A new proteinase 3 substrate with improved selectivity over human neutrophil elastase.

We report the synthesis and enzymatic studies on a new proteinase 3 intermolecular quenched substrate with enhanced selectivity over neutrophil elastase. Using combinatorial chemistry methods, we were able to synthesize the hexapeptide library with the general formula ABZ-Tyr-Tyr-Abu-X1'-X2'-X3'-Tyr(3-NO2)-NH2 using the mix and split method. The iterative deconvolution of such a library allowed us to obtain the sequence ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO2)-NH2 with a high specificity constant (kcat/KM=1534×10(3)M(-1)s(-1)) and superior selectivity over neutrophil elastase and other neutrophil-derived serine proteases. Moreover, using the obtained substrate, we were able to detect a picomolar concentration of proteinase 3 (PR3). Incubation of the above-mentioned substrate with neutrophil lysate resulted in a strong fluorescent signal that was significantly reduced in the presence of a PR3 selective inhibitor.

Hybrid analogues of SFTI-1 modified in P1 position by ß- and γ-amino acids and N-substituted ß-alanines.

A series of compounds containing either non-proteinogenic ß-/γ-amino acids or N-substituted ß-alanine residues (ß-peptoid units) in P1 specificity position were synthesized based on the structure of sunflower trypsin inhibitor 1 (SFTI-1). The compounds were synthesized on a solid support; the N-substituted ß-alanines (ßNhlys and ßNhphe) were introduced into a peptidomimetic chain via a two-step approach using acryloyl chloride and an appropriate primary amine. The inhibitory activities were characterized in vitro against bovine α-chymotrypsin or bovine ß-trypsin. Three analogues displayed activity comparable to fully proteinogenic counterparts-monocyclic SFTI-1 and [Phe(5)]SFTI-1. Moreover, all active peptidomimetics were resistant toward proteolytic degradation, even after 24-h incubation at room temperature.

Substrate specificity and inhibitory study of human airway trypsin-like protease.

Human airway trypsin-like protease (HAT), also referred to as TMPRSS11D, is an important physiological enzyme with the main activity pronounced in an airway. In this work we have described the substrate specificity and selectivity study of the protease, performed by the combinatorial approach. Fluorogenic/chromogenic tetrapeptide library was used for this purpose. The most efficiently hydrolyzed substrates' sequences that we selected were ABZ-Arg-Gln-Asp-Arg(Lys)-ANB-NH(2). The most active inhibitor with C-terminal Arg residue underwent detectable proteolysis action in the presence of 35pM of HAT. Based on the selected sequences the two peptide aldehydes were synthesized and (Abz-Arg-Gln-Asp-Arg(Lys)-H) were found to be an effective HAT inhibitor, working in nanomolar range with inhibition constant 54nM and 112nM, respectively.

Relationship between copper(ii) complexes with FomA adhesin fragments of F. nucleatum and colorectal cancer. Coordination pattern and ability to promote ROS production.

The copper(ii) ion binding of the Ac-KGHGNG-NH2 and Ac-PTVHNE-NH2 fragments of FomA adhesin from Fusobacterium nucleatum was studied using potentiometry, UV-Vis, CD, EPR and DFT techniques. The coordination pattern was described in a wide range of pH values. Ligands begin interactions with metal ions using imidazole nitrogen. At pH 6.8 (a value typical of the large intestine environment), the metal ion was coordinated by the 3N donor atoms {Nim, 2 × N-amide} in both cases. However, the copper(ii) ion was bound more effectively by the Ac-PTVHNE-NH2 peptide. The formation of reactive oxygen species (ROS) was studied by UV-Vis and fluorescence spectroscopy, as well as gel electrophoresis in the presence of H2O2 and/or ascorbic acid. The complexes generated ROS in the highest amounts among all compounds. Moreover, they stimulated the CT26 cell line (mouse colon carcinoma) to produce ROS which lead to oxidative stress. It was also determined that such radicals took part in the plasmid degradation mechanism.

New cyclosporin A analogue: synthesis and immunosuppressive activity.

A synthetic analogue of cyclosporine A, in which an unusual amino acid (4R)-N-methyl-4-butenyl-4-methyl-L-threonine (MeBmt) is replaced with L-threonine (Thr), was synthesized by the solid phase method. Its activity in the humoral response to sheep red blood cells in vitro and in vivo in mice was practically the same as that of cyclosporine A used as a standard, whereas the analogue studied exerted a significantly stronger effect in the delayed type hypersensitivity to sheep red blood cells in mice.

Modifications outside the proteinase binding loop in Cucurbita maxima trypsin inhibitor III (CMTI-III) analogues change the binding energy with bovine beta-trypsin.

Five 26-peptide analogues of the trypsin inhibitor [Pro18]CMTI-III containing Leu or Tyr in position 7 and Val or Tyr in position 27: 1 (Leu7, Tyr27), 2 (Tyr7, Val27), 3 (Tyr7, Tyr27), 4 (Leu7, Val27) and 5 (Leu7, Ala18, Tyr27) were synthesized by the solid-phase method. Analogues 1-4 displayed Ka with bovine beta-trypsin of the same order of magnitude as the wild CMTI-III inhibitor, whereas for analogue 5, this value was lower by about 3 orders of magnitude. This indicated that for the analogues with Pro (but not with Ala) in position 18, the side-chain interactions between positions 7 and 27 did not play a critical role for the stabilization of the active structure. In addition, these results also suggest that Tyr7 is involved in an additional aromatic interaction with position 41 of the enzyme.

Effect of scyliorhinin I and synthetic scyliorhinin I derivatives at mammalian tachykinin NK1, NK2 and NK3 receptors.

The dogfish tachykinin peptide scyliorhinin I and a number of its analogues substituted in position 7 were tested in bioassays for tachykinin NK1, NK2 and NK3 receptors. Scyliorhinin I behaved as a full agonist at tachykinin NK1 receptors of the guinea-pig ileum longitudinal muscle and at NK2 receptors of the rabbit pulmonary artery and hamster trachea. In these three preparations scyliorhinin I was as potent agonist as substance P methylester and neurokinin A, respectively. Evidence for activation of tachykinin NK1 and NK2 receptors by scyliorhinin I was obtained by using the selective tachykinin antagonists FK 888, MEN 10,376 and L 659,877. Scyliorhinin I was poorly active as an agonist at NK3 receptors of the rat portal vein. Among scyliorhinin I analogues, [beta-(2-naphthyl)-Ala7]scyliorhinin I, [Val7]scyliorhinin I and [Ile7]scyliorhinin I were 3-25 times weaker than scyliorhinin I itself at NK1 and NK2 receptors. [Phe7]scyliorhinin I, [Phe(F)7]scyliorhinin I and [Phe(Cl)7]scyliorhinin I were as potent as scyliorhinin I at NK1 receptors in the guinea-pig ileum, while they showed 10-30 times lower affinity than scyliorhinin I for NK2 receptors. The present results are discussed in relation to the importance of position 7 in determining the potency and selectivity of scyliorhinin I analogues at tachykinin receptors.

C-Terminal glycine is crucial for hyperalgesic activity of nociceptin/orphanin FQ-(1-6).

A C-terminal analog of the hexapeptide orphanin FQ/nociceptin-(1-6), [Ala(6)]-orphanin FQ/nociceptin-(1-6), and a pentapeptide orphanin FQ/nociceptin-(1-5) were tested in vivo for their analgesic/hyperalgesic activity in the hot-plate test with rats. Replacement of the C-terminal glycine by L-alanine (Phe-Gly-Gly-Phe-Thr-Ala) in orphanin FQ/nociceptin-(1-6) abolished the hyperalgesic potency of native orphanin FQ/nociceptin-(1-6) (Phe-Gly-Gly-Phe-Thr-Gly), but analgesic activity was retained and was diminished by naloxone. Removal of the C-terminal amino acid (glycine or alanine) from orphanin FQ/nociceptin-(1-6) caused a significant loss of analgesic activity. It is anticipated that glycine plays a crucial role in the biphasic activity of orphanin FQ/nociceptin-(1-6). This may suggest the existence of a mechanism for terminating the biological action of orphanin FQ/nociceptin.

Determination of nociceptin-orphanin FQ metabolites by capillary LC-MS.

Nociceptin-orphanin FQ (OFQ/N) is a newly discovered peptide involved in pain transmission. The method is described to identify metabolic pathway of this neuropeptide in the spinal cord of rats using capillary size-exclusion liquid chromatography coupled to electrospray ionization mass spectrometry. The applied technique is rapid and selective, and allows for simultaneous measurement and quantitation of several fragments in the incubation mixture.

Orphanin FQ/nociceptin inhibits morphine withdrawal.

The influence of orphanin FQ/nociceptin (OFQ/N) on the morphine-withdrawal symptom was investigated. Withdrawal syndrome was induced in the morphine-dependent rats by an intraperitoneal (i.p.) injection of 2 mg/kg naloxone hydrochloride--an opioid receptors antagonist. Wet-dog shakes were used as a measure of the abstinence syndrome. Intraventricular injections of OFQ/N (5-20 microg/animal) caused significant inhibition of the withdrawal signs at doses between 15-20 microg, in the morphine-dependent rats. OFQ/N alone did not change behavior of the morphine-dependent animals. The obtained results indicate that OFQ/N can inhibit the morphine withdrawal symptoms induced by naloxone.

Potentiometric and spectroscopic studies of the Cu(II) complexes of Ala-Arg8-vasopressin and oxytocin: two vasopressin-like peptides.

The results are reported of a potentiometric and spectroscopic study of the H+ and Cu2+ complexes of Ala-Arg8-vasopressin (Ala-AVP) and oxytocin at 25 degrees C and an ionic strength of 0.10 mol dm-3 (KNO3). The coordination chemistry of oxytocin and Cu(II) has been shown to be virtually identical to that of Arg8-vasotocin, forming unusually stable complexes with four nitrogen coordination (4N complexes) below pH 7. Spectroscopic evidence suggests weak interaction between Cu(II) and the sulphur atom of the -Cys6- residue in the 2N species (pH congruent to 6) but this is absent in the 4N complex. Evidence is also presented for perturbation of electronic transitions within the aromatic ring of the Tyr residue by Cu(II). While the physiological potency of Ala-AVP is very high, its coordination chemistry differs significantly from that of Arg8-vasopressin. With Cu(II) it forms complexes of similar stability to those with tetraglycine, demonstrating that the addition of an alanyl residue to the amino-terminal of the peptide destroys the conformation which is particularly favorable for rapid 4N coordination.

A potentiometric and spectroscopic study of the proton and copper(II) complexes of methionine enkephalin and some related ligands.

The results are reported of a potentiometric and spectrophotometric study of the proton and copper(II) complexes of methionine enkephalin and four related pentapeptides which all show greater biological activity than their parent enkephalin. Measurements were carried out at 25 degrees C and I = 0.10 mol dm-3 (KNO3). All the ligands studied form stable copper(II) complexes comparable to those formed by pentaglycine, with the peptide chain locked in a folded conformation by NNN or NNNN coordination to the metal ion. There is no indication of bonding through the tyrosine-phenolate oxygen atoms or the methionine sulfurs.

Substrate specificity of human matriptase-2.

Human matriptase-2 is an enzyme that belongs to the family of type II transmembrane serine proteases. So far there is a limited knowledge regarding its specificity and protein substrate(s). One of the identified natural substrates is hemojuvelin, a protein involved in the control of iron homeostasis. In this work, we describe the synthesis and evaluation of internal quenched substrates using a combinatorial approach. The iterative deconvolution of two libraries to define the specificity of matriptase-2 yielded to the identification of the substrate ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 with a k(cat)/K(m) value of 4.5 × 10(5) M(-1) × s(-1), i.e. the highest specificity constant reported so far for matriptase-2.

Nociceptin and its natural and specifically-modified fragments: Structural studies.

The vibrational structures of Nociceptin (FQ), its short bioactive fragments, and specifically-modified [Tyr¹]FQ (1-6), [His¹]FQ (1-6), and [His(1,4)]FQ (1-6) fragments were characterized. We showed that in the solid state, all of the aforementioned peptides except FQ adopt mainly turn and disordered secondary structures with a small contribution from an antiparallel ß-sheet conformation. FQ (1-11), FQ (7-17) [His¹]FQ (1-6), and [His(1,4)]FQ (1-6) have an α-helical backbone arrangement that could also slightly influence their secondary structure. The adsorption behavior of these peptides on a colloidal silver surface in an aqueous solution (pH = ∼8.3) was investigated by means of surface-enhanced Raman scattering (SERS). All of the peptides, excluding FQ (7-17), chemisorbed on the colloidal silver surfaces through a Phe4 residue, which for FQ, FQ (1-11), FQ (1-6), [Tyr¹]FQ (1-6), and [His¹]FQ (1-6) lies almost flat on this surface, while for FQ (1-13) and FQ (1-13)NH2 adopts a slightly tilted orientation with respect to the surface. The Tyr¹ residue in [Tyr¹]FQ (1-6) does not interact with the colloidal silver surface, suggesting that the Tyr¹ and Phe4 side chains are located on the opposite sides of the peptide backbone, which can be also true for His¹ and Phe4 in [His¹]FQ (1-6). The lone pair of electrons on the oxygen atom of the ionized carbonyl group of FQ (1-13) and FQ (7-17) appears to be coordinated to the colloidal silver nanoparticles, whereas in the case of the remaining peptides, it only assists in the adsorption process, similar to the --NH4 group. We also showed that upon adsorption, the secondary structure of these peptides is altered.

Is the nociceptin (NOP) receptor involved in attenuation of the expression of sensitization to morphine-induced hyperlocomotion in mice?

In the present study we investigated whether synthetic agonists of the nociceptin (NOP) receptors, Ro 64-6198 [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one] and Ro 65-6570 (8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one), influence the expression of sensitization to the locomotor stimulant effect of morphine in mice. Sensitization was produced by five repeated administrations of morphine (10 mg/kg, s.c.) at 3-day intervals. Seven days after the last morphine injection, Ro 64-6198 (1 and 3 mg/kg, i.p.) and Ro 65-6570 (3 and 6 mg/kg, i.p.) were given immediately before the challenge dose of morphine (10 mg/kg, s.c.). Both substances inhibited the expression of sensitization to the locomotor stimulant action of morphine. However, the selective NOP receptor antagonist, [Nphe1]NC(1-13)NH2 (30 nmol, i.c.v.) did not reverse the inhibitory effect of the Ro-compounds. Therefore, our results suggest that the NOP receptor may not be critical for the influence of Ro-compounds on the morphine-induced sensitization, or the observed effect may be attributed to one functional subset of this receptor, stimulation of which is not blocked by [Nphe1]NC(1-13)NH2.

Chromogenic substrates of bovine beta-trypsin: the influence of an amino acid residue in P1 position on their interaction with the enzyme.

The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design and synthesize a series of trypsin chromogenic substrates modified in position P1: Ac-Ala-Val-Abu-Pro-X-pNA, where X = Orn, Lys, Arg, Har, Arg(NO(2)), Cit, Hci, Phe(p-CN), Phe(p-NH(2)); pNA = p-nitroanilide. The most active compounds (as determined by specificity constant k(cat)/K(m)) were peptides with the Arg and Lys residues in the position discussed. Changes in the length and the decrease of the positive charge of the amino acid residue side chain in position P(1) resulted in the decrease or loss of the affinity towards bovine beta-trypsin. Among peptides containing amino acid residues with uncharged side chains in position P1, only one with p-cyano-l-Phe revealed activity. These results correspond well with trypsin inhibitory activity of CMTI-III analogues modified in the equivalent position, indicating the same type of interaction between position P1 of the substrate or inhibitor and S1 site specificity of trypsin.

Opiate-like peptides. Part VIII. Methylamides and dimethylamides of [D-Leu5]-enkephalin and [D-Ala2, D-Leu5]-enkephalin. Synthesis and analgesic activity.

Syntheses of [D-Ala2, D-Leu5]-enkephalin, methyl ester of [D-Ala2, D-Leu3]-enkephalin, methylamides and dimethylamides of [D-Leu5]-enkephalin and [D-Ala2, D-Leu5]-enkephalin are described together with their analgesic activity determined on the basis of four analgesic tests: the hot-plate method, the reaction to electric stimulus, the tail immersion test and the frequency of writhing syndrome test. The neurotoxicity was estimated by the rota-rod test. The most pronounced analgesic effect was induced by compound: [D-Ala2, D-Leu5]-enkephalin, [D-Ala2, D-Leu5-OMe]-enkephalin and [D-Ala2, D-Leu5-NMe2]-enkephalin. In the tail immersion test all analogs did not exhibit analgesic activity.

Design, chemical synthesis and kinetic studies of trypsin chromogenic substrates based on the proteinase binding loop of Cucurbita maxima trypsin inhibitor (CMTI-III).

A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P(4) on the substrate-enzyme interaction, kinetic parameters of newly synthesized substrates with bovine beta-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P(4) significantly enhanced the substrate specificity (k(cat)/K(m)) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine beta-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme.

Opiate-like peptides. Part V. Aminoalkylamides of Met-enkephalin and [D-Ala]2-Met-enkephalin: synthesis and analgesic activity.

Four analogs of Met-enkephalin were synthetized using DPPA method. Their analgesic activity was estimated by the hot plate method, the neurotoxic effect being also determined. One of the analogs was 16 times as potent as Met-enkephalin, whereas with the three remaining the neurotoxic effect preceded the expected analgesic activity.

New active analogues of Cucurbita maxima trypsin inhibitor III (CMTI-III) modified in the non-contact region.

Four new analogues of trypsin inhibitor CMTI-III(3-28) = [desArg1,desVal2,desGly29]CMTI-III which was recently shown to be fully active, were synthesized by the solid-phase method. The introduction of glycine in position 9 (peptide 1) and Gly-Pro-Gly (peptide 2) and Gly-Pro-Asn (peptide 3) in the regions 17-19 and 23-25, respectively, did not change the antitrypsin activity of all modified peptides. All of these substitutions are presumed to be outside the trypsin-binding loop as judged from the X-ray structure of the complex between beta-trypsin and the related inhibitor CMTI-I. Also the fourth analogue which was substituted in all the positions mentioned, exhibited the full activity.