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A set of benzoxazolyl-L-alanine derivates along with the MCA moiety (donors of fluorescence) were introduced into a proteinase 3 (PR3) substrate with a C-terminal ANB-N[H.sub.2] that serves as a fluorescence acceptor. Five substrates with general formula X-Tyr-Tyr-Abu-ANB-N[H.sub.2] were synthesized, and their kinetic parameters against proteinase 3 were determined. The highest [k.sub.cat]/ [K.sub.M] value, 1.5 x [10.sup.6] [M.sup.-1][s.sup.-1] ], was obtained for (Pyr)Box-Ala-Tyr-Tyr-Abu-N[H.sub.2] where (Pyr)Box-Ala stands for N-methylpyrrole benzoxazole-L-alanine. Titration of this peptide with proteinase 3 resulted in measurable fluorescence at an enzyme amount equal to 29 pmol. This substrate was selected used to detect quantifiable levels of proteinase 3 in serum samples, including those of normal subjects. For all c-ANCA-positive samples (diagnosed Wegener granulomatosis), a significant increase of PR3 concentration was observed. Wegener granulomatosis is a severe autoimmune disease causing inflammation of the blood vessels. Our results clearly show that this substrate can be used for the construction of a very reliable, inexpensive, and easy to use diagnostic test for PR3 determination. 10.1021/ac1004103

With only 14 amino acid residues, the trypsin inhibitor SFTI-1 is the smallest naturally occurring serine proteinase inhibitor. It consists of two cyclic fragments (with head-to-tail cyclization and a disulfide bridge). In our previous paper, we showed that the removal of the disulfide bridge produced 2.4-fold lower activity. Here, we present the total conformational analysis of the [Abu(3, 11)]-SFTI-1 analog by means of 2D NMR spectroscopy in conjunction with theoretical methods. The peptide was synthesized by Fmoc SPPS. It was cyclized with PyBop and DIPEA in DMF. The NMR studies were performed in DMSO-d(6) at 303 K. Conformations of the peptide studied were calculated by the following three approaches: distance geometry (DG), molecular dynamics (MD) and determination of the statistical weights of conformations. The first two algorithms use a CHARMM force field, whereas the last uses an ECEPP/3 force field. Our calculations resulted in three sets of conformers with 7, 9 and 6 representatives, respectively. All our results were compared with published ones. It was found that the peptide has an ill-defined structure. Despite its conformational flexibility, the binding loop (3-11 fragment) displayed geometry similar to the corresponding fragments of the other SFTI-1 analogs and to the inhibitor itself. Furthermore, the peptide bond between the Ile7 and Pro8 residues adopts cis geometry, which is essential for inhibitory activity., (Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.)

Combinatorial chemistry approach was applied to design chromogenic substrates of human beta-tryptase. The most active substrate, Ala-Ala-Pro-Ile-Arg-Asn-Lys-ANB-NH(2), was selected from among over 9 million heptapeptides. The amide of 5-amino-2-nitrobenzoic acid (ANB-NH(2)) attached at the C-terminus served as a chromophore. In order to determine the optimal length of the tryptase substrate, a series of N-terminally truncated fragments of this substrate was synthesized. Pro-Ile-Arg-Asn-Lys-ANB-NH(2), with the determined value of the specificity constant (k(cat)/K(M)) above 9 x 10(6) M(-1) s(-1), appeared to be the most specific substrate of tryptase. This substrate was twice as active as the parent heptapeptide substrate. We postulate that the optimal size of the pentapeptide substrate for the interaction with human beta-tryptase is associated with the unique structure of this proteinase, comprising four almost identical monomer subunits arranged in a square flat ring with its substrate pockets faced inside, forming a tetramer with a central pore that can be penetrated by this short peptide., (Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.)

The primary specificity residue of a substrate or an inhibitor, called the P(1) residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S(1) pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine alpha-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P(1) with the sequence Ac-Phe-Ala-Thr-X-Anb(5,2)-NH(2) were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO(2)), 4-aminophenylalanine - Phe(p-NH(2)), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonyl-phenylalanine - Phe(p-COOMe), 4-cyanophenylalanine - Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (k(cat), K(M) and k(cat)/K(M)) of the peptides synthesized with bovine alpha-chymotrypsin were determined. The highest value of the specificity constant k(cat)/K(M), reaching 6.0 x 10(5) [M(-1)xs(-1)], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO(2))-Anb(5,2)-NH(2). The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of k(cat) more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO(2)) in position P(1) and potent chymotrypsin inhibitors were obtained. The dissociation constant (K(i)) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO(2))-CHO, amounted to 1.12 x 10(-8) M.

Specificity of human cathepsin G was explored using combinatorial chemistry methods. Deconvolution of a tetrapeptide library, where 5-amino-2-nitrobenzoic acid served as a chromophore attached at the C-terminus, yielded the active sequence Phe-Val-Thr-Tyr-Anb(5,2)-NH(2). This sequence was used for a second-generation library with the general formula Ac-Phe-Val-Thr-X-Anb(5,2)-NH(2), where position X was replaced with several amino acids: L-pyridyl- alanine (Pal), 4-nitro-L-phenylalanine (Nif), 4-amino-L- phenylalanine (Amf), 4-carboxy-L-phenylalanine (Cbf), 4-guanidine-L-phenylalanine (Gnf), 4-methyloxycarbonyl- L-phenylalanine (Mcf), 4-cyano-L-phenylalanine (Cyf), Phe, Tyr, Arg and Lys. Specificity ligand parameters, k(cat) and K(M), with human cathepsin G were determined for all chromogenic substrates synthesized. The highest value of the specificity constant (k(cat)/K(M)) was obtained for a substrate with the Gnf residue in position P(1). This peptide was 10 times more active than the second most active substrate which contained the Amf residue. The following order of potency was established: Gnf > > Amf > Tyr = Phe > Arg= Lys > Cyf. Substrate specificity for cathepsin G is greatly enhanced when an aromatic side chain and a strong positive charge are incorporated in residue P(1).

Human prion protein (hPrP) fragments encompassing the 91-120 region, namely hPrP92-100 (SP1), hPrP106-113 (SP2), hPrP91-120 (LP1), and hPrP91-114 (LP2), were considered for delineation of the Cu(II)-binding site(s). NMR and EPR spectroscopy results obtained from LP1 or LP2 were compared with those obtained from SP1 and SP2. The coexistence of two binding sites, one centered at His96 and the other at His111, was evidenced and ratified by ESI mass spectrometry at low and high metal:peptide ratios. While room-temperature NMR spectroscopy data were consistent with the binding site centered on His111 being approximately fourfold stronger than that centered on His96, low-temperature EPR spectroscopy results yielded evidence for the opposite trend. This disagreement, which has also occurred in the literature, was clarified by temperature-dependent molecular dynamics runs that demonstrated Met112 approaching the metal at room temperature, a process that is expected to stabilize the His111-centered binding site through hydrophobic shielding of the metal coordination sphere.

Conformational studies of nociceptin (NC-NH2), its fully active fragment, NC(1-13)-NH2, and two significantly less potent fragments, NC(1-13)-OH and NC(1-11)-OH, were conducted in water and TFE solutions by the employment of circular dichroism, and in DMSO-d6 by 2DNMR spectroscopy in conjunction with theoretical conformational analysis. The conformations of all thepeptides studied were calculated taking two approaches. The first assumes multiconformational equilibrium of the peptide studied, which is characterized by a set of conformations (and their statistical weight values)obtained from a global conformational analysis using three methods: the electrostatically driven Monte-Carlo (EDMC) with the ECEPP/3 force field, the simulated annealing (SA) protocols in the AMBER and CHARMM force fields. The second approach incorporates the interproton distance and dihedral angle constraints into the starting conformation. Calculations were performed using the distance geometry and SA protocol in the CHARMM force field implemented in the X-PLOR program. The CD experiments indicated that for the active peptides, hydrophobic solvents induced a significantly higher (compared with those remaining)content order, probably a helical structure. Unfortunately, as a result of the conformational flexibility of thepeptides, the analysis of conformations obtained with both approaches and different force fields did not alllow the selection of any structural elements of the NC peptides that might be connected with their bioactivity. The only common element found in most conformations of the active peptides was a helical character of fragment 8-13, which allowed the side chains of basic amino acid residues to be exposed to the outside of the molecule and probably to interact with the ORL1 receptor.

Neuropeptide gamma is one of the largest members of the tachykinin family of peptides, exhibiting strong agonistic activity towards the NK-2 tachykinin receptor. This peptide was synthesized by the solid-phase method using the Fmoc chemistry. Circular-dichroism spectroscopy (CD) investigations of this peptide were performed in phosphate buffer, in the presence of sodium dodecylsulphate (SDS) micelles and trifluoroethanol (TFE) solutions and in DMSO-d6 using the 2D NMR technique in conjunction with two different theoretical approaches. The first assumes multiconformational equilibrium of the peptide studied characterized by the values of statistical weights of low-energy conformations. These calculations were performed using three different force fields ECEPP/3, AMBER4.1 and CHARMM (implemented in the X-PLOR program). The second method incorporates interproton distance and dihedral angle constraints into the starting conformation using the Simulated Annealing algorithm (X-PLOR program). The CD experiments revealed that although the peptide studied is flexible in polar solvents, a tendency to adopt a helical structure was observed in the hydrophobic environment. The NMR data (NOE effects) indicate a helical or reverse structure in the Ile7-His12 fragment of the peptide studied in DMSO-d6 solution. The results obtained cannot be interpreted in terms of a single conformation. Most of the conformations obtained with the ECEPP/3 force field possess a high content of a helical structure. None of the conformers, obtained with the AMBER4.1 and CHARMM force fields, can be considered as the dominant one. In all conformations several beta-turns were detected and in some cases gamma-turns were also found. But in fact, it is rather difficult to select the position of the secondary element(s) present in the structure of NPgamma in solution. All conformers calculated with the X-PLOR program (with using NMR derived distance and torsion angle constraints) are stabilized by several beta-turns. Common structural motives are a type IV beta-turn in the Gln6-His12 fragment. All conformations obtained using two approaches adopt very similar turn shapes in the middle region of molecule and a random structure on the N- and C-terminal fragments.

The in vivo effects of three new analogs of somatostatin (ASS-51, ASS-52 and ASS-53 analogs) on GH, insulin and glucagon were studied in WKY rats. The solid phase method was used for the synthesis of ASS. Octreotide and ASS were given iv. in a dose of 0.05 &mgr;g/kg per animal in a time-dependent manner. ASS-52 and ASS-53 were longer acting and more potent somatostatin analogs when compared to octreotide in producing the inhibition of GH. ASS-51 was found to be the most potent and selective inhibitor of insulin and glucagon release. Our results show that the increased inhibitory effect and the higher selectivity of the new somatostatin analogs may result from the differences in their chemical structure. ASS-52 is most active in inhibiting GH release. The mechanism by which ASS-52 inhibits preferentially GH release may involve the opioid system and the activation of GABA-ergic receptors. In studies in vitro ASS-52 inhibited GH release from pituitary cells" culture.