Existing methods for the synthesis and screening of large numbers of peptides are limited by their inability to generate and screen the requisite number (millions) of individual peptides and/or their inability to generate unmodified free peptides in quantities able to interact in solution. We have circumvented these limitations by developing synthetic peptide combinatorial libraries composed of mixtures of free peptides in quantities which can be used directly in virtually all existing assay systems. The screening of these heterogeneous libraries, along with an iterative selection and synthesis process, permits the systematic identification of optimal peptide ligands. Starting with a library composed of more than 34 million hexa-peptides, we present here the precise identification of an antigenic determinant recognized by a monoclonal antibody as well as the straightforward development of new potent antimicrobial peptides.

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The ubiquitous serine endoprotease furin has been implicated in the activation of bacterial toxins and viral glycoproteins as well as in the metastatic progression of certain tumors. Although high molecular mass bioengineered serpin inhibitors have been well characterized, no small nontoxic nanomolar inhibitors have been reported to date. Here we describe the identification of such inhibitors using positional scanning amidated and acetylated synthetic L-and D-hexapeptide combinatorial libraries. The results indicated that L-Arg or L-Lys in all positions generated the most potent inhibitors. However, further investigation revealed that the peptide terminating groups hindered inhibition. Consequently, a series of non-amidated and acetylated polyarginines was synthesized. The most potent inhibitor identified, nona-L-arginine, had a K i for furin of 40 nM. The K i values for the related convertases PACE4 and prohormone convertase-1 (PC1) were 110 nM and 2.5 M, respectively. Although nona-L-arginine was cleaved by furin, the major products after a 6-h incubation at 37°C were hexaand hepta-L-arginines, both of which retained the great majority of their potency and specificity against furin. Hexa-D-arginine was as potent and specific a furin inhibitor as hexa-L-arginine (K i values of hexa-D-arginine: 106 nM, 580 nM, and 13.2 M for furin, PACE4, and PC1, respectively). PC2 was not inhibited by any polyarginine tested; indeed, PC2 showed an increase in activity of up to 140% of the control in the presence of L-polyarginines. Data are also presented that show extended subsite recognition by furin and PC2. Whereas N-terminal acetylation was found to reduce the inhibitory potency of the L-hexapeptide LLRVKR against furin 8-fold, Cterminal amidation reduced the potency <2-fold. Conversely, N-terminal acetylation increased the potency against PC2 nearly 3-fold, whereas C-terminal amidation of the same peptide increased the potency by a factor of 1.6. Our data indicate that non-acetylated, poly-D-arginine-derived molecules may represent excellent lead compounds for the development of therapeutically useful furin inhibitors.

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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.

Polyarginine-containing peptides represent potent inhibitors of furin, a mammalian endoprotease that plays an important role in metabolism, activation of pathogenic toxins, and viral proliferation. The therapeutic use of D-polyarginines is especially interesting because they are not cleaved by furin and possess inhibitory potency almost equal to L-polyarginines. In this study we attempted to determine the important elements within polyarginines that contribute to effective inhibition. Structure-function analyses of polyarginine peptides showed that inhibition by polyarginine-containing peptides appeared to depend on the total number of basic charges of the positively charged inhibitors bound to the negatively charged substrate binding pocket; peptide positioning did not appear to be rigorously determined. Screening of L-and D-decapeptide positional scanning combinatorial peptide libraries indicated a preference for basic residues in nearly all positions, similar to previous results with hexapeptide libraries. Length and terminal modification studies showed that the most potent D-polyarginine tested was nona-D-arginine (D9R) amide with a K i of 1.3 nM. D9R amide was shown to protect RAW264.7 cells against anthrax toxemia with an IC 50 of 3.7 M. Because of its high stability, specificity, low toxicity, small molecular weight, and extremely low K i against furin, D9R amide or its derivatives may represent promising compounds for therapeutic use.Furin is a mammalian subtilisin/Kex2p-like endoprotease that is involved in the processing of many precursor proteins (reviewed in Refs. 1-3). The enzyme has a ubiquitous tissue distribution and cycles between the trans-Golgi network, the cell surface, and the endosomes. Furin plays a role in embryogenesis and homeostasis (4) and is also responsible for processing bacterial toxin precursors and virus envelope glycoprotein precursors (5, 6). Because of its involvement in bacterial and viral pathogenesis, furin represents an attractive target for therapeutic drugs.Polyarginines are known to be potent, small inhibitors of furin. L6R (hexa-L-arginine), 1 for example, exhibits the low inhibition constant (K i ) of 114 nM (7), and the D-forms of these polyarginines were also shown to be inhibitory. Moreover, D6R amide has been shown to block the activation of Pseudomonas aeruginosa exotoxin A (8) and to protect against anthrax toxemia both in vivo and in vitro (9).The structure of mouse furin has been recently determined (10) and reveals that the active site of the enzyme contains an extended substrate-binding groove that is lined with many negatively charged residues: these include Asp-258 and Asp-306 (surrounding the S1 subsite); Asp-154 and Asp-191, which form the surface of the S2 pocket; Glu-236 and Glu-264 (S4 subsite); Glu-257 and Glu-264 (Glu-264 takes part in forming the S4 and S5 subsites); and Glu-230 and Asp-233 (S6 subsite). No basic residues are present in the general area between the S5 and S1 subsites; basic residues are found only on the outer edge of the S1Ј sub...

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