Actions of bradykinin and related peptides on rabbit coeliac artery rings.

1. Rabbit coeliac artery rings were mounted in tissue baths containing Krebs solution at 37 degrees C in order to determine whether their response to bradykinin is initiated by B1- or B2-receptors. Tension was recorded isometrically. 2. Phenylephrine contracted the tissue. Subsequent addition of bradykinin or des Arg10-kallidin caused relaxation which was not dependent on an intact endothelium. Des Arg10-kallidin, a B1-receptor selective agonist, was more potent than bradykinin. 3. [beta-Thienyl alanyl6,9, D-Phe8]-kallidin and [Leu9]-des Arg10-kallidin antagonized bradykinin and des Arg10-kallidin. [Leu9]-des Arg10-kallidin, a B1-receptor selective antagonist, was more potent than [Thi6,9, D-Phe8]-kallidin, a less selective drug that acts on both B1- and B2-receptors. 4. Kinin-induced relaxation was reversibly antagonized by ibuprofen (a cyclo-oxygenase inhibitor) and by 5-(N,N-hexamethylene)amiloride (an inhibitor of Na+/H+ exchange). Ibuprofen caused a parallel shift to the right of a semi-logarithmic plot of the agonist concentration-effect relationship, whereas the amiloride analogue depressed the maximum response and reduced the slope. 5. We conclude that bradykinin and des Arg10-kallidin relax rabbit coeliac artery by combining with B1-receptors. The response is mediated by a cyclo-oxygenase product and may be influenced by cellular Na+/H+ exchange.     

Probing the role of proline in peptide hormones. NMR studies of bradykinin and related peptides

The use of NMR methods to study conformational and dynamic aspects of the proline residues in the nonapeptide bradykinin is reviewed. NMR analyses involve considerations of bistable equilibria which include the cis/trans conformational heterogeneity of the imide bond, the cis'/trans' regions of conformational stability which characterize rotation about the C alpha-CO bond (dihedral angle psi), and the interconversion of the pyrrolidine ring of proline between puckered C gamma-endo and C gamma-exo conformations. These conformational features are all characterized by different kinetic behavior, are interdependent with peptide bond conformation, and exhibit sensitivity to amino acid substitutions. Thus, the substitution of Gly6 for Ser6 increases the fractional cis probability of the sixth peptide bond from 0.1 to 0.35. Substitutions of alpha-aminoisobutyric acid (AIB) residues for proline introduce conformational constraints analogous to those in cis' proline. Correlations of pyrrolidine ring conformation and dynamics with the cis/trans ratio of the imide bond have also been observed in model systems. Conformational and activity analyses of [AIB7]-bradykinin provided a stimulus for the development of the first bradykinin antagonist by Stewart and Vavrek (Vavrek RJ and Stewart JM, Competitive antagonists of bradykinin. Peptides 6: 161-164, 1985).     

Structure-activity studies of carbamate and other esters: agonists and antagonists to nicotine

A number of aromatic, cycloalkyl, and heterocyclic carbamic acid esters, thiocarbamic acid esters, and carboxylic acid esters of di- and trial-kylaminoalkyl and heterocyclic amino alcohols have been synthesized and tested for their pharmacologic and receptor binding characteristics at the nicotine receptor. Receptor binding was measured in rat brain membranes using (-)-3H-nicotine or 3H-methylcarbamylcholine as radioligands. The compounds were tested for their ability to produce seizures and prostration and to antagonize the nicotine-induced prostration and seizures as well as the hypertensive action of nicotine in rats. Among the potent agonists were the N-methylcarbamyl and N-methylthiocarbamyl esters of choline (trimethylaminoethanol), with the tertiary amino derivatives between considerably less potent than the quaternary. Potent antagonists included trimethylaminoethyl benzoate, 3-quinuclidinyl benzoate, and trimethylaminoethyl esters of phenyl and phenylthiocarbamic acids. One of the most potent antagonists to nicotine was alpha-lobeline.     

Structure-activity studies on C-terminal hirudin peptides containing sulfated tyrosine residues

To clarify the role of the negative charge of the C-terminal region of hirudin, we chemically synthesized the C-terminal peptide of hirudin variant-1 (HV-1), HV-1-(54-65), and its analogs, [E61Y,E62Y]HV-1-(54-65) and [E62Y]HV-1-(54-65), and then sulfated the Tyr residue(s) in these peptides by both enzymic and chemical methods. Enzymic O-sulfation of Tyr residues in the peptides by use of sulfotransferase isolated from Eubacterium A-44 allowed us to produce four kinds of the sulfated peptide, whose C-terminal sequences were -PEY(SO₃H)YLQ, -PYY(SO₃H)YLQ, -PYYY(SO₃H)LQ and -PYY(SO₃H)Y(SO₃H)LQ. On the other hand, all Tyr residues in the peptides were successfully sulfated by chemical reaction with N, N′-dicyclohexylcarbodiimide in the presence of sulfuric acid. Based on the analysis of structure-activity relationships of these sulfated peptides for thrombin inhibition, the Tyr62 and Tyr63 bisulfated peptide GDFEEIPEY(SO₃H)Y(SO₃H)LQ was found to be the most potent inhibitor of thrombin among the products tested. No increase in potency was observed by further substitution of Glu61 with Tyr(SO₃H). The inhibitory activity by substitution with Tyr(SO₃H) at position 63 was greater than that obtained by the substitution at position 62. © Munksgaard 1996.     

ACTH-like peptides and opiate receptors in the rat brain: Structure-activity studies

The present study aims at further identifying the interaction of ACTH-like peptides and rat brain opiate receptors in vitro. The sequence ACTH_4–10 is crucial with respect to affinity since it is the shortest sequence to inhibit the binding of [³H]-dihydromorphine and [³H]-naltrexone to these receptors. A second active site seems to be localized in the N-terminal part of ACTH_11–24. This structure—activity relationship is compared to that observed for these peptides on the adrenal cortex and behavior.     

Structure-activity relationship of reversibly lipidized peptides: studies of fatty acid-desmopressin conjugates

Purpose. To synthesize a series of reversible fatty acid-desmopressin (DDAVP) conjugates and to study their structure-activity relationship as anti-diuretic drugs. Methods. Seven fatty acid conjugates of DDAVP were prepared using various reversible lipidization reagents as described in our previous reports. All products were purified by acid precipitation and/or size-exclusion chromatography. Reversed-phase HPLC was used to evaluate their purity and lipophilicity. The anti-diuretic efficacy of these fatty acid conjugates was assessed in vasopressin-deficient Brattleboro rats. Four selected conjugates, i.e., DPA, DPH, DPD and DPP (acetic, hexanoic, decanoic, and palmitic acid conjugate, respectively), along with DDAVP itself were used in Caco-2 cell uptake studies and their degradation and the regeneration of active DDAVP were investigated using an in vitro liver slice metabolic system coupled with a HPLC assay. Results. All fatty acid-DDAVP conjugates were more lipophilic than DDAVP as examined by HPLC analyses. When cysteine was used as the linker, the capacity index (k, a measure of lipophilicity) of the conjugates was linearly correlated with the number of carbons in the fatty acid chain. The anti-diuretic activity of the conjugates was correlated with the length of the fatty acid chain, with C10 as the minimal requirement for possessing the enhanced anti-diuretic activity. Among the seven fatty acid conjugates, palmitic acid conjugate was the most potent DDAVP derivative. Removal of carboxyl group from the cysteine linker completely abolished the enhancement of the activity. The extent of cellular uptake also positively correlated with the lipophilicity of the conjugates. The metabolism of DDAVP, DPH, DPD, and DPP by liver slices all followed first order kinetics with half-life of 0.30, 0.01, 0.06 and 3.44 hr, respectively. The degradation rates of DPH and DPD in the liver slice incubation were much faster than that of DDAVP and therefore an accumulation of regenerated DDAVP in the media was observed. In contrast, DPP was metabolized much slower than DDAVP and, consequently, no significant accumulation of regenerated DDAVP could be detected. Conclusion. Conjugation of DDAVP with fatty acids increased the lipophilicity and the anti-diuretic activity of this peptide drug. The anti-diuretic activity of lipidized DDAVP was dependent on the chain length of the fatty acid, as well as the structure of the linker in the conjugate. The preservation and enhancement of the in vivo anti-diuretic activity of the conjugates is most likely due to a combination of an improved pharmacokinetic behavior and a concurrent regeneration of active DDAVP in tissues.     

Structure-activity relationship studies on cyclic RGD peptides utilizing novel alkene dipeptide isosteres

A structure-activity relationship study was performed on cyclic RGD peptides using a combination of multisubstituted alkene dipeptide isosteres. To clarify the effects on bioactivity of a valine N-methyl group in the cyclo(-Arg-Gly-Asp-D-Phe-MeVal-) peptide developed by Kessler's group, novel D-Phe-Val-type isosteres with methyl-substituting groups on the olefin were designed and synthesized. Syntheses of D-Phe-psi[(E)-CH=CMe]-Val-type isosteres were carried out in essentially identical fashion to the previously reported preparation of psi[(E)-CH=CH]-type congeners. Alternatively, D-Phe-psi[(E)-CMe=CX]-Val-type isosteres (X=H or Me) were synthesized via stereoselective alkylation of beta-(1,3-oxazolidin-2-on-5-yl)-alpha,beta-enoates using organocopper reagents. The resulting four isosteres were utilized in either solution- or solid-phase peptide synthesis to afford the cyclic RGD peptidomimetics, cyclo(-Arg-Gly-Asp-D-Phe-psi[(E)-CX=CX]-Val-) (X=H or Me). alpha(V)beta(3) and alpha(IIb)beta(3) integrin antagonistic activities of the peptidomimetics along with Kessler's peptides were comparatively evaluated. In addition, structural calculations of these compounds by simulated annealing/energy minimization using dihedral and distance restraints derived from (1)H-NMR data in DMSO gave insight into the effects of the valine N-methyl group as well as the D-phenylalanine carbonyl oxygen.     

GABA-A agonists and GABA uptake inhibitors: Structure-activity relationships

Muscimol is a potent but non-selective GABA-A agonist. Structure-activity studies on the (S)- and (R)-forms of chiral muscimol analogues have disclosed a high degree of agonist stereoselectivity of the GABA-A receptors. THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a specific GABA-A agonist, which has been the subject of clinical studies in different groups of patients. Even minor alterations of the structure of THIP result in substantial or complete loss of GABA-A agonist activity. 4-PIOL (5-(4-piperidyl)isoxazol-3-ol) shows in vivo GABA-A agonist activity on spinal neurones, whereas the in vitro pharmacological effects in brain tissue preparations are consistent with a GABA-A antagonist profile of 4-PIOL in the brain. Whereas nipetcotic acid and related GABA uptake inhibitors are substrates for the neuronal and glial transport carrier, the glia-selective GABA uptake inhibitors THPO (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol) and probably also THAO (5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol) are not being transported by the glial uptake carrier. Introduction of the DPB (4,4-diphenyl-3-butenyl) or BEE (benzhydryl ethyl ether) substituents on the basic nitrogen atoms of GABA uptake inhibitors, including nipecotic acid and THPO, results in markedly more potent inhibitors. However, unlike THPO, N-DPB-THPO interacts non-selectively with neuronal and glial GABA uptake, and, in contrast to nipecotic acid, N-DPB-nipecotic acid (SKF-89976-A) has been shown not to be transported by the neuronal or glial GABA carriers. Whereas N-DPB- and N-BEE-GABA are weak inhibitors of synaptosomal GABA uptake, N-methylation of these compounds gives potent uptake inhibitors.     

Structure-activity relationships of the delta-opioid-selective agonists, deltorphins.

Deltorphins are naturally occurring peptides with high affinity and selectivity for delta-opioid receptors. They share with dermorphin, another mu-selective opioid agonist, the same N-terminal tripeptide Tyr-D-Xaa-Phe, where D-Xaa is a D-Ala or a D-Met residue. This common sequence appears to be essential for the best fitting of the peptides to both mu- or delta-opioid sites. We studied the changes in receptor affinity and selectivity and in biological potency of deltorphins due to shortening of the sequence, C-terminal deamidation or single amino acid substitutions. The results support the view that a code addressing the molecule towards delta-opioid sites is expressed in the C-terminal region of these peptides. This addressing domain confers high delta-selectivity to the ligand in the following two ways: (i) increased affinity for delta-sites; (ii) decreased affinity for mu-sites. The sequence of the C-terminal tripeptide appears to be responsible for the high delta-affinity of the molecules. Negatively charged side chains inhibit mu-binding and enhance delta-selectivity.     

Structure-activity studies on nociceptin analogues: ORL1 receptor binding and biological activity of cyclic disulfide-containing analogues of nociceptin peptides.

Nociceptin/Orphanin FQ is an endogenous peptide ligand for the opioid receptor-like 1 (ORL1) receptor. To investigate the structural and conformational requirements of the nociceptin (NC)-receptor interaction, six cyclic analogues containing Cys disulfide linkages were designed and synthesized. Analogues cyclized at the N-terminal part, cyclo[Cys(0), Cys(7)]NC(1-13)-NH(2) (2) and cyclo[Cys(0), Cys(11)]NC(1-13)-NH(2) (4), and their corresponding linear peptides had very low activities in both the receptor binding and the GTP gamma S functional assays using human ORL1 transfected cell membranes. On the contrary, analogues cyclized at the C-terminal parts by the disulfide linkages at positions 6-10, 7-11, 7-14, and 10-14 sustained relatively high potencies in both assays. Notably, cyclo[Cys(10), Cys(14)]NC(1-14)-NH(2) (12) was found to be a potent NC agonist nearly as active as the parent peptide or NC. The maximum efficacy (Emax) of the C-terminally cyclized analogues and their linear counterparts in the GTP gamma S functional assay showed more than 94% (vs NC as 100%), suggesting that these analogues are full agonists. Analogue 12 is the first conformationally constrained NC analogue with almost full activity, and thus may serve to analyze the bioactive conformations of NC at the receptor site as well as serving as a template for more potent NC agonists.     

Structure-activity relationship study: short antimicrobial peptides

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an α-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an α-helical structure correlated with the antimicrobial activity and a β-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of l -amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the β-sheet structure retained antifungal activity in the same condition.     

Structure-activity studies on alpha-conotoxins

Conotoxins are small bioactive highly structured peptides from the venom of marine cone snails (genus Conus). Over the past 50 million years these molluscs have developed a complex venom cocktail for each species that is comprised of 100-2000 distinct cysteine- rich peptides for prey capture and defence. This review focuses on an important and well-studied class of conotoxins, the α- conotoxins. These α-conotoxins are potent and selective antagonists of various subtypes of the nicotinic acetylcholine receptors (nAChRs). Key structure-activity relationship studies are presented to illustrate the common motifs, structural features and pharmacophores that define this interesting peptide class. Additionally, their synthesis, chemical modifications, the development of more selective and stable analogues and their therapeutic potential are discussed.     

Metabolism of bradykinin agonists and antagonists by plasma aminopeptidase P.

In addition to angiotensin I converting enzyme (ACE; EC 3.4.15.1) and carboxypeptidase N (CPN; EC 3.4.17.3), other peptidases contribute to bradykinin (BK) degradation in plasma. Rat plasma degraded BK by hydrolysis of the N-terminal Arg1-Pro2 bond, and the characteristics of hydrolysis are consistent with identification of aminopeptidase P (APP; EC 3.4.11.9) as the responsible enzyme. BK and BK[1-5] N-terminal hydrolysis was optimal at neutral pH, was inhibited by 2-mercaptoethanol, dithiothreitol, o-phenanthroline and EDTA, but was unaffected by the aminopeptidase inhibitors amastatin, puromycin and diprotin A, the endopeptidase-24.11 inhibitors phosphoramidon and ZINCOV, and the ACE and CPN inhibitors captopril and D,L-mercapto-methyl-3-guanidinoethylthiopropanoic acid (MERGETPA), respectively. Although kallidin (Lys-BK) was not metabolized directly by APP, conversion to BK by plasma aminopeptidase M (EC 3.4.11.2) resulted in subsequent degradation by APP. BK analogs containing N-terminal Arg1-Pro2 bonds, including [Tyr8-(OMe)] BK and [Phe8 psi(CH2NH)Arg9]BK (B2 agonists), des-Arg9-BK and [D-Phe8]des-Arg9-BK (B1 agonists), and [Leu8]des-Arg9-BK (B1 antagonist), were degraded by APP with Km and Vmax values comparable to those found for BK (Km = 19.7 +/- 2.6 microM; Vmax = 12.1 +/- 1.2 nmol/min/mL). In contrast, B2 antagonists containing D-Arg0 N-termini, including D-Arg[Hyp3,Thi5.8,D-Phe7]BK and D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, were resistant to APP-mediated hydrolysis. These data support a role for plasma aminopeptidase P in the degradation of circulating kinins, and a variety of B2 and B1 kinin agonists and antagonists. However, APP does not participate in the degradation of D-Arg0-containing antagonists.     

Opioid peptides. Structure-activity relationships of dermorphin endothiotetrapeptides. VII

We describe the synthesis and preliminary in vitro and in vivo pharmacological tests of five endothiotetrapeptide analogues of the opioid heptapeptide dermorphin H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser.-NH2. The modification obtained by substituting Phet for Phe3 or Glyt for Gly4 provided further analogues with significant opioid activity.     

Design and synthesis of potent bradykinin agonists containing a benzothiazepine moiety.

A bradykinin analogue (H-Arg-Pro-Pro-Gly-Phe-Ser-D-BT-Arg-OH, 3) in which the Pro-Phe dipeptide was replaced by the (3S)[amino]-5-(carbonylmethyl)-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety has been synthesized. The same modification was performed on the potent bradykinin B(2) receptor antagonist HOE 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH), in which the -D-Tic-Oic- moiety was replaced by D-BT to yield H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-BT-Arg-OH, 1 (JMV1116). These compounds were examined in vitro for their binding affinity toward bradykinin B(1) and B(2) receptors as well as for their ability to interfere with bradykinin-induced contraction of both human umbilical vein and rat uterus. The two compounds 3 and 1 competed with [(3)H]bradykinin binding to the human cloned B(2) receptor giving K(i) values of 13 +/- 2 and 0.7 +/- 0.1 nM, respectively. Unexpectedly, both compounds were full bradykinin B(2) receptor agonists on the human umbilical vein (pD(2) = 6.60 +/- 0.07 for 3 and 6.80 +/- 0.08 for 1) and rat uterus (pD(2) = 7.20 +/- 0.09 for 3 and 7.50 +/- 0.09 for 1) preparations with the same efficacy as bradykinin. In addition 1 induced a concentration-dependent phosphoinositide production in CHO cells expressing the human cloned B(2) receptor. These data provide evidence for a bioactive conformation of bradykinin constrained at the dipeptide Pro-Phe.