cancer xenografts by positron emission tomography, which allowed clear tumor visualization. The ML-peptide has potent effects to block the further progression of prostate cancer cells, however, the uptake mechanism of this peptide is not clear and whether it is PACE4-dependent. We therefore tested the uptake and efflux rates of the ML-peptide peptide in prostate cancer cells and correlated these with PACE4 expression levels in wild type and stable PACE4-knockdown cell lines. In addition to prostate cancer cell lines LNCaP, DU145 and PC3, we tested HepG2, Huh7 and HT1080 cells, which also express PACE4. To measure peptide uptake, the ML peptide was modified through the addition a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) moiety to strongly chelate radiometal such as 64Cu. Our study shows that uptake of the ML-peptide is correlated with expression levels, but more importantly can be blocked in PACE4-knockdown cell lines, demonstrating a PACE4-dependant uptake mechanism. We also correlated uptake with the anti-proliferative effects of the ML-peptide and showed that entry into these cell lines predicted the effects of the ML-peptide on the growth of these cells. These results confirm the notion that ML-peptide entry within cell is an important requirement to exert growth inhibition properties, and further provide a mechanism for that entry. This study demonstrates further support for the use of the ML-peptide or its analogs as potential drugs in prostate cancer, as well as any other PACE4-dependent tumors. These indications of peptide uptake within tumor could allows PACE4-status to be determined by positron emission tomography and may be of great theranostics uses in the context of pharmacological intervention with PACE4 inhibitors. Citation Format: Frédéric Couture, Kevin Ly, Christine Levesque, Anna Kwiatkowska, Roxane Desjardins, Brigitte Guérin, Robert Day. PACE4-dependent cellular uptake and retention of the Multi-Leucine peptide inhibitor into cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1707. doi:10.1158/1538-7445.AM2015-1707

PACE4 inhibitors and their peptidomimetic analogs block prostate cancer tumor progression through quiescence induction, increased apoptosis and impaired neovascularisation.

Prostate cancer is the leading cancer in North American men. Current pharmacological treatments are limited to anti-androgen strategies and the development of new therapeutic approaches remains a challenge. As a fundamentally new approach, we propose the inhibition of PACE4, a member of the proprotein convertases family of enzymes, as a therapeutic target in prostate cancer. We developed an inhibitor named the Multi-Leu peptide, with potent in vitro anti-proliferative effects. However, the Multi-Leu peptide has not been tested under in vivo conditions and its potency under such conditions is most likely limited, due to the labile characteristics of peptides in general. Using a peptidomimetic approach, we modified the initial scaffold, generating the analog Ac-[DLeu]LLLRVK-Amba, which demonstrates increased inhibitory potency and stability. The systemic administration of this peptidomimetic significantly inhibits tumor progression in the LNCaP xenograft model of prostate cancer by inducing tumor cell quiescence, increased apoptosis and neovascularization impairment. Pharmacokinetic and biodistribution profiles of this inhibitor confirm adequate tumor delivery properties of the compound. We conclude that PACE4 peptidomimetic inhibitors could result in stable and potent drugs for a novel therapeutic strategy for prostate cancer.

Design, Synthesis, and Structure–Activity Relationship Studies of a Potent PACE4 Inhibitor

PACE4 plays an important role in the progression of prostate cancer and is an attractive target for the development of novel inhibitor-based tumor therapies. We previously reported the design and synthesis of a novel, potent, and relatively selective PACE4 inhibitor known as a Multi-Leu (ML) peptide. In the present work, we examined the ML peptide through detailed structure-activity relationship studies. A variety of ML-peptide analogues modified at the P8-P5 positions with leucine isomers (Nle, DLeu, and DNle) or substituted at the P1 position with arginine mimetics were tested for their inhibitory activity, specificity, stability, and antiproliferative effect. By incorporating d isomers at the P8 position or a decarboxylated arginine mimetic, we obtained analogues with an improved stability profile and excellent antiproliferative properties. The DLeu or DNle residue also has improved specificity toward PACE4, whereas specificity was reduced for a peptide modified with the arginine mimetic, such as 4-amidinobenzylamide.