Discovery of a novel, potent, and specific family of factor Xa inhibitors via combinatorial chemistry.

A series of low molecular weight peptide inhibitors of factor Xa, unrelated to any previously described, was identified by screening a combinatorial peptide library composed of L-amino acids. The minimal inhibitory sequence is a tripeptide, L-tyrosinyl-L-isoleucyl-L-arginyl, which competitively inhibits the hydrolysis of small chromogenic substrates by factor Xa but binds in an orientation which prevents a productive nucleophilic attack by serine 195 of the catalytic triad on the carbonyl carbon of the carboxyterminal arginine. The initial leads identified in an octamer combinatorial peptide library ranged in potency from 4 to 15 microM. These peptides were modified into peptide mimetics with a greater than 1000-fold increase in potency while retaining unusual selectivity for factor Xa over the related serine proteases thrombin, factor VIIa/tissue factor, plasmin, activated protein C, kallikrein, and trypsin. One of the most potent analogues, SEL 2711, with a Ki of 0.003 microM for factor Xa and 40 microM for thrombin, is active in in vitro and ex vivo coagulation assays, suggesting the potential application of these inhibitors in anticoagulant therapy.

High-volume cellular screening for anticancer agents with combinatorial chemical libraries: a new methodology.

A single-step cancer cell cytotoxic assay system for anticancer drug discovery has been developed which facilitates rapid screening of large combinatorial chemical libraries synthesized using the 'one-bead-one-compound' (OBOC) methodology. Each OBOC library bead incorporates two orthogonally cleavable linkers that release the bead-bound compound at a different pH. The assay utilizes high concentrations of tumor cells mixed directly with OBOC beads and plated in soft agarose containing tissue culture medium. One of the orthogonal linkers is cleaved at neutral pH in tissue culture releasing an aliquot of compound to diffuse at a relatively high local concentration into the soft agarose immediately surrounding the bead. Active compounds are identified visually from a clear ring of tumor cell lysis which forms within 48 h around just the rare bead releasing a cytotoxic compound. The bead releasing a cytotoxin is then plucked from the agar and the remaining compound still linked to the bead can be released for structural analysis, followed by compound resynthesis and confirmatory testing. This assay system has been successfully applied to identification of lead cytotoxic compounds from model peptidic and non-peptidic combinatorial chemical libraries. Use of this methodology may facilitate anticancer drug discovery.

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Library of libraries: approach to synthetic combinatorial library design and screening of "pharmacophore" motifs.

Construction of synthetic combinatorial libraries is described that allows for the generation of a library of motifs rather than a library of compounds. Peptide libraries based on this strategy were synthesized and screened with model targets streptavidin and anti-beta-endorphin antibody. The screens resulted in observation of expected motifs providing evidence of the effectiveness of the suggested approach.

One-bead-one-structure combinatorial libraries.

Combinatorial libraries employing the one-bead-one-compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect "coding" methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and "scaffold" libraries, together with a new approach for the discovery of a peptide binding motif using a "library of libraries," are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries.

Sequence-dependent modification of Trp by the Pmc protecting group of Arg during TFA deprotection.

The extent of transfer of the Pmc protecting group from the guanidino group of arginine to the side chain of tryptophan depends on the spacial distance of these side chains. When these two amino acids are separated by one amino acid, the transfer of the Pmc protecting group is the most pronounced, and it cannot be completely prevented by the use of currently utilized scavenger mixtures. The extent of this side reaction also depends on the amino acid separating the arginine and tryptophan residues and position of tryptophan within the peptide chain as well as on the type of the solid-phase carrier.

Spontaneous chemical degradation of substance P in the solid phase and in solution.

The instability of the undecapeptide substance P (SP), a neuropeptide implicated in several physiological processes, was occasionally observed when the peptide was stored in the solid state or in solution. The aim of the present study was to identify the decomposition products of SP stored as lyophilized peptide or in aqueous neutral solution. The main pathway of the decomposition of SP acetate consists of the subsequent release of N-terminal dipeptides via their diketopiperazines, cyclo(Arg-Pro) and cyclo(Lys-Pro). In contrast to the decomposition of the acetate of SP, the hydrochloride and trifluoroacetate salts were found to be considerably more stable. Under the studied conditions the release of N-terminal dipeptides dominates over other possible routes of spontaneous modifications, such as S-oxidation and deamidation.

The use of hydrogen-deuterium exchange to facilitate peptide sequencing by electrospray tandem mass spectrometry.

The utility of hydrogen-deuterium exchange for sequencing peptides by mass spectrometry is demonstrated. The number of exchangeable hydrogens in a peptide is readily obtained by electrospray analysis of the peptide dissolved in deuterated solvents. This information can be used, in conjunction with published computer algorithms for interpreting peptide mass spectra, to reduce significantly the number of candidate sequences that fit the experimental data. This information, when combined with fragment-ion information in the mass spectrum, greatly increases the reliability of sequence determination.

[Atrial natriuretic peptides. IV. Synthesis and study of shortened analogs]. / Atrial'nye natriiureticheskie peptidy. IV. Sintez i izuchenie ukorochennykh analogov.

New analogues of atrial peptides of rat were synthesized by classical methods of peptide chemistry in solution. They contain a D-amino acid residue in the C-terminal part and a residue of mercaptopropionic acid in the N-terminal part of the molecule. Biological activity of the new analogues was studied.

Rearrangement, racemization and decomposition of peptides in aqueous solution.

We reported earlier that peptides containing glycine as the third amino acid from the amino end undergo sequence rearrangement of the first two amino acid residues. In the course of this experimental verification of the suggested reaction mechanism, we found extensive racemization of the amino acid residue in position 1. Racemization is preferred over rearrangement in peptides containing amino acids different from glycine in position 3. We demonstrate that this reaction can be used for the selective labeling of peptides. Using model peptides, we suggest a mechanism that explains both the rearrangement and racemization of these peptides in aqueous solution. This mechanism is based on formation of a diketopiperazine-like (DKP-like) structure by attack of the N-terminal amino group on the amide carbonyl group of the second residue in the peptide chain. This tetrahedral intermediate, which contains a secondary amino group derived from the amide bond between the second and third amino acid residue, can (i) decompose with the formation of diketopiperazine and a shortened peptide sequence; (ii) form a bicyclic structure by transanular attack on the first amino acid carbonyl group in the DKP-like ring by the newly formed amino group, leading to the rearranged product; and (iii) form a bicyclic structure by transanular attack of the newly formed hydroxyl group on the carbonyl group in the DKP-like ring, leading to the race-mixed product.

[Structure of the carbohydrate chain of riboflavin-binding glycoprotein from hen egg white. Neutral oligosaccharides of the hybrid type]. / Struktura uglevodnykh tsepei riboflavinsviazyvaiushchego glikoproteinabelka kurinogo iaitsa. Neitral'nye oligosakharidy gibrid tipa.

Reductive cleavage of the riboflavin-binding glycoprotein from hen egg white with LiBH4/tert-BuOH followed by NaBH4 treatment gave rise to oligosaccharide alditols. After fractionation by HPLC two individual oligosaccharide alditols of a hybrid type were isolated. Their structures were proved by 1H NMR 500 MHz spectroscopy and methylation analysis. One of the oligosaccharides has earlier been found in ovalbumin, whereas the other is identified in glycoproteins for the first time.

[The structure of carbohydrate chains of riboflavin-binding glycoprotein from chicken egg protein. III. 1H-NMR spectroscopy (500 MHz) of neutral fucosylated oligosaccharides]. / Struktura uglevodnykh tsepei riboflavinsviazyvaiushchego glikoproteina belka kurinogo iaitsa. III. Spektroskopiia 1H-IaMR (500 MHz) neitral'nykh fukozilirovannykh oligosakharidov.

Using LiBH4/ButOH treatment, oligosaccharides were cleaved off the hen egg white riboflavin-binding glycoprotein. HPLC led to the isolation of four fucose-containing oligosaccharide alditols, whose structure was elucidated by means of 1H NMR 500 MHz spectroscopy. The main fucosylated oligosaccharide, also present in hen ovomucoid, was found to be a biantennary carbohydrate chain of N-acetyllactosamine type.

[Structure of carbohydrate chains of the riboflavin-binding glycoprotein of chicken egg protein. II. 1H-NMR (500 MHz) spectroscopy of the major neutral oligosaccharides]. / Struktura uglevodnykh tsepei riboflavinsviazyvaiushchego glikoproteina belka kurinogo iaitsa. II. Spektroskopiia 1H-YaMR (500 MGts) glavnykh neitral'nykh oligosakharidov.

Reductive cleavage of riboflavin-binding glycoprotein from hen egg white (RF-GPw) with LiBH4/tert-BuOH followed by NaBH4/NaOH treatment gave rise to oligosaccharide alditols, fractionated by a successive HPLC on muBondapak C18 and Zorbax NH2 columns. Seven main individual oligosaccharide alditols were isolated and their structure was investigated by 1H NMR 500-MHz spectroscopy. The structure and relative content of the main oligosaccharide chains were proved to be identical in RF-GPw and ovomucoid. Structure of polypeptide chains and their molecular weight, number of glycosylation sites and their structure had little or no effect on the glycosylation pattern in both glycoproteins. HPLC of the oligosaccharide alditols from another egg white glycoprotein, ovotransferrin, also revealed its high microheterogeneity and close resemblance to those of ovomucoid and RF-GPw.

[A study of enzymatic hydrolysis of splenopentin and its analogs in human serum]. / Issledovanie fermentativnogo gidroliza splenopentina i ego analogov v syvorotke krovi chekloveka.

Degradation of immunoactive peptide splenopentin and its analogue N-acetylsplenopentin in human serum has been investigated by 1H NMR spectroscopy. It is shown that degradation of splenopentin occurs due to hydrolysis of peptide bonds in its N-terminal part, whereas in N-acetylsplenopentin peptide bonds in C-terminal part of the molecule are cleaved. Degradation pathways and life times of these peptides in human serum are established.

The flexible region of protein L12 from bacterial ribosomes studied by proton nuclear magnetic resonance.

The dimeric protein L7/L12 from bacterial ribosomes has a highly elongated and flexible structure. We have, using 1H NMR methods, analyzed the extent of the flexible region and also the size of the organized structures of the molecule. A number of mutants of the protein as well as monomeric and dimeric forms of the protein and a COOH-terminal fragment have been used for the identification of certain resonances. Thus, residues 37-50 were found to be highly mobile whereas the amino-terminal and COOH-terminal regions are organized into folded domains. The flexibility between the domains and its relation to functional properties of the protein are discussed.

[1H-NMR study of protein L7/L12 from bacterial ribosomes]. / Issledovanie belka L7/L12 iz bakterial'nykh ribosom metodom 1H-IaMR.

The 500 MHz 1H-NMR spectra of dimeric protein L12 from ribosomes shows a limited number of unusually sharp signals at room temperature. This is interpreted as evidence for substantial segmental flexibility of the region in the protein molecule. We have analysed the extent of the flexible region and also the size of the organized structures of the molecule. Thus residues 37-50 were found to be highly mobile whereas the N-terminal and C-terminal region are organized into folded domains.

[Atrial natriuretic peptides. II. Synthesis of N-terminal fragments]. / Atrial'nye natriiureticheskie peptidy. II. Sintez N-kontsevykh fragmentov.

N-terminal fragments of atrial natriuretic peptides have been synthetized by classical methods of peptide chemistry in solution and characterized by various physicochemical methods. The choice of the scheme and methods of synthesis is discussed.

[Atrial natriuretic peptides. I. Synthesis of C-terminal fragments]. / Atrial'nye natriiureticheskie peptidy. I. Sintez C-kontsevykh fragmentov.

C-terminal fragments of atrial natriuretic peptides have been synthesized by classical methods of peptide chemistry in solution and characterized by various physico-chemical methods. The choice of the scheme and methods of synthesis is discussed.

[Synthesis of endorphins]. / Sintez éndorfinov.

alpha-, beta-, gamma- and (des-Tyr1)-gamma-endorphins were synthesised by classical methods of peptide chemistry. Optical purity of these peptides was controlled by GLC and NMR. Data on the opioid activity of the peptides synthesised are presented.