Jon R. Appel

Polyarginines Are Potent Furin Inhibitors

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 syntheticl- and d-hexapeptide combinatorial libraries. The results indicated that l-Arg orl-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 aK i for furin of 40 nm. TheK i values for the related convertases PACE4 and prohormone convertase-1 (PC1) were 110 nm and 2.5 μm, respectively. Although nona-l-arginine was cleaved by furin, the major products after a 6-h incubation at 37 °C were hexa- and 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 μm 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 ofl-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 thel-hexapeptide LLRVKR against furin 8-fold, C-terminal 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.

Exploring immunological specificity using synthetic peptide combinatorial libraries

The definition of epitopes for human B and T cells is fundamental for the understanding of the immune response mechanism and its role in the prevention and cause of human disease. This understanding can be applied to the design of diagnostics and synthetic vaccines. In recent years, the understanding of the specificity of B and T cells has been advanced significantly by the development and use of combinatorial libraries made up of thousands to millions of synthetic peptides. The use of this approach has had four major effects: first, the definition of high affinity ligands both for T cells and antibodies; second, the application of alternative means for identifying immunologically relevant peptides for use as potential preventive and therapeutic vaccines; third, a new appreciation of the requirements for TCR interactions with peptide-MHC complexes in immunogenicity; fourth, the establishment of new principles regarding the level of cross-reactivity in immunological recognition.

Advances in the use of synthetic combinatorial chemistry: mixture-based libraries.

The conceptual and technical approaches that led to the explosive growth of combinatorial chemistry began approximately 20 years ago. In the past decade, combinatorial chemistry has continued to expand with new chemistries, technological improvements and, most importantly, a clear demonstration of its utility in the identification of active compounds for research and drug-discovery programs. This article describes the conceptual and practical breakthroughs that have been critical for the development of synthetic combinatorial methods and includes the most recent developments and applications of mixture-based combinatorial libraries.

Identification of Inhibitors of Prohormone Convertases 1 and 2 Using a Peptide Combinatorial Library

A positional scanning synthetic peptide combinatorial library containing approximately 52 million hexapeptides was used to identify potential inhibitory peptides for recombinant mouse prohormone convertase 1 (PC1) and PC2 and to provide information on the specificity of these enzymes. The library surveys revealed that a P6 Leu, a P4 Arg, a P2 Lys, and a P1 Arg were most inhibitory against PC1, and a P6 Ile and a P4 Arg were most inhibitory against PC2. Using information derived from the library surveys, hexapeptide sets were synthesized and screened for inhibition of PC1 and PC2. The data obtained revealed the preference of both enzymes for a P3 Val. At P5, many substitutions were well tolerated. PC1 and PC2 proved to differ mainly in the selectivity of their S6 subsites. In PC1, this subsite displayed a strong preference toward occupation by Leu; theK i value for peptide Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 28 times lower than that for peptide Ac-Ile-Ile-Arg-Val-Lys-Arg-NH2. In contrast, PC2 discriminated little between Leu and Ile at P6, as evidenced by the small (1.5-fold) difference in K i values for these two peptides. Several hexapeptides synthesized as a result of the screen were found to represent potent inhibitors of PC2 (withK i values in the submicromolar range) and, particularly, of PC1 (with K i values in the low nanomolar range). The most potent inhibitor, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2, proved to be the same peptide for both enzymes and inhibited PC1 and PC2 in a competitive, fast-binding manner with K i values of 3.2 and 360 nm, respectively. The four most potent peptide inhibitors of PC1 and PC2 were also tested against soluble human furin and found to exhibit a different rank order of inhibition; for example, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 440-fold less potent against furin than against PC1, with a K i of 1400 nm.

Short Polybasic Peptide Sequences Are Potent Inhibitors of PC5/6 and PC7: Use of Positional Scanning-Synthetic Peptide Combinatorial Libraries as a Tool for the Optimization of Inhibitory Sequences

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.

Functional importance of amino acid residues making up peptide antigenic determinants

The functional importance of each amino acid residue making up the antigenic determinants of three different peptide-mAb interactions was determined using complete series of substitution analogs of the three immunizing synthetic peptides. Fingerprint substitution profiles for the three different antigenic determinants were obtained separately by direct and competitive ELISA. Competitive ELISA was found to offer the advantage of being able to measure the concn of each peptide substitution analog necessary to inhibit antibody binding to the original peptide. In this manner, the relative functional contribution to antibody binding of each amino acid residue making up the antigenic determinant was determined and termed the relative positional importance factor (RPIF). Each antigenic determinant was found to contain one very highly specific residue (i.e., highest RPIF) that was, on average, the least replaceable with any of the natural L-amino acids (the average decrease in recognition ranged 250- to 28,000-fold). At the other extreme, two or three positions in each antigenic determinant were found to be only weakly involved in recognition. These positions were considered redundant since the average decrease in recognition of the substitution analogs for these positions was found to be 20-fold or less. The remaining antigenic determinant residues exhibited the fine specificity common to antigen-antibody interactions in that only relatively conservative substitutions for these residues were recognized by their respective antibodies. It is of interest that the positional arrangement of specific and nonspecific residues were different for each of the three antigenic determinants examined.

Synthetic peptide combinatorial libraries (SPCLs): identification of the antigenic determinant of β-endorphin recognized by monoclonal antibody 3E7

The use of synthetic peptide combinatorial libraries (SPCLs), each composed of tens of millions of peptides, is described here for the identification of bioactive peptides. The identification of optimal peptide sequences is achieved through the screening of SPCLs in solution, each element of which is composed of more than 10(5) nonsupport-bound peptides in approximately equimolar representation, along with an iterative synthesis and screening process. Using an SPCL composed in total of 52 128 400 nonacetylated hexapeptides, along with an iterative selection process based on competitive ELISA, we identified the antigenic determinant of beta-endorphin recognized by monoclonal antibody (mAb) 3E7. These results will be compared with the results found by others investigating mAb 3E7 using different peptide library approaches.

The Multi-Leu Peptide Inhibitor Discriminates Between PACE4 and Furin And Exhibits Antiproliferative Effects On Prostate Cancer Cells

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.

Exploring antibody polyspecificity using synthetic combinatorial libraries

SummaryExtensive mapping studies for seven antigen-antibody interactions have been carried out using both individual analogs and peptide libraries. With competitive ELISA, these studies have revealed that monoclonal antibodies exhibit a broad range of specificities, from antibodies that recognize only conservative substitutions for 1–2 positions of the antigenic determinant, to antibodies that recognize sequences that are completely unrelated to the parent antigen with comparable affinities. Synthetic combinatorial libraries, containing millions of peptide sequences, permit a more systematic and rapid evaluation of the extent of multiple-binding specificities of monoclonal antibodies than individual analogs. The peptide libraries used here comprise mixtures of compounds having specifically defined positions and mixture positions. The same diversity of sequences in different formats, which differ by the numbers of positions singularly defined and different locations defined within the sequence, can be examined. Comparison of the screening results, selection criteria of the most active mixtures, and different approaches used for the deconvolution of active individual compounds are discussed. Synthetic combinatorial libraries greatly facilitate the understanding of antigen-antibody interactions at the amino acid level and will assist in the development of improved immunodiagnostics.

Mapping the detailed specificity of a calcium-dependent monoclonal antibody through the use of soluble positional scanning combinatorial libraries: Identification of potent calcium-independent antigens

SummaryThe detailed specificity of monoclonal antibody M1, which has been reported to bind in a calcium-dependent manner to the ‘FLAG’ sequence DYKDDDDK-NH2, was examined using soluble hexa- and decapeptide positional scanning synthetic combinatorial libraries (PS-SCLs) made up of 52×106 and 4×1012 different sequences, respectively. To study the influence of calcium on the specificity of this antigen-antibody interaction, each PS-SCL was screened in the presence and absence of calcium using a competitive ELISA. Overall, peptide mixtures had greater inhibitory activity against mAb M1 binding to FLAG in the absence of calcium. A total of 16 individual hexapeptides were identified, all of which contained the motif -DYK_K_-, and were recognized by mAb M1 in the absence of calcium with 50-to 100-fold higher affinity than the FLAG octapeptide (IC50=273 nM). On average, the same set of peptides bound 10-fold less effectively in the presence of calcium. Upon screening the decapeptide PS-SCL in the absence of calcium, lysine was also more active in the fifth position than the original aspartic acid. Based on the screening results, 24 individual decapeptides were prepared and were found to have activities 10- to 100-fold higher than the FLAG octapeptide in the absence of calcium. The specificity of lysine at the fifth position in the antigen-antibody interaction was further examined by synthesizing and assaying substitution analogs at this position for the octapeptide and hexapeptide forms of the FLAG sequence, as well as for two hexapeptides identified from the PS-SCL. Truncation analog analysis was also carried out on the FLAG octapeptide to determine optimal antigen length for antibody binding. Overall, lysine at the fifth position could be substituted with ornithine with no significant loss in activity, and peptide length was not a critical factor for antibody binding in the absence of calcium. Also, the octapeptide having lysine at the fifth position in place of the aspartic acid had the same activity in the presence or absence of calcium. This study demonstrates the ease and effectiveness of PS-SCLs over individual peptide analogs for the examination of the degree of cross-reactivity for a given monoclonal antibody as well as for the identification of novel, high-affinity peptides.