Ser Phe Research Articles

Anticoagulant Oligonucleotide-Peptide Conjugates: Identification of Thrombin Aptamer Conjugates with Improved Characteristics.

Oligonucleotide–peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3–Ser–Phe–Asn (SFN) or T3–Tyr–Trp–Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.

Inhibition of arterial thrombosis by a protease-activated receptor 1 antagonist, FR171113, in the guinea pig

The antiplatelet and antithrombotic effects of FR171113, 3-(4-chlorophenyl)-2-(2,4-dichlorobenzoylimino)-5-(methoxycarbonyl methylene)-1,3-thiazolidin-4-one, a non-peptide protease-activated receptor 1 (PAR1) antagonist, were evaluated in guinea pigs. FR171113 inhibited Ser–Phe–Leu–Leu–Arg–Asn–NH2 (a synthetic PAR1 agonist peptide)-induced and thrombin-induced aggregation of guinea pig platelets in a concentration-dependent manner in vitro (IC50=1.5 and 0.35 μM, respectively). Subcutaneous administration of FR171113 (0.1–3.2 mg/kg) produced a dose-dependent inhibition of platelet aggregation ex vivo. The ED50 value of FR171113 for platelet aggregation was 0.49 mg/kg s.c. However, FR171113 did not have an inhibitory effect on ADP- or collagen-induced platelet aggregation in vitro and ex vivo. One hour after FR171113 treatment at 1.0 mg/kg s.c., significant inhibition of arterial thrombosis without a prolongation of thrombin time or coagulation time was seen in the FeCl3-induced carotid artery thrombosis model in guinea pigs. Furthermore, FR171113 did not prolong bleeding time even at 32 mg/kg s.c., which is a much higher dose than that required in the thrombosis model. These observations indicate that FR171113 has desirable antiplatelet effects both in vitro and in vivo and that its in vivo antithrombotic activity is efficacious without causing a prolongation of bleeding time.

A complex heterozygous mutation of His373Leu and Asp487–Ser488–Phe489 deletion in human cytochrome P450c17 causes 17α-hydroxylase/17,20-lyase deficiency in three Chinese sisters

Cytochrome P450c17 deficiency is one of the rare forms of enzyme disorders in steroid biosynthesis, resulting from defects in 17α-hydroxylase and 17,20-lyase activities. The disease is caused by the mutations in CYP17 gene, inherited in an autosomal recessive pattern. We reported a Chinese family with three sisters suffering from P450c17 deficiency based on their clinical features and molecular genetics. The patients were found to be compound heterozygotes with two different mutations. Screened by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), a heterozygous point mutation His373Leu was detected in the exon 6 of CYP17 gene which was proved to be derived from paternal allele. The other allele contained nine-base pair deletion, located in exon 8, eliminating codons 487–489 (Asp–Ser–Phe) near the carboxy-terminus of P450c17. The mother and the brother have been demonstrated to be carriers of deletion mutation through restriction enzyme analysis. Both mutations have been reported previously in Asia. This is the first report of the molecular genetic study of 17α-hydroxylase/17,20-lyase deficiency in mainland China with a novel compound heterozygous mutation.

Differentiated reactivity of whole blood neonatal platelets to various agonists

Neonatal platelets have been occasionally reported to show a reduced response to various agonists. The molecular mechanism(s) of such a depressed reactivity remains unclear. To further address this problem we studied neonatal platelet activation with thrombin, TRAP (thrombin receptor activating peptide, Ser–Phe– Leu–Leu–Arg–Asn–Pro–Asn–Asp–LysTyrGlu and ADP in 42 healthy 1–2 day old neonates using a whole peripheral blood flow cytometry. The neonates did not show an increased fraction of P-selectinpositive circulating platelets, whereas the expression of GPIb (glycoprotein Ib) in resting neonatal platelets was significantly lower compared to adults. Neonatal platelets were significantly less reactive than adult platelets to thrombin and TRAP, especially at lower agonist concentrations, but not to ADP or when incubated for 1 h at room temperature. Activation of neonatal platelets with agonists resulted in a marked alterations in the expression of P-selectin, whereas the internalization of GPIb was not affected. The reduced neonatal platelet sensitivity to thrombin and TRAP was accompanied by significantly reduced ATIII (antithrombin III) and increased prothrombin fragment F 1+2 in neonatal plasma. We conclude that various receptor systems potentially able to bind thrombin are relatively insensitive in neonatal platelets. The novelty of our work is that neonatal platelet hyposensitivity is not a generalized phenomenon, but concerns only selected agonists and selected receptor systems.

In vitro antiplatelet profile of FR171113, a novel non-peptide thrombin receptor antagonist

Synthetic peptides (5 to 14 amino acids), identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage by thrombin, act as thrombin receptor agonist peptides. Whilst thrombin receptor antagonist peptides are known, non-peptide thrombin receptor antagonists have yet to be described. In the present study, we compared the antiplatelet effects of 3-(4-chlorophenyl)-2-(2,4-dichlorobenzoylimino)-5-(methoxycarbonyl methylene)-1,3-thiazolidin-4-one (FR171113), a novel non-peptide thrombin receptor antagonist, with the known thrombin receptor antagonist 3-mercapto-propionyl-Phe–Cha–Cha–Arg–Asn–Pro–Asn–Asp–Lys–Tyr-OH (C186-65), and argatroban, a specific protease inhibitor of thrombin. FR171113 and C186-65 inhibited thrombin-induced platelet aggregation (IC 50=0.29 μM and 15 μM, respectively) and Ser–Phe–Leu–Leu–Arg–Asn-NH 2 [a synthetic thrombin receptor agonist peptide (TRAP-6)] induced platelet aggregation (0.15 μM and 20 μM, respectively) in human washed platelets. Argatroban potently inhibited thrombin-induced platelet aggregation (IC 50=3.5 nM), but did not inhibit TRAP-6-induced aggregation even at 100 μM. In contrast, these compounds did not show inhibitory effects on ADP- and collagen-induced aggregation in human platelet-rich plasma even at 100 μM. FR171113 caused a parallel shift to the right of the concentration–response curve describing aggregation induced by TRAP-6. The Schild plot of the data had a slope of −0.840 ( r=0.98) and the p A 2 was 7.29. In protease activity studies using a chromogenic substrate, argatroban inhibited thrombin protease activity in a dose-dependent manner, whereas FR171113 and C186-65 were inactive, even at 100 μM. Additionally, only argatroban displayed dose-dependent prolongation of thrombin time, activated partial thromboplastin time and prothrombin time. FR171113 and C186-65 showed no effects, even at a concentration of 100 μM. These results suggest that FR171113 has a similar mode of action to C186-65, but with more potent antiplatelet activity. In conclusion, FR171113 is suggested to be the first example of a non-peptide thrombin receptor antagonist.

The Role of Arginine in Thrombin Receptor Tethered-Ligand Peptide in Intramolecular Receptor Binding and Self-Activation

Abstract Synthetic heptapeptide of the human thrombin receptor tethered-ligand peptide, H–Ser–Phe–Leu–Leu–Arg–Asn–Pro–NH2 (SFLLRNP), activates fully the thrombin receptor without thrombin. The functional role of Arg-5 was examined using a series of analogs having amino acid substitutions at position 5 in this assays was to assess the abilities to hydrolyze phosphoinositide in human neuroblastoma SH-EP cells and to aggregate the human platelet. The replacement of Arg-5 by Ala reduced the activity (9% activity of the parent peptide) in the PI-turnover assay, and abolished completely the platelet aggregation activity. SFLL/Lys/NP was also active, but moderately: 36% in PI-turnover and 12% in platelet aggregation. These results indicated that the electrostatic interaction of the Arg-guanidino group is important for a peptide to interact with the receptor. When citrulline or glutamine was placed at position 5 instead of arginine, the resulting SFLL/citrulline/NP and SFLL/Gln/NP were found to be potent in both assays. Since citrulline and glutamine possess a side chain which can serve as hydrogen donor and/or acceptor, the receptor activation of these peptides appears to be due to hydrogen bonding at this position. The molecular mechanisms to explain both electrostatic and hydrogen-bonding interactions were postulated based on the structural modeling of seven-transmembrane domain thrombin receptor.

Transferred nuclear Overhauser effect spectroscopy study of a peptide from the PapG pilus subunit bound by the Escherichia coli PapD chaperone

Interaction of the Escherichia coli PapD chaperone with the synthetic peptide PapG308-314 (Thr–Met–Val–Leu–Ser–Phe–Pro), corresponding to the seven C-terminal residues of the PapG pilus subunit, was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The observation of cross-peaks corresponding to either intraresidue or sequential C αH/NH and C βH/NH TRNOEs and the absence of sequential NH i/NH i+1 TRNOEs indicate that the peptide binds to PapD in an extended conformation. In addition, line-broadening effects gave information of the peptide's mode of interaction with PapD. These observations were in excellent agreement with a recent crystal structure of a PapG peptide complexed with PapD.

Different Roles of Two Consecutive Leucine Residues in a Receptor-Tethered Ligand Peptide (SFLLRNP) in Thrombin Receptor Activation

Abstract A synthetic peptide, H–Ser–Phe–Leu–Leu–Arg–Asn–Pro–NH2, which corresponds to a ligand peptide tethered to a human thrombin receptor, was able to activate the thrombin receptor with no thrombin. In order to inspect the structural requisites of two consecutive leucines (Leu-3 and Leu-4) in receptor activation, two sets of analogs with substitutions at either position 3 or 4 were synthesized and evaluated for their ability to hydrolyze phosphoinositide in human neuroblastoma SH-EP cells. The replacement of Leu-4 by Ala drastically decreased the activity of the parent peptide (only about 4% activity), while that of Leu-3 retained about 50% activity. A similar result was obtained when replaced by Gly. Substitution by Phe for Leu-3 sustained full activity. Although the Leu-4/Phe substitution exhibited a slight reduction in activity, Leu-4/Ile substitution unexpectedly diminished the activity (20%). These results suggested that two consecutive leucines have different roles in receptor activation; i.e., Leu-3 behaves something like a connection of peptide units to construct a bioactive conformation and Leu-4 acts like a structural element essential for interactions with receptors.

Optimum Cross-linking Spacer Length of Dimeric Neurokinin B Analogs for Interaction with NK-1 Tachykinin Receptors

Abstract A series of dimeric analogs of neurokinin B (NKB) COOH-terminal heptapeptide were synthesized in order to find an optimum length of cross-linking spacer for bivalent interaction with tachykinin receptors. Dimerization was carried out at the NH2-terminus of heptapeptide with succinyl bis[(Gly)n–OH], in which the number of glycine varies from 0 to 4. Dimers D–(Gly)n–NKB4—10, namely succinyl bis[(Gly)n–Asp–Ser–Phe–Val–Gly–Leu–Met–NH2] (n = 0—4), were almost inactive in the assay using rat vas deferens, indicating that they have no interaction with NK-2 receptor subtype. In contrast, these dimers were very active in guinea pig ileum (GPI) containing all of NK-1, 2, and 3 receptors. Relative activity was highest when the cross-linking spacer with oligoglycine of n = 2, and decreased sharply for dimers with shorter and longer chain lengths (n = 0 and 1; 3 and 4). In order to specify receptor subtype (NK-1 or NK-3) in GPI to which dimers bind, dimers were examined under the conditions that NK-1 receptors are desensitized by substance P methyl ester. All dimers exhibited drastically diminished contractile activity, indicating that dimers exclusively interact with NK-1. This was further confirmed by blocking the response of NK-3 receptors with atropine, which had no effect on the contractile activity of dimers. The results suggested that dimers interact bivalently with NK-1 receptors by bridging adjacent two binding sites.

Opioid Activities of Morphiceptin Analogs Derived from Human β-Casein

Abstract Four tetrapeptide amides with the N-terminal Tyr–Pro sequence were synthesized as possible opioid agonists that can be produced by degradation of human β-casein. When these peptides were tested for their ability to bind to the μ and δ opioid receptors in rat brain, only H–Tyr–Pro–Phe–Val-NH2 was active, showing the 60% increased affinity for the μ receptors as compared with morphiceptin (H–Tyr–Pro–Phe–Pro–NH2) derived from bovine β-casein. It was highly μ-selective, as well as morphiceptin, with the μ/δ-selectivity ratio of 285. Other three analogs, H–Tyr–Pro–Ser–Phe–NH2, H–Tyr–Pro–Val–Arg–NH2 and H–Tyr–Pro–Val–Pro–NH2, were almost completely inactive. These results suggested that, for the morphiceptin-like tetrapeptide amides, the presence of Phe at position 3 is essential to elicit an activity to bind to the μ opioid receptors. Conformational considerations by measuring the CD spectra indicated that the sequence of tetrapeptide amide Tyr–Pro–Xxx–Pro(or Val)–NH2 is an important structural requirement to interact with the μ opioid receptors.

Gastrin-releasing peptide from the intestine of the elasmobranch fish, Scyliorhinus canicula (common dogfish)

The concentration of gastrin-releasing peptide in the intestine of the elasmobranchian fish, Scyliorhinus canicula, measured with an antiserum directed against the COOH-terminal region of porcine gastrin-releasing peptide, was higher than the concentrations measured in mammalian intestines. The immunoreactivity was resolved by gel permeation chromatography into two peaks with the approximate elution volumes of porcine gastrin-releasing peptide and bombesin/neuromedin C. The primary structure of the larger peptide was established as Ala Pro Val Glu Asn Gln Gly Ser Phe Pro Lys Met Phe Pro Arg Ser His (Trp) Ala Val Gly (His Leu Met · NH 2). Residues in parentheses are only tentatively assigned. Chromatographic evidence and the presence of the arginyl residue at position 15 in the peptide suggest that the smaller molecular form of gastrin-releasing peptide may be identical to mammalian neuromedin C. Amphibian bombesin was not identified in the dogfish gut.

Amino acid sequence of the K-peptide generated by limited proteolysis of glucose dehydrogenase from Bacillus megaterium by proteinase K

Proteolysis of glucose dehydrogenase from Bacillus megaterium with proteinase K apparently generated two fragments. The small fragment, designated as K-peptide, was sequenced and its covalent structure was determined as SerSerGluAlaSerTyrValThrGlyIleThrLeuPhe AlaAspGlyGlyMetThrGlnTyrProSerPheGluAla GlyArgGly. The sequence analysis showed that the K-peptide consists of two identical fragments, one of which is lacking one serine residue at the aminoterminus.

Isolation and amino acid sequence of a mating pheromone produced by mating type α cells of Saccharomyces kluyveri

A peptide, termed α sk2 pheromone, was isolated from culture filtrates of mating type α cells of Saccharomyces kluyveri as a mating pheromone, having both shmoo inducing (in a cells of S. kluyveri and S. cerevisiae) and agglutinability inducing (in a cells of S. cerevisiae) actions. The amino acid sequence of α sk2 pheromone was determined as H—Trp—His—Trp—Leu—Ser—Phe—Ser—Lys—Gly—Glu—Pro—Met—Tyr-OH by mass spectrometry, sequence analysis and enzymatic digestions.

ChemInform Abstract: THE SYNTHESIS OF PEPTIDES RELATED TO A CONSERVED SEQUENCE FOUND IN HISTONE H‐1 AND H‐5. THEIR ABILITY TO ACT AS SUBSTRATES AND INHIBITORS OF EXOGENOUS PROTEIN KINASES

Two peptides, H-Gly Ser Phe Lys-NH2 and H-Gly Ser Phe Lys Leu-NH2, whose sequence may be found in a conserved and phosphorylated site in histone H-1 have been synthesized. Their ability to act as s...

The synthesis of peptides related to a conserved sequence found in histone H-1 and H-5. Their ability to act as substrates and inhibitors of exogenous protein kinases

Two peptides, H-Gly Ser Phe Lys-NH2 and H-Gly Ser Phe Lys Leu-NH2, whose sequence may be found in a conserved and phosphorylated site in histone H-1 have been synthesized. Their ability to act as substrates for several protein kinase preparations have been examined. In addition, their ability to inhibit the action of several protein kinases of muscle origin was also tested.