A novel method for selection of chymotrypsin inhibitors from a phage peptide library

A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library. In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates. After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing. These peptides share a consensus sequence motif as (S/T)RVPR(R/H). Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin. The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure.

Single-chain antibodies produced by phage display against the C-terminal 19 kDa region of merozoite surface protein-1of Plasinodium yoelii reduce parasite growth following challenge

Antibodies have the potential to be therapeutic reagents for malaria. Here we describe the production of a novel phage antibody display library against the C-terminal 19 kDa region of the Plasmodium yoelii YM merozoite surface protein-1 (MSP1(19)). In vivo studies against homologous lethal malaria challenge show an anti-parasite effect in a dose dependent manner, and analysis by plasmon resonance indicates binding to the antigen is comparable to the binding of a protective monoclonal antibody. The data support the lack of a need for any antibody Fc-related function and hold great significance for the development of a therapeutic reagent for malaria. (C) 2002 Elsevier Science Ltd. All rights reserved.

A gradient descent algorithm for minimizing amino acid coupling reactions when synthesizing cyclic-peptide libraries

Combinatorial chemistry has become an invaluable tool in medicinal chemistry for the identification of new drug leads. For example, libraries of predetermined sequences and head-to-tail cyclized peptides are routinely synthesized in our laboratory using the IRORI approach. Such libraries are used as molecular toolkits that enable the development of pharmacophores that define activity and specificity at receptor targets. These libraries can be quite large and difficult to handle, due to physical and chemical constraints imposed by their size. Therefore, smaller sub-libraries are often targeted for synthesis. The number of coupling reactions required can be greatly reduced if the peptides having common amino acids are grouped into the same sub-library (batching). This paper describes a schedule optimizer to minimize the number of coupling reactions by rotating and aligning sequences while simultaneously batching. The gradient descent method thereby reduces the number of coupling reactions required for synthesizing cyclic peptide libraries. We show that the algorithm results in a 75% reduction in the number of coupling reactions for a typical cyclic peptide library.

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters

Treatment of human cancers with an inherent antigen-processing defect due to a loss of peptide transporters (TAP-1 and TAP-2) and/or MHC class I antigen expression remains a considerable challenge. There is now an increasing realization that tumor cells with down-regulated expression of TAP and/or MHC class I antigens display strong resistance to cytotoxic T lymphocyte (CTL)mediated immune control, and often fail to respond to the conventional immunotherapeutic protocols based on active immunization with tumor-associated epitopes (TAE) or adoptive transfer of tumor-specific T cells, In the present study, we describe a novel approach based on immunization with either genetically modified tumor cells or naked DNA vectors encoding TAE fused to an endoplasmic reticulum (ER) signal sequence (ER-TAE) which affords protection against challenge by melanoma cells with down-regulated expression of TAP-1/2 and MHC class I antigens. In contrast, animals immunized with a vaccine based on TAE alone showed no protection against tumor challenge. Although MHC-peptide tetramer analysis showed a similar frequency of antigen-specific CTL in both ER-TAE- and TAE-immunized mice, functional analysis revealed that CTL activated following immunization with ER-TAE displayed significantly higher avidity for TAE when compared to animals immunized with the TAE alone, These observations provide a new strategy in anti-cancer vaccine design that allows activation of a highly effective and well-defined CTL response against tumors with down-regulated expression of TAP and MHC class I antigens.

The role of the cyclic peptide backbone in the anti-HIV activity of the cyclotide kalata B1

The plant cyclotides, the largest known family of circular proteins, have tightly folded structures and a range of biological activities that lend themselves to potential pharmaceutical and agricultural applications. Based on sequence homology, they are classified into the bracelet and Mobius subfamilies. The bracelet subfamily has previously been shown to display anti-HIV activity. We show here that a member of the Mobius subfamily, kalata B1, also exhibits anti-HIV activity despite extensive sequence differences between the subfamilies. In addition, acyclic permutants of kalata B1 displayed no anti-HIV activity, suggesting that this activity is critically dependent on an intact circular backbone. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Self-condensation of a thiazole-peptide bearing a 21-membered loop into a library of giant macrocycles with multiple orthogonal loops

Tetrapeptide analogue H-[Glu-Ser-Lys(Thz)]-OH, containing a turn-inducing thiazole constraint, was used as a template to produce a 21-membered structurally characterized loop by linking Glu and Lys side chains with a Val-Ile dipeptide. This template was oligomerized in one pot to a library (cyclo-[1](n), n = 2-10) of giant symmetrical macrocycles (up to 120-membered rings), fused to 2-10 appended loops that were carried intact through multiple oligomerization (chain extension) and cyclization (chain terminating) reactions of the template. A three-dimensional solution structure for cyclo-[1](3) shows all three appended loops projecting from the same face of the macrocycle. This is a promising approach to separating pepticle motifs over large distances.

The cyclotide family of circular miniproteins: Nature's combinatorial peptide template

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The struclural features that contribute to their remarkable stability are described ill this review. (c) 2006 Wiley Periodicals, Inc.

p-cresol as a reversible acylium ion scavenger in solid-phase peptide synthesis

In this work we have defined the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. Contrary to the results in previous reports, we found that both p-cresol and p-thiocresol predominantly form. aryl esters under typical cleavage conditions. Initially we investigated a number of small peptides containing either a single glutamate residue or a C-terminal long-chain amino acid which allowed us to unambiguously characterize the scavenged side products. Whereas, the p-cresol esters are stable at 0 degrees C they rearrange irreversibly at higher temperatures (5-20 degrees C) to form aryl ketones. By contrast, p-thiocresol esters do not undergo a Fries rearrangement but readily undergo further additions of p-thiocresol to form ketenebisthioacetals and trithio ortho esters, even at low temperatures. Importantly, we found by LC/MS and FT-ICR MS analysis that peptides containing p-cresol esters at glutamyl side chains are susceptible to amidation and fragmentation reactions at these sites during standard mild base workup procedures. The significance of these side reactions was further demonstrated in the synthesis of neutrophil immobilization factor, a 26-residue peptide, containing four glutamic acid residues. The side reactions were largely avoided by mild hydrogen peroxide-catalyzed hydrolysis which converted the p-cresol adducts to the free carboxylic acids in near quantitative yield. The choice of p-cresol as a reversible acylium ion scavenger when coupled with the simple workup conditions described is broadly applicable to Boc/benzyl peptide synthesis and will significantly enhance the quality of peptides produced.

alpha-conotoxin EpI, a novel sulfated peptide from Conus episcopatus that selectively targets neuronal nicotinic acetylcholine receptors

We have isolated and characterized ol-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus, The peptide was classified as an cy-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has ho mology to sequences of previously described cu-conotoxins, particularly PnIA, PnIB, and ImI, However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry, Native EpI was shown to coelute with synthetic EpI, The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides, The activities of synthetic EpI and its unsulfated analogue [Tyr(15)]EpI were similar. Both peptides caused competitive inhibition of nicotine action on bovine adrenal chromaffin cells (neuronal nicotinic ACh receptors) but had no effect on the rat phrenic nerve-diaphragm (muscle nicotinic ACh receptors), Both EpI and [Tyr(15)]EpI partly inhibited acetylcholine-evoked currents in isolated parasympathetic neurons of rat intracardiac ganglia, These results indicate that EPI and [Tyr(15)]EpI selectively inhibit alpha 3 beta 2 and alpha 3 beta 4 nicotinic acetylcholine receptors.

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands, As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.

Solution structure of amyloid beta-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?

The three-dimensional solution structure of the 40 residue amyloid beta-peptide, A beta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles, In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of A beta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6.0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for A beta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of A beta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the Literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease.

Solution structure of methionine-oxidized amyloid beta-peptide (1-40). Does oxidation affect conformational switching?

The solution structure of A beta(1-40)Met(O), the methionine-oxidized form of amyloid beta-peptide A beta(1-40), has been investigated by CD and NMR spectroscopy. Oxidation of Met35 may have implications in the aetiology of Alzheimer's disease. Circular dichroism experiments showed that whereas A beta(1-40) and A beta(1-40)Met(O) both adopt essentially random coil structures in water (pH 4) at micromolar concentrations, the former aggregates within several days while the latter is stable for at least 7 days under these conditions. This remarkable difference led us to determine the solution structure of A beta(1-40)Met(O) using H-1 NMR spectroscopy. In a water-SDS micelle medium needed to solubilize both peptides at the millimolar concentrations required to measure NMR spectra, chemical shift and NOE data for A beta(1-40)Met(O) strongly suggest the presence of a helical region between residues 16 and 24. This is supported by slow H-D exchange of amide protons in this region and by structure calculations using simulated annealing with the program XPLOR. The remainder of the structure is relatively disordered. Our previously reported NMR data for A beta(1-40) in the same solvent shows that helices are present over residues 15-24 (helix 1) and 28-36 (helix 2), Oxidation of Met35 thus causes a local and selective disruption of helix 2. In addition to this helix-coil rearrangement in aqueous micelles, the CD data show that oxidation inhibits a coil-to-beta-sheet transition in water. These significant structural rearrangements in the C-terminal region of A beta may be important clues to the chemistry and biology of A beta(1-40) and A beta(1-42).

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-H-3]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin I. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin I using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an cc-helical secondary structure. (C) 1998 Elsevier Science B.V, All rights reserved.