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Supplementary material and methods relating to cell culture, cell treatments and transfections, proliferation assays, Western blot analysis etc. and Supplementary Figure Legends for Supplementary Figures 1-8

PACE4 mRNA splicing analysis and primer design

SILAC secretome data

Primer sequences and antibodies specifications

Inhibition of PACE4, a proprotein convertase that is overexpressed in prostate cancer, has been shown to block cancer progression in an androgen-independent manner. However, the basis for its overexpression and its growth-inhibitory effects are mitigated and uncertain. Here, we report that PACE4 pre-mRNA undergoes DNA methylation–sensitive alternative splicing of its terminal exon 3′ untranslated region, generating an oncogenic, C-terminally modified isoform (PACE4-altCT). We found this isoform to be strongly expressed in prostate cancer cells, where it displayed an enhanced autoactivating process and a distinct intracellular routing that prevented its extracellular secretion. Together, these events led to a dramatic increase in processing of the progrowth differentiation factor pro-GDF15 as the first PACE4 substrate to be identified in prostate cancer. We detected robust expression of PACE4-altCT in other cancer types, suggesting that an oncogenic switch for this proenzyme may offer a therapeutic target not only in advanced prostate cancer but perhaps also more broadly in human cancer. Cancer Res; 77(24); 6863–79. ©2017 AACR.

Supplementary data and material regarding the alternative splicing detailed analysis by PCR and IHC, decitabine efficacy, supplementary cell line testing by overexpression and silencing and substrate screening by western blots

The processes regulating the generation of proteins from the early translation events to the final biologically active products are complex and tightly controlled [...]

Introduction: Patients with nevoid basal cell carcinoma syndrome (NBCCS), also termed Gorlin syndrome, develop numerous basal cell carcinomas (BCCs). Current treatment options include systemic hedgehog pathway inhibitors, with significant side effects when used long term, and multiple surgeries with the risk of scarring and sometimes even disfigurement over the long term. We have developed an intralesional treatment for BCC based on the intracellular delivery of an antisense oligonucleotide (ASO) targeting Gli1, the main protein active in the hedgehog pathway responsible for BCC. To test the efficacy of our treatment, we developed an inducible NBCCS-like mouse model, genetically modified at the level of the expression of the Ptch1 and p53 genes in the skin. Methods: The inducible mouse model uses Ptch1- and P53-knockdown mice irradiated with UVB three times a week until BCCs appear. These BCCs were treated once or twice a week with injections of an antisense oligonucleotide (ASO) targeting the production of the Gli1 protein, with and without the use of a peptide (the “Feldan shuttle") designed to introduce the ASO into the tumor cells. The evolution of the tumors was monitored several times a week with caliper measurements and photography. Following the treatments, the tumor site tissue was harvested and analyzed by both routine (H&E) and specialized (IHC, IF) histologic methods . Results: Intralesional treatment of BCCs in the inducible mouse model using the ASO and peptide combination (FLD103) resulted in rapid and marked tumor regression, and sometimes complete eradication, over a few weeks. In addition, healthy perilesional skin appeared unaffected by the treatment, demonstrating targeted treatment specificity. The healed skin appeared grossly normal. Histologic examination of the treated sites after recovery showed no residual BCC as well as complete healing of the epidermis, dermis and subcutaneous tissue. We also observed that intralesional treatment with the ASO alone is not associated with tumor regression and in fact permits continued tumor growth. Conclusion: We have demonstrated the utility of a specific intralesional treatment for BCC in a genetically inducible mouse model. This alternative and novel treatment produces rapid results with normal healing of the treated site. This treatment could offer an attractive option for patients with multiple BCCs who wish to avoid systemic treatments or multiple surgeries.

The field of portable healthcare monitoring devices has an urgent need for the development of real-time, noninvasive sensing and detection methods for various physiological analytes. Currently, transdermal sensing techniques are severely limited in scope (i.e., measurement of heart rate or sweat composition), or else tend to be invasive, often needing to be performed in a clinical setting. This study proposes a minimally invasive alternative strategy, consisting of using dissolving polymeric microneedles to deliver naked eye-invisible functional fluorescent ratiometric microneedle tattoos directly to the skin for real-time monitoring and quantification of physiological and pathological parameters. Reactive oxygen species are overexpressed in the skin in association with various pathological conditions. Here, one demonstrates for the first time the microneedle-based delivery to the skin of active fluorescent sensors in the form of an invisible, ratiometric microneedle tattoo capable of sensing reactive oxygen species in a reconstructed human-based skin disease model, as well as an in vivo model of UV-induced dermal inflammation. One also elaborates a universal ratiometric quantification concept coupled with a custom-built, multiwavelength portable fluorescence detection system. Fully realized, this approach presents an opportunity for the minimally invasive monitoring of a broad range of physiological parameters through the skin.

The aim of this paper is to study the impact of thermodynamics models on the CO2 storage capacity of an aquifer, by using modelling and simulation of transport phenomena in porous media. The aquifer is represented here by a simplified three phase isothermal non-reacting medium composed by an inert solid phase and two binary (CO2 and H2O) fluid phases. The mathematical description is classically developed by the volume averaging method. For each phase, the conservation equations of mass, momentum and energy alongside thermodynamic laws and boundary conditions are first written. They are then integrated over a representative elementary volume in order to establish the description at the local scale. In a thermodynamic point of view, two kinds of models have been implemented and compared. The first one assumes that the gas and liquid phases are ideal. On the contrary, in the second approach, non-ideal thermodynamics is taken into account by calculating an activity coefficient (γ-φ approach) for the liquid phase and a fugacity coefficient (PengRobinson approach) for the gas phase. Results show, among other things, that the amount of CO2 dissolved in the liquid phase is significantly reduced with the non-ideal approach compared to the ideal case. These results highlight the interest of considering non-ideal phases in more complex models.

Bacteriocins are receiving increased attention as potent candidates in food preservation and medicine. Although the inhibitory activity of bacteriocins has been studied widely, little is known about their gastrointestinal stability and toxicity toward normal human cell lines. The aim of this study was to evaluate the gastrointestinal stability and activity of microcin J25, pediocin PA-1, bactofencin A and nisin using

Phosphatase and tensin homolog (PTEN) tumor suppressor protein loss is common in prostate cancer (PCa). PTEN loss increases PI3K/Akt signaling, which promotes cell growth and survival. To find secreted biomarkers of PTEN loss, a proteomic screen was used to compare secretomes of cells with and without PTEN expression. We showed that PTEN downregulates Prorenin Receptor (PRR) expression and secretion of soluble Prorenin Receptor (sPRR) in PCa cells and in mouse. PRR is an accessory protein required for assembly of the vacuolar ATPase (V-ATPase) complex. V-ATPase is required for lysosomal acidification, amino acid sensing, efficient mechanistic target of Rapamycin complex 1 (mTORC1) activation, and β-Catenin signaling. On PCa tissue microarrays, PRR expression displayed a positive correlation with Akt phosphorylation. Moreover, PRR expression was required for proliferation of PCa cells by maintaining V-ATPase function. Further, we provided evidence for a potential clinical role for PRR expression and sPRR concentration in differentiating low from high Gleason grade PCa. Overall, the current study unveils a mechanism by which PTEN can inhibit tumor growth. Lower levels of PRR result in attenuated V-ATPase activity and reduced PCa cell proliferation.

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.

The spurious acquisition and optimization of a furin cleavage site in the SARS-CoV-2 spike protein is associated with increased viral transmission and disease, and has generated intense interest in the development and application of therapeutic furin inhibitors to thwart the COVID-19 pandemic. This review summarizes the seminal studies that informed current efforts to inhibit furin. These include the convergent efforts of endocrinologists, virologists, and yeast geneticists that, together, culminated in the discovery of furin. We describe the pioneering biochemical studies which led to the first furin inhibitors that were able to block the disease pathways which are broadly critical for pathogen virulence, tumor invasiveness, and atherosclerosis. We then summarize how these studies subsequently informed current strategies leading to the development of small-molecule furin inhibitors as potential therapies to combat SARS-CoV-2 and other diseases that rely on furin for their pathogenicity and progression.

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.

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.

The monitoring of lymphatic drainage is of great importance, particularly in the context of the early detection and diagnosis of several diseases. Existing methods of imaging and monitoring lymphatic drainage can be costly and require trained personnel, posing problems for at-home or point-of-care monitoring. Recently, an alternative approach has been proposed, consisting of using microneedles to deliver a near-infrared (NIR) fluorescent tattoo to the skin, which can be monitored with traditional laboratory-based fluorescence detectors. In this work, we present further development of this approach, using a specifically designed NIR-fluorescent probe and rational optimization of microneedle properties and the spatial location of the NIR dye within the microneedles. Moreover, we demonstrate that this method is compatible with a custom-made portable fluorescence measurement device and able to discriminate between drainage and lack of drainage in vivo in rats.

Paired basic amino acid cleaving enzyme 4 (PACE4), a serine endoprotease of the proprotein convertases family, has been recognized as a promising target for prostate cancer. We previously reported a selective and potent peptide-based inhibitor for PACE4, named the multi-Leu peptide (Ac-LLLLRVKR-NH2 sequence), which was then modified into a more potent and stable compound named C23 with the following structure: Ac-dLeu-LLLRVK-Amba (Amba: 4-amidinobenzylamide). Despite improvements in both in vitro and in vivo profiles of C23, its selectivity for PACE4 over furin was significantly reduced. We examined other Arg-mimetics instead of Amba to regain the lost selectivity. Our results indicated that the replacement of Amba with 5-(aminomethyl)picolinimidamide increased affinity for PACE4 and restored selectivity. Our results also provide a better insight on how structural differences between S1 pockets of PACE4 and furin could be employed in the rational design of selective inhibitors.

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.

// Christine Levesque 1, 2 , Frederic Couture 1, 2 , Anna Kwiatkowska 1, 2 , Roxane Desjardins 1, 2 , Brigitte Guerin 2, 3 , Witold A. Neugebauer 2 , Robert Day 1, 2 1 Department of Surgery/Urology Division and Faculte de Medecine et Sciences de la Sante, Universite de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada 2 Institut de Pharmacologie de Sherbrooke, Faculte de Medecine et Sciences de la Sante, Universite de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada 3 Department of Nuclear Medicine and Radiobiology and Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Faculte de Medecine et Sciences de la Sante, Universite de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada Correspondence to: Robert Day, e-mail: Robert.day@usherbrooke.ca Keywords: PACE4, Multi-Leu peptide, Prostate Cancer, Proprotein convertases, Peptide inhibitors Received: November 16, 2014 Accepted: December 14, 2014 Published: February 19, 2015 ABSTRACT 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.

Since the discovery of furin, numerous reports have studied its role in health and diseases, including cancer, inflammatory and infectious diseases. This interest has led to the development of both large protein- and peptide-based inhibitors aiming to control furin activity to treat these disorders. The most recent advances include the development of potent peptidomimetic furin inhibitors, considerably expanding the field of therapeutic applications.In this review, the use of furin or its inhibitors for therapeutic conditions is described through the patent literature since 1994. Only compounds with biological efficacy or augmented properties demonstrated within the patent literature or the associated publications concerning their claimed uses are discussed.Considering the diseases that may benefit from furin inhibition, several patents detail the use of the restricted number of furin inhibitors. However, there have been recent reports of new scaffolds, and even the use of furin itself, as a therapeutic agent. Despite considerable evidence of in vivo efficacy, limited confirmation from clinical trials supports or refutes the further use of these compounds in a therapeutic context. The most advanced application is the use of furin knockdown in the generation of an autologous cancer vaccine, which has initiated clinical trials.

This article has been withdrawn by the authors. Figs 1C, 2A, and 2E contained some inadvertently mislabeled data. The authors state that the mislabeling does not affect the conclusions of the article.

The potential of PACE4 as a pharmacological target in prostate cancer has been demonstrated as this proprotein convertase is strongly overexpressed in human prostate cancer tissues and its inhibition, using molecular or pharmacological approaches, results in reduced cell proliferation and tumor progression in mouse tumor xenograft models. We developed a PACE4 high-affinity peptide inhibitor, namely, the multi-leucine (ML), and sought to determine whether this peptide could be exploited for the targeting of prostate cancer for diagnostic or molecular imaging purposes. We conjugated a bifunctional chelator 1,4,7-triazacyclononane-1,4,7- triacetic acid (NOTA) to the ML peptide for copper-64 (64Cu) labeling and positron emission tomography (PET)– based prostate cancer detection. Enzyme kinetic assays against recombinant PACE4 showed that the NOTA-modified ML peptide displays identical inhibitory properties compared to the unmodified peptide. In vivo biodistribution of the 64Cu/NOTA-ML peptide evaluated in athymic nude mice bearing xenografts of two human prostate carcinoma cell lines showed a rapid and high uptake in PACE4-expressing LNCaP tumor at an early time point and in PACE4-rich organs. Co-injection of unlabeled peptide confirmed that tumor uptake was target-specific. PACE4-negative tumors displayed no tracer uptake 15 minutes after injection, while the kidneys, demonstrated high uptake due to rapid renal clearance of the peptide. The present study supports the feasibility of using a 64Cu/NOTA-ML peptide for PACE4-targeted prostate cancer detection and PACE4 status determination by PET imaging but also provides evidence that ML inhibitor–based drugs would readily reach tumor sites under in vivo conditions for pharmacological intervention or targeted radiation therapy.

Proprotein convertases are a family of kexin-like serine proteases that process proteins at single and multiple basic residues. Among the predicted and identified PC substrates, an increasing number of proteins having functions in cancer progression indicate that PCs may be potential targets for antineoplastic drugs. In support of this notion, we identified PACE4 as a vital PC involved in prostate cancer proliferation and progression, contrasting with the other co-expressed PCs. The aim of the present study was to test the importance of PCs in ovarian cancer cell proliferation and tumor progression. Based on tissue-expression profiles, furin, PACE4, PC5/6 and PC7 all displayed increased expression in primary tumor, ascites cells and metastases. These PCs were also expressed in variable levels in three model ovarian cell lines tested, namely SKOV3, CAOV3 and OVCAR3 cells. Since SKOV3 cells closely represented the PC expression profile of ovarian cancer cells, we chose them to test the effects of PC silencing using stable gene-silencing shRNA strategy to generate knockdown SKOV3 cells for each expressed PC. In vitro and in vivo assays confirmed the role of PACE4 in the sustainment of SKOV3 cell proliferation, which was not observed with the other three PCs. We also tested PACE4 peptide inhibitors on all three cell lines and observed consequent reduced cell proliferation which was correlated with PACE4 expression. Overall, these data support a role of PACE4 in promoting cell proliferation in ovarian cancer and provides further evidence for PACE4 as a potential therapeutic target.

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.

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.

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.

Better understanding of the distinct and redundant functions of the proprotein convertase (PC) enzyme family within pathophysiological states has a great importance for potential therapeutic strategies. In this study, we investigated the functional redundancy of PCs in prostate cancer in the commonly used androgen-sensitive LNCaP and the androgen-independent DU145 human cell lines. Using a lentiviral-based shRNA delivery system, we examined in vitro and in vivo cell proliferation characteristics of knockdown cell lines for the endogenous PCs furin, PACE4, and PC7 in both cell lines. Of the three PCs, only PACE4 was essential to maintain a high-proliferative status, as determined in vitro using XTT proliferation assays and in vivo using tumor xenografts in nude mice. Furin knockdowns in both cell lines had no effects on cell proliferation or tumor xenograft growth. Paradoxically, PC7 knockdowns reduced in vitro cellular proliferation but had no effect in vivo. Because PCs act within secretion pathways, we showed that conditioned media derived from PACE4 knockdown cells had very poor cell growth-stimulating effects in vitro. Immunohistochemistry of PACE4 knockdown tumors revealed reduced Ki67 and higher p27KIP levels (proliferation and cell cycle arrest markers, respectively). Interestingly, we determined that the epidermal growth factor receptor signaling pathway was activated in PC7 knockdown tumors only, providing some explanations of the paradoxical effects of PC7 silencing in prostate cancer cell lines. We conclude that PACE4 has a distinct role in maintaining proliferation and tumor progression in prostate cancer and this positions PACE4 as a relevant therapeutic target for this disease.

There is increasing interest in the therapeutic targeting of proteases for the treatment of important diseases. Additionally new protein-based therapeutic strategies have the potential to widen the available treatments against these pathologies. In the last decade, accumulated evidence has confirmed that the family of proteases known as proprotein convertases (PCs) are potential targets for viral infections, osteoarthritis, cancer and cardiovascular disease, among others. Nevertheless, there are still many unanswered questions about the relevance of targeting PCs in a therapeutic context, especially regarding the anticipated secondary effects of treatment, considering the observed embryonic lethality of some PC knockout mice. In this review, the benefits of PCs as pharmacological targets will be discussed, with focus on concepts and strategies, as well as on the state of advancement of actual and future inhibitors.

The electrochemical behavior of the substituted nitrocyclopropanes 3 (n = 1) as well as of the nitrophenylcyclopropanes 7 (n = 1) have been studied at a Pt electrode by cyclic voltammetry, microcoulometry, and preparative electrolysis in DMF-(n-Bu)4PF6. Electroreduction of 8 consumes one electron to give radical anion 8•−, which undergoes a homolytic C-N bond cleavage leading to the formation of the corresponding cyclopropyl radical and the nitrite ion. Electroreduction of 11 consumes 2 electrons. The radical anion 11•− is formed first followed by C-C bond cleavage (homolytic and/or heterolytic) of the cyclopropane ring leading to a distonic radical anion 5•−. The latter is reduced to the corresponding distonic dianion, which is protonated in the aqueous work up.

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.

The overexpression as well as the critical implication of the proprotein convertase PACE4 in prostate cancer progression has been previously reported and supported the development of peptide inhibitors. The multi-Leu peptide, a PACE4-specific inhibitor, was further generated and its capability to be uptaken by tumor xenograft was demonstrated with regard to its PACE4 expression status. To investigate whether the uptake of this inhibitor was directly dependent of PACE4 levels, uptake and efflux from cancer cells were evaluated and correlations were established with PACE4 contents on both wild type and PACE4-knockdown cell lines. PACE4-knockdown associated growth deficiencies were established on the knockdown HepG2, Huh7, and HT1080 cells as well as the antiproliferative effects of the multi-Leu peptide supporting the growth capabilities of PACE4 in cancer cells.

Our recent studies have provided direct evidence for the critical role of PACE4 in the progression of prostrate cancer, identifying this enzyme as a promising target to design novel and effective treatments [1]. Moreover, we developed a potent PACE4 inhibitor with considerable selectivity (20-fold over furin) known as the Multi-Leu (ML) peptide [2]. In order to improve its pharmacological profile, we performed structure-activity relationship (SAR) studies and determined that the incorporation of the decarboxylated arginine mimetic (4-amidinobenzylamide, Amba) at the P1 position led to a more potent and stable analog [3]. Unfortunately, this inhibitor suffered from a reduced selectivity towards PACE4. To restore its specificity profile, we used a positional-scanning approach and synthesized peptide libraries by substituting each amino acid residue in the leucine core of our inhibitor. These studies revealed that we are able to enhance the specificity profile (3-fold) and preserve the inhibitory activity as well as antiproliferative properties of our inhibitor by incorporating a leucine isomer – L-isoleucine into its structure (Maluch, et al., unpublished data). Based on these results, we decided to perform further SAR studies aiming to improve the specificity and activity of our MLAmba inhibitor. We focused on the leucine core (P8-P5) and its modification with unnatural amino acid residues possessing hydrophobic character (Figure 1). First we evaluated the impact of a single substitution (from the P8 to P5 position) on the inhibitory activity of the resulting peptides, and then we combined the most promising modifications. In this work, we present the synthesis and biological evaluation of a new series of MLAmba analogs.

Proprotein convertases (PCs) are crucial in the processing and entry of viral or bacterial protein precursors and confer increased infectivity of pathogens bearing a PC activation site, which results in increased symptom severity and lethality. Previously, we developed a nanomolar peptide inhibitor of PCs to prevent PC activation of infectious agents. Herein, we describe a peptidomimetic approach that increases the stability of this inhibitor for use in vivo to prevent systemic infections and cellular damage, such as that caused by influenza H5N1 and Shiga toxin. The addition of azaβ(3)-amino acids to both termini of the peptide successfully prevented influenza hemagglutinin 5 fusogenicity and Shiga toxin Vero toxicity in cell-based assays. The results from a cell-based model using stable shRNA-induced proprotein convertase knockdown indicate that only furin is the major proprotein convertase required for HA5 cleavage.

Gene silencing strategies targeting mRNA are suitable methods to validate the functions of specific genes. In this chapter, we sought to compare two knockdown strategies for the study of proprotein convertases and proliferation in prostate cancer cells. We used both SOFA-HDV ribozyme and lentiviral-mediated shRNA delivery system to reduce PACE4 mRNA levels and validate its implication in the proliferation of DU145 prostate cancer cells. The cellular effects of PACE4 knockdown were assessed (1) in vitro using two tetrazolium salts (MTT and XTT assays) and (2) in vivo using a tumor xenograft approach in immunodeficient mice (Nu/Nu). Our results confirm the unique role of the proprotein convertase PACE4 in prostate cancer cell proliferation while demonstrating advantages and disadvantages of each approach. Achieving target validation in an effective manner is critical, as further development using a drug development approach is highly laborious and requires enormous resources.