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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.

Positional scanning-synthetic peptide combinatorial libraries (PS-SPCLs) are powerful molecular tools to identify enzyme substrate and potent inhibitory sequences and also to provide crucial information about active site determinants. PS-SPCLs have been surveyed for furin, proprotein convertase (PC)2, PC1/3, and PACE4 and proven efficient to identify potent peptidyl inhibitors in the low nanomolar range for furin and PC1/3. We report herein the screenings of nonamidated and acetylated hexapeptide PS-SPCLs for PC5/6A and PC7. The L-configuration library surveys distinctively revealed that L-Arg, L-Lys, and sometimes L-His in all six positions would generate the most potent inhibitors for both enzymes. Based on this clear polybasic preference, L-poly-Arg peptides ranging from four to nine residues were assayed. Inhibitory potency of these polybasic peptides increased with chain length, making nona-l-Arg a potent nanomolar inhibitor of PC5/6A and PC7 (Ki of 150 and 120 nM). PC5/6 and PC7 inhibition by nona-l-arginine was equivalent to that of furin (Ki of 114 nM) (J Biol Chem 275: 36741-36749, 2000). Nona-d-arginine was a more potent inhibitor of PC5/6 and PC7 than its levorotatory version (Ki of 19 and 81 nM), reminiscent of furin (Ki of 1.3 nM) (J Biol Chem 279:36788-36794, 2004). Our data indicate that certain poly-arginine peptides represent potent inhibitors targeting PCs of the constitutive secretory pathway (furin, PC5/6, and PC7). We conclude that basic residues within PC peptide inhibitors might be responsible for targeting PCs in general and for inhibitory potency, but that select amino acid changes will be necessary to acquire true specificity toward a single PC.

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF–EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor–PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83) 1 (PA63) 6 or (PA83) 2 (PA63) 5 heteroheptamer with LF–EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.

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.

Polypeptide protease inhibitors are often found to inhibit targets with which they did not coevolve, as in the case of high-affinity inhibition of bacterial subtilisin by the leech inhibitor eglin c. Two kinds of contacts exist in such complexes: ( i ) reactive site loop-active site contacts and ( ii ) interactions outside of these that form the broader enzyme-inhibitor interface. We hypothesized that the second class of “adventitious” contacts could be optimized to generate significant increases in affinity for a target enzyme or discrimination of an inhibitor for closely related target proteases. We began with a modified eglin c, Arg-42–Arg-45–eglin, in which the reactive site loop had been optimized for subtilisin-related processing proteases of the Kex2/furin family. We randomized 10 potential adventitious contact residues and screened for inhibition of soluble human furin. Substitutions at one of these sites, Y 49 , were also screened against yeast Kex2 and human PC7. These screens identified not only variants that exhibited increased affinity (up to 20-fold), but also species that exhibited enhanced selectivity, that is, increased discrimination between the target enzymes (up to 41-fold for furin versus PC7 and 20-fold for PC7 versus furin). One variant, Asp-49–Arg-42–Arg-45–eglin, exhibited a K i of 310 pM for furin and blocked furin-dependent processing of von Willebrand factor in COS-1 cells when added to the culture medium of the cells. The exploitation of adventitious contact sites may provide a versatile technique for developing potent, selective inhibitors for newly discovered proteases and could in principle be applied to optimize numerous protein–protein interactions.

The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

Following protein biosynthesis, some of the most important cellular mechanisms that generate biological diversity are the enzymatically driven post-translational modifications that ultimately lead to the formation of bioactive molecules. Within the secretory pathway, a multitude of precursor proteins are thus modified resulting in hormones, neuropeptides, growth factors, receptors and even enzymes. These modifications include cleavage at specific sites through endo- or exo-peptidase action, amidation, glycosylation and sulfation. In recent years, an important family of these processing enzymes was discovered and characterized. The so-called proprotein convertases are the products of seven distinct genes and function as endopeptidases that cleave protein precursors C-terminal to basic residue sites. They are structurally related to the bacterial subtilisin family of enzymes and are thus referred to as the subtilisin-like proprotein convertases (SPCs). Many studies have examined the inhibition of this family of enzymes, through the search of endogenous inhibitors or through the development of peptidyl, non-peptidyl or protein inhibitors. Some potent inhibitors have been discovered or engineered. While it is certain that potent inhibitors could serve as important tools to further elucidate the specific functions of each SPC, it has also been suggested that such inhibitors may be developed into lead compounds that could have important therapeutic applications. This review examines the progress made in regards to endogenous and engineered inhibitors and evidence for possible uses as molecular tools or in therapeutic applications. It is noted that although important inhibitory potencies have often been reported, there is generally insufficient evidence to demonstrate high levels of specificity. It is thus suggested that an important short-term challenge before the field will be a better understanding of the catalytic specificity of each SPC.

The SPCs (subtilisin-like pro-protein convertases) are a family of enzymes responsible for the proteolytic processing of numerous precursor proteins of the constitutive and regulated secretory pathways. SPCs are themselves synthesized as inactive zymogens. Activation of SPCs occurs via the intramolecular autocatalytic removal of the prodomain. SPC prodomains have been proposed as templates in the development of potent and specific SPC inhibitors. In this study, we investigated the specificity and potency of complete prodomains and short C-terminal prodomain peptides of each SPC on highly purified, soluble enzyme preparations of human SPC1, SPC6, and SPC7. Progress curve kinetic analysis of prodomain peptides and complete prodomains showed competitive inhibitory profiles in the low nanomolar range. Complete prodomains were 5-100 times more potent than C-terminal prodomain peptides, suggesting that N-terminal determinants are involved in the recognition process. However, complete prodomains and prodomain peptides exhibit only a partial specificity toward their cognate enzyme. Ala-scan structure activity studies indicated the importance of basic residues in the P(4), P(5), and P(6) positions for inhibition of SPC1. In contrast, hydrophobic residues in P(6) and P(7), as well as basic residues in P(4) and P(5), were critical for inhibition of SPC7. Our data demonstrated that the use of prodomains as specific inhibitors acting in trans would be of limited usefulness, unless modified into more specific compounds.

Endothelial cells (ECs) secrete numerous bioactive peptides that are initially synthesized as inactive precursor proteins. One of these, proendothelin-1 (proET-1), undergoes proteolysis at specific pairs of basic amino acids. Here, we wished to examine the role of mammalian convertases in this event. Northern blot analysis shows that only furin and PC7 are expressed in ECs. In vitro cleavage of proET-1 by furin or PC7 demonstrated that both enzymes efficiently and specifically process proET-1. These data reveal that furin and PC7 have similar specificities towards proET-1 and suggest that both enzymes may participate in the maturation of proET-1 in ECs.

Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor-alpha converting enzyme (TACE) processing/activation. Using the furin-deficient LoVo cells, as well as the furin-proficient synoviocytes and HT1080 cells expressing the furin inhibitor alpha(1)-PDX, we demonstrate that furin activity alone is not sufficient for effective maturation and activation of the TACE enzyme. Data from in vitro and in vivo cleavage assays indicate that PACE-4, PC5/PC6, PC1 and PC2 can directly cleave the TACE protein and/or peptide. PC inhibition in macrophages reduced the release of soluble TNF-alpha from transmembrane pro-TNF-alpha. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation.