The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region

Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH₂, where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k_cat/K_M = 275,000 M⁻¹ s⁻¹), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.     

A simple method for selection of trypsin chromogenic substrates using combinatorial chemistry approach

A tetrapeptide combinatorial library, considered as chromogenic substrates of bovine beta-trypsin, was synthesized by the solid phase method. The peptides contain an analog of p-nitroanilide, obtained by attaching 5-amino-2-nitrobenzoic acid (Anb(5,2)) to the C-termini. Deconvolution of the peptide library, performed in solution using an iterative method, yielded four efficient trypsin substrates. The most active one, Phe-Val-Pro-Arg-Anb(5,2)-NH(2), appeared to be 125-fold more active than Bz-D,L-Arg-pNA (BAPNA) used as a reference compound. The reported method of designing trypsin chromogenic substrate libraries is straightforward. Such p-nitroanilides may be useful for the investigation of any protease substrate specificity.     

Development of sensitive cathepsin G fluorogenic substrate using combinatorial chemistry methods

In the current work, a synthesis of new sensitive fluorescence substrates of cathepsin G is reported. The substrate sequence was selected using combinatorial chemistry methods. The starting structure of chromogenic cathepsin G substrate Ac-Phe-Val-Thr-Gnf-ANB-NH(2), where Gnf stands for 4-guanidine-l-phenylalanine, was modified by replacing the acetyl moiety with a residue of 7-methoxycoumarin-4-yl acetic acid (Mca) that served as a fluorescence donor. An amide of amino benzoic acid (ANB-NH(2)) was used as an acceptor. This peptide, exhibiting effective fluorescence resonance energy transfer (FRET) phenomena, was used as a starting structure to construct the library Mca-Phe-Val-Thr-Gnf-X(1)-X(2)-ANB-NH(2), where in both variable X positions all proteinogenic amino acid residues except Cys were introduced. Deconvolution of such a library, performed by the iterative method in solution, revealed prime site preferences of cathepsin G. Finally, the most susceptible sequence, Mca-Phe-Val-Thr-Gnf-Ser-Trp-ANB-NH(2), was selected. The determined value of the specificity constant (k(cat)/K(M) = 252 x 10(3)M(-1)xs(-1)) was two orders of magnitude higher than that obtained for the parent compound. By the use of this substrate, we were able to detect as little as 70 pM of the enzyme studied.     

Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure.

A small peptide library of monocyclic SFTI-1 trypsin inhibitor from sunflower seeds modified in positions P(1) and P(4)' was synthesized using a portioning-mixing method. The peptide library was deconvoluted by the iterative approach in solution. Two trypsin ([Met(9)]-SFTI-1 and [Arg(5), Abu(9)]-SFTI-1), one chymotrypsin ([Phe(5)]-SFTI-1) and one human elastase ([Leu(5), Trp(9)]-SFTI-1) inhibitors were selected and resynthesized. The values of their association equilibrium constants (K(a)) with target enzymes indicate that they are potent inhibitors. In addition, the last two analoges belong to the most active inhibitors of this size. The results obtained show that the conserved Pro(9) residue in the Bowman-Birk inhibitor (BBI)s is not essential for inhibitory activity.     

Detection of l-alanylaminopeptidase activity in microorganisms using chromogenic self-immolative enzyme substrates

Three potential chromogenic enzymatic probes, each possessing a self-immolative spacer unit, were synthesised for the purpose of detecting l-alanylaminopeptidase activity in microorganisms. An Alizarin-based probe was the most effective, allowing several species to generate strongly coloured colonies in the presence of metal ions.     

A new proteinase 3 substrate with improved selectivity over human neutrophil elastase

We report the synthesis and enzymatic studies on a new proteinase 3 intermolecular quenched substrate with enhanced selectivity over neutrophil elastase. Using combinatorial chemistry methods, we were able to synthesize the hexapeptide library with the general formula ABZ-Tyr-Tyr-Abu-X₁′-X₂′-X₃′-Tyr(3-NO₂)-NH₂ using the mix and split method. The iterative deconvolution of such a library allowed us to obtain the sequence ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO₂)-NH₂ with a high specificity constant (k_cat/K_M = 1534 × 10³ M⁻¹ s⁻¹) and superior selectivity over neutrophil elastase and other neutrophil-derived serine proteases. Moreover, using the obtained substrate, we were able to detect a picomolar concentration of proteinase 3 (PR3). Incubation of the above-mentioned substrate with neutrophil lysate resulted in a strong fluorescent signal that was significantly reduced in the presence of a PR3 selective inhibitor.     

The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase

The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. Accurate determination of the dimer dissociation constant and the role of the N-finger (residues 1-7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissociation constant of the wild-type protein was determined to be 14.0microM by analytical ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and analytical ultracentrifugation. A single mutation of M6A was found to be critical to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K(i) of 2.20mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.     

One-step affinity purification of recombinant urokinase-type plasminogen activator receptor using a synthetic peptide developed by combinatorial chemistry

Several lines of evidence have pointed to a role of urokinase-type plasminogen activator receptor (uPAR) as a modulator of certain biochemical processes that are active during tumor invasion and metastasis. Consequently, the structure and function of this receptor have been studied extensively, using recombinantly produced uPAR that has been purified by either affinity chromatography using its cognate ligand, the urokinase-type plasminogen activator (uPA), or a monoclonal anti-uPAR antibody (R2), or by hydroxyapatite. Here, we present a new method for the efficient one-step affinity purification of recombinant uPAR exploiting a high-affinity synthetic peptide antagonist (AE152). The corresponding parent peptide was originally identified in a random phage-display library and subsequently subjected to affinity maturation by combinatorial chemistry. This study compares the affinity purification of a soluble, recombinant uPAR using the monoclonal antibody R2 or the peptide AE152 immobilized on Sepharose. The two affinity ligands perform equally well in purifying uPAR from Drosophila melanogaster Schneider 2 cell culture medium and yield products of comparable purity, activity, and stability as judged by SDS-PAGE, size exclusion chromatography and surface plasmon resonance analysis. The general availability of peptide synthesis renders the present AE152-based affinity purification of uPAR more accessible than the traditional protein-based affinity purification strategies. In this way, large amounts of recombinant uPAR can conveniently be purified for further structural and functional studies.     

Development of a herbicide biosensor using a peptide receptor screened from a combinatorial library

The purpose of this study was to screen for peptides that bind herbicides with a chlorinated aniline chemical structure. A tetrapeptide library was constructed using a solid phase split synthesis approach. Peptide beads were suspended in a buffer containing fluorescent-labeled dichloroaniline (DCA) as the bait. Eighteen fluorescent peptide beads were selected which bound to the bait after two rounds of staining screenings. The beads were then stained and suspended in a solution containing an excess of DCA and five quenched peptide beads were subsequently selected that recognized the DCA moiety. The screened peptides had many sequence similarities. The binding affinity of the screened peptides to herbicides was analyzed using surface plasmon resonance (SPR). N′-(3,4-dichlorophenyl)-N,N-dimethylurea [3-(3,4-dichlorophenyl)-1,1-dimethylurea] solution was injected over the peptide immobilized SPR chip. The SPR signal was found to increase in proportion to the DCMU concentration, whereas no signal was obtained from the negative control, 2-(2-methyl-4-chlorophenoxy) propionic acid (MCPP). From these results it is suggested that the screened peptide selectively recognizes the chemical structure of DCA.     

Peroxyl-oxidized erythrocyte membrane band 3 protein with anion transport capacity is degraded by membrane-bound proteinase

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.     

Design of inhibitors using a combinatorial library for HIV-Nef and human SH3 domain interaction

The HIV-1 Nef protein has the ability to down regulate important molecules at the immune synapse. These include class I and class II (Human Leukocyte Antigen) HLA on the Antigen Presenting Cells (APC). The receptors in these molecules consist of SH-3 domain and their interaction with the HIV-1 Nef is critical. Therefore, it is important to inhibit this HIV-Nef and human SH3 domain interaction. Thus, we used a combinatorial library to screen for molecules to inhibit this interaction. The exercise identified a group of top ranking compounds for further consideration.     

Spectrophotometric determination of peptide transport with chromogenic peptide mimetics

A spectrophotometric assay to determine peptide transport has been developed. Using two chromogenic peptide mimetics, L-phenylalanyl-L-2-sulfanilylglycine (PSG) and L-phenylalanyl-L-3-thiaphenylalanine (PSP), the peptide transport patterns in individual cell species can be evaluated effectively. After the addition of PSG to a HeLa cell suspension, sulfanilic acid accumulated progressively inside, but not outside, the cells, demonstrating that PSG was transported wholly intact. The addition of PSP to the same cell suspension was followed immediately by extracellular thiophenol production. Measurement of the rate of thiophenol release thereby provided direct determination of PSP transport. The thiophenol release was consistent with Michaelis-Menten kinetics, with a K(m) of 0.016 mM and a V(max) of 5.07 nmol/min (1 x 10(6) cells/ml, pH 7.4, 37 degrees C). The resulting kinetic constants estimated were in agreement with values determined by single-substrate enzyme kinetics. Using PSP, transport kinetics of various dipeptides was examined by competitive spectrophotometry. As a result, dipeptides tested could be ranked in order of kinetic power for their transport.     

Construction of ethanol-tolerant yeast strains with combinatorial library-selected peptides

Combinatorial yeast libraries were constructed by transformation of expression plasmids containing artificially synthesized random sequences into Saccharomyces cerevisiae MT8-1 and IFO10150. Approximately 200 yeast strains with enhanced ethanol tolerance were obtained from yeast libraries by incubation in 10% ethanol for 24 h. Following separate evaluation of their ethanol tolerance, the 10 clones with the highest values were selected. After 3 h incubation in 12.5% ethanol, whereas most of the control cells died, the clone with the highest tolerance from the MT8-1 library, M-1, showed approximately 40% cell viability, and the corresponding clone from the IFO10150 library, I-12, 48% viability. The half-life of M-1 cells was 20 times greater than that of control cells. Three of the library-selected peptides endowing with ethanol tolerance were identified as Gly-Thr-Arg-Leu-His pentapeptides. Four seemed to be extremely hydrophobic, and three of these were predicted to be transmembrane peptides. The three other peptides seemed to be more hydrophilic than standard yeast proteins. The results of the study show that yeast strains with fairly high ethanol tolerance can be successfully constructed by directed selection from yeast libraries expressing combinatorial peptides.     

Identification of inorganic crystal-specific sequences using phage display combinatorial library of short peptides: A feasibility study

The feasibility of using a combinatorial phage display library of decapeptides to identify ligands which can interact with the surface of a crystal was assessed using geological calcium carbonate as a model. Two relatively strong binding clones were identified by ELISA, sequenced and the encoded oligopeptides were prepared by solid phase synthesis and their properties compared with those of casein hydrolysate.     

Kinetics of enzyme-catalyzed oligopeptide cleavage monitored by capillary zone electrophoresis: Comparison to spectrophotometric and HPLC methods

Summary A capillary zone electrophoresis method has been developed for monitoring the rate of cleavage of basic oligopeptides. This method is particularly applicable to monitoring the cleavage rate of peptides where the specificity of the proteinase dictates against the presence of bulky chromophores. The kinetic parameters determined for the cleavage of a related chromophoric substrate were directly comparable. The kinetic analysis of intrinsically or mutationally induced inefficient enzymes can be facilitated by this approach.     

Inhibition of Aβ42 aggregation using peptides selected from combinatorial libraries

Increasing evidence suggests that the aggregation of the small peptide Aβ42 plays an important role in the development of Alzheimer's disease. Inhibiting the initial aggregation of Aβ42 may be an effective treatment for preventing, or slowing, the onset of the disease. Using an in vivo screen based on the enzyme EGFP, we have searched through two combinatorially diverse peptide libraries to identify peptides capable of inhibiting Aβ42 aggregation. From this initial screen, three candidate peptides were selected and characterized. ThT studies indicated that the selected peptides were capable of inhibiting amyloid aggregation. Additional ThT studies showed that one of the selected peptides was capable of disaggregating preformed Aβ42 fibers. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to epsilon-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.     

Identifying peptide ligands for cell surface receptors using cell-growth-on-bead assay and one-bead one-compound combinatorial library

A cell-growth-on-bead assay was used to screen one-bead, one-compound combinatorial libraries for peptide ligands that attract attachment and growth of human lung cancer cells. A consensus cyclic peptide sequence, d-cys-l-asn-l-gly-l-arg-l-gly-l-glu-l-gln-d-cys (cNGRGEQc), was identified as a ligand for a cell surface receptor, 31 integrin. This peptide ligand and its analogs have the potential to be used as diagnostic and therapeutic agents for lung cancer.     

Docking of combinatorial peptide libraries into a broadly cross-reactive human IgM

A monoclonal IgM cryoglobulin with diverse binding behavior was isolated from a patient (Mez) with Waldenstr?m's macroglobulinemia. It gave very high titers in the binding of combinatorially synthesized libraries of peptides ranging in size from two to eight residues. The crystal structure of Mez Fv revealed that the binding site was divided into two cavities of unequal volumes with dimensions and chemical properties that were compatible with the binding of peptides. Access to this unique combination of structural information and peptide binding data led us to carry out Mez-peptide docking simulations to gain insight into the Mez binding propensities. In the present article, the results for docking of five peptide libraries are combined with discussions of the methods and approximations involved in the docking process. We analyze the origins of peptide binding affinity for Mez IgM in terms of its cross-reactivity and its structural preferences.