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.

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 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.Proteolytic processing is a post-translational modification by which a cell diversifies and controls the protein products of its genes. In mammalian species, endoproteolytic activation of many secretory protein precursors is carried out by the SPCs

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.

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR2GL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1 were more selective for furin. Peptides with P1 Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.

Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor-K K converting enzyme (TACE) processing/activation. Using the furin-de¢cient LoVo cells, as well as the furin-pro¢cient synoviocytes and HT1080 cells expressing the furin inhibitor K K 1 -PDX, we demonstrate that furin activity alone is not su⁄cient for e¡ective 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-K K from transmembrane pro-TNF-K K. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation. ß

Invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP) functions in cancer cells as an oncogene and as a mediator of proteolytic events on the cell surface. To exert its functional activity, MT1-MMP requires proteolytic removal of the prodomain sequence. There are two potential furin cleavage motifs, R 89 -R-P-R-C 93 and R 108 -R-K-R-Y 112 , in the prodomain sequence of MT1-MMP. Our data suggest an important role of furin and related proprotein convertases (PCs) in mediating both the activation of MT1-MMP and the levels of functionally active MT1-MMP at the surface of cancer cells. We have determined that the peptide sequence that spans the first cleavage site is susceptible to furin and PC5/6, whereas the second sequence is susceptible to furin and also to PC5/6, PC7 and PACE4. In the structure of the MT1-MMP proenzyme, the R 89 -R-P-R-C 93 site, however, is inaccessible to PCs. Our studies also demonstrated a direct functional link between the activation and the uptake rate of the proenzyme and the enzyme of MT1-MMP. Thus, the uptake rate of the latent MT1-MMP proenzyme noticeably exceeded that of the active enzyme. We conclude that furin and related PCs are the essential components of the specialized cellular machinery that controls the levels of the functionally active, mature, MT1-MMP enzyme on the cell surface to continually support the potency of pericellular proteolysis.

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 loopactive 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 Kex2͞furin 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.