Identification of novel antifungal nonapeptides through the screening of combinatorial peptide libraries based on a hexapeptide motif

Four sets of mixture based nonapeptide libraries derived from an antifungal hexapeptide pharmacophore Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH(2) (II) have been synthesized. The three C-terminal positions 7, 8 and 9 were subject to randomization using 19 genetically coded amino acids. They were then screened for their antifungal activity against Candida albicans and Cryptococcus neoformans in order to quantify inhibition at each step of the nonapeptide sublibrary deconvolution. The studies led to the identification of several novel nonapeptides with potent antifungal activity. Two of the nonapeptides exhibited approximately 17-fold increase in the activity in comparison to the lead hexapeptide motif His-D-Trp-D-Phe-Phe-D-Phe-Lys-NH(2) (I) against C. albicans.     

Identification of novel peptide ligands for the cancer‐specific receptor mutation EFGRvIII using a mixture‐based synthetic combinatorial library

We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer‐specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in several kinds of cancer, in particular, ovarian, glioblastomas, and breast cancer, but not in normal tissue. The library consisted of six individual positional sublibraries in the format, H‐O_1–6XXXXX‐NH₂, O being one of the 19 proteinogenic amino acids (cysteine omitted) and X an equimolar mixture of these. The library consisted of 114 mixtures in total. Using a biotin‐streptavidin assay, the binding of each sublibrary to NR6M, NR6W‐A, and NR6 cells was tested. These cells express EGFRvIII, EGFR, and neither of the receptors, respectively. The result from each sublibrary was examined to identify the most active amino acid residue at each position. On the basis of this knowledge, eight peptides were synthesized and tested for binding to EGFRvIII. We identified one peptide, H‐FALGEA‐NH₂, that showed more selective binding to the mutated receptor than the EGFRvIII specific peptide PEPHC1. This study demonstrates the value of using mixture‐based combinatorial positional scanning libraries for the identification of novel peptide ligands targeted against the cancer‐specific EGFRvIII. Our best candidate H‐FALGEA‐NH₂ will be radioactively labeled and evaluated as an imaging agent for positron emission tomography investigation for diagnosis, staging, and monitoring of therapy of various types of cancer. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 201–206, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Identification of novel peptide agonists from a random peptide library for a 5-oxo-ETE receptor, a receptor for bioactive lipids

A combinatorial peptide library contains an enormous combination of amino acid sequences and drug candidates, but an effective screening strategy to identify a variety of bioactive peptides has yet to be established. In this article, a random hexapeptide library was screened to identify novel peptide ligands for a 5-oxo-ETE receptor (OXER), which is a G-protein-coupled receptor for bioactive lipids, by using an OXER-Gi1alpha fusion protein. We successfully identified 2 hexapeptides-Ac-HMQLYF-NH2 and Ac-HMWLYF-NH(2)-that exhibited agonistic activity. Although the corresponding affinities were relatively low (EC50 values of 146 and 6.7 microM, respectively), the activities were confirmed by other independent cell-based assay methods, namely, intracellular calcium mobilization and cell chemotaxis. This study demonstrates that a combinatorial peptide library may be screened using a [35S]GTPgammaS binding assay with G-protein-coupled receptor (GPCR)-Galpha fusion proteins, in general, and that of peptide ligands can be obtained even for nonpeptide receptors.     

The antimicrobial activity of hexapeptides derived from synthetic combinatorial libraries

S.E. BLONDELLE, E. TAKAHASHI, K.T. DINH AND R.A. HOUGHTEN. 1995. A series of peptides identified through the use of synthetic hexapeptide combinatorial libraries (represented by the formula Ac-RRWWCO-NH₂) were examined for their antimicrobial activity against five different micro-organisms. Their toxicity was also evaluated in an in vitro haemolytic assay. The peptides showed activity against the five micro-organisms, although higher activities were found against Gram-positive bacteria. Both growth inhibition and cell viability assays were carried out to demonstrate the bactericidal activities of these peptides against two of the micro-organisms tested. The dimeric cystine forms of these peptides were shown to have biological activities identical to the monomeric forms.     

Identification of chymotrypsin inhibitors from a second-generation template assisted combinatorial peptide library.

In an earlier study (McBride JD, Freeman N, Domingo GJ, Leatherbarrow RJ. Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library. J. Mol. Biol. 1996; 259: 819-827) we described a resin-bound cyclic peptide library, constructed based on the sequence of the anti-tryptic reactive site loop of Bowman Birk Inhibitor (BBI), a proteinase inhibitor protein. This library was used to identify re-directed chymotrypsin inhibitors with Ki values as low as 17 nM. We have now extended this work by constructing an enhanced library in which a further position, at the P4 site of the inhibitor, has been randomized. This new library has variation at three target locations (P4, P1 and P2) within the inhibitory loop region, producing 8,000 variants. Screening this library allowed selection of new inhibitor sequences with Ki values as low as 3.4 nM. The success of this approach is reflected by the fact that the inhibition constant given by the selected peptide sequence is slightly lower than that reported against chymotrypsin for the most studied full length BBI protein, Soybean BBI 2-IV.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Identification of FAK substrate peptides via colorimetric screening of a one‐bead one‐peptide combinatorial library

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is associated with regulating cellular functions such as cell adhesion and migration and has emerged as an important target for cancer research. Short peptide substrates that are selectively and efficiently phosphorylated by FAK have not been previously identified and tested. Here we report the synthesis and screening of a one‐bead one‐peptide combinatorial library to identify novel substrates for FAK. Using a solid‐phase colorimetric antibody tagging detection platform, the peptide beads phosphorylated by FAK were sequenced via Edman degradation and then validated through radioisotope kinetic studies with [γ‐³²P] ATP to derive Michaelis–Menton constants. The combination of results gathered from both colorimetric and radioisotope kinase assays led to the rational design of a second generation of FAK peptide substrates. Out of all the potential peptide substrates evaluated, the most active was GDYVEFKKK with a K_M = 92 μM and a V_max = 1920 nmol/min/mg. Peptide substrates discovered within this study may be useful diagnostic tools for future kinase investigations and may lead to novel therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Alkaline protease inhibitor: a novel class of antifungal proteins against phytopathogenic fungi.

A Streptomyces sp., which produces an alkaline protease inhibitor (API) exhibiting antifungal activity has been isolated from soil. The protein has been purified to homogeneity. The molecular characterization has revealed that it is a dimer (M(r) 28 kDa) with five disulphide linkages and has a pI of 3.8. API is a competitive type of inhibitor with a K(i) value of 2.5 x 10(-9) M. The inhibitor is stable over a pH range of 6 to 12 and a temperature range of 40 to 95 degrees C. API exhibits antifungal activity (in vitro) against phytopathogenic fungi such as Fusarium, Alternaria, and Rhizoctonia and also against Trichoderma, a saprophytic fungus. The antifungal activity of API appears to be associated with its ability to inhibit the fungal serine alkaline protease(s), which is indispensable for its growth. Retardation of the rate of fungal spore germination, as well as hyphal extention, was observed in the presence of API. Both the protease inhibitory and the antifungal activity were abolished on treatment of API with DTT (5 mM), suggestive of a common site for both the activities. This is the first report on API as a potential biocontrol agent against phytopathogenic fungi.     

The use of synthetic peptide combinatorial libraries for the identification of bioactive peptides.

The systematic preparation of synthetic peptide combinatorial libraries (SPCLs), each composed of tens of millions of peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal peptide sequences has been achieved through the screening in solution of SPCLs, each element of which is composed of more than 100,000 nonsupport-bound peptides in equimolar representation, along with an iterative synthesis and screening process. Examples are presented in which an SPCL, composed in total of 52,128,400 acetylated hexa-peptides, is used along with an iterative selection process to precisely identify the antigenic determinant of a peptide recognized by a monoclonal antibody using competitive enzyme-linked immunosorbent assay. This same library was also used to develop highly potent antimicrobial peptides in bacterial growth inhibition assays. A separate non-acetylated SPCL was used to screen and identify high affinity peptide ligands using an opiate radio-receptor binding assay.     

Determination of the sequence specificity of XIAP BIR domains by screening a combinatorial peptide library

Inhibitor of apoptosis (IAP) proteins regulate programmed cell death by inhibiting members of the caspase family of proteases. The X-chromosome-linked IAP (XIAP) contains three baculovirus IAP repeat (BIR) domains, which bind directly to the N-termini of target proteins including those of caspases-3, -7, and -9. In the present study, we defined the consensus sequences of the motifs that interact with the three BIR domains in an unbiased manner. A combinatorial peptide library containing four random residues at the N-terminus was constructed and screened using BIR domains as probes. We found that the BIR3 domain binds a highly specific motif containing an alanine or valine at the N-terminus (P1 position), an arginine or proline at the P3 position, and a hydrophobic residue (Phe, Ile, and Tyr) at the P4 position. The BIR2-binding motif is less stringent. Although it still requires an N-terminal alanine, it tolerates a wide variety of amino acids at P2-P4 positions. The BIR1 failed to bind to any peptides in the library. SPR analysis of individually synthesized peptides confirmed the library screening results. Database searches with the BIR2- and BIR3-binding consensus sequences revealed a large number of potential target proteins. The combinatorial library method should be readily applicable to other BIR domains or other types of protein modular domains.     

Identification of candidate IgG biomarkers for Alzheimer's disease via combinatorial library screening

The adaptive immune system is thought to be a rich source of protein biomarkers, but diagnostically useful antibodies remain unknown for a large number of diseases. This is, in part, because the antigens that trigger an immune response in many diseases remain unknown. We present here a general and unbiased approach to the identification of diagnostically useful antibodies that avoids the requirement for antigen identification. This method involves the comparative screening of combinatorial libraries of unnatural, synthetic molecules against serum samples obtained from cases and controls. Molecules that retain far more IgG antibodies from the case samples than the controls are identified and subsequently tested as capture agents for diagnostically useful antibodies. The utility of this method is demonstrated using a mouse model for multiple sclerosis and via the identification of two candidate IgG biomarkers for Alzheimer's disease.     

Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library

Aims:  The aim of this study was to demonstrate the application of environmental sample pre-enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s).
Methods and Results:  A positive recombinant clone (UVCL29), conferring an esterase phenotype was identified from a shotgun gene library. The complete sequence of the 3·0 kb DNA insert from the pUVCL29 recombinant plasmid was obtained using primer-walking strategies. Nucleotide sequence analysis revealed a complete 945 bp open reading frame (ORF1). Translational analysis of the ORF1 showed a protein of 314 amino acids (named EstAM) with a predicted molecular weight of 34 kDa. EstAM's primary structure showed a classical (–G–D–S–A–G–) motif, corresponding with the generally conserved (G–x–S–x–G) esterase signature motif. Identity searches indicated that EstAM has high sequence similarity with esterases from family IV. EstAM was successfully expressed in Escherichia coli in a biologically active form. Partial purification was achieved using a one-step Pro-PurTM IMAC column. Biochemical characterization revealed that EstAM has a temperature optimum of 40°C.
Conclusion:  Based on its substrate profile, EstAM was classified as a carboxylesterase because of its preference for short p -nitrophenyl ester substrates.
Significance and Impact of the Study:  This study is a demonstration of the successful application of environmental sample pre-enrichment technology in accessing novel esterases from a mining environment.     

Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits

A hexapeptide of amino acid sequence Ac-Arg-Lys-Thr-Trp-Phe-Trp-NH2 was demonstrated to have antimicrobial activity against selected phytopathogenic fungi that cause postharvest decay in fruits. The peptide synthesized with either all D- or all L-amino acids inhibited the in vitro growth of strains of Penicilium italicum, P. digitatum, and Botrytis cinerea, with MICs of 60 to 80 microM and 50% inhibitory concentration (IC50) of 30 to 40 microM. The inhibitory activity of the peptide was both sequence- and fungus-specific since (i) sequence-related peptides lacked activity (including one with five residues identical to the active sequence), (ii) other filamentous fungi (including some that belong to the genus Penicllium) were insensitive to the peptide's antifungal action, and (iii) the peptide did not inhibit the growth of several yeast and bacterial strains assayed. Experiments on P. digitatum identified conidial germination as particularly sensitive to inhibition although mycelial growth was also affected. Our findings suggest that the inhibitory effect is initially driven by the electrostatic interaction of the peptide with fungal components. The antifungal peptide retarded the blue and green mold diseases of citrus fruits and the gray mold of tomato fruits under controlled inoculation conditions, thus providing evidence for the feasibility of using very short peptides in plant protection. This and previous studies with related peptides indicate some degree of peptide amino acid sequence and structure conservation associated with the antimicrobial activity, and suggest a general sequence layout for short antifungal peptides, consisting of one or two positively charged residues combined with aromatic amino acid residues.     

Sequence-selective peptide detection by small synthetic chemosensors selected from an encoded combinatorial chemosensor library.

Synthetic chemosensors hold great potential in many diagnostic applications. In this study, we describe the design and preparation of the first encoded combinatorial library of chemosensors for tripeptides. Subsequent screening of the library resulted in the discovery of novel chemosensors able to distinguish between random tripeptides.     

De novo synthetic route to a combinatorial library of peptidyl nucleosides

A stereoselective synthetic route has been developed for the combinatorial synthesis of a structurally unique class of C-4' side chain modified peptide-linked nucleosides. The synthetic strategy and approach involves initial synthesis of a strategically functionalized amino butenolide template, utilizing L-serine as a chiral starting material. Subsequent transformation of the above lactone to C4' aminoalkyl substituted nucleosides, followed by the peptidic coupling of the C4' side chain amine with various amino acids completed the syntheses of the target peptidyl nucleosides. Employing the above route, and utilizing a combination of easily available nucleobases (4) and amino acids (6) as the two diversity elements, synthesis of a 24-member combinatorial library of the title peptide-linked nucleosides has been accomplished.     

Chitinolytic actinomycetes from a Brazilian tropical soil active against phytopathogenic fungi

Five Streptomyces spp. isolated from a Brazilian forest soil showed endochitinase activity in the alkaline range with optima between 40 and 50°C. Three were highly active against three phytopathogenic fungi by in vitro experiments. Preliminary experiments suggested that the isolates may belong to a new taxon.     

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.