Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.     

Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins

In vitro display technologies, such as mRNA display and DNA display are powerful tools to screen peptides and proteins with desired functions from combinatorial libraries in the fields of directed protein evolution and proteomics. When screening combinatorial libraries of polypeptides (phenotype), each of which is displayed on its gene (genotype), the problem remains, how best to recover the genotype moiety whose phenotype moiety has bound to the desired target. Here, we describe the use of a photocleavable 2-nitrobenzyl linker between genotype (DNA or mRNA) and phenotype (protein) in our DNA and mRNA display systems. This technique allows rapid and efficient recovery of selected nucleic acids by simple UV irradiation at 4 degrees C for 15 min. Further, we confirmed that the photocleavable DNA display and mRNA display systems are useful for in vitro selection of epitope peptides, recombinant antibodies, and drug-receptor interactions. Thus, these improved methods should be useful in therapeutics and diagnostics, e.g., for screening high-affinity binders, such as enzyme inhibitors and recombinant antibodies from random peptide and antibody libraries, as well as for screening drug-protein interactions from cDNA libraries.     

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5 min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.     

Nociceptin/orphanin FQ receptor ligands

Nociceptin (NC), alias Orphanin FQ (OFQ) is a heptadecapeptide structurally related to opioid peptides, especially Dynorphin A, which, however, does not interact with classic opioid receptors. NC selectively activates its own receptor (OP(4)), which has been shown to be insensitive to the naturally occurring opioid peptides as well as to a large number of non-peptide opioid receptor ligands, including naloxone. Thus, the NC/OP(4) system represents a new peptide-based signaling pathway, which is pharmacologically distinct from the opioid systems. The pharmacological tools available for investigating NC actions are at present rather limited and include: 1) peptide ligands obtained from structure activity studies performed using NC(1-13)NH(2) as a template or discovered by screening peptide combinatorial libraries; 2) nonpeptide ligands that are either molecules already known to interact with classic opioid receptors or novel molecules designed and synthesized as selective ligands of the OP(4) receptor. In the present paper the functional data obtained from both in vitro and in vivo studies with each relevant OP(4) receptor ligand will be analyzed and discussed comparing the advantages and disadvantages of each molecule. We hope that the present work will aid investigators, working in the NC/OP(4) field, in the choice of the pharmacological tools suitable for their experiments.     

Electrospray Mass Spectrometry and Multiple Sequence Analysis of Synthetic Peptide Libraries

The determination of the composition and purity of synthetic combinatorial libraries of free peptides requires analytical techniques that are especially suited for the challenging task of mixture analysis. Qualitative and semi-quantitive information on the composition of complex mixtures can be obtained by electrospray mass spectrometry with a maximum of reliability, accuracy, and fastness. Especially in combination with tandem mass spectrometry, contaminations with by-products (e.g., peptides with uncleaved side-chain protecting groups) can be easily revealed. Multiple sequence analysis based on Edman degradation allows a quantitative determination of the composition of libraries as well as the determination of coupling efficiencies during the synthesis of a library.     

Do inhalation general anesthetic drugs induce the neuronal release of endogenous opioid peptides?

The antagonism of some effects of inhalation general anesthetic agents by naloxone suggests that there may be an opioid component to anesthetic action. There is evidence that this opioid action component is due to neuronal release of endogenous opioid peptides. The strongest evidence is provided by studies that monitor changes in the concentration of opioid peptides in the perfused brain following inhalation of the anesthetic. Indirect or circumstantial evidence also comes from studies of anesthetic effects on regional brain levels of opioid peptides, antagonism of selected anesthetic effects by antisera to opioid peptides and anesthetic-induced changes radioligand binding to opioid receptors. It is likely that some inhalation general anesthetics (e.g., nitrous oxide) can induce neuronal release of opioid peptides and that this may contribute to certain components of general anesthesia (e.g., analgesia). More definitive studies utilizing in vivo microdialysis or autoradiography in selected areas of the brain during induction and successive states of general anesthesia have yet to be conducted.     

Light directed massively parallel on-chip synthesis of peptide arrays with t-Boc chemistry

Peptide and peptidomimetic molecule arrays are emerging powerful tools for parallel screening of binding in proteomics and pharmaceutical discovery research. Up to now the common method of preparing peptide arrays was based on spotting on glass using a library of presynthesized peptides. However, due to the large number of monomers (amino acids) it is not possible to have combinatorial libraries which include all combinations of natural and synthetic amino acids. We describe a very flexible on-chip oligopeptide synthesis method which uses the well developed t-Boc based solid state synthesis chemistry. A very high degree of flexibility is achieved by using light photo generated acids and maskless projection lithography for spatially directed deprotection. Use of microfluidic chips enables moderately high densities, short reaction times and off-the-shelf chemicals. Examples are given from synthesis of metal ion binding peptides and epitope binding assays.     

Active-site specificity of digestive aspartic peptidases from the four species of Plasmodium that infect humans using chromogenic combinatorial peptide libraries

Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009; Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1' library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1'-Gln-(Xaa). In both cases, P1 and P1' were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1' residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatography-mass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1'-Phe pools revealed the favored amino acids at the P3, P2, P2', and P3' positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.     

Solid phase synthesis of mixture-based acyclic and heterocyclic small molecule combinatorial libraries from resin-bound polyamides.

The development of soluble mixture-based heterocyclic combinatorial libraries derived from amino acids and peptides is described. Starting with a "toolbox" of various chemical transformations, including alkylations, reductions, acylations, and the use of a variety of bifunctional reagents, the "libraries from libraries" concept has been expanded to encompass the development of more than fifty positional scanning combinatorial libraries each composed of tens of thousands of low molecular weight acyclic and heterocyclic compounds.     

mRNA display: ligand discovery,interaction analysis and beyond

In vitro peptide and protein selection using mRNA display enables the discovery and directed evolution of new molecules from combinatorial libraries. These selected molecules can serve as tools to control and understand biological processes, enhance our understanding of molecular interactions and potentially treat disease in therapeutic applications. In mRNA display, mRNA molecules are covalently attached to the peptide or protein they encode. These mRNA-protein fusions enable in vitro selection of peptide and protein libraries of >10(13) different sequences. mRNA display has been used to discover novel peptide and protein ligands for RNA, small molecules and proteins, as well as to define cellular interaction partners of proteins and drugs. In addition, several unique applications are possible with mRNA display, including self-assembling protein chips and library construction with unnatural amino acids and chemically modified peptides.     

Automatic Edman microsequencing of peptides containing multiple unnatural amino acids

It is now routine using automatic Edman microsequencing to determine the primary structure of peptides or proteins containing natural amino acids; however, a deficiency in the ability to readily sequence peptides containing unnatural amino acids remains. With the advent of synthetic peptide chemistry, combinatorial chemistry, and the large number of commercially available unnatural amino acids, there is a need for efficient and accurate structure determination of short peptides containing many unnatural amino acids. In this study, 35 commercially available alpha-unnatural amino acids were selected to determine their elution profile on an ABI protein sequencer. Using a slightly modified gradient program, 19 of these 35 PTH amino acids can be readily resolved and distinguished from common PTH amino acids at low picomole levels. These unnatural amino acids in conjunction with the 20 natural amino acids can be used as building blocks to construct peptide libraries, and peptide beads isolated from these libraries can be readily microsequenced. To demonstrate this, we synthesized a simple tripeptide "one-bead one-compound" combinatorial library containing 14 unnatural and 19 natural amino acids and screened this library for streptavidin-binding ligands. Microsequencing of the isolated peptide-beads revealed the novel motif Bpa-Phe(4-X)-Aib, wherein X = H, OH, and CH3.     

Smart peptide libraries are accessible via enzymatic modifications

Summary The minireview summarizes the recent preparation of the following unusually modified combinatorial peptide collections useful for diagnostics and screening in drug finding. Tissue transglutaminase catalyzes cross couplings with transamidation between Gln and Lys peptide chains resulting in libraries with isopeptide bonds. The enzyme is involved in the triggering of autoantigenic B- and T-cell epitopes of coeliac disease. The microbial enzyme EpiD involved in lantibiotic biosynthesis catalyzes oxidative decarboxylation of C-terminal cysteine residues in peptide libraries transforming peptidyl-cysteines to peptide (2-mercaptovinyl)amides. Novel backbone modified peptide libraries are prepared using oxazole and thiazole building blocks carrying amino acid side chains. These amino acids have been found in many biologically active natural products from marine and microbial organisms such as microcin B17. Dityrosine and isodityrosine linked peptide dimer libraries are accessible by oxidative phenol coupling using horseradish peroxidase. Such structural elements are found for example in the polycyclic glycopeptide antibiotics of the vancomycin type. Microstructured layers of linear and cyclic peptide libraries are generated on transducer surfaces for cellular assays, sensor developments and even chiral recognition. Examples include a light-directed and microstructured electrochemical polymerization of phenol labelled peptides.     

Mimotopes of the nicotinic receptor binding site selected by a combinatorial peptide library

Peptide libraries allow selecting new molecules, defined as mimotopes, which are able to mimic the structural and functional features of a native protein. This technology can be applied for the development of new reagents, which can interfere with the action of specific ligands on their target receptors. In the present study we used a combinatorial library approach to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. On the basis of amino acid sequence comparison of different alpha-bungarotoxin binding receptors, we designed a 14 amino acid combinatorial synthetic peptide library with five invariant, four partially variant, and five totally variant positions. Peptides were synthesized using SPOT synthesis on cellulose membranes, and binding sequences were selected using biotinylated alpha-bungarotoxin. Each variant position was systematically identified, and all possible combinations of the best reacting amino acids in each variant position were tested. The best reactive sequences were identified, produced in soluble form, and tested in BIACORE to compare their kinetic constants. We identified several different peptides that can inhibit the binding of alpha-bungarotoxin to both muscle and neuronal nicotinic receptors. Peptide mimotopes have a toxin-binding affinity that is considerably higher than peptides reproducing native receptor sequences.     

Identification of peptide superagonists for a self-K-ras-reactive CD4+ T cell clone using combinatorial peptide libraries and mass spectrometry.

The proliferative responses of a human CD4+ T cell clone 29.15.2, reactive with a self-K-ras-derived peptide (3EYKLVVVGAGGVGKSALT20), were tested using a set of X9 combinatorial peptide libraries containing the flanking residues (EYKLVXXXXXXXXXSALT, where X indicates random amino acids). Certain peptide libraries, such as EYKLVXXXXXXM XXSALT and EYKLVXXXXXXXH XSALT, stimulated a marked proliferation of 29.15.2. However, no combinations of substitutions tested, such as EYKLVXXXXXXMH XSALT, exhibited additive effects. We subsequently synthesized peptides with degenerate sequences (a mixture of 480 species), where each position is composed of the wild-type (wt) residue or of amino acids that induced the proliferation of 29.15.2, in positional scanning. Interestingly, one fraction of degenerate peptides, separated by reverse-phase HPLC, stimulated much higher proliferation than did the wt; in addition, the retention time of this fraction was distinct from that of the wt. Mass spectrometry analysis of this fraction and flanking fractions identified five peptide species that exhibit strong signals in a manner that parallels the antigenic activity. Finally, 17 candidate peptide sequences were deduced from mass spectrometry and hydrophobicity scoring results, of which two peptides (EYKLVVVGAGGML KSALT and EYKLVVVGAGGMI KSALT) did induce 52- and 61-fold stronger proliferation, respectively, compared with the wt. These findings indicate that: 1) synthetic peptides that carry "the best" residue substitution at each position of combinatorial peptide libraries do not always exhibit superagonism, and 2) such a drawback can be overcome with the use of mass spectrometry. This approach provides new perspectives for the accurate and efficient identification of peptide superagonists.     

Structural-functional relations of short analogs of enkephalin

The similarity of action of narcotic analgesics and opioid peptides is due to activation of a common opiate receptor as the primary step in initiating biochemical chains responsible for diverse morphine-like effects. The most widely used assays for opioid and analgesic activities are presented and evaluated. Approximately 180 short enkephalin analogues (di-, tri- and tetrapeptides), described in the literature, are systematized and their opioid and systemic analgesic activities compared with methionine-enkephalin and morphine as the reference compounds, respectively. The analysis of structure-opioid activity relationships among these enkephalin analogues substantiates the hypothesis that only a limited N-terminal region of the peptide molecule is essential for the binding of opioid peptides to the subclass of opiate receptors interacting with narcotic alkaloids (mu-receptors). An attempt has been made to identify minimal structural elements responsible for the mu-receptor activation. Shortening of the molecule and modification of its elements are examined with regard to the mu- and delta-receptor selectivity. It is emphasized that the aromatic structure of the C-terminal region of the peptide is not obligatory for the mu-receptor binding. Modifications of short enkephalin analogues which might confer them antagonistic properties are reviewed. The correlation between the ability of short enkephalin analogues to interact with mu-receptors and their antinociceptive properties is discussed along with some structural features pertinent to the analgesic effect after systemic administration of peptides. On the basis of this analysis, peptides containing no more than four amino acids are considered as the most probable morphine-like analgesics.     

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.     

Use of combinatorial peptide libraries for T-cell epitope mapping

T lymphocytes play important roles not only in infectious diseases and autoimmunity, but also in immune responses against tumors. For many of these disorders, the relevant target antigens are not known. Designing effective methods that allow the search for T-cell epitopes is therefore an important goal in the areas of infectious diseases, oncology, vaccine development, and numerous other biomedical specialties. So far, the strategies used to examine T-cell recognition have been based largely on mapping T-cell epitopes with overlapping peptides from known proteins or with entire proteins, e.g., from a specific virus, bacterium, or human tissue. These approaches are tedious and have a number of limitations. It is, for example, almost impossible to isolate T cells that infiltrate an organ or infectious site and identify their specificity unless one already has a concept as to which antigens may be relevant. During recent years, a number of laboratories have developed less biased approaches that employ either the selection of putative T-cell epitopes based on the prediction of binding to certain major histocompatibilty complex (MHC) molecules and peptide or protein libraries that have been generated in expression systems, e.g. phage, or rely on combinatorial peptide chemistry. The latter technique has been refined by a number of laboratories including ours. Bead-bound or, preferably, positional scanning synthetic and soluble combinatorial peptide libraries allow the identification of T-cell epitopes within complex mixtures of proteins even for T cells that have been expanded from an organ infiltrate with a polyclonal stimulus. The practical steps that are involved in the latter method are described in this article.     

Successful identification of novel agents to control infectious diseases from screening mixture-based peptide combinatorial libraries in complex cell-based bioassays

Mixture-based peptide synthetic combinatorial libraries (SCLs) represent a valuable source for the development of novel agents to control infectious diseases. Indeed, a number of studies have now proven the ability of identifying active peptides from libraries composed of thousands to millions of peptides in cell-based biosystems of varying complexity. Furthermore, progressing knowledge on the importance of endogenous peptides in various immune responses lead to a regain in importance for peptides as potential therapeutic agents. This article is aimed at providing recent studies in our laboratory for the development of antimicrobial or antiviral peptides derived from mixture-based SCLs using cell-based assays, as well as a short review of the importance of such peptides in the control of infectious diseases. Furthermore, the use of positional scanning (PS) SCL-based biometrical analyses for the identification of native optimal epitopes specific to HIV-1 proteins is also presented.