Multipin Peptide Synthesis Research Articles

Abstract 215: An Agonistic Human Monoclonal Autoantibody to the β2-adrenergic Receptor: Implications in Postural Hypotension

Functional autoantibodies directed to the autonomic receptors have been increasingly recognized to contribute to the pathophysiology of various cardiovascular diseases. However, to date, no human monoclonal autoantibodies to these receptors with agonist activity have been available. We have recently reported a subgroup of patients with idiopathic postural hypotension who have no known etiology are associated with vasodilatory autoantibodies to the β2-adrenergic receptor (β2AR). Using standard hybridoma techniques, we produced a β2AR-activating monoclonal autoantibody C5F2 from the peripheral blood lymphocytes of a patient with idiopathic postural hypotension and high ELISA antibody titers to a peptide corresponding to the second extracellular loop (ECL2) of β2AR. C5F2 was of the IgG3 isotype and recognized an epitope located at the N-terminus of β2AR ECL2 as determined by epitope mapping using multipin peptide synthesis. Confocal immunofluorescence revealed C5F2 binding to the β2AR in Chinese hamster ovary (CHO) cells transfected with human β2AR and rat cremaster arterioles, both of which were inhibited by presabsorption with the β2AR ECL2 peptide. Functionally, the C5F2 IgG stimulated cyclic AMP production in β2AR-expressing CHO cells in a dose-dependent manner (0.005-5 mg/ml). C5F2-induced cyclic AMP activation was specifically blocked by both the β2AR ECL2 peptide and β2AR selective antagonist ICI-118551. In an isolated rat cremaster arteriole assay, infusion of the C5F2 IgG produced a potent vasodilation which was inhibited by the β2AR ECL2 peptide and β2AR blocker ICI-118551 (from 67.8±5.8% to 9.3±5.1% and 4.4±2.1% of maximal dilatory response, respectively), indicating that antibody activation of vascular β2AR may contribute to systemic vasodilation. These data support the concept that circulating agonistic autoantibodies serve as vasodilators and may cause or exacerbate orthostasis by altering the compensatory postural vascular response. The availability of this first human monoclonal autoantibody to the β2AR provides opportunities to identify previously unrecognized causes and new pharmacological management of postural hypotension.

The antigenic binding site(s) of antibodies to factor XII associated with the antiphospholipid syndrome

Phospholipid binding proteins, including factor XII (FXII), are known to be targeted by antiphospholipid antibodies (aPA). Factor XII antibodies (FXIIab) have been described in some patients with the antiphospholipid syndrome (APS) and have been shown to lead to reduced levels of FXII. The antigenic binding site(s) and the pathophysiological effects of FXIIab are unknown. In an attempt to elucidate the binding site of these antibodies, immobilized plasma kallikrein was used to cleave FXII into its 52-kDa heavy-chain (HCFXII) and 28-kDa light-chain (LCFXII) components. Plasma samples from 12 female patients with definite APS and FXIIab were investigated for the presence of antibodies to FXII, HCFXII and LCFXII. All but one patient's plasma reacted to FXII, HCFXII and LCFXII in a similar manner. One patient gave markedly reduced positivity to HCFXII and LCFXII, suggesting that the FXIIab in this patient had a higher affinity for the intact FXII molecule. To further investigate the antigenic binding site(s) of FXII, 150 biotinylated peptides of the known FXII sequence were synthesized using a Multipin(TM) peptide synthesis procedure. The IgG and IgM fractions of the 12 patients' plasma were purified by affinity chromatography. The synthesized peptides were captured on streptavidin plates and individual patients' purified FXIIab assayed against the peptides in a modified enzyme-linked immunosorbent assay (ELISA). Two regions were identified as possible antigenic binding site(s) for FXIIab: one in the growth factor domain and the other in the catalytic domain.

The identification of immunodominant linear epitopes of dengue type 2 virus capsid and NS4a proteins using pin-bound peptides

We have used multi-pin peptide synthesis strategy to identify B-cell epitopes on two small dengue virus proteins, capsid and NS4a. We have identified several linear, immunodominant epitopes on both these proteins. Almost all these epitopes mapped to regions predicted to be hydrophilic based on Kyte and Doolittle profiles. Of the capsid epitopes identified in this study, the most immunogenic ones mapped to the C-terminal α4 helix, which lies on the solvent-exposed surface of the capsid dimer. The capsid epitopes were dengue-specific in that they could recognize antibodies in dengue virus-, but not yellow fever virus (YFV)- or Japanese encephalitis virus (JEV)-immune sera. This study has demonstrated the presence of anti-NS4a antibodies in dengue-patient sera definitively, for the first time, using authentic NS4a-derived pin-bound peptides as capture antigens. All the NS4a epitopes mapped to the amino-terminal third of the NS4a molecule. Our study suggests that the immunodominant epitopes of these two dengue proteins might have the potential to be used as a part of a recombinant multi-epitope protein containing carefully chosen E and NS1 epitopes for the detection of dengue infections with a high degree of sensitivity and specificity.

A Novel CRM1-mediated Nuclear Export Signal Governs Nuclear Accumulation of Glyceraldehyde-3-phosphate Dehydrogenase following Genotoxic Stress

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.

Multiple parallel synthesis of peptides on SynPhase™ grafted supports

The Multipin peptide synthesis approach originated as an immunological tool for epitope mapping. However, continuing evolution of the basic technology has permitted the synthesis of peptides at scales up to 25 μmol per modular grafted surface. At this loading, the methodology can no longer be considered just a screening tool and is now used for the synthesis of micromoles of peptide and other small drug-like molecules for high throughput compound screening. Recent developments such as the introduction of novel grafted polymeric surfaces, new linkers, as well as novel cleavage formats has extended the scope of applications for modular grafted surfaces. This review summarizes the important achievements over the last 15 years of applications of the Multipin synthesis approach and also introduces a new modular grafted surface for peptide and small molecule synthesis called the SynPhase Lantern.

The roles of the N‐terminal portions of various tachykinins in promoting salivation

In order to determine the active sites for salivation of various tachykinins, the regulatory roles of the N-terminal portion of various newly-synthesized tachykinins were studied after i.p. injection of rats using the submandibular glands as model organs. N-shortened oligopeptides from kassinin, eledoisin, neurokinins A (NKA) and NKB were synthesized by the multipin peptide synthesis method. Amino acids were eliminated one by one to form octa- to undeca-peptides adjoining the inactive or less active heptapeptides and various heptapeptides, in which an amino acid in position 8 (Xaa8), numbering as in an undecapeptide, was replaced with Tyr, Phe, Ile or Val. The N-terminal amino acids in positions 1 to 4 could be activators or inhibitors, depending on whether the C-terminal heptapeptide was inactive or less active. The Xaa8 residue, in combination with amino acids in positions 5 and 6, seemed to be very important in determining the sialogogic activity of a heptapeptide. The discrimination between NKA and NKB appeared due to the N-terminal amino acid sequence in positions I to 4 including Phe or Ser in position 6. It is concluded that the N-terminal amino acids in positions I to 4 serve as either activators or inhibitors depending upon the sialogogic activity of the C-terminal heptapeptide, in which particular amino acids in positions 5, 6 and 8 regulate its activity.

Investigation of the epitopes of human profilaggrin derived peptide targeted by antibodies of patient with rheumatoid arthritis

Anti-filaggrin antibodies (AFA), directed against the 37-40 kD epidermal protein filaggrin are one of the most specific markers of rheumatoid arthritis (RA) and include anti-keratin antibodies (AKA) and anti-perinuclear factor (APF). Although the antigen triggering autoimmune response to filaggrin related proteins has not been identified, recent studies confirmed that citrulline is essential constituent of protein related antigenic determinats recognised by RA specific autoantibody population. The aim of our study was to identify epitopes of filaggrin derived-peptides targeted by RA specific antibodies to provide further information about the nature of the initial autoantigenic substance. Citrullin containing peptides of human profilaggrin region (amio acid residues 306-324) derived from known cDNA and on the basis of the data published by Shellekens were synthetised by the multipin peptide synthesis on solid support and were reacted in situ by patient sera. Two 19-mer peptides were prepared with single cit-rullin substitution at position 312 or 321 respectively and four additional ones with simultanious replacements of two Arg by Cit. Shortened versions of the 306SHQESTCitGRSGRSGRSGR324 peptidewere also produced by removal of 1-6 amino acid residues from its N-terminal and the 14 -mer truncated one was further shortened from its C-terminal as well. The reactivities of these peptides with RA sera and healthy controls were determined. The results showed that substitution of arginine 312 by citrulline plays major role in the anigenicity of filaggrin-derived sequences. Peptides not containing Cit in position 312 almost lost their ability to bind antibodies from RA sera. Replacement of one additional Arg by Cit in different positions did not improved the antigenicity. When the peptide with Cit in position 312 were shortened from its N-terminal, the 14-mer one showed the highest reactivity. Further shortening of this sequence from its C-terminal showed that TXGRS motif seems to be essential to comprise the autoanigenic epitope. In conclusion our results provide further evidence that not simply the presence of citrullin but also the nature of its surronding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies.

Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding.

To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.

A monoclonal antibody against the X protein of hepatitis B virus: fine mapping of its epitope and application in a quantitative ELISA of the X protein in sera of hepatitis B patients.

A HBx-specific mouse monoclonal antibody was developed and its epitope mapped to a hydrophilic segment 94HKRTLGL100 using the multipin peptide synthesis technique. A sensitive ELISA with a threshold of 5 to 10 ng was developed to identify the HBx-positive hepatitis B cases and measure the levels of HBx in sera. The same patient sera were also analyzed for the presence of anti-HBx using the purified recombinant antigen. HBx was present in 23% of the cases (15/65) whereas only 14% of the cases (9/65) were positive for anti-HBx. The mean value of HBx in acute hepatitis sera was higher (522 ng/ml) than in cirrhosis cases (48 ng/ml). PCR amplification of the S gene showed that all 15 HBx-positive cases were also positive for the viral DNA.

Systematic Mapping of Potential Binding Sites for Shc and Grb2 SH2 Domains on Insulin Receptor Substrate-1 and the Receptors for Insulin, Epidermal Growth Factor, Platelet-derived Growth Factor, and Fibroblast Growth Factor

Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways.

Mapping of cyclosporin A binding sites in cyclophilin A by using synthetic peptides

In order to map cyclosporin A (CsA) binding sites of cyclophilin (CyP), we synthesized the complete set of overlapping 157 octapeptides corresponding to human CyP A using the multi-pin peptide synthesis system. The pin-coupled synthetic octapeptides were examined in terms of binding ability to CsA by a modification of the enzyme-linked immunosorbent assay. Significant binding of CsA was detected with 35 synthetic N α-acetylated octapeptides possessing the N-terminal amino acids corresponding to the residues in positions 24–26, 42–44, 69–73, 75, 76, 89–91, 102, 116, 124–131, 144–151 and 152 in human CyP A, respectively. Other eight octapeptides showed moderate CsA binding activity. The distinct binding of octapeptides covering the C-terminal region of the CyP A was particularly significant. These data are to be compared with the information provided by X-ray and NMR studies on the CsA binding sites and furnish thus a test of the reported method. The present study also gave added insight into the CsA interaction sites of CyP.

Immunochemical characterisation and epitope mapping of a novel fimbrial protein (Pg-II fimbria) of Porphyromonas gingivalis

Mouse monoclonal antibody (mAb) Pgf-II specific for a 72-kDa major cell-surface protein (72K-CSP) derived from Porphyromonas gingivalis OMZ 409 was prepared. Immunoblotting analysis revealed that mAb Pgf-II reacted with 72K-CSP but not with 41-kDa fimbrial subunit protein (41K-fimbrilin) derived from P. gingivalis 381. Electron microscopic observation revealed that P. gingivalis OMZ 409 possessed peritrichous, thin fimbriae on their surface. Immunogold electron microscopy also demonstrated that mAb Pgf-II bound to the 72K-CSP examined with the gold particles arranged along the fibril array originating from the cell surface of the bacteria. These findings suggested that P. gingivalis 72K-CSP was identifiable as another fimbriae (termed Pg-II fimbriae) different from the fimbriae (termed Pg-I fimbriae) composed of a 41K-fimbrilin. Using multipin peptide synthesis technology, 102 sequential overlapping peptides covering the entire 514 amino-acid stretch of Pg-II fimbriae were synthesised. Seven immunodominant regions within Pg-II fimbrial protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected.

Immunochemical characterisation and epitope mapping of a novel fimbrial protein (Pg-II fimbria) of Porphyromonas gingivalis.

Mouse monoclonal antibody (mAb) Pgf-II specific for a 72-kDa major cell-surface protein (72K-CSP) derived from Porphyromonas gingivalis OMZ 409 was prepared. Immunoblotting analysis revealed that mAb Pgf-II reacted with 72K-CSP but not with 41-kDa fimbrial subunit protein (41K-fimbrilin) derived from P. gingivalis 381. Electron microscopic observation revealed that P. gingivalis OMZ 409 possessed peritrichous, thin fimbriae on their surface. Immunogold electron microscopy also demonstrated that mAb Pgf-II bound to the 72K-CSP examined with the gold particles arranged along the fibril array originating from the cell surface of the bacteria. These findings suggested that P. gingivalis 72K-CSP was identifiable as another fimbriae (termed Pg-II fimbriae) different from the fimbriae (termed Pg-I fimbriae) composed of a 41K-fimbrilin. Using multipin peptide synthesis technology, 102 sequential overlapping peptides covering the entire 514 amino-acid stretch of Pg-II fimbriae were synthesised. Seven immunodominant regions within Pg-II fimbrial protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected.

Sonication-assisted cleavage of hydrophobic peptides. Application in multipin peptide synthesis

High-power sonication enables hydrophobic peptides, that would otherwise cleave with poor efficiency, to cleave and elute from the solid support in good yield. The method is demonstrated in conjunction with the multipin method of multiple synthesis using a diketopiperazine-forming handle with cleavage at pH 8.3.

The potential protective immune responses to synthetic peptides containing conserved epitopes of Porphyromonas gingivalis fimbrial protein

The immunodominant and T-cell epitopes within the fimbrial subunit protein (fimbrilin) of Porphyromonas gingivalis strain 381 were analysed by multi-pin peptide synthesis technology. Six regions with immunodominant epitopes within a sequence of 337 amino acids that reacted with the serum of patients with adult periodontitis were detected. T cells from mice immunised with P. gingivalis fimbriae exhibited proliferative responses to P. gingivalis fimbriae or to six 10-mer synthetic peptides from the amino-acid sequence of the fimbrillin. Three synthetic peptides that contained the regions responsible for the immunodominant epitopes as well as those which coincided with a T-cell epitope of P. gingivalis fimbrial molecules--FP38 (142-161), FP381(202-221) and FP381(216-243)--were selected and synthesised. When guinea-pigs were immunised with fimbriae or one of the three synthetic peptide segments and an adjuvant in Freund's incomplete adjuvant, enhanced production of the antigen-specific IgG antibodies was induced in the serum of the animals. Furthermore, of the three synthetic peptides tested, FP381(202-221) produced the greatest protective immune response in guinea-pigs infected with P. gingivalis and this was more effective than the native fimbrial protein.

Anti-M Monoclonal Antibodies Cross-Reacting With Variant Mg Antigen: An Example of Modulation of Antigenic Properties of Peptide by Its Glycosylation

Some monoclonal antibodies (MoAbs) directed against blood group M-related epitope of glycophorin A (GPA) were found to agglutinate rare variant erythrocytes carrying GPA of Mg type. In contradistinction to normal GPA-M or -N, the N-terminal portion of GPA-Mg is not glycosylated. Therefore, the multipin peptide synthesis was used for testing the specificity of the cross-reacting MoAbs. Among several anti-M and anti-N MoAbs tested, only three anti-M (E3, E6, 425/2B) agglutinated Mg erythrocytes and showed binding to the synthetic octapeptides corresponding to N-terminal sequences of GPA-M (SSTTGVAM), GPA-N (LSTTEVAM), and GPA-Mg (LSTNEVAM). Testing multiple peptide analogs (window and replacement analysis) showed that these MoAbs were specific for peptidic epitope in which Met8 and Val6 were the most essential amino acid residues. The amino acid replacements Ser<-->Leu1 or Gly<-->Glu5 (M v N) and Thr4<-->Asn4 (M and N v Mg) had no or negligible effect on the reaction of synthetic peptides with the MoAbs. However, when Ser2, Thr3, and Thr4 carry O-linked sialooligosaccharides (normal GPA-M or -N), the MoAbs recognize Gly5- and sialic acid-dependent blood group M-related epitope. An interesting finding concerning anti-M/Mg MoAbs described here is the fact that glycosylation of amino acid residues adjacent to the most important part of peptidic epitope not only differentially modulates the proper exposure of peptidic epitope, but also alters the requirement for some amino acid residues present within the epitope. Pathologic conditions, including hematologic disorders, are often accompanied by alterations in protein glycosylation, resulting not only from differences in the structure of antigen polypeptide chain, but also from changes in specificity or expression of enzymes involved in glycosylation. Our present findings draw attention to possibility of the bidirectional modulation of protein antigenicity by glycosylation and may be helpful in interpretation of some results obtained with MoAb used for diagnostic or other purposes.

Anti-M monoclonal antibodies cross-reacting with variant Mg antigen: an example of modulation of antigenic properties of peptide by its glycosylation

Some monoclonal antibodies (MoAbs) directed against blood group M- related epitope of glycophorin A (GPA) were found to agglutinate rare variant erythrocytes carrying GPA of Mg type. In contradistinction to normal GPA-M or -N, the N-terminal portion of GPA-Mg is not glycosylated. Therefore, the multipin peptide synthesis was used for testing the specificity of the cross-reacting MoAbs. Among several anti- M and anti-N MoAbs tested, only three anti-M (E3, E6, 425/2B) agglutinated Mg erythrocytes and showed binding to the synthetic octapeptides corresponding to N-terminal sequences of GPA-M (SSTTGVAM), GPA-N (LSTTEVAM), and GPA-Mg (LSTNEVAM). Testing multiple peptide analogs (window and replacement analysis) showed that these MoAbs were specific for peptidic epitope in which Met8 and Val6 were the most essential amino acid residues. The amino acid replacements Ser&lt;--&gt;Leu1 or Gly&lt;--&gt;Glu5 (M v N) and Thr&lt;--&gt;Asn4 (M and N v Mg) had no or negligible effect on the reaction of synthetic peptides with the MoAbs. However, when Ser2, Thr3, and Thr4 carry O-linked sialooligosaccharides (normal GPA-M or -N), the MoAbs recognize Gly5- and sialic acid- dependent blood group M-related epitope. An interesting finding concerning anti-M/Mg MoAbs described here is the fact that glycosylation of amino acid residues adjacent to the most important part of peptidic epitope not only differentially modulates the proper exposure of peptidic epitope, but also alters the requirement for some amino acid residues present within the epitope. Pathologic conditions, including hematologic disorders, are often accompanied by alterations in protein glycosylation, resulting not only from differences in the structure of antigen polypeptide chain, but also from changes in specificity or expression of enzymes involved in glycosylation. Our present findings draw attention to possibility of the bidirectional modulation of protein antigenicity by glycosylation and may be helpful in interpretation of some results obtained with MoAb used for diagnostic or other purposes.

Multiple peptide synthesis on acid‐labile handle derivatized polyethylene supports

Using the multipin peptide synthesis approach, a range of peptides with native amide and carboxylate C-termini were generated using an acid-labile approach. Polyethylene crowns grafted with hydroxyethylmethacrylate (HEMA) polymer were functionalized with either 4-hydroxymethylphenoxyacetic acid for the generation of peptide-carboxylate or p-[(R,S)-alpha-[1-(9H-fluoren-9-yl)methoxyformamido]-2,4-dim ethoxy- benzyl]phenoxyacetic acid for peptide-amide. A range of known peptide hormone sequences and other peptides with native C-termini were assembled by sequential incorporation of N alpha-Fmoc protected amino acids. Peptides were sidechain deprotected and cleaved from crowns with TFA/scavengers within 2 mL centrifuge tubes, and isolated by a series of ether/petrol wash and centrifugation steps. In this way it was possible to avoid a cleavage and isolation botteneck, allowing rapid processing of large numbers of peptides.

Systematic study of substance P analogs

The multipin peptide synthesis technique, a method for simultaneous multiple peptide synthesis, was developed for large-scale screening of oligopeptides [Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002]. A modification of the technique allows the peptides assembled on polyethylene pins to be cleaved in their native amide form and reconstituted into physiologically compatible solutions. In this study, the suitability of these peptides for quantitative receptor binding assay was evaluated. Substance P and 18 analogs, including a set of N-terminal truncated substance P and a set of naturally occurring substance P analogs, were synthesized by the multipin methods. An average yield of 20 +/- 3 nmol of peptide per pin was obtained. The purity of the peptides was estimated to be ca. 90%. The binding activities of these peptides were determined in a competition assay against 125I-BHSP binding to a rat brain synaptosome preparation. The rank order of the affinities of these peptides depicted a typical pharmacological profile of central NK1 receptor. The IC50 values obtained were also in good agreement with data reported by other groups using similar experimental conditions, except that bulk synthesized peptides were used. This study demonstrates that the peptides synthesized with the multipin technique are suitable for quantitative receptor studies, particularly for a high-volume screening of bioactive peptides.

Systematic study of substance P analogs

Recent developments in multiple peptide synthesis have made it feasible to synthesize and screen large numbers of peptide analogs simultaneously. We report here a model study of large scale screening of stereoisomers of substance P with systematic D-amino acid replacements. Such studies are useful in exploring conformational requirements of peptide-receptor interaction and to provide empirical information for peptide drug design. 512 stereoisomers of SP were prepared by the multipin peptide synthesis method. Receptor binding affinities of these analogs were estimated by an iterative competition assay. Results obtained form a comprehensive database on the stereochemical requirement of SP binding to central NK1 receptor. Data from analogs with single D-amino acid replacement are consistent with those previously reported showing that SP binding is highly sensitive to the chirality change of the C-terminal residues (Gln6-Leu10), but less sensitive to the chirality change of the N-terminal residues (Arg1-Gln5). A qualitative analysis of the database by comparison of series of peptide pairs revealed a repeated pattern of affinity change with D-amino acid replacement, suggesting a largely additive binding activity of SP from each residue. On the other hand, possible intramolecular interactions between some N-terminal and C-terminal residues to form an optimal binding conformation were also found. A set of 189 peptides with IC50 values less than 5 microM was subjected to an empirical QSAR analysis using a linear additive model. The relative contribution coefficients obtained agreed with the observation that the predominant contribution comes from the C-terminal residues, suggesting considerable independency of each residue on binding to NK1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)