Developing peptide based capture agents for diagnostics and therapeutics

Iterative in situ click chemistry (IISCC) is a robust general technology for development of high throughput, inexpensive protein detection agents. In IISCC, the target protein acts as a template and catalyst, and assembles its own ligand from modular blocks of peptides. This process of ligand discovery is iterated to add peptide arms to develop a multivalent ligand with increased affinity and selectivity. The peptide based protein capture agents (PCC) should ideally have the same degree of selectivity and specificity as a monoclonal antibody, along with improved chemical stability. We had previously reported developing a PCC agent against bovine carbonic anhydrase II (bCAII) that could replace a polyclonal antibody. To further enhance the affinity or specificity of the PCC agent, I explore branching the peptide arms to develop branched PCC agents against bCAII. The developed branched capture agents have two to three fold higher affinities for the target protein. In the second part of my thesis, I describe the epitope targeting strategy, a strategy for directing the development of a peptide ligand against specific region or fragment of the protein. The strategy is successfully demonstrated by developing PCC agents with low nanomolar binding affinities that target the C-terminal hydrophobic motif of Akt2 kinase. One of the developed triligands inhibits the kinase activity of Akt. This suggests that, if targeted against the right epitope, the PCC agents can also influence the functional properties of the protein. The exquisite control of the epitope targeting strategy is further demonstrated by developing a cyclic ligand against Akt2. The cyclic ligand acts as an inhibitor by itself, without any iteration of the ligand discovery process. The epitope targeting strategy is a cornerstone of the IISCC technology and opens up new opportunities, leading to the development of protein detection agents and of modulators of protein functions.

A biologically active peptide in the skin of lampreys (Eudontomyzon danfordi vladykovi). [Translation from: Z.Naturforsch. (B), 26(10), 1021-1023, 1971. ]

In recent years interest in the production and description of kinin-type substances has been greatly intensified. So, for example, bradykinin, phyllokinin, physalaemin, ranatensin and caerulein could be extracted from the skin of amphibians as well as. eledoisin out of the salivary glands of Eledon moschata. An examination of lampreys seemed to us particularly profitable in the search for the incidence of further kinins. Ammocoetes of different sizes and also adults of both sexes of the species Eudontomyzon danfordi vladykovi were studied in this research. This species is found in many tributaries of the Danube. Skin extracts were tested on on isolated rat uterus, rat duodenum, guinea pig ileum and rabbit jejunum, further tests were done in order to determine a peptide character of the biologically active substance.

The structure specificity of alpha-chymotrypsin. I. Polypeptides as substrates. II. N-acylated peptide esters as substrates. III. Some reactive esters of N-acylated amino acids as substrates

The structural specificity of α-chymotrypsin for polypeptides and denatured proteins has been examined. The primary specificity of the enzyme for these natural substrates is shown to closely correspond to that observed for model substrates. A pattern of secondary specificity is proposed.

A series of N-acetylated peptide esters of varying length have been evaluated as substrates of α-chymotrypsin. The results are interpreted in terms of proposed specificity theories.

The α-chymotrypsin-catalyzed hydrolyses of a number of N-acetylated dipeptide methyl esters were studied. The results are interpreted in terms of the available specificity theories and are compared with results obtained in the study of polypeptide substrates. The importance of non-productive binding in determining the kinetic parameters of these substrates is discussed. A partial model of the locus of the active site which interacts with the R’₁CONH- group of a substrate of the form R’₁CONHCHR₂COR’₃ is proposed.

Finally, some reactive esters of N-acetylated amino acids have been evaluated as substrates of α-chymotrypsin. Their reactivity and stereo-chemical behavior are discussed in terms of the specificity theories available. The importance of a binding interaction between the carboxyl function of the substrate and the enzyme is suggested by the results obtained.

The protein, peptide and free amino-acid contents of some species of Gracilaria from south-east coast of India

Four species of Gracilaria are investigated for their free amino-acid contents, as well as amino-acid constituents in the proteins and the peptides, using quantitative paper chromatographic technique. Amino-acid constituents of different species of Gracilaria differ only in amount, while free amino-acids and the amino-acids in the peptides vary both in quality and quantity. A number of amino-acids recorded as protein constituents have even escaped detection in the peptides, while in the free state they occur either in all the species or in some only except homocystine. Moreover, some amino-acids occur exclusively in the free state.

A novel bradykinin-related peptide from skin secretions of toad Bombina maxima and its precursor containing six identical copies of the final product

Amphibian skin contains rich bradykinin-related peptides, but the mode of biosynthesis of these peptides is unknown. In the present study, a novel bradykinin-related peptide, termed bombinakinin M, was purified from skin secretions of the Chinese red bell

An anionic antimicrobial peptide from toad Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide. named maximin H5. was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2,. Containing three aspartate residues and no basic amino acid residues. maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides. only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5. while the other bacteria] and fungal strains tested ere resistant to it. The presence of metal ions. like Zn2+ and Mg2+, did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians, The results provide the first evidence that. together kith cationic antimicrobial peptides. anionic antimicrobial peptides may also exist naturally as part of the innate defense system. (C), 2002 Elsevier Science (USA). All rights reserved.

A novel proline rich bombesin-related peptide (PR-bombesin) from toad Bombina maxima

A novel bombesin-related peptide was isolated from skin secretions of Chinese red belly toad Bombina maxima. Its primary structure was established as pGlu-Lys-Lys-Pro-Pro-Arg-Pro-Pro-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH2. The amino-terminal (N-terminal) 8-residue segment comprising four prolines and three basic residues is extensively different from bombesins from other Bombina species. The peptide was thus named proline rich bombesin (PR-bombesin). PR-bumbesin was found to elicit concentration-dependent contractile effects in the rat stomach strip, with both increased potency and intrinsic activity as compared with those of [Leu(13)]bombesin. Analysis of different bombesin cDNA structures revealed that an 8 to 14- nucleotide fragment replacement in the peptide coding region (TGGGGAAT in the cDNAs of multiple bombesin forms from Bombina orientalis and CACCCCGGCCACCC in the cDNA of PR-bombesin) resulted in an unusual Pro-Pro-Arg-Pro-Pro motif in the N-terminal part of PR-bombesin. (C) 2002 Elsevier Science Inc. All rights reserved.

An opioid peptide from synganglia of the tick, Amblyomma testindinarium

An opioid peptide, which shares similarity with mammalian hemorphins, has been identified from the synganglia (central nervous system) of the hard tick, Amblyomma testindiarium. Its primary sequence was established as LVVYPWTKM that contains a tetrapeptide sequence Tyr-Pro-Trp-Thr of hemorphin-like opioid peptides. By hot-plate bioassay, the purified peptide and synthetic peptide displayed dose-related antinociceptive effect in mice, as observed for other hemorphin-like opioid peptides. This is the first opioid peptide identified from ticks. Ticks may utilize the opioid peptide in their strategy to escape host immuno-surveillance as well as in inhibiting responses directed against themselves. (c) 2004 Elsevier Inc. All rights reserved.

A novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequen

A novel algesic peptide derived from skin secretions of the frog Amolops loloensis

Several algesic agents including bradykinin and tachykinin have been identified from skin secretions of amphibians. They may act in defensive roles against aggressors. In this study, a novel peptide named Amolos with an amino acid sequence of FLPIVGAKL an

Bombinakinin M gene associated peptide, a novel bioactive peptide from skin secretions of the toad Bombina maxima

A novel 28-amino acid peptide, termed bombinakinin-GAP, was purified and characterized from skin secretions of the toad Bombina maxima. Its primary structure was established as DMYEIKQYKTAHGRPPICAPGEQCPIWV-NH2, in which two cysteines form a disulfide bond. A FASTA search of SWISS-PROT databank detected a 32% sequence identity between the sequences of the peptide and a segment of rat cocaine- and amphetamine-regulated transcript (CART). Intracerebroventricular (i.c.v.) administration of the peptide induced a significant decrease in food intake in rats, suggesting that it played a role in the control of feeding by brain. Analysis of its cDNA structure revealed that this peptide is coexpressed with bombinakinin M, a bradykinin-related peptide from the same toad. Bombinakinin-GAP appears to be the first example of a novel class of bioactive peptides from amphibian skin, which may be implicated in feeding behavior. (C) 2003 Elsevier Science Inc. All rights reserved.

An antimicrobial peptide with antimicrobial activity against Helicobacter pylori

An antimicrobial peptide named odorranain-HP was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLRASSVWGRKYYVDLAGCAKA. By BLAST search, odorranain-HP had si