Cutting back on pro-protein convertases: the latest approaches to pharmacological inhibition
Authors: Martin Fugère, Robert Day
Journal: Trends in Pharmacological Sciences (2005)

The secretory pathway in cells possesses an elaborate set of endoproteolytic enzymes that carry out a crucial step in protein precursor maturation. This step is proteolytic activation by cleavage at specific pairs of basic residues. These enzymes, named pro-protein convertases (PCs), are responsible for generating bioactive peptides and activating several enzymes and growth factors that are implicated in many important physiological events. PCs have roles in several pathologies including viral infections and cancers and, thus, are promising targets for therapeutic applications. Recent structural and homology-modeling studies demonstrate more similarity than expected at the catalytic site of the seven PCs, which makes the development of selective drugs to target individual PCs frustrating. Based on this information, we review the latest strategies to inhibit PCs, which might lead to the development of specific compounds. The secretory pathway in cells possesses an elaborate set of endoproteolytic enzymes that carry out a crucial step in protein precursor maturation. This step is proteolytic activation by cleavage at specific pairs of basic residues. These enzymes, named pro-protein convertases (PCs), are responsible for generating bioactive peptides and activating several enzymes and growth factors that are implicated in many important physiological events. PCs have roles in several pathologies including viral infections and cancers and, thus, are promising targets for therapeutic applications. Recent structural and homology-modeling studies demonstrate more similarity than expected at the catalytic site of the seven PCs, which makes the development of selective drugs to target individual PCs frustrating. Based on this information, we review the latest strategies to inhibit PCs, which might lead to the development of specific compounds. The biology of pro-protein convertases (PCs) is fundamental to the cell infrastructure and to the proper coordination of the entire mammalian physiology. Through their endoproteolytic actions on inactive precursor proteins in the secretory pathway, PCs generate essential bioactive peptides, including hormones and neuropeptides (e.g. adrenocorticotropic hormone and insulin), that have vital roles in homeostatic balance and the regulation of life functions. They also activate growth factors and differentiation factors, proteins in the extracellular matrix and plasma, enzymes, receptors, viral-coat proteins and bacterial toxins. The PC family of enzymes is encoded by seven genes (see Box 1 and Table 1 for nomenclature), of which the archetype furin is the most studied (reviewed in [1Thomas G. Furin at the cutting edge: from protein traffic to embryogenesis and disease.Nat. Rev. Mol. Cell Biol. 2002; 3: 753-766Crossref PubMed Scopus (1000) Google Scholar, 2Rockwell N.C. Thorner J.W. The kindest cuts of all: crystal structures of Kex2 and furin reveal secrets of precursor processing.Trends Biochem. Sci. 2004; 29: 80-87Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]). Mammalian PCs are related to the yeast kexin and bacterial subtilisins, and Figure 1 presents the structural features of PCs. PCs are Ca2+-dependent serine proteases that were identified originally based on their ability to cleave precursor proteins at the peptide bond C-terminal to paired basic residues, such as K-R↓ and R-R↓. However, the cleavage-recognition motifs are now known to extend N-terminal to the cleavage site and often include additional arginine residues (e.g. R-x-[K/R]-R↓).The newly adopted PC nomenclatureThe seven members of the PC family are classified as MEROPS clan SB family S8 (http://merops.sanger.ac.uk). They are related to the bacterial family of degradative subtilisins (IUBMB EC 3.4.21.62) and, to some extent, are structurally and functionally analogous to the yeast kexin (EC 3.4.21.61). The family of PCs has several confusing nomenclatures. A consensus nomenclature was agreed at the Gordon Research Conference: Pro-protein Processing, Trafficking and Secretion (2004) and the officially adopted nomenclature of the pro-protein convertases is now furin (EC 3.4.21.75), PC2 (EC 3.4.21.93), PC1/3 (EC 3.4.21.94), PACE4, PC4, PC5/6 and PC7. The corresponding genes that encode these PCs are designated officially as PCSK type 1–PCSK type 7. Table 1 presents the names attributed to each PC and the corresponding official nomenclature.Table 1PC nomenclatureaAbbreviations: LPC, lymphoma pro-protein convertase; PACE, paired basic amino acids-converting enzyme; PCSK, pro-protein convertase subtilisin/kexin type; SPC, subtilisin-like (or subtilase-like) pro-protein convertase.Gene designation (human)EnzymeAlternative enzyme namesPCb'Pro-hormone convertase' is also used in the literature but is an inadequate appellation because pro-proteins other than pro-hormones are also matured by PCs.SPCPCSK3FurinPACE, PC1, SPC1PCSK2PC2SPC2PCSK1PC1/3PC1, PC3, SPC3PCSK6PACE4PACE4, SPC4PCSK4PC4SPC5PCSK5PC5/6PC5, PC6, SPC6PCSK7PC7PC8, LPC, SPC7a Abbreviations: LPC, lymphoma pro-protein convertase; PACE, paired basic amino acids-converting enzyme; PCSK, pro-protein convertase subtilisin/kexin type; SPC, subtilisin-like (or subtilase-like) pro-protein convertase.b 'Pro-hormone convertase' is also used in the literature but is an inadequate appellation because pro-proteins other than pro-hormones are also matured by PCs. Open table in a new tab The seven members of the PC family are classified as MEROPS clan SB family S8 (http://merops.sanger.ac.uk). They are related to the bacterial family of degradative subtilisins (IUBMB EC 3.4.21.62) and, to some extent, are structurally and functionally analogous to the yeast kexin (EC 3.4.21.61). The family of PCs has several confusing nomenclatures. A consensus nomenclature was agreed at the Gordon Research Conference: Pro-protein Processing, Trafficking and Secretion (2004) and the officially adopted nomenclature of the pro-protein convertases is now furin (EC 3.4.21.75), PC2 (EC 3.4.21.93), PC1/3 (EC 3.4.21.94), PACE4, PC4, PC5/6 and PC7. The corresponding genes that encode these PCs are designated officially as PCSK type 1–PCSK type 7. Table 1 presents the names attributed to each PC and the corresponding official nomenclature. During the past few years, several important pathologies have been linked with PC-like activity in the cell-secretory pathway and at the cell membrane. The aim is to develop inhibitors that target individual PCs because not every PC is implicated in a given disease state. In this article, we review the involvement of PCs in various pathologies, which justifies their targeting for therapeutic applications. Although increasing evidence of similarity between the various PCs raises the issue of whether specific compounds that target individual PCs can be developed, five major approaches that are described in this article appear to make this possible. There is increasing interest in PCs as novel targets for drug design because they are proteolytically selective enzymes, the tissue and cellular localization patterns of which have been studied extensively [3Bergeron F. et al.Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications.J. Mol. Endocrinol. 2000; 24: 1-22Crossref PubMed Scopus (167) Google Scholar]. In addition, they have well-defined cellular tasks and are crucial for the initiation and progress of many important diseases, most prominently in several viral infections and cancers. For example, furin and PC7 are required for the activation of HIV-1 viral coat protein glycoprotein 160 (gp160) to gp120 and gp41 to produce virions that are effective in receptor binding and membrane fusion during infection [4Kibler K.V. et al.Polyarginine inhibits gp160 processing by furin and suppresses productive human immunodeficiency virus type 1 infection.J. Biol. Chem. 2004; 279: 49055-49063Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar]. A similar PC-mediated processing requirement for viral spread and cytopathicity also occurs with the Epstein-Barr virus gB glycoprotein [5Johannsen E. et al.Proteins of purified Epstein-Barr virus.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16286-16291Crossref PubMed Scopus (358) Google Scholar], the hepatitis B virus e antigen [6Messageot F. et al.Proteolytic processing of the hepatitis B virus e antigen precursor. Cleavage at two furin consensus sequences.J. Biol. Chem. 2003; 278: 891-895Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar] and the severe acute respiratory syndrome coronavirus spike glycoprotein [7Bergeron E. et al.Implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus.Biochem. Biophys. Res. Commun. 2005; 326: 554-563Crossref PubMed Scopus (68) Google Scholar]. These roles make drugs that target PCs potential antiviral agents. In the area of cancer, ongoing work is clarifying the pivotal role of PCs in growth regulation and tumor progression by activating cancer-promoting factors such as stromelysin-3 [8Bassi D.E. et al.The proprotein convertases furin and PACE4 play a significant role in tumor progression.Mol. Carcinog. 2000; 28: 63-69Crossref PubMed Scopus (76) Google Scholar], vascular endothelial growth factor c (VEGF-C) [9Siegfried G. et al.The secretory proprotein convertases furin, PC5, and PC7 activate VEGF-C to induce tumorigenesis.J. Clin. Invest. 2003; 111: 1723-1732Crossref PubMed Scopus (201) Google Scholar], insulin-like growth factor 1 receptor (IGFr1) [10Stawowy P. et al.Proprotein convertases regulate insulin-like growth factor 1-induced membrane-type 1 matrix metalloproteinase in VSMCs via endoproteolytic activation of the insulin-like growth factor-1 receptor.Biochem. Biophys. Res. Commun. 2004; 321: 531-538Crossref PubMed Scopus (30) Google Scholar, 11Khatib A.M. et al.Inhibition of proprotein convertases is associated with loss of growth and tumorigenicity of HT-29 human colon carcinoma cells: importance of insulin-like growth factor-1 (IGF-1) receptor processing in IGF-1-mediated functions.J. Biol. Chem. 2001; 276: 30686-30693Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar], transforming growth factor β (TGF-β) [12Dubois C.M. et al.Evidence that furin is an authentic transforming growth factor-beta1-converting enzyme.Am. J. Pathol. 2001; 158: 305-316Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar] and membrane type 1 matrix metalloproteinase (MMP-1) [13Mercapide J. et al.Inhibition of furin-mediated processing results in suppression of astrocytoma cell growth and invasiveness.Clin. Cancer Res. 2002; 8: 1740-1746PubMed Google Scholar]. In addition, PC-inhibition studies are beginning to provide direct evidence of their roles in tumorigenesis and invasiveness [13Mercapide J. et al.Inhibition of furin-mediated processing results in suppression of astrocytoma cell growth and invasiveness.Clin. Cancer Res. 2002; 8: 1740-1746PubMed Google Scholar, 14Wick W. et al.BCL-2-induced glioma cell invasiveness depends on furin-like proteases.J. Neurochem. 2004; 91: 1275-1283Crossref PubMed Scopus (41) Google Scholar, 15Bassi D.E. et al.Furin inhibition results in absent or decreased invasiveness and tumorigenicity of human cancer cells.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10326-10331Crossref PubMed Scopus (142) Google Scholar]. Evidence of the diverse roles of PCs in other pathologies is also accumulating. Bacterial pathogens such as Pseudomonas aeruginosa exotoxin A and the protective antigen of Bacillius anthracis tripartite toxin require furin-mediated cleavage for cytotoxicity, which can be reduced and blocked by PC inhibitors [16Sarac M.S. et al.The furin inhibitor hexa-D-arginine blocks the activation of Pseudomonas aeruginosa exotoxin A in vivo.Infect. Immun. 2002; 70: 7136-7139Crossref PubMed Scopus (72) Google Scholar, 17Sarac M.S. et al.Protection against anthrax toxemia by hexa-D-arginine in vitro and in vivo.Infect. Immun. 2004; 72: 602-605Crossref PubMed Scopus (75) Google Scholar]. PCs have a role in neurodegenerative and aging maladies such as Alzheimer's disease through the activation of pro-β site amyloid-precursor-protein-cleaving enzyme (BACE1) [18Pinnix I. et al.Convertases other than furin cleave beta-secretase to its mature form.FASEB J. 2001; 15: 1810-1812Crossref PubMed Scopus (28) Google Scholar] and its novel binding partner BRI3 [19Wickham L. et al.beta-Amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI: binding partners processed by furin.J. Neurochem. 2005; 92: 93-102Crossref PubMed Scopus (36) Google Scholar], and in arthritis through the processing of aggrecanase-1 [20Wang P. et al.Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi network.J. Biol. Chem. 2004; 279: 15434-15440Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar] and tumor necrosis factor α-converting enzyme (TACE) [21Srour N. et al.TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity.FEBS Lett. 2003; 554: 275-283Crossref PubMed Scopus (89) Google Scholar]. Coordinated interplay between PC2 and PC1/3 has been uncovered recently in the production of peptides derived from cocaine and amphetamine-regulated transcript precursor (proCART) [22Dey A. et al.Biological processing of the cocaine and amphetamine-regulated transcript precursors by prohormone convertases, PC2 and PC1/3.J. Biol. Chem. 2003; 278: 15007-15014Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar], which are believed to potentiate anorexia. Thus, we conclude that the potential of PC inhibitors as novel therapeutic agents is considerable and warrants further analysis. To date, no PC-oriented therapies are available. To achieve this goal, new insights are needed into the molecular determinants that are responsible for the proteolytic activities of individual PCs. Strategically, the mainstream of PC inhibitor development is to target their catalytic site. This is based on the fact that PCs are highly selective proteinases that cleave an uncommon sequence motif in proteins (e.g. R-x-K-R). However, the most potent inhibitors known to date, which target the catalytic site, are not significantly specific for a single PC. Comparing inhibitors of PCs shows that many have high potency but are not specific for one PC (Figure 2). In addition, PCs are often coexpressed within cells, so that all cells express a 'cocktail' of at least two PCs. Thus, poorly selective PC inhibitors will affect multiple cellular functions, and not only the pathological processes that are targeted. The first approaches to developing specific PC inhibitors were based on our understanding of the molecular determinants of selectivity of cleavage by PCs. The cleavage sites of numerous furin substrates were aligned to assess these molecular determinants [23Nakayama K. Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins.Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (714) Google Scholar]. However, subsequent cleavage studies show that many substrates are also processed by other PCs. This has been emphasized recently by studies in transgenic inducible-knockout mice, which lack furin in the liver [24Roebroek A.J. et al.Limited redundancy of the proprotein convertase furin in mouse liver.J. Biol. Chem. 2004; 279: 53442-53450Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar]. Known substrates of furin in the liver (e.g. albumin, α5 integrin, lipoprotein-receptor-related protein, vitronectin, α1-microglobulin and bikunin) are either processed or partially processed in these mice, which indicates redundancy with other PCs in the liver. These data also underline the potential undesirable effects of nonspecific PC inhibitors (that inhibit several PCs), which would block the processing of more proteins than desired if applied therapeutically. Uniquely specific PC inhibitors have the potential advantage that they can be used in a combination therapy approach, should simultaneous blocking of two PCs be required. Currently, the development of PC inhibitors is based on: (i) newly acquired structural and modeling information about PCs; (ii) peptides that are optimized by combinatorial methods; (iii) engineering polypeptide protease inhibitors that have not co-evolved with PCs but are used as stable scaffolds on which to graft the appropriate modifications to specifically target PCs; (iv) endogenous, co-evolutionary inhibitors; and (v) small non-peptidyl compounds and derivatives. Each of these approaches is discussed below. Understanding of the molecular basis of pro-protein processing by PCs and the design of specific inhibitors have both benefited from the recent elucidation of the three-dimensional conformation of the kinetically trapped furin [Protein Data Bank (PDB) (http://www.rcsb.org/pdb/) entry 1P8J, molecule A] [25Henrich S. et al.The crystal structure of the proprotein processing proteinase furin explains its stringent specificity.Nat. Struct. Biol. 2003; 10: 520-526Crossref PubMed Scopus (304) Google Scholar] and of the yeast homolog kexin (PDB entry 1ot5, chain A) [26Holyoak T. et al.2.4 A resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor.Biochemistry. 2003; 42: 6709-6718Crossref PubMed Scopus (91) Google Scholar]. The crystal structure of furin provides crucial information on the spatial arrangement of the two domains that are implicated in PC activity: the catalytic domain, which contains the active site that is responsible for substrate-specific recognition and processing; and the P domain, which is responsible for enzyme stability (Figure 3a). The crystal structure of furin in a covalently bound complex with the suicide inhibitor decanoyl-RVKR-chloromethylketone (dec-RVKR-cmk) [25Henrich S. et al.The crystal structure of the proprotein processing proteinase furin explains its stringent specificity.Nat. Struct. Biol. 2003; 10: 520-526Crossref PubMed Scopus (304) Google Scholar] has led to the identification of the reactive amino acids that make up the S1–S4 subsite pockets *The catalytic site of proteases, including PCs, is defined by subsite positions (e.g. S3, S2 and S1), which correspond to the residues of a binding substrate or inhibitor (P3-P2-P1). Thus, the substrate residue at position P1 binds the S1-subsite pocket of the protease. Each subsite can be composed of several amino acids. Substrate and inhibitor residues and subsites on the C-terminal side of the cleavage site are defined by primes (Pn′ and Sn′). The processing of substrates occurs at the P1-P1′ peptidyl bond (e.g. Pn-P3-P2-P1↓P1′-P2′-P3′-Pn′) [49Schechter I. Berger A. On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain.Biochem. Biophys. Res. Commun. 1968; 32: 898-902Crossref PubMed Scopus (351) Google Scholar].. This structural information permits modeling and docking analysis [27Kacprzak M.M. et al.Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-D-arginine.J. Biol. Chem. 2004; 279: 36788-36794Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 28Henrich S. et al.Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity.J. Mol. Biol. 2005; 345: 211-227Crossref PubMed Scopus (136) Google Scholar], which has led to the extrapolation of further subsites, including the S5, S6 and S1′ subsite pockets (Figure 3b, Table 2). Apart from S3 and S1′, all subsite pockets of the active site of furin contain several protophilic carboxylates and carboxamides, which explains the stringent preference for either arginyl or lysyl functionality groups in substrates and inhibitors (Table 2).Table 2Important PC subsite residuesaNumbering of the amino acids starts at the first amino acid (methionine) of the signal peptide sequence. Triad residues and S1–S4 subsites residues are deduced from the crystal structure of furin complexed with dec-RVKR-cmk. S5, S6 and S1′ are extrapolated by sequence-alignment analysis and confirmed by homology modeling. Residues in bold and underlined contrast with the active site of furin. Refer to the footnote for subsite nomenclature.PCS6S5S4S3S2S1TriadS1′FurinE230, D233E257, D264E236, D264L227, T254D154, D191D258, D306D153, H194, S368R193, R197, H364PC2P245, D249T273, D280E252, D280L242, W270D169, F206D274, D321D168, H209, S385S208, R212, H381PC1/3I244, D247N271, E278E250, E278L241, W268D168, E205D272, D320D167, H208, S382K207, R211, H378PACE4D283, D286D310, D317E289, D317L280, W307D207, D244D311, D359D206, H247, S421K256, R250, H417PC4A233, D236E260, D267E239, D267L220, W257D157, D193D271, D309D156, H197, S371R196, R200, H367PC5/6D249, D252D276, D283E255, D283L246, W273D173, E210D277, D325D172, H213, S385K212, R216, H381PC7P264, D267D291, D298E270, D298L261, W288D188, G225D292, D340D187, H228, S406H227, R231, H402a Numbering of the amino acids starts at the first amino acid (methionine) of the signal peptide sequence. Triad residues and S1–S4 subsites residues are deduced from the crystal structure of furin complexed with dec-RVKR-cmk. S5, S6 and S1′ are extrapolated by sequence-alignment analysis and confirmed by homology modeling. Residues in bold and underlined contrast with the active site of furin. Refer to the footnote for subsite nomenclature. Open table in a new tab Based on homology modeling, the catalytic domains of the seven PCs are highly homologous (50–70%) [28Henrich S. et al.Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity.J. Mol. Biol. 2005; 345: 211-227Crossref PubMed Scopus (136) Google Scholar] and the level of identity is even greater for the residues of the catalytic-cleft subsites (Table 2). For example, for all PCs, the S1 subsite that interacts with the P1 arginine residue of a substrate is composed of two acidic aspartic acid residues (Table 2) and three residues of the catalytic triad (aspartic acid, histidine and serine). In addition, nine additional amino acids that provide the steric environment to the P1 lateral chain are identical in all PCs, as illustrated by the comparison of furin and PC7 (Figure 3c,d). The subsite identities go beyond the S1 subsite, and are also found from S2 to S5 (Table 2). However, differences increase away from the catalytic triad, at the S6 subsite pocket and beyond. These observations lower expectations for the possible development of short peptide inhibitors of PCs (four–five residues) that are specific for individual PCs. However, the differences observed at the S6 subsite indicate that specific, short-peptide inhibitors require at least six residues. Beyond the S6 subsite, further differences might be exploited to design specific inhibitors. As yet, these differences are not well identified but some attempts have been made. For example, a recent study demonstrates that exploiting the catalytic-site proximity of so-called 'adventitious contacts' can lead to more-specific inhibitors [29Komiyama T. et al.Optimization of protease-inhibitor interactions by randomizing adventitious contacts.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8205-8210Crossref PubMed Scopus (38) Google Scholar]. The mapping of proximal sites is also aided by solution-structure studies of the PC1/3 pro-domain [30Tangrea M.A. et al.Solution structure of the pro-hormone convertase 1 pro-domain from Mus musculus.J. Mol. Biol. 2002; 320: 801-812Crossref PubMed Scopus (39) Google Scholar] and subsequent docking analysis with PCs. Unlike dec-RVKR-cmk, which maps only four subsite positions [28Henrich S. et al.Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity.J. Mol. Biol. 2005; 345: 211-227Crossref PubMed Scopus (136) Google Scholar], pro-domain-docking analysis demonstrates distinctions between furin and the other PCs at the S5-subsite and S6-subsite pockets (Table 2). Further structural studies of the crystallization of furin and other PCs complexed with different inhibitors are needed to provide a better understanding of the catalytic site of PCs. The inhibitory properties of a small-peptide inhibitor can be optimized in vitro using positional scanning of synthetic peptide combinatorial libraries (PS-SPCLs) [31Apletalina E. et al.Identification of inhibitors of prohormone convertases 1 and 2 using a peptide combinatorial library.J. Biol. Chem. 1998; 273: 26589-26595Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 32Fugere M. Day R. Inhibitors of the subtilase-like pro-protein convertases (SPCs).Curr. Pharm. Des. 2002; 8: 549-562Crossref PubMed Scopus (30) Google Scholar]. An optimal sequence, determined by screening PS-SPCLs can then be embedded into polypeptides to achieved higher specificity and potency. Such combinatorial libraries of degenerate mixtures of small-peptide inhibitors have been used successfully to compile the most potent sequence for inhibiting furin (nona-arginine) [33Cameron A. et al.Polyarginines are potent furin inhibitors.J. Biol. Chem. 2000; 275: 36741-36749Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar], and to identify a previously unknown sequence, LLRVKR, which potently inhibits PC1/3 [31Apletalina E. et al.Identification of inhibitors of prohormone convertases 1 and 2 using a peptide combinatorial library.J. Biol. Chem. 1998; 273: 26589-26595Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 34Basak A. et al.Inhibitory specificity and potency of proSAAS-derived peptides toward proprotein convertase 1.J. Biol. Chem. 2001; 276: 32720-32728Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar]. This latter sequence was identified in a novel, endogenous protein precursor, proSAAS, that has a tissue-distribution pattern that resembles closely that of PC1/3 [35Fricker L.D. et al.Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing.J. Neurosci. 2000; 20: 639-648Crossref PubMed Google Scholar, 36Lanoue E. Day R. Coexpression of proprotein convertase SPC3 and the neuroendocrine precursor proSAAS.Endocrinology. 2001; 142: 4141-4149Crossref PubMed Scopus (28) Google Scholar]. The development of nona-arginine and LLRVKR as PC inhibitors demonstrates the strength of PS-SPCLs in identifying potent inhibitory peptide sequences. In the case of the polyarginine sequence, peptides ranging from four to nine residues are potent inhibitors of furin in both enantiomeric configurations [33Cameron A. et al.Polyarginines are potent furin inhibitors.J. Biol. Chem. 2000; 275: 36741-36749Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar]. The most potent polyarginine peptide is d-nona-arginine (Ki=1 nM) [27Kacprzak M.M. et al.Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-D-arginine.J. Biol. Chem. 2004; 279: 36788-36794Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar]. Interestingly, all l- and d-polyarginine peptides are also potent inhibitors of PC5/6 and PC7 (M. Fugère et al., unpublished), which highlights the difficulty in achieving substantial specificity using the active site exclusively. From a therapeutic perspective, d-polyarginines are interesting as lead compounds because they cannot be cleaved by PCs and are thus more stable. d-Peptide therapeutics are also less prone to other proteolytic activities such as carboxypeptidases. d-hexa-arginine blocks the activation of P. aeruginosa exotoxin A [16Sarac M.S. et al.The furin inhibitor hexa-D-arginine blocks the activation of Pseudomonas aeruginosa exotoxin A in vivo.Infect. Immun. 2002; 70: 7136-7139Crossref PubMed Scopus (72) Google Scholar] and d-nona-arginine partially protects macrophages and mice against anthrax toxemia [17Sarac M.S. et al.Protection against anthrax toxemia by hexa-D-arginine in vitro and in vivo.Infect. Immun. 2004; 72: 602-605Crossref PubMed Scopus (75) Google Scholar, 27Kacprzak M.M. et al.Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-D-arginine.J. Biol. Chem. 2004; 279: 36788-36794Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar]. l-Hexa-arginine is an efficient inhibitor of the processing of gp160 (env) at the REKR551 sequence in vitro and ex vivo, and effectively suppresses HIV-1 infection of T cells and macrophages [4Kibler K.V. et al.Polyarginine inhibits gp160 processing by furin and suppresses productive human immunodeficiency virus type 1 infection.J. Biol. Chem. 2004; 279: 49055-49063Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar]. Both furin and PC7, the two principal PCs that are expressed by HIV-host cells, are the best candidates for processing the precursor of HIV-1 gp160 [37Moulard M. Decroly E. Maturation of HIV envelope glycoprotein precursors by cellular endoproteases.Biochim. Biophys. Acta. 2000; 1469: 121-132Crossref PubMed Scopus (118) Google Scholar]. If polyarginine peptides do not exhibit undesirable interactions with other biological processes they might become key components for a combined therapy that targets furin, PC5/6 and PC7 simultaneously. Conversely, introducing these polyarginine peptide sequences into engineered polypeptides might achieve greater specificity while retaining high potency. Astute bioengineering of protease inhibitors that did not co-evolve with PCs has also led to the development of potent PC inhibitors. The first successful example is α1-antitrypsin, wh