SAR studies of novel 5-substituted 2-arylindoles as nonpeptidyl GnRH receptor antagonists

The discovery of the potency-enhancing effect of 5-substitutions on the novel 2-arylindoles as nonpeptidyl GnRH receptor antagonists led to the identification of several analogues with high affinities on the GnRH receptor. The syntheses and SARs of these 5-substituted-2-arylindole analogues are reported.     

Structure-function studies of N-acyl derivatives of tryptophan that function as specific cholecystokinin receptor antagonists

Benzotript, N-p-chlorobenzoyl-L-tryptophan, is a specific cholecystokinin receptor antagonist. In the present study we used dispersed pancreatic acini to examine tryptophan as well as several different N-acyl derivatives of tryptophan for their abilities to function as cholecystokinin receptor antagonists. L-Tryptophan, D-tryptophan as well as each acyl derivative tested inhibited cholecystokinin-stimulated amylase secretion and outflux of ⁴⁵Ca and there was a good correlation between the ability of a particular agent to inhibit the action of cholecystokinin on acinar function and its ability to inhibit binding of ¹²⁵I-labeled cholecystokinin to pancreatic acini. Results with butyloxycarbonyl-L-tryptophan indicated that the inhibition of the action of cholecystokinin caused by L-tryptophan and various acyl derivatives is specific, competitive and fully reversible. In functioning as a cholecystokinin receptor antagonist the relative potencies of the agents tested were: carbobenzoxyl-L-tryptophan >benzotript >benzoyl-L-tryptophan = butyloxycarbonyl-L-tryptophan >acetyl-L-tryptophan >L-tryptophan. In inhibiting the actions of cholecystokinin, native as well as N-acyl derivatives of D-tryptophan were equipotent with the corresponding compound containing L-tryptophan. Although L-tryptophan inhibited the actions of cholecystokinin, L-phenylalanine, L-methionine or L-aspartic acid, even when tested at concentrations as high as 3 mM, did not alter the action of cholecystokinin on pancreatic acini. The antagonism of the actions of cholecystokinin was not restricted to N-acyl derivatives of L-tryptophan because butyloxycarbonyl-L-methionine and butyloxycarbonyl-L-phenylalanine but not butyloxycarbonyl-L-aspartic acid also antagonized the actions of cholecystokinin. These results demonstrate that both the nature of the N-Prmacyl group and the amino acid residue are important determinants of the affinity of the antagonist for the cholecystokinin receptor. For derivatives of L-tryptophan, the more hydrophobic the N-acyl moiety, the greater the affinity of the derivative for the cholecystokinin receptor.     

Structure-function relationships of the NMDA receptor antagonist peptide, conantokin-R

Conantokin-R (con-R) is a gamma-carboxyglutamate-containing 27-residue neuroactive peptide present in the venom of Conus radiatus, and acts as a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor. This peptide features a single disulfide bond, a type of structural element found in most classes of conotoxins, but not in other conantokins. The NMDA receptor antagonist activity of chemically synthesized con-R was determined through an assay involving inhibition of the spermine-enhanced binding of the NMDA receptor channel blocker, [(3)H]MK-801, to rat brain membranes, and yielded an IC(50) of 93 nM. This value represents a 2-5 times better potency than con-G or con-T, the other two characterized conantokins. Circular dichroism (CD) analysis of the metal-free form of con-R is indicative of a low alpha-helical content. There is an increase in alpha-helicity upon the addition of divalent cations, such as Ca(2+), Mg(2+), or Zn(2+). Isothermal titration calorimetry experiments showed one detectable Mg(2+) binding site with a K(d) of 6.5 microM, and two binding sites for Zn(2+), with K(d) values of 150 nM and 170 microM. Residue-specific information of the conformational state of con-R was obtained by two-dimensional (1)H-NMR. Analyses of the alpha-proton chemical shifts, NOE patterns, and hydrogen exchange rates of the peptide indicated an alpha-helical conformation for residues 1-19. Synthetic con-R-derived peptide variants, containing deletions of 7 and 10 amino acid residues from the carboxy-terminus of the wild-type peptide, displayed unaltered cation binding and NMDA receptor antagonist properties. The alpha-helical secondary structures of the two truncation peptides were more stable than full-length con-R, as evidenced by CD measurements and reduced backbone hydrogen exchange rates. These results provide experimental evidence that the structural elements common to the three conantokins thus far identified are the primary determinants for receptor function and cation binding/secondary structure stability.     

Investigation of the 4-O-alkylamine substituent of non-peptide quinolone GnRH receptor antagonists.

Synthesis and in vitro activity of the enantiomers of quinolone GnRH antagonist (+/-)-1 are reported. Chiral amino alcohols were prepared from the appropriate cyclic D- or L-amino acids by the Amdt-Eistert homologation followed by reduction of the resulting esters. Incorporation of these pharmacophores was achieved via a novel Mitsunobu alkylation of 4-hydroxyquinolones. The key amine pharmacophore for binding to the rat GnRH receptor was most active in the S-configuration. Ring size was not important for potency with 4, 5, 6, and 7-membered ring amines exhibiting similar potency.     

Structure-function studies on the cyclic peptide MT-II, lactam derivative of alpha-melanotropin.

The alanine-substituted and the retro, enantio, and retro-enantio analogs of MT-II, a potent agonist at melanocortin (MC) receptors, were prepared by solid-phase synthesis and evaluated for their ability to bind and activate human MC3, MC4, and MC5 receptors. Replacement of His with Ala resulted in [Ala6]-MT-II with affinity and agonist potency at human MC3, MC4, and MC5 receptors similar to MT-II. Substitution of Arg with Ala gave compound 100-fold less potent than MT-II, but replacement of Phe or Trp with Ala led to inactive compounds (at the micromolar concentrations). The significant drop of potency of the retro, enantio, and retro-enantio analogs of MT-II, demonstrated a crucial role of side-chain topology, and to a lesser degree, of peptide backbone in interactions of MT-II with the melanocortin receptors. The nuclear magnetic resonance analysis of MT-II suggested involvement of Phe and Arg residues in H-bonds stabilizing the bent conformations of the peptide backbone.     

Interaction of linear and cyclic peptide antagonists at the human B(2) kinin receptor

The ligand receptor interactions involving the C-terminal moiety of kinin B(2) receptor antagonists Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7gamma-10alpha)) and a series of analogs modified in position 10 were investigated by radioligand-binding experiments at the wild type (WT) and at the Ser(111)Ala and Ser(111)Lys mutant human kinin B(2) receptors. Icatibant and [Lys(10)]-Icatibant maintained the same high affinity towards the three receptors. For Icatibant-NH(2), [Ala(10)]-Icatibant, MEN 11270 and [Glu(10)]-MEN 11270, the changes in affinity at the WT and Ser(111)Lys receptors indicated that the presence of a net positive or negative charge at the C-terminal moiety of these peptides caused a decrease in affinity to the WT receptor and that Ser(111) residue is in proximity of the side chain of residue 10. The changes in affinity measured with [desArg(10)]-Icatibant and [desArg(10)]-Icatibant-NH(2), moreover, confirmed that a C-terminal charge compensation between the positive charge of Arg(10) side chain and the C-terminal free carboxylic function favours a high affinity interaction.     

MCH receptor peptide agonists and antagonists

Melanin-concentrating hormone (MCH) is an important neuropeptide hormone involved in multiple physiological processes. Peptide derivatives of MCH have been developed as tools to aid research including potent radioligands, receptor selective agonists, and potent antagonists. These tools have been used to further understand the role of MCH in physiology, primarily in rodents. However, the tools could also help elucidate the role for MCHR1 and MCHR2 in mediating MCH signaling in higher species.     

Modification of the pyridine moiety of non-peptidyl indole GnRH receptor antagonists

The synthesis of a number of indole GnRH antagonists is described. Oxidation of the pyridine ring nitrogen, combined with alkylation at the two position, led to a compound with an excellent in vitro activity profile as well as oral bioavailability in both rats and dogs.     

Substituted indole-5-carboxamides and -acetamides as potent nonpeptide GnRH receptor antagonists.

The 2-aryltryptamine class of GnRH receptor antagonists has been modified to incorporate carboxamide and acetamide substituents at the indole 5-position. With either a phenol or methanesulfonamide terminus on the N-aralkyl side chain, potent binding affinity to the GnRH receptor was achieved. A functional assay for GnRH antagonism was even more sensitive to structural modification and revealed a strong preference for branched tertiary amides.     

GnRH analogues--agonists and antagonists

GnRH analogues have achieved widespread clinical use for the control of reproduction in animals. Over 2000 analogues of GnRH have been developed and tested over the last 30 years. Paradoxical anti-fertility effects are seen when the more potent agonists are delivered continuously to animals. The evaluation of agonist potency depends largely on the model used and wide varying potencies are reported for the same agonist. The design of analogues has centered on improving the receptor-binding and subsequent activation for agonists. Antagonists have been produced with strong receptor binding but without activation. Deslorelin is classified as a superagonist, with a potency perhaps 100 times that of GnRH. The interactions between agonist potency, dose and duration of treatment largely determine whether pro- or anti-fertility effects are induced. Due to the peptide nature of the synthetic analogues oral administration and potential gastrointestinal enzymatic degradation poor bioavailability results necessitating a parenteral delivery system. Some GnRH antagonists have been associated with significant histamine release, inhibiting their widespread use. More recently, antagonists have been developed that avoid this side effect without compromising potency. However the GnRH antagonist development has lagged behind that of the agonists, in part related to their high cost of production. In conclusion, GnRH agonists have achieved widespread clinical use in animals for controlling reproduction in either pro- or anti-fertility roles, yet antagonist development has been slower.     

Identification of neutral 4-O-alkyl quinolone nonpeptide GnRH receptor antagonists

A series of neutral, nonbasic quinolone GnRH antagonists were prepared via Mitsunobu alkylation of protected and unprotected 4-hydroxy quinolone intermediates. The synthetic route was improved by utilization of unique reactivity and convergency afforded by the use of mono and bis-trimethylsilylethyl protected quinolones. Potent neutral GnRH antagonists were identified, including ether and lactam derivatives, that show similar in vitro binding affinity and functional activity as compared to the earlier basic 4-aminoalkyl quinolone series of nonpeptide GnRH antagonists.     

Molecular basis for selectivity of high affinity peptide antagonists for the gastrin-releasing peptide receptor.

Few gastrointestinal hormones/neurotransmitters have high affinity peptide receptor antagonists, and little is known about the molecular basis of their selectivity or affinity. The receptor mediating the action of the mammalian bombesin (Bn) peptide, gastrin-releasing peptide receptor (GRPR), is an exception, because numerous classes of peptide antagonists are described. To investigate the molecular basis for their high affinity for the GRPR, two classes of peptide antagonists, a statine analogue, JMV594 ([d-Phe(6),Stat(13)]Bn(6-14)), and a pseudopeptide analogue, JMV641 (d-Phe-Gln-Trp-Ala-Val-Gly-His-Leupsi(CHOH-CH(2))-(CH(2))(2)-CH(3)), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimeric or mutated receptors were transiently expressed in Balb/c 3T3. Only substitution of the fourth extracellular (EC) domain of the GRPR by the comparable NMBR domain markedly decreased the affinity for both antagonists. Substituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 amino acid differences between the GRPR and NMBR in this domain were exchanged. The substitutions of Thr(297) in GRPR by Pro from the comparable position in NMBR, Phe(302) by Met, and Ser(305) by Thr decreased the affinity of each antagonist. Simultaneous replacement of Thr(297), Phe(302), and Ser(305) in GRPR by the three comparable NMBR amino acids caused a 500-fold decrease in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagonist. Receptor modeling showed that each of these three amino acids faced inward and was within 5 A of the putative binding pocket. These results demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagonists. They demonstrate that Thr(297), Phe(302), and Ser(305) of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-pi interactions and hydrogen bonding are important for their high affinity interaction.     

Complementary base-pairing properties of cyclized and linear oligonucleotides

Oligouridylates of varying chain lengths were synthesized by polynucleotide phosphorylase and cyclized by RNA ligase. Over chain lengths from 7 to 15, the bindings of the cyclized and linear oligomers to polyadenylate were measured on the basis of differential migration of bound and free oligomers on a gel exclusion column. Binding of the cyclized oligomers was found to be far weaker than that of their linear counterparts of equal length. Such a general reduction in base-pairing capacity due to the cyclized conformation, by limiting the strength of unintended base-pairing without obstructing the possible development of strong specific base-pairing, may represent an advantage important to the function and evolution of loop structures in tRNA and other RNA molecules.     

Characterization of the bioactive form of linear peptide antagonists at the δ-opioid receptor

The stereochemical requirements for δ-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The δ-opioid receptor analogues with significant affinity, Tyr-Tic-NH₂ (TI-NH₂), Tyr-Tic-Phe-OH (TIP), Tyr-Tic-Phe-NH₂(TIP-NH₂), Tyr-Tic-Phe-Phe-OH (TIPP), Tyr-Tic-Phe-Phe-NH₂) (TIPP-NH₂), and the low affinity δ-opioid peptides Tyr-Pro-Phe-Pro-NH₂ (morphiceptin) and Tyr-Phe-Phe-Phe-NH₂ (TPPP-NH₂), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic simulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH₂ and TIPP-NH₂, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr¹-D -Pen²-Gly³-Phe⁴-D -Pen⁵) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high δ-opioid affinity L -Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr¹ and Tic² residues. Moreover, both the Tic² and Phe³ residues could mimic the hydrophobic interactions with the receptor of the Phe⁴ moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di-and tripeptides. The low δ-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition. © 1996 John Wiley & Sons, Inc.     

Structure-function studies of canine cardiac sarcolemmal membranes. I. Estimation of receptor site densities

A novel method for the estimation of receptor site densities in purified canine cardiac sarcolemmal vesicles is described. Canine sarcolemmal vesicles, purified by the method of Jones et al. (Jones, L.R., Maddock, S.W. and Besch, H.R. (1980) J. Biol. Chem. 255, 9971-9980) had high (Na+ + K+)-ATPase specific activity (127 +/- 1.9 mumol Pi/mg per h). Total phospholipid content, estimated by measurements of total phosphorus and total fatty acid contents, was 3.09 mumol/mg. Saturation isotherms for several receptor ligands gave the following values for Kd and Bmax: ouabain 32.6 +/- 2.7 nM, 365 +/- 59 pmol/mg; quinuclidinyl benzilate 0.055 +/- 0.010 nM, 5.8 +/- 0.7 pmol/mg; dihydroalprenolol 4.6 +/- 1.0 nM, 2.2 +/- 0.2 pmol/mg; and nitrendipine 0.21 +/- 0.04 nM, 0.93 +/- 1.04 pmol/mg. Membrane phospholipid surface area per ligand-binding sites was estimated from the Bmax values for each receptor ligand utilizing 3.09 mumol phospholipid/mg and 60 A2 as the average surface area occupied by each phospholipid molecule. The following receptor site densities per micrometer 2 phospholipid surface were obtained: ouabain, 400; quinuclidinyl benzilate, 6; dihydroalprenolol, 2; and nitrendipine, 1. As the surface area contributed by protein was estimated to be less than 20% of the lipid surface area, these values must be reduced by approx. 20% to estimate site densities per micrometer 2 membrane surface. These data demonstrate much lower beta-adrenergic and muscarinic receptor density compared to that of Na+ pump sites.     

Docking of linear peptide antagonists into the human V(1a) vasopressin receptor. Identification of binding domains by photoaffinity labeling.

A novel photoactivatable linear peptide antagonist selective for the V(1a) vasopressin receptor, [(125)I][Lys(3N(3) Phpa)(8)]HO-LVA, was synthesized, characterized, and used to photolabel the human receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85-90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [(125)I]3N(3)Phpa-LVA (Phalipou, S., Cotte, N. , Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem. 272, 26536-26544). To identify contact sites between the new photoreactive analogue and the V(1a) receptor, the labeled receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted receptor region spanning the first extracellular loop. The results established that sequence Asp(112)-Pro(120) could be considered as the smallest covalently labeled fragment with [(125)I][Lys(3N(3)Phpa)(8)]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [(125)I]3N(3)Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V(1a) receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.     

Structure-function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor protein.

Expression of the 37 kDa laminin receptor precursor protein (37LRP) correlates directly with increased invasiveness and the metastatic potential of tumors. The 37LRP matures to a 67 kDa protein which facilitates the binding of cancer cells to basement membranes. The palindrome peptide sequence LMWWML, corresponding to the 173-178-residue stretch of the human 37LRP sequence, has been identified as the laminin-1-binding site. Peptides from 37LRP of species that contain this palindrome-bind laminin-1 with high affinity. Nuclear magnetic resonance (NMR) conformational studies have been undertaken on a synthetic 15-residue peptide (KGAHSVGLMWWMLAR) containing the palindrome to establish the structural basis of this activity. To further correlate the structural data with laminin-1-binding function, analogous structural studies were conducted for a similar peptide (RGKHSIGLIWYLLAR) lacking the palindrome, originating from 37LRP sequence of Saccharomyces cerevisiae and exhibiting low laminin-1-binding affinity. Finally, in vitro cell invasion assays were performed to investigate the possibility that the laminin-1-binding affinity of the peptides influences their inhibitory activity.     

Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin.

Indolicidin is a cationic 13 amino acid peptide amide produced in the granules of bovine neutrophils with the sequence H-ILPWKWPWWPWRR-NH2. Indolicidin is both antimicrobial and, to a lesser extent, haemolytic. In order to systematically investigate structure-function relationships, the solid-phase synthesis of indolicidin and 48 distinct analogues are reported, as well as the characterization of their respective biological properties. Peptides synthesized and characterized include analogues with modified terminal functions, truncations from either terminus, an alanine scan to determine the role of each individual amino acid, specific amino acid exchanges of aromatic, charged and structural residues and several retro-, inverso- and retroinverso-analogues. Together, characterization of these analogues identifies specific residues involved in antimicrobial or haemolytic activity and suggests a core structure that may form a scaffold for the further development of peptidomimetic analogues of indolicidin.     

Structure-function relationships of hirulog peptide interactions with thrombin.

Using hirudin as a model, a novel class of bivalent thrombin inhibitors has been designed and characterized (Maraganore et al. (1990) Biochemistry 29, 7095-7101). These peptides, designated 'hirulogs', interact with both thrombin's catalytic center and its anion-binding exosite for fibrinogen recognition. In order to investigate structure-activity relationships in hirulog peptides, a number of peptide and peptidomimetic derivatives with alterations in catalytic-site binding and anion-binding exosite binding moieties were prepared. Replacements or modifications in the catalytic site and exosite binding moieties were achieved with the consequences of maintaining or improving antithrombin activity. In addition to showing improved affinity for thrombin, some derivatives with Ki's in the sub-nanomolar range showed increased anticoagulant activities. These findings highlight the versatility of hirulog peptides in their bivalent interactions with thrombin.