Design, synthesis and bioassays of antagonists of LHRH which have high antiovulatory activity and release negligible histamine

Potent antagonists of the luteinizing hormone releasing hormone have been achieved which release negligible histamine. [N-Ac-D-2-Nal1, D-pClPhe2, D-3-Pal3, NicLys5, D-NicLys6, ILys8, D-Ala10]-LHRH showed 100%AOA/1 microgram and 36%/0. 5 micrograms; ED50 greater than 300. [N-Ac-D-2-Nal1,D-pClPhe2,D-3-Pal3, PicLys5, D-PicLys6, ILys8, D-Ala10]-LHRH showed 100% AOA/0.5 micrograms and 40%/0.25 micrograms; ED50, 93 +/- 11, and is the most potent of 52 new peptides. These antagonists feature designs with weakly basic acylated D-Lys6, notably D-NicLys6 and D-PicLys6 and alkylated Lys8 or Orn8, e.g., ILys8 and IOrn8, and NicLys5 and PicLys5. Concepts included balanced overall basicity, superiority of ILys8 and IOrn8 which are sequence dependent and sensitivity of positions 5 and 6 for potency.     

Increased potency of antagonists of the luteinizing hormone releasing hormone which have D-3-Pal in position 6

Ever increasing potency is an international goal for antagonists of the luteinizing hormone releasing hormone (LHRH). [N-Ac-D-2-Nal1,D-pClPhe2, D-3-Pal3,Ser4,Arg5,D-3-Pal6,Leu7,Arg8,Pro9,D- Ala10]-NH2 caused 60%/125 ng inhibition of ovulation in the rat, and appears to be the most potent antagonist yet described. Strategy of design was the replacement of D-Arg6 with D-3-Pal6 and of Tyr5 with Arg5. Replacing Arg5 with His5 reduced activity by 50% at 250 ng. Both the Arg5 and His5 analogs showed 100% inhibition of ovulation at 0.5 microgram. Of ten pairs of analogs with D-3-Pal6 and D-Arg6, 3/10 with D-3-Pal6 were more potent than those with D-Arg6. Histamine release was less for the D-3-Pal6 peptides of three pairs.     

Antagonists of luteinizing hormone releasing hormone with novel unnatural amino acids at position six

Five new antagonists of luteinizing hormone releasing hormone (LHRH) containing novel unnatural amino acids at position six are reported. They are very effective in the rat antiovulatory assay. Using saline as vehicle, antagonist-[N-Ac-D-2-Nal1, D-4-Cl-Phe2, D-3-Pal3, Arg5, D-A26, D-Ala10]-LHRH inhibited ovulation completely at 1 micrograms/rat and three of the other antagonists showed some antiovulatory activity at 0.5 micrograms/rat.     

Antagonists of the luteinizing hormone releasing hormone (LHRH) with emphasis on the TRP7 of the salmon and chicken II LHRH's

The sequences of four naturally occurring luteinizing hormone releasing hormones (LHRH's) differ only in positions 5, 7 and 8. Salmon and chicken II LHRH's have Trp7; porcine/ovine (P/O) and chicken I LHRH's have Leu7. The receptor for P/O LHRH might effectively bind certain antagonists with Trp7. Thirteen antagonists having Trp7 and eight antagonists with other substitutions in position 7 were synthesized. One of the thirteen antagonists with the natural Trp7, [N-Ac-D-2-Nal1,D-pClPhe2,D-3-Pal3,D-Arg6,Trp7,D- Ala10]-LHRH, not only maintained activity, but had increased potency (ca. 58%; 90% antiovulatory activity/250 ng; rats) in comparison with the companion analog with the natural Leu7 of P/O LHRH. The other twelve Trp7-antagonists had lower potency.     

Antagonists of the luteinizing hormone releasing hormone with pyridyl-alanines which completely inhibit ovulation at nanogram dosage

[N-Ac-D-2-Nal1,pCl-D-Phe2,D-3-Pal3,D-Arg6,D-Ala10]-LHRH caused 100% and 57% inhibition of ovulation in rats, s.c., at 500 and 250 ng, respectively, and 56%, per os at 500 micrograms. [N-Ac-3,4-diC1-D-Phe1,pC1-D-Phe2,D-3-Pal3,D-Arg6,D-Ala10]-LHRH inhibited ovulation, s.c., 82% at 500 ng, and 63%, per os at 500 micrograms. These analogs are the most effective reported inhibitors of ovulation. The new introduction of pyridyl-alanines can be superior substituents. For pairs of analogs, relationships are: D-3-Pal (beta-(3-pyridyl)-D-alpha-alanine) in position 3 is superior to D-Trp3, D-2-Pal3 and D-4-Pal3. D-Arg6 was superior to D-3-Pal6 and D-4-Pal6 was superior by 2-fold to D-Arg6. D-Ala10 was superior to Gly10 and D-Abu10.     

Involvement of amino acids flanking Glu7.32 of the gonadotropin-releasing hormone receptor in the selectivity of antagonists

The Glu/Asp(7.32) residue in extracellular loop 3 of the mammalian type-I gonadotropin-releasing hormone receptor (GnRHR) interacts with Arg(8) of GnRH-I, conferring preferential ligand selectivity for GnRH-I over GnRH-II. Previously, we demonstrated that the residues (Ser and Pro) flanking Glu/Asp(7.32) also play a role in the differential agonist selectivity of mammalian and non-mammalian GnRHRs. In this study, we examined the differential antagonist selectivity of wild type and mutant GnRHRs in which the Ser and Pro residues were changed. Cetrorelix, a GnRH-I antagonist, and Trptorelix-2, a GnRH-II antagonist, exhibited high selectivity for mammalian type-I and non-mammalian GnRHRs, respectively. The inhibitory activities of the antagonists were dependent on agonist concentration and subtype. Rat GnRHR in which the Ser-Glu-Pro (SEP) motif was changed to Pro-Glu-Val (PEV) or Pro-Glu-Ser (PES) had increased sensitivity to Trptorelix-2 but decreased sensitivity to Cetrorelix. Mutant bullfrog GnRHR-1 with the SEP motif had the reverse antagonist selectivity, with reduced sensitivity to Trptorelix-2 but increased sensitivity to Cetrorelix. These findings indicate that the residues flanking Glu(7.32) are important for antagonist as well as agonist selectivity.     

Comparison of spantide II and CP-96,345 for blockade of tachykinin-evoked contractions of smooth muscle.

CP-96,345, a quinuclidine, is a potent inhibitor of substance P for the NK1 receptor of bovine brain, but has reduced potency for the corresponding receptor of the rat and mouse, and none for NK2 or NK3 receptors. A related quinuclidine showed similar but lower potency than CP-96,345 for NK1. CP-96,345 was more potent than the spantide I of 1984, D-Arg1,Pro2,Lys3,Pro4,Gln5,Gln6,D-Trp7,Phe8,D-Trp9, Leu10,Leu11,NH2. Our continued designs for antagonists of substance P led to spantide II in 1990 which is: D-NicLys1,Pro2,3-Pal3,Pro4,D-Cl2Phe5,Asn6,D-Trp7 ,Phe8,D-Trp9,Leu10,Nle11-NH2. The pA2 values of spantide II and CP-96,345 for guinea pig taenia coli were 7.6 and 6.8, respectively. The pIC50 values for blockade of tachykinin-mediated neurotransmission in the rabbit iris sphincter were 6.1 and 5.4, respectively. Spantide II was nearly 10 times more potent than CP-96,345 in these two assays.     

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described. 2. Bk is rapidly inactivated by neuronal perikarya (4.2 +/- 0.6 fmol/min/cell body). 3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages. 4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15. 5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond. 6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases. 7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Conformational studies of two bradykinin antagonists by using two-dimensional NMR techniques and molecular dynamics simulations

The solution conformations of two potent antagonists of bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9), [Aca(-1),DArg0,Hyp3,Thi5,DPhe7,(N-Bzl)Gly8]BK (1) and [Aaa(-1),DArg0,Hyp3,Thi5,(2-DNal)7,Thi8]BK (2), were studied by using 2D NMR spectroscopy in DMSO-d6 and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe7-(N-Bzl)Gly8 peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a beta-turn structure for the Hyp3-Gly4 residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe7 and (N-Bzl)Gly8 in analogue 1 suggests type VI beta-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb beta-turn comprising residues Ser6-Arg9 and the betaI or betaII-turn involving the Pro2-Thi5 fragment, whereas peptide 2 shows the tendency towards the formation of type I beta-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9. Moreover, the side chain of DArg0 is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.     

The influence of basic residues on the substrate specificity of protein kinase C

The substrate specificity of protein kinase C has been examined using a series of synthetic peptide analogs of glycogen synthase, ribosomal protein S6, and the epidermal growth factor receptor. The glycogen synthase analog peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala10 was phosphorylated at Ser7 with a Km of 40.3 microM. Peptide phosphorylation was strongly dependent on Arg4. When lysine was substituted for Arg4 the Km was increased approximately 20-fold. Addition of basic residues on either the NH2-terminal or COOH-terminal side of the phosphorylation site of the glycogen synthase peptide improved the kinetics of peptide phosphorylation. The analog Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys was phosphorylated with a Km of 4.1 microM. Substitution of Ser7 with threonine increased the apparent Km to 151 microM. The truncated peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val8 was phosphorylated with similar kinetic constants to the parent peptide, however, deletion of Val8 increased the apparent Km to 761 microM. The ribosomal peptide S6-(229-239) was phosphorylated with a Km of approximately 0.5 microM predominantly on Ser236 and is one of the most potent synthetic peptide substrates reported for a protein kinase. The apparent Km for S6 peptide phosphorylation was increased by either deletion of the NH2-terminal 3 residues Ala229-Arg-231 or by substitution of Arg238 on the COOH-terminal side of the phosphorylation site with alanine. This analog peptide, [Ala238]S6-(229-239) was phosphorylated with an approximate 6-fold reduction in Vmax and a switch in the preferred site of phosphorylation from Ser236 to Ser235. These results support the concept that basic residues on both sides of the phosphorylation site can have an important influence on the kinetics of phosphorylation and site specificity of protein kinase C.     

Position of Pro and Ser near Glu7.32 in the extracellular loop 3 of mammalian and nonmammalian gonadotropin-releasing hormone (GnRH) receptors is a critical determinant for differential ligand selectivity for mammalian GnRH and chicken GnRH-II

A Glu/Asp7.32 residue in the extracellular loop 3 of the mammalian GnRH receptor (GnRHR) is known to interact with Arg8 of mammalian GnRH (mGnRH), which may confer preferential ligand selectivity for mGnRH than for chicken GnRH-II (cGnRH-II). However, some nonmammalian GnRHRs also have the Glu/Asp residue at the same position, yet respond better to cGnRH-II than mGnRH. Amino acids flanking Glu/Asp7.32 are differentially arranged such that mammalian and nonmammalian GnRHRs have an S-E/D-P motif and P-X-S/Y motif, respectively. We presumed the position of Ser7.31 or Pro7.33 of rat GnRHR as a potential determinant for ligand selectivity. Either placing Pro before Glu7.32 or placing Ser after Glu7.32 significantly decreased the sensitivity and/or efficacy for mGnRH, but slightly increased that for cGnRH-II in several mutant receptors. Among them, those with a PEV, PES, or SES motif exhibited a marked decrease in sensitivity for mGnRH such that cGnRH-II had a higher potency than mGnRH, showing a reversed preferential ligand selectivity. Chimeric mGnRHs in which positions 5, 7, and/or 8 were replaced by those of cGnRH-II revealed a greater ability to activate these mutant receptors than mGnRH, whereas they were less potent to activate wild-type rat GnRHR than mGnRH. Interestingly, a mutant bullfrog type I receptor with the SEP motif exhibited an increased sensitivity for mGnRH but a decreased sensitivity for cGnRH-II. These results indicate that the position of Pro and Ser near Glu7.32 in the extracellular loop 3 is critical for the differential ligand selectivity between mammalian and nonmammalian GnRHRs.     

Comparison of the effect of several gonadotropin releasing hormone antagonists on luteinizing hormone secretion, receptor binding and ovulation

The biological activity of three gonadotropin releasing hormone (GnRH) antagonists was evaluated in the following assays: suppression of GnRH-mediated luteinizing hormone (LH) secretion by cultured pituitary cells, suppression of the spontaneous LH release by ovariectomized rats, blockade of ovulation in regularly cycling females and inhibition of binding of a potent radiolabeled agonist to rat pituitary membrane homogenates. The peptides were: [Ac-delta 3Pro1,4FDPhe2, DTrp3,6]-GnRH (Antagonist 1); [Ac-delta 3Pro1,4FDPhe2,DNAL(2)3,6]-GnRH (Antagonist 2); and [Ac-DNAL(2)2,4FDPhe2,DTrp3,DArg6]-GnRH (Antagonist 3). All three antagonists exhibited similarly high potency in suppressing LH secretion in vitro, while Antagonist 1 was the most active peptide in the radioreceptor assay. When administered by gavage, Antagonist 3 exhibited the highest potency to inhibit LH secretion in gonadectomized rats and to block ovulation. Comparison of the oral versus the subcutaneous mode of administration of these analogs indicates that less than 1% is absorbed after gavage. However, these data demonstrate that the intragastric administration of GnRH antagonists can lower gonadotropin secretion and interfere with reproductive functions.     

Synthesis and biological evaluation in vitro of selective,high affinity peptide antagonists of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1

Human melanin-concentrating hormone (hMCH) and many of its analogues are potent but nonspecific ligands for human melanin-concentrating hormone receptors 1 and 2 (hMCH-1R and hMCH-2R). To differentiate between the physiological functions of these receptors, selective antagonists are needed. In this study, analogues of Ac-Arg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), a high affinity but nonselective agonist at hMCH-1R and hMCH-2R, were prepared and tested in binding and functional assays on cells expressing these receptors. In the new analogues, 5-aminovaleric acid (Ava) was incorporated in place of the Leu(9)-Gly(10) and/or Arg(14)-Pro(15) segments of the disulfide ring. Several of these compounds turned out to be high affinity antagonists selective for hMCH-1R. Moreover, even at micromolar concentrations, they were devoid of agonist potency at both hMCH receptors and not effective as hMCH-2R antagonists. For example, peptide 14, Gva(6)- cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Ava(14,15)-Cys(16))-NH(2), (Gva = 5-guanidinovaleric acid), was a full competitive hMCH-1R antagonist (IC(50) = 14 nM, K(B) = 0.9 nM) with more than 1000-fold selectivity over hMCH-2R. Examination of various compounds with Ava in positions 9,10 and/or 14,15 revealed that the Leu(9)-Gly(10) and Arg(14)-Pro(15) segments of the disulfide ring are the principal structural elements determining hMCH-1R selectivity and ability to act as a hMCH-1R antagonist.     

Effect of thyrotropin-releasing hormone on immune functions of peripheral blood mononuclear cells

The tripeptide thyrotropin-releasing hormone (TRH) works as a hypothalamic hormone, but is found also outside the brain in intrinsic nerve fibers of the gastrointestinal tract. There is evidence that TRH modulates the activity of immunocompetent cells, although there are only very few data on TRH-mediated immune effector functions. Since we could recently show that TRH inhibits monocyte activities we were also interested in other possible TRH modulated immune functions. Peripheral blood mononuclear cells (PBMC) from ten healthy subjects were cultured for 7 days and pulsed with 0.125 and 0.250 microgram/ml Pokeweed mitogen (PWM). 10(-12) to 10(-6) M TRH was added simultaneously with PWM. Lymphocyte proliferation [(3H]thymidine incorporation), interferon-gamma (IFN-gamma) activity (RIA) and immunoglobulin activities (IgG, IgM, IgA; ELISA) were determined in the supernatants. We could demonstrate a TRH-dependent decrease in PWM-pulsed IgG activity with significant (alpha = 0.05) values at 10(-8) and 10(-10) M (-29 +/- 6%/-16 +/- 3% for PWM 0.125 microgram/ml and -17 +/- 9%/-11 +/- 9% for PWM 0.250 microgram/ml). This inhibitory effect could be abolished by an anti-TRH antiserum. There was no TRH effect on IgM and IgA activities, IFN-gamma activity and lymphocyte proliferation compared with the PWM stimulated values alone. The described TRH effect on the polyclonal IgG response by PBMC gives further evidence for a functional link between the immune system and the endocrine system, although its underlying mechanism is not yet clear.     

The importance of the N-terminal beta-turn in bradykinin antagonists

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.     

Dehydrophenylalanyl analogs of bradykinin: Synthesis and biological activities

We have synthesized three analogs of the potent vasodilator peptide bradykinin, ArgProProGlyPhe SerProPheArg (BK), containing dehydrophenylalanine (Δ^zPhe) in place of the phenylalanyl residues at positions 5 and/or 8. The analogs, [Δ^zPhe⁵]BK, [Δ^zPhe⁸]BK, and [Δ^zPhe^5,8]BK, were assayed for their effects on isolated smooth muscle tissues and on the systemic arterial blood pressure of rats. In these assays [Δ^zPhe⁵]BK showed considerably high biological activities, particularly in terms of its blood pressure-lowering effects, being over 23 times more potent than BK when given intravenously. [Δ^zPhe⁸]BK was less potent than BK and [Δ^zPhe^5,8]BK had effects comparable to those of BK. All three synthetic analogs appear to be more resistant than BK to enzymic degradation during passage through the pulmonary vascular bed.     

Beta-turns induced in bradykinin by (S)-alpha-methylproline.

The ability of (S)-alpha-methylproline (alpha-MePro) to stabilise reverse-turn conformations in the peptide hormone bradykinin (BK = Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) has been investigated. Two BK analogues containing alpha-MePro at position 3 or position 7 were synthesised and their conformations in aqueous solution investigated by NMR spectroscopy. Whereas BK is largely disordered on the NMR time scale both analogues showed ROE connectivities in 2D-ROESY spectra indicative of reverse-turn conformations at both Pro2-Phe5 and Ser6-Arg9, whose formation appears to be cooperative. Some potential applications of alpha-MePro as a reverse-turn mimetic in the construction of synthetic peptide libraries is discussed.     

Identification of coding polymorphisms in human circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS in a multi-ethnic screening panel.

STUDY OBJECTIVE: In this study, the exonic regions of the circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS were re-sequenced and coding changes identified in a panel of 95 individuals varying in ethnicity. STUDY PARTICIPANTS: DNA screening panel consisting of 95 DNA samples (17 American Caucasians, 17 African Americans, 8 Ashkenazi Jews, 8 Chinese, 8 Japanese, 5 Mexican Indians, 8 Mexicans, 8 Northern Europeans, 8 Puerto Ricans, and 8 South Americans) selected from the Coriell Institute Human Variation Panel. RESULTS: In addition to coding changes already identified in the database dbSNP, novel coding changes were identified, including PER1: Pro37Ser, Pro351Ser, Gln988Pro, Ala998Thr; PER2: Leu83Arg, Leu157Leu, Thre174Ile, Phe400Phe, Pro822Pro, Ala828Thr, Ala861Val, Phe876Leu, Val883Met, Val903Ile, Ala923Pro; PER3: Pro67Pro, Val90Ile, His638His, Ala820Ala, Leu929Leu; ARNTL: Arg166Gln, Ser459Phe; CLOCK: Ala34Ala, Ser208Cys, Phe233Phe, Ser632Thr, Ser816Ser; TIMELESS: Met870Val and CRY2: His35His. No coding polymorphisms were identified in CRY1. CONCLUSIONS: Considerable genetic variation occurs within the coding region of the genes regulating circadian rhythm. Many of the non-synonymous coding polymorphisms could affect protein structure/function with the potential to affect molecular regulation of the sleep/wake cycle. Many of the potential functional effects could be ethnic group specific.     

Degradation of brain natriuretic peptide by neutral endopeptidase: species specific sites of proteolysis determined by mass spectrometry

Brain natriuretic peptide (BNP) from 3 different species was cleaved by neutral endopeptidase (NEP) and the products separated by HPLC. The newly formed products were identified by fast atom bombardment or nebulizer-assisted electrospray mass spectrometry to elucidate the sites of proteolysis. Porcine BNP was cleaved at the Arg8-Leu9 and Ser14-Leu15 bonds. Rat BNP was cleaved at the Arg23-Leu24 and Arg30-Leu31 bonds. Human BNP was cleaved at the Pro2-Lys3, Met4-Val5 and Arg17-Leu18 bonds. The Cys-Phe bond which is present in all species of BNP is not cleaved by NEP.