Synthesis of neurotensin(9-13) analogues exhibiting enhanced human neurotensin receptor binding affinities

Recent evidence is consistent with neurotensin (NT)(8-13) adopting a Type I beta-turn conformation while binding the NT receptor, which would place the cationic side-chains of Arg(8) and Arg(9) in close proximity. This was the basis for the design, synthesis and analysis of truncated NT(9-13) analogues 1-5 with dicationic position 9 side-chains to emulate the functions of the 8 and 9 side-chains of NT(8-13).     

Preparation and receptor binding affinities of cyclic C-terminal neurotensin (8-13) and (9-13) analogues.

Cyclic analogues of neurotensin (NT) C-terminal fragments NT(8-13) and NT(9-13) were produced via intramolecular nucleophilic substitution of the Tyr(11) phenoxide anion on a 6-bromohexanoyl side chain substituted at position 8 or 9 and tested for NT receptor binding affinity.     

Synthesis and biological studies of novel neurotensin(8-13) mimetics

Novel neurotensin (NT) (8–13) (Arg⁸-Arg⁹-Pro¹⁰-Tyr¹¹-Ile¹²-Leu¹³) mimetics 3, 4 were designed by adopting all intrinsic functional groups of the native neurotensin(8–13) and using a substituted indole as a template to mimic the pharmacophore of NT(8–13). Biological studies at subtype 1 of the NT receptor showed that 3 has a 55 and 580 nM binding affinity at rat and human neurotensin receptors, respectively. As a comparison, compounds 5 and 6 were also synthesized. The binding difference between 3, 4 and 5, 6 argues the importance of the carboxylic group in achieving higher potency NT(8–13) mimetics.     

Verification of both the sequence and conformational specificity of neurotensin in binding to mast cells

Neurotensin (NT) analogs, modified at Arg⁸ and Arg⁹, were used to assess the role of Arg in NT binding to mast cells. [D-Arg⁸]- and [D-Arg⁹]-NT bound 4–5 times better than NT, whereas [D-Arg^8,9]-NT had the same binding affinity as NT. Binding of [Ala⁸]-NT was not parallel to NT and exhibited a dissociation constant 38-fold lower than NT while [Ala⁹]-NT had 32% binding. C-terminal peptides, NT_8–13 and NT_9–13, had about 65% binding. These data suggest that Arg⁸ plays a greater role than Arg⁹ in the binding to mast cell NT receptors. Reduction of the disulfide bond in [Cys^2,13]-NT produced an analog 4-times more potent than NT, while the cyclized form had only 3% binding. Thus, a linear peptide with a free C-terminus appears to be required for binding.     

Partial alanine scan of mast cell degranulating peptide (MCD): importance of the histidine- and arginine residues.

The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcepsilonRIalpha binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His13 by Ala o ccurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg7 and His8 resulted in an approximately 40 fold increase, and for Arg16 in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcepsilonRIalpha binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala8 (for His) and Ala16 (for Arg) showed the same binding affinities as MCD, whereas analog Ala7 (for Arg) and analog Ala13 (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.     

Glucagon antagonists: contribution to binding and activity of the amino-terminal sequence 1-5, position 12, and the putative alpha-helical segment 19-27

Glucagon binding to and recognition by its cell surface receptor is the necessary first step in the cascade of events leading to the activation of adenylate cyclase by the hormone. It has long been presumed that glucagon adopts an ordered conformation upon binding to its membrane-bound receptor. A recent model of this three-dimensional structure based on biophysical data, predicts beta-turns at positions 2-5, 10-13, and 15-18, and an alpha-helical region between residues 19-27. Our approach in the design of antagonists of glucagon was to elucidate the steric and electronic features that stabilize these secondary structures to obtain analogs that bind with high affinity to the receptor but do not activate adenylate cyclase. Nineteen glucagon analogs incorporating structural changes at the amino-terminal sequence 1-5, at positions 9 and 12, and at the carboxyl-terminal helical region were synthesized. Des-His1-[Glu9]glucagon amide was recently shown to be a competitive inhibitor. Our synthetic studies in combination with this modification have resulted in seven new glucagon antagonists. The implications for the structural and conformational properties required for binding and activity of glucagon and the glucagon peptide family are discussed.     

Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs

Neurotensin(8-13) is a hexapeptide with subnanomolar affinity to the neurotensin receptor 1 which is expressed with high incidence in several human tumor entities. Thus, radiolabeled neurotensin(8-13) might be used for tumor targeting. However, its application is limited by insufficient metabolic stability. The present study aims at improving metabolic stability by the synthesis of multimeric neurotensin(8-13) derivatives rather than commonly employed chemical modifications of the peptide itself. Thus, different dimeric and tetrameric peptides carrying C- or N-terminal attached neurotensin(8-13) moieties have been synthesized and their binding affinity toward the neurotensin receptor has been determined. The results demonstrate that branched compounds containing neurotensin(8-13) attached via its C-terminus only show low receptor affinities, whilst derivatives with neurotensin(8-13) attached via the N-terminus show IC50 values in the nanomolar range. Moreover, within the multimeric neurotensin(8-13) derivatives with neurotensin(8-13) attached via the N-terminus an increasing number of branching units lead to higher binding affinities toward the neurotensin receptor.     

Synthesis and biological activity of dynorphin-(1-13) and analogs substituted in positions 8 and 10

Dynorphin-(1-13) (Dyn-(1-13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid-phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [3H]-ethylketocyclazocine (EKC, kappa ligand), [3H]-[D-Ala2, MePhe4-Gly-ol5]-enkephalin (DAGO, mu ligand) and [3H]-[D-Ser2, Thr6]-Leu-enkephalin (DSLET, delta ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22-fold increase in its affinity for the kappa receptor ([3H]-EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn-(1-13)). On the other hand, the ability of [Ala8] Dyn-(1-13) to displace the binding of [3H]-DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the mu receptor was not greatly affected ([3H]-DAGO displacement; Ki of 0.44 nM as compared with 0.50 nM for Dyn-(1-13)). Replacement of position 8 by D-Ala caused similar changes in the activity of the peptide but the increase in its affinity for the kappa site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D-Pro10]-Dyn-(1-13) was equipotent to [Ala8]-Dyn-(1-13) in the GPI but its affinity for the mu binding site was decreased by a factor of 2.7 as compared with Dyn-(1-13).(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the binding environment of alpha-factor in its G protein-coupled receptor using fluorescence spectroscopy.

Mating in Saccharomyces cerevisiae is induced by the interaction of alpha-factor (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) with its cognate G protein-coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of alpha-factor in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group was placed at the alphaN-terminus and in side-chains at positions 1, 3, 4, 6, 7, 12 and 13 were synthesized and assayed for biological activity and receptor affinity. Eleven of the analogs retained 6-60% of the biological activity of the alpha-factor, as judged using a growth arrest assay. The binding affinities depended on the position of NBD attachment in the peptide and the distance of the tag from the backbone. Derivatization of the positions 3 and 7 side-chains with the NBD group resulted in analogs with affinities of 17-35% compared with that of alpha-factor. None of the other NBD-containing agonists had sufficient receptor affinity or strong enough emission for fluorescence analysis. The position 3 and 7 analogs were investigated using fluorescence spectroscopy and collisional quenching by KI in the presence of Ste2p in yeast membranes. The results showed that the lambda max of NBD in the position 7 side-chain shifted markedly to the blue (510 nm) when separated by 4 or 6 bonds from the peptide backbone and that this probe was shielded from quenching by KI. In contrast, separation by 3, 5, 10 or more bonds resulted in lambda max ( approximately 540 nm) and collisional quenching constants consistent with increasing degrees of exposure. The NBD group in the position 3 side-chain was also found to be blue shifted (lambda max=520 nm) and shielded from solvent. These results indicate that the position 7 side-chain is likely interacting with a pocket formed by extracellular domains of Ste2p, whereas the side-chain of Trp3 is in a hydrophobic pocket possibly within the transmembrane region of the receptor.     

Histamine release by neurotensin in the rat hindquarter: structure-activity studies

Histamine releasing effects of neurotensin (NT) and several NT fragments and structural analogues were measured in the rat perfused hindquarter. The results show that the chemical groups responsible for histamine release are located in the C-terminal sequence Arg9-Pro10-Tyr11-Ile12-Leu13-OH. Both the spatial configuration and positive charge of Arg8 and Arg9 appear to contribute to the histamine releasing effect of NT. Optimization of the histamine releasing effect of NT requires both a free C-terminal carboxyl group and the presence in position 11 of NT of an aromatic residue, with the L-configuration, bearing an heteroatom capable of hydrogen bonding with the receptor. The results indicate that the structural requirements of NT to induce histamine release from the rat perfused hindquarter are similar to those involved in other peripheral biological actions of NT.     

Orientation of d-tubocurarine in the muscle nicotinic acetylcholine receptor-binding site

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist, d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR gamma-subunit. Because data for mutations at residue gammaTyr(117) were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of gammaTyr(117) with both the 2-N and the 13'-positions of dTC. Examination of a dTC analog with a negative charge at the 13'-position failed to reveal electrostatic interaction with charged side-chain substitutions at gamma117, but the effects of side-chain substitutions remained consistent with proximity of Tyr(117) to the cationic 2-N of dTC. The apparent interaction of gammaTyr(117) with the 13'-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-pi electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to gammaTyr(117). This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR.     

Synergistic effect of basic residues at positions 14-15 of nociceptin on binding affinity and receptor activation

Nociceptin is an endogenous ligand that activates a G protein-coupled receptor ORL1 and contains two indispensable Arg-Lys (RK) dipeptide units at positions 8-9 and 12-13. By replacing an additional RK unit at positions 6-7, 10-11, 14-15, or 16-17, of the peptide we have identified the analog, [RK(14-15)]nociceptin as a superagonist. In fact, this peptide exhibits 3-fold higher binding affinity and 17-fold greater potency in a functional GTPgammaS-binding assay compared to wild-type nociceptin. Here, we have further investigated the role of basic residues in position 14-15. The replacement of three other possible basic dipeptides, KR, RR, and KK, into nociceptin at positions 14-15 resulted in similar enhancements of binding affinity (3-5-fold) and biological potency (10-12-fold in the GTPgammaS assay). However, when only a single basic residue (Arg or Lys) was replaced in either position 14 or 15, all the resulting analogs showed moderate enhancements of binding and biological activity (2-4-fold in both). These results indicate that the addition of basic charges in positions 14 and 15 enhance in a synergistic fashion the interaction of nociceptin with the receptor and only the simultaneous presence of two adjacent basic residues yields an optimal effect. This suggests that specific electrostatic interactions between both amino acids present in 14-15 and corresponding residues in the receptor are responsible for the enhancement of nociceptin activity.     

Receptor fragment approach to the binding between CCK8 peptide and cholecystokinin receptors: a fluorescence study on type B receptor fragment CCK(B)-R (352-379)

Fluorescence titrations in a membrane mimetic solvent system allowed us to estimate that the dissociation constant of the bimolecular complex between CCK8 peptide and cholecystokinin type B receptor fragment CCK(B)-R (352-379) is in the micromolar range. When considered in the context of the full receptor/ligand model, these experiments demonstrate that the receptor fragment chosen on the basis of previous structural studies represents a reliable model system to monitor the ability of CCK8 or CCK8 analogs to bind the cholecystokinin receptor. Together with previous studies, this confirms that the receptor fragment approach adopted to define the binding mode of the CCK8 fragment of cholecystokinin with its two receptors, CCK(A) and CCK(B,) can be used to characterize the binding from the equilibrium standpoint. In this context, fluorescence spectroscopy proves to be the favored technique to measure dissociation constants in the nanomolar to micromolar range.     

Mimicking the membrane-mediated conformation of dynorphin A-(1-13)-peptide: circular dichroism and nuclear magnetic resonance studies in methanolic solution.

The structural requirements for the binding of dynorphin to the kappa-opioid receptor are of profound clinical interest in the search for a powerful nonaddictive analgesic. These requirements are thought to be met by the membrane-mediated conformation of the opioid peptide dynorphin A-(1-13)-peptide, Tyr1-Gly2-Gly3-Phe4-Leu5-Arg6-Arg7-Ile8-Arg9-Pro10- Lys11-Leu12-Lys13. Schwyzer has proposed an essentially alpha-helical membrane-mediated conformation of the 13 amino acid peptide [Schwyzer, R. (1986) Biochemistry 25, 4281-4286]. In the present study, circular dichroism (CD) studies on dynorphin A-(1-13)-peptide bound to an anionic phospholipid signified negligible helical content of the peptide. CD studies also demonstrated that the aqueous-membraneous interphase may be mimicked by methanol. The 500- and 620-MHz 1H nuclear magnetic resonance (NMR) spectra of dynorphin A-(1-13)-peptide in methanolic solution were sequence-specifically assigned with the aid of correlated spectroscopy (COSY), double-quantum filtered phase-sensitive COSY (DQF-COSY), relayed COSY (RELAY), and nuclear Overhauser enhancement spectroscopy (NOESY). 2-D CAMELSPIN/ROESY experiments indicated that at least the part of the molecule from Arg7 to Arg9 was in an extended or beta-strand conformation, which agreed with deuterium-exchange and temperature-dependence studies of the amide protons and analysis of the vicinal spin-spin coupling constants 3JHN alpha. The results clearly demonstrated the absence of extensive alpha-helix formation. chi 1 rotamer analysis of the 3J alpha beta demonstrated no preferred side-chain conformations.     

Importance of the C-terminal phenylalanine of gastrin for binding to the human CCK(2) receptor.

The importance of the C-terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK2 receptor were assessed using analogs of [Leu15]G(11-17). The following peptides were synthesized, Ac[Leu15]G(11-17), Ac[Leu15]G(11-16)NH2, [Leu15]G(11-17), [Leu15,Ala17]G(11-17), [Leu15,Abu17]G(11-17), [Leu15,Val17]G(11-17), [Leu15,Leu17]G(11-17), [Leu15,Cha17]G(11-17), [Leu15,Trp17]G(11-17), [Leu15,Tic17]G(11-17), [Leu15, d-Phe17]G(11-17) and [Leu15,p-X-Phe17]G(11-17), where X = F, Cl, Br, I, OH, CH3, NH2 and NO2. Competition binding experiments with [3H]CCK-8 were performed using human CCK2 receptors stably expressed in CHO cells. Phe17 was shown to be important for binding. A hydrophobic side-chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side-chain either in the g+ or g- conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d-Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des-Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK2 receptor.     

The C region of human insulin-like growth factor (IGF) I is required for high affinity binding to the type 1 IGF receptor

We have produced and characterized the binding properties of three structural analogs of human insulin-like growth factor I (hIGF-I). These analogs are [1-62]hIGF-I, an analog lacking the carboxyl-terminal 8-amino acid D region of hIGF-I; [1-27, Gly4, 38-70]hIGF-I, an analog in which residues 28-37 of the C region of hIGF-I are replaced by a 4-reside glycine bridge; and [1-27,Gly4,38-62]hIGF-I, an analog with the C region glycine replacement and a D region deletion. The removal of the D region of hIGF-I has little effect on binding to the type 1 and type 2 insulin-like growth factor (IGF) receptors. [1-62]hIGF-I has 2-fold higher affinity for the insulin receptor and 4-fold higher affinity for IGF serum-binding proteins. The replacement of the C region of hIGF-I with a four-glycine span results in a 30-fold loss of affinity for the type 1 IGF receptor. However this analog has near normal affinity for the type 2 IGF receptor, the insulin receptor, and IGF serum-binding proteins. Incorporating the C region glycine replacement and the D region deletion into one analog does not affect binding to either the type 2 receptor or to IGF serum-binding proteins. As predicted from the single deletion analogs [1-27,Gly4,38-62]hIGF-I has reduced affinity for the type 1 IGF receptor (approximately 40-fold) and increased affinity for the insulin receptor (5-fold). These data indicate that determinants in the C region of hIGF-I are involved in maintaining high affinity binding to the type 1 IGF receptor and that neither the C region nor the D region are required for high affinity binding to the type 2 IGF receptor or to IGF serum-binding proteins.     

Purin-8-ones as corticotropin-releasing hormone (CRH-R1) receptor antagonists

A series of purin-8-ones was prepared and discovered to have excellent binding affinity to the CRH-R1 receptor. Structure-activity studies focused on amine side-chain optimization, urea substitution and pyridyl isostere incorporation. Thus, the highly potent purin-8-ones show promise as a new class of potential anxiolytics and/or antidepressants.     

Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus)

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.     

Inactivation of Neurotensin by Rat Brain Synaptic Membranes Partly Occurs Through Cleavage at the Arg8-Arg9 Peptide Bond by a Metalloendopeptidase

One of the primary inactivating cleavages of neurotensin (NT) by rat brain synaptic membranes occurs at the Arg8-Arg9 peptide bond, leading to the formation of NT1-8 and NT9-13. The involvement at this site of a recently purified metalloendopeptidase was demonstrated by the use of its specific inhibitor, N-[1(R,S)-carboxy-2-phenylethyl]-alanylalanylphenylalanine-p-amino -benzoate, which exerts an inhibition on NT1-8 formation with an IC50 (0.6 microM) close to its Ki for the purified metalloendopeptidase (1.94 microM). Furthermore, we established the role of a postproline dipeptidyl-aminopeptidase in the secondary processing of NT9-13 formation.     

Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic alpha-helix

Salmon calcitonin (sCT) forms an amphipathic helix in the region 9-19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161-174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu(23),Ala(24)-sCT in which Pro(23) and Arg(24) were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic alpha-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9-19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.