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The binding affinity of short chain somatostatin (SRIF) analogues at the five human SRIF receptors (sst) was determined to identify sterically constrained somatostatin receptor subtype 1 (sst(1)) selective scaffolds. Des-AA(1,2,4,13)-[d-Trp(8)]SRIF (2) retained high binding affinity at all receptors but sst(1), Des-AA(1,2,4,5)-[d-Trp(8)]SRIF (3) at sst(4) and sst(5), and Des-AA(1,2,4,5,13)-[d-Trp(8)]SRIF (4) at sst(2) and sst(4) (AA = amino acid). Des-AA(1,2,4,12,13)-[d-Trp(8)]SRIF (6) was potent and sst(4)-selective (>25-fold); Des-AA(1,2,5,12,13)-[d-Trp(8)]SRIF (7) and Des-AA(1,2,4,5,12,13)-[d-Trp(8)]-SRIF (9, ODT-8) were most potent at sst(4) and moderately potent at all other receptors. Dicyclic SRIF agonists of the sst(1)-selective Des-AA(1,5)-[Tyr(2),d-Trp(8),IAmp(9)]SRIF, (14, sst(1) IC(50) = 14 nM) were prepared in which a lactam bridge introduced additional conformational constraint (IAmp = 4-(N-isopropyl)-aminomethylphenylalanine). Cyclo(7-12)Des-AA(1,5)-[Tyr(2),Glu(7),d-Trp(8),IAmp(9),hhLys(12)]SRIF (31) (sst(1) IC(50) = 16 nM) and cyclo(7-12) Des-AA(1,2,5)-[Glu(7),d-Trp(8),IAmp(9),m-I-Tyr(11),hhLys(12)]SRIF (45) (sst(1) IC(50) = 6.1 nM) had equal or improved affinities over that of the parent 14. Binding affinity was decreased in all other cases with alternate bridging constraints such as cyclo (6-11), cyclo (6-12), and cyclo (7-11). Compound 45 is an agonist (EC(50) = 8.8 nM) in the adenylate cyclase assay.

We present evidence that members of the corticotropin releasing factor (CRF) family assume distinct structures when interacting with the CRF(1) and CRF(2) receptors. Predictive methods, physicochemical measurements, and structure-activity relationship studies have suggested that CRF, its family members, and competitive antagonists such as astressin [cyclo(30-33)[DPhe(12),Nle(21),Glu(30),Lys(33),Nle(38)]hCRF((12-41))] assume an alpha-helical conformation when interacting with their receptors. We had shown that alpha-helical CRF((9-41)) and sauvagine showed some selectivity for CRF receptors other than that responsible for ACTH secretion(1) and later for CRF2.(2) More recently, we suggested the possibility of a helix-turn-helix motif around a turn encompassing residues 30-33(3) that would confer high affinity for both CRF(1) and CRF(2)(2,4) in agonists and antagonists of all members of the CRF family.(3) On the other hand, the substitutions that conferred ca. 100-fold CRF(2) selectivity to the antagonist antisauvagine-30 [[DPhe(11),His(12)]sauvagine((11-40))] did not confer such property to the corresponding N-terminally extended agonists. We find here that a Glu(32)-Lys(35) side chain to side chain covalent lactam constraint in hCRF and the corresponding Glu(31)-Lys(34) side chain to side chain covalent lactam constraint in sauvagine yield potent ligands that are selective for CRF(2). Additionally, we introduced deletions and substitutions known to increase duration of action to yield antagonists such as cyclo(31-34)[DPhe(11),His(12),C(alpha)MeLeu(13,39),Nle(17),Glu(31),Lys(34)]Ac-sauvagine((8-40)) (astressin(2)-B) with CRF(2) selectivities greater than 100-fold. CRF receptor autoradiography was performed in rat tissue known to express CRF(2) and CRF(1) in order to confirm that astressin(2)-B could indeed bind to established CRF(2) but not CRF(1) receptor-expressing tissues. Extended duration of action of astressin(2)-B vs that of antisauvagine-30 is demonstrated in the CRF(2)-mediated animal model whereby the inhibition of gastric emptying of a solid meal in mice by urocortin administered intraperitoneally at time zero is antagonized by the administration of astressin(2)-B but not by antisauvagine-30 at times -3 and -6 h while both peptides are effective when given 10 min before urocortin.

We report the electrospray mass spectra and tandem mass (MS/MS) spectra of a series of chemically-stable somatostatin (SRIF) and gonadotropin-releasing hormone (GnRH) analogs which incorporate acylated or alkylated/acylated aminoglycine residues (betidamino acids) into their structure . In the case of SRIF, we compare the betidamino acid-containing analogs with the equivalent peptide without the betide-type linkage. We observe significant differences in the fragmentation patterns of each class of compound. We extend this study by comparing a variety of different betidamino acid-containing GnRH analogs to investigate the effects of different acyl groups, chirality and methylation of the side-chain nitrogen of amino glycine on the gas-phase stability of these protonated peptide molecules.

In three earlier papers, the structures and biological potencies of numerous mono- and dicyclic antagonists of GnRH were reported. Among these, two families, each containing two to four members were identified that had very high antagonist potencies in an antiovulatory assay (within a factor of 2 of those of the most potent linear analogues) and high affinities (K(i) 20-fold) was observed with the substitution of [DAsp(3)] in 14 by [DGlu(3)] in 15 (K(i) = 23 nM). Dicyclic analogues possessing the (4-10) cycle and selected (1-6), (2-6), and (2-8) cycles led to analogues that were inactive at doses of 500 microgram/rat or larger. Two analogues with (1-8/4-10) cycles (16, K(i) = 1.1 nM) or (3-8/4-10) cycles (22, K(i) = 17 nM) showed full antiovulatory potency at 250 microgram/rat. None of these substitutions yielded analogues potent enough (>80% inhibition of ovulation at 5 microgram/rat or less and K(i) < 0.5 nM) to be candidates for structural analysis by NMR. On the other hand, four dicyclic (1, 1'-5/4-10) analogues met this criterion: dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DTrp(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (32, K(i) = 0.22 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DNal(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (34, K(i) = 0.38 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(betaAla),DCpa(2), DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (40, K(i) = 0.15 nM), and dicyclo(1, 1'-5/4-10)[Ac-Glu(1)(Gly), DCpa(2),DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (41, K(i) = 0.24 nM). Since they differed slightly in terms of the (1,1'-5) bridge length (21 and 22 atoms) and bridgehead configuration, we may hypothesize that they assume similar bioactive conformations that satisfy a very discriminating receptor, since many other closely related analogues were significantly less potent.

In an earlier report we identified specific modifications and substitutions of corticotropin releasing factor (CRF) that led to the discovery of antagonists with extended duration of action as compared to that of astressin {cyclo(30-33)[DPhe(12),Nle(21),Glu(30), Lys(33),Nle(38)]hCRF((12)(-)(41))}. These additional modifications included elongation of the peptide chain by three residues at the N-terminus, its acetylation, and the [CalphaMeLeu(27)] substitution to yield cyclo(30-33)[DPhe(12), Nle(21),CalphaMeLeu(27),Glu(30), Lys(33),Nle(38)]Ac-hCRF((9)(-)(41)), which was found to be longer acting than astressin (Rivier, J.; et al. J. Med. Chem. 1998, 41, 5012-5019). To further increase the efficiency (potency, duration of action, and bioavailability) of this family of antagonists, we introduced two or more CalphaMe-leucine residues at positions shown in earlier studies to be favorable (Hernandez, J.-F.; et al. J. Med. Chem. 1993, 36, 2860-2867). Whereas the introduction of CalphaMe-leucine residues at positions 27 and either 18 (11), 37 (17), or 40 (19) resulted in dramatic increases in duration of inhibitory action in the adrenalectomized (adx) rat after intravenous injection, the same substitution at positions 27 and either 15 (7, 8), 17 (9), 19 (12, 13), or 41 (20) led to short acting analogues. Other substitutions by CalphaMeLeu at positions 27 and either 10 (4), 13 (5), 14 (6), 21 (14), 24 (15), 36 (16), or 38 (18) yielded analogues with duration of action intermediate between those mentioned above. Cyclo(30-33)[DPhe(12), Nle(21), CalphaMeLeu(27),Glu(30),Lys(33),Nle(38), CalphaMeLeu(40)]Ac-hCRF((9)(-)(41)) (astressin B, 19) was one of the most efficacious analogues of this series (4 h inhibition of ACTH secretion at 25 microgram/adx rat). It was found to be even longer acting via subcutaneous administration in either an aqueous (24 h inhibition of ACTH secretion at 100 microgram/adx rat) or lipid milieu (DMSO/peanut oil,24 h inhibition of ACTH secretion at 30 microgram/adx rat) than after intravenous administration (12 h inhibition of ACTH secretion at 100 microgram/adx rat). We concluded that Calpha-methylation at some positions may favor a bioactive conformation while also preventing degradation and/or elimination, resulting in significant extension of duration of action.

This investigation examined receptor subtype specificity and possible modulation by GABAa receptor ligands of NPY-induced behavioral responses to stressful stimuli. First, a series of NPY receptor agonists were examined for their potential effects on punished responding in a conflict test modified for incremental shock. NPY, peptide YY (PYY) and NPY Y1 receptor agonists [Leu31,Pro34]-NPY and [Gly6, Glu26,Lys26,Pro34]-NPY produced increases in punished responding in the conflict test. No significant effects on unpunished responding were noted. The pattern of responding was similar to that observed with the benzodiazepine agonist chlordiazepoxide. Neither pancreatic peptide (PP) nor the Y2 agonists NPY13-36 or [Glu2,32,Ala6,Dpr27,Lys28]-NPY significantly altered punished or unpunished responding. Of significance, the atypical Y1 agonist [Cys7,21,Pro34]-NPY produced negligible effects on punished responding, consistent with the presence of a subclass of Y1 receptors. Second, the anxiolytic effects of NPY were subjected to treatments that block actions at the GABAa receptor complex. The increase in punished responding produced by NPY was not altered by administration of the benzodiazepine antagonist flumazenil and only partially blocked by the picrotoxinin receptor ligand isopropylbicyclophosphate (10 and 15 microg/kg). These findings further support the hypothesis that the pharmacologic substrates for the anxiolytic-like actions of NPY may be mediated by the Y1 receptor subtype and suggest that these actions are independent of either the benzodiazepine or picrotoxinin binding sites of the GABA/benzodiazepine receptor complex.

Separation of analogues of neuropeptide Y (NPY) in which a single d -amino acid replaced the corresponding naturally occurring residue was performed by chromatographic techniques to ensure the quality of the synthetic peptides to be used for structural and biological studies. Of the 35 compounds, 28 were easily separated (α = 1.02–2.76) from native NPY by standard reversed-phase high-performance chromatography (RP-HPLC) methods using a Vydac C18 column and a gradient buffer system developed in our laboratory comprised of triethylammonium phosphate (TEAP) at pH 2.25 and acetonitrile at 40°C. The identical diastereomers could be separated on the same solid support and by using 0.1% trifluoroacetic acid (TFA) as the mobile phase modifier, however separation factors were smaller and retention times were longer. Three of the remaining seven unresolved analogues were separated (α = 1.02–1.96) by changing the solid-phase support to Vydac diphenyl derivatized silica and a buffer system consisting of 0.1% TFA and acetonitrile. Of the four remaining unresolved analogues, only two could be separated by capillary zone electrophoresis (CZE) in 0.1 M sodium phosphate at pH 2.5, but all four were finally resolved by changing the electrophoretic buffer to 0.1 M TEAP buffer at pH 2.5. Migration times of the diastereomers differed by 0.2–2.0 min from that of the natural NPY. In addition to confirming the uniqueness of each isomer, this investigation demonstrated the expansive utility and high efficiency of the TEAP buffer system for both RP-HPLC and CZE as well as the difference in selectivity produced by the TEAP and TFA buffers in RP-HPLC. The conditions described here have broad applications for the analysis and preparative separation of synthetic and native peptides.

To further elucidate the minimum bioactive conformation of neuropeptide Y (NPY), a series of truncated and conformationally constrained analogues has been prepared. The synthesis and purification of these peptides was achieved using routine laboratory strategies and techniques. Parent molecules consisted of the native NPY N-terminal 1-4 and C-terminal 25-36 segments, having the residue 5-24 core replaced by either a single flexible omega-aminoalkanoic acid, or a more rigid Pro-Gly or Pro-DAla sequence which was expected to constrain a putative turn, and allow the N- and C-termini to align. Cross-linking between residues 2 and 27 through lactamization using side-chain length and chirality suggested by computer simulations, resulted in cyclo-(2/27)-des-AA7-24[Glu2,Gly6,DDpr27]NPY that exhibited very high affinity (Ki = 0.3 versus 0.3 nM for NPY) for the Y2 receptor using SK-N-BE2 human neuroblastoma cells, yet very low affinity for the Y1 receptor using SK-N-MC human neuroblastoma cells (Ki = 130 versus 2.0 nM for NPY). The added constraint resulting from bridging in this analogue as well as in others suggested that the combination of the deletion of residues 5-24 and the introduction of an internal ring produced exclusive selectivity for the Y2 receptor with little or no loss of affinity. The tolerance of structural recognition was further demonstrated as a second ring was introduced which was expected to constrain the amphiphilic alpha-helix, resulting in the full Y2 agonist dicyclo (2/27,28/32)-des-AA7-24 [Glu2,32,DAla6,DDpr27,Lys28]NPY. Improvement of Y1 binding activity was achieved only by including more residues (des-AA10-17) in the central PP-fold region, while allowing limited flexibility of the termini. Although the length of the bridge seemed to have little effect on binding potency, changes in the location of and chirality at the bridgehead resulted in analogues with different binding affinities. Combination of optimum structural modifications resulted in cyclo-(7/21)-des-AA10-18[Cys7,21]NPY, an analogue shortened by 25% but retaining comparable binding properties to that of native NPY at Y1 and Y2 receptor types (Ki = 5.1 and 1.3 nM, respectively).

It has been shown that NPY and select C-terminal fragments of NPY that evoke a hypotensive response upon intraarterial administration in the rat also cause mast cell degranulation and histamine release in vitro. Additionally, elevation of plasma histamine levels has been observed concomitant with the hypotensive effect of NPY and various C-terminal fragments. In order to investigate whether the hypotensive response to NPY18-36 is correlated to this observed elevation of histamine in vivo, we sought to characterize the structural requirements for each activity. We conducted a systematic replacement of each amino acid in NPY18-36 by its D-isomer. Additionally, various modifications were made to the N- or C-terminii of NPY18-36. The following rank order of potency was obtained for the hypotensive action of these analogues of NPY18-36 relative to NPY18-36. Only one analogue ([D-Tyr21]NPY18-36) exhibited significantly enhanced potency. Eleven analogues of NPY18-36, ([D-Thr32]-, [D-Arg35]-, [D-Ile31]-, [D-Leu30]-, [D-Tyr27]-, [D-Ser22]-, [D-Tyr36]-, [D-Gln34]-, [D-Asn29]-, [D-Ala23]-, and [D-Arg33]NPY18-36) were equipotent with NPY18-36. Four analogues ([D-His26]-, [D-Ile28]-, and [D-Ala18]NPY18-36 and -NPY18-27) had reduced potency (10-80%) while eight analogues ([D-Arg19]-, [D-Tyr20], [D-Leu24]-, [D-Arg25]-, [Ac-Ala18]-, [Me-Ala18]-, [desamino-Ala18]NPY18-36 and NPY18-36 free acid) failed to produce a significant hypotensive response (less than 10%) at the doses tested. The sensitivity of NPY18-36 to chiral inversion of single residues or other modifications at the N-terminus suggested the presence of a conformationally well defined N-terminal pharmacophore. Additionally, five NPY18-36 analogues were tested for elevation of plasma histamine levels. The rank order of potency ([D-Thr32]NPY18-36 = [D-Tyr21]NPY18-36 much greater than NPY18-36 greater than [D-Ala18]NPY18-36 greater than [Ac-Ala18]NPY18-36) was correlated with each analogue's potency at evoking a hypotensive response. In contrast, NPY1-36 failed to evoke an elevation in plasma histamine levels despite its hypotensive effects. Hence, we conclude that the magnitude of the hypotensive response evoked by an NPY18-36 analogue is primarily a function of its ability to elevate plasma histamine levels. However, the mechanism underlying NPY1-36-evoked hypotension appears to be different.

The ability of MALDI-MS to analyze photolabile arylazido peptide derivatives was investigated. Peptides containing UV-labile p-azidobenzoyl groups were subjected to MALDI-MS analysis in a variety of matrices. As standard MALDI-MS employs a UV laser (337 nm), we investigated conditions that would allow detection of the intact molecule ions for these light-sensitive peptides. When using α-cyano-4-hydroxycinnamic acid (ACHC) or 2,5 dihydroxybenzoic acid (DHB) as the matrix, photoinduced degradation products were prevalent. In contrast, when employing the matrix sinapinic acid, the intact molecule ion corresponding with the azido peptide was the predominant signal. The protection of photolabile azido derivatives correlates with the UV absorbance properties of the matrix employed, i.e., sinapinic acid, which exhibits a strong absorbance near 337 nm, most efficiently protects the azido derivative from photodegradation.

In the pursuit of potent analogues of neuropeptide Y (NPY) that are selective for the Y 1 receptor subtype, two lactam bridge scans of a centrally truncated parent compound were synthesized. A single lactam bridge (γ-carboxyl of Glu to ∈-amino of Lys) extending from residues i to i + 3 or i to i + 4 of the proposed α-helical region (residues 25-31 of NPY) was introduced in desAA 7-24 [Gly 6 ]NPY. Cyclogues (contraction of cyclic analogues), which were approximately one-half the size of native NPY, were initially screened for binding affinity at two discrete NPY receptor types using human neuroblastoma cell membranes, SK-N-MC and SK-N-BE2. Exploitation of the subtle differences present on each receptor type allowed for the identification of cyclogues which bound specifically to Y 1 receptors with increased affinity when compared to the corresponding linear parent analogue, while one short Y 1 specific cyclogue, des-AA 2,3,5,7-24 cyclo-(26/29)[Gly 6 ,Glu 26 ,Lys 29 ,Pro 34 ]NPY, bound with K i = 16 nM. Other cyclogues showed distinct preference for Y 2 receptors and bound in the low-nanomolar range. Functionally, the compounds inhibited the norepinephrine-stimulated accumulation of cAMP indicating that all acted as agonists with varying potencies.

In an effort to gain insight into the bioactive conformation of neuropeptide Y upon interaction with its receptors, all single-point D-amino acid substituted NPY analogues were prepared, and their Y1 and Y2 receptor binding affinities were evaluated using the human neuroblastoma cell lines, SK-N-MC and SK-N-BE2, respectively. Solid-phase synthesis (Boc strategy) followed by preparative HPLC purification produced analogues of high purity that were characterized by RP-HPLC, AAA, LSIMS, CZE, and optical rotation. Of the 37 isomers (a naturally occurring glycine at position 9 was replaced by Ala and D-Ala), Y1 receptor binding was most perturbed by chiral inversion of residues at the C-terminus (residues 20, 27, 29-35, Ki > or = 300 nM). Substitutions at residues 2-5, 28, and 36 had Ki values ranging from 40 to 260 nM. Substitutions at all other positions yielded analogues with affinities ranging from 1.5 to 20 nM. Binding affinities to the Y2 class of receptors all measured in the low or sub-nanomolar concentrations, with the exception of C-terminally modified isomers (residues 30-35). Only [D-Arg33]- and [D-Gln34]NPY displayed no measurable binding affinity to Y2 receptors at the highest concentration tested (1000 nM). Representative analogues were selected on the basis of their binding affinities and position in the sequence for structural analysis using circular dichroism (CD) spectroscopy. Of the nine peptide evaluated ([D-Pro5]-, [Ala9]-, [D-Glu10]-, [D-Asp11]-, [D-Ala18]-, [D-Tyr20]-, [D-Tyr27]-, and [D-Arg33]NPY), only [D-Tyr27]NPY expressed a definitive correlation between loss of binding affinity and disruption of secondary structure by having the propensity to form beta-sheets at the expense of alpha-helical content. It was concluded that although the incorporation of a single D-amino acid within the sequence of NPY may confer a conformational perturbation, the receptor interaction was only affected when certain critical residues were modified, findings that provide a basis for the identification of the binding pharmacophore of NPY.

Publisher Summary This chapter discusses large-scale synthesis of gonadotropin-releasing hormone antagonists for clinical investigations. Once a new peptide has been discovered and its biological activities identified, a logical extension is to design analogs for the study and understanding of its structure–activity relationships and to identify molecules that are either more potent, longer acting, or competitive antagonists. Either an agonist or an antagonist of a given peptide may be examined as a potential candidate for the treatment of diseases arising from defects in the normal pathways that the native peptide governs. Such synthetic peptides and their analogs can be synthesized and purified in large-enough quantities for their successful and safe use in a clinical setting. The chapter discusses the development of two antagonists of gonadotropin-releasing hormone (GnRH): (1) Ac- d -2-Nal- d -Cpa- d -3-Pal-Ser-Arg- d -2-amino-5-oxo-5-(4-methoxyphenyl) pentanoic acid-Leu-Arg-Pro- d -Ala-NH 2 (Nal-Glu antagonist), which has already been tested extensively in humans; and (2) Ac- d -2-Nal- d -Cpa- d -3-Pal-Ser-Lys(atz)- d -Lys(atz)-Leu-ILys-Pro- d -Ala-NH 2 (Azaline), a member of the most recent generation of potent, water-soluble GnRH antagonists with little or no histamine-releasing activity.