Identification of B cell and T cell epitopes using synthetic peptide combinatorial libraries.

Synthetic combinatorial libraries (SCLs) are collections of very large numbers of synthetic compounds, in which all possible combinations of the burlding blocks used are represented. The development and verification of the utility of combinatorial libraries represent a dramatic advance in the drug discovery process by greatly reducing the time needed to identify new drug leads. Positional scanning (PS) SCLs (,) represent a modified format of the origmal synthetic combinatorial libraries described by this laboratory (). In contrast to the original libraries, which required several iterative syntheses to identify individual active compounds, this library format provides mformation on the substituent responsible for activity at each varied position withm a structure. Therefore, only a single subsequent synthesis is required. The screening of PS-SCLs, in most instances, permits the identification of the most active substituents at each position of a compound in a single assay. Thus PS-SCLs serve to reduce further the time required to identify new drug leads. PS-SCLs are composed of individual positional SCLs, in which a single position is defined with one substituent while the remaining positions are composed of mixtures of substituents. The defined position is "walked" through the entire sequence of the PS-SCL. Therefore, the number of positional SCLs is equal to the number of residues in each compound of the PS-SCL. It should also be noted that each posrtional SCL, although addressing a single position of the sequence, represents the same collection of individual compounds For example, a hexapeptide, or a compound with six positions, can be represented as: O1XXXXX, XO2,XXXX, XXO3XXX, XXO4,XX, XXXXO5X, or XXXXXO6.

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(1-6)XXXXX-NH(2), 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-NH(2), 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-NH(2) 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.

The 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 x 10(6) and 4 x 10(12) 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.

Combinatorial library synthesis and screening methods, which enable the rapid identification of highly active compounds, have revolutionized basic research and drug discovery. A number of different combinatorial approaches based on the principles of solid phase synthesis have been used to generate enormous molecular diversities, including peptides, peptidomimetics, and small organic molecules. The advantage of the various combinatorial approaches compared to traditional drug synthesis and screening methods is the very large number of compounds that can be simultaneously synthesized and rapidly screened in biological assays.

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

BackgroundImmunotherapy offers many potential advantages to traditional chemotherapy, including high specificity and low toxicity. One of the goals of immunotherapy is to generate tumor-specific cytotoxic lymphocytes (CTLs), which have the...

BackgroundImmunotherapy offers many potential advantages to traditional chemotherapy, including high specificity and low toxicity. One of the goals of immunotherapy is to generate tumor-specific cytotoxic lymphocytes (CTLs), which have the...

The use of water-soluble synthetic peptide combinatorial libraries permits the systematic examination of tens to hundreds of millions of peptides in existing microdilution assays. In the present study, we prepared and determined the antistaphylococcal activities of two new synthetic peptide combinatorial libraries (one N-acetylated, the other not) composed of tetrapeptides having one position defined and the remaining three positions made up of mixtures of L-, D-, and unnatural amino acids (a total of 58 different amino acids). These libraries, when used in conjunction with an iterative selection process, allowed for the development of a series of individually defined tetrapeptides with high levels of activity against Staphylococcus aureus. The activities of the final individual peptides against two additional strains of gram-positive bacteria (methicillin-resistant S. aureus and Streptococcus sanguis), a gram-negative bacterium (Escherichia coli), as well as the yeast Candida albicans were also determined. Cell viability assays showed that the identified peptides are bacteriostatic against both S. aureus and E. coli.

Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4(+) T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.

159 - Identification of Antimelanogenic Peptides through Positional Scanning of a Synthetic Peptide Combinatorial Library

Graves' disease is characterized by the overproduction of thyroid hormones due to the persistent stimulation of TSH receptor by autoantibodies. To determine the epitopes recognized by the autoantibodies, an enzyme-linked immunosorbent assay was developed that uses the human TSH receptor extracellular domain attached to plastic wells. The total IgG from some of the Graves' patients interacted with the bound TSH receptor (TSHR) at a significantly higher level than that in normal individuals. The IgG preparation that showed the highest binding activity was used for the identification of peptide sequences that prevent binding of Graves' IgG to TSHR from positional scanning synthetic peptide combinatorial libraries. A hexapeptide mixture, X1X2FDDA (X1 is a mixture of E, M, and Y; X2 is a mixture of E, H, and T), was found to be effective for inhibiting the binding of Graves' IgG to the TSHR. Further fractionation of X1X2FDDA showed that the following three sequences were highly effective: EEFDDA, ETFDDA, and EHFDDA. The second position of the three peptides did not appear to be important. The peptides also inhibited the cAMP synthesis induced by IgG of four of eight patients with Graves' disease tested. The synthesis of cAMP by TSH was also inhibited by the peptides to some extent. The peptide sequences most likely mimic a part of the conformational epitopes recognized by at least one class of Graves' IgG.

High-Throughput Screening of Novel Peptide Inhibitors of an Integrin Receptor from the Hexapeptide Library by Using a Protein Microarray Chip

Adrenomedullin (AM) and calcitonin gene‐related peptide (CGRP) have diverse functions in the cardiovascular, lymphatic, and nervous systems including regulation of vascular tone, cardioprotection, and pain transmission. These peptides signal through three heterodimeric receptors comprising the class B GPCR, the calcitonin receptor‐like receptor (CLR), and one of three receptor activity‐modifying proteins (RAMP1‐3) that determine ligand selectivity. CLR‐RAMP complexes are proven or promising drug targets for several diseases, however, the moderate binding affinities and short plasma half‐lives of AM and CGRP limit their utility as therapeutics. Here, we used synthetic peptide combinatorial library and rational structure‐guided design approaches to develop AM and CGRP variant peptides with dramatically enhanced receptor affinities. The binding of the variants to purified RAMP‐CLR extracellular domain (ECD) complexes was characterized and a high‐resolution crystal structure of an AM variant bound to RAMP2‐CLR ECD explained its enhanced affinity. Incorporation of the variants into different length AM and CGRP scaffolds yielded picomolar affinity antagonists or agonists that exhibited substantially prolonged cAMP signaling after ligand washout. These activities were demonstrated in a model cell line transiently expressing the receptors and in primary human cells or immortalized cell lines that naturally express the receptors. This work provides a suite of AM and CGRP variants that will be valuable pharmacological tools and may have promise as peptide therapeutics with long receptor residence times. In addition, the properties of the variants support a mechanism for RAMP function in which direct RAMP‐peptide contacts and allosteric modulation of CLR cooperate to determine ligand selectivity.Support or Funding InformationThis work was supported by grants NIH R01GM104251 and OCAST HR16‐005 (AAP) and AHA predoctoral fellowship 18PRE33990152 (JMB).

Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a "synthetic peptide combinatorial library." We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an IC50 of 2 microM. The peptide is active on IN proteins from other retroviruses such as HIV-2, feline immunodeficiency virus, and Moloney murine leukemia virus, supporting the notion that a conserved region of IN is targeted. The hexapeptide was also tested in the disintegration reaction. This phosphoryl-transfer reaction can be carried out by the catalytic core of IN alone, and the peptide HCKFWW was found to inhibit this reaction, suggesting that the hexapeptide acts at or near the catalytic site of IN. Identification of an IN hexapeptide inhibitor provides proof of concept for the approach, and, moreover, this peptide may be useful for structure-function analysis of IN.