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Degarelix is a potent very long-acting GnRH antagonist after subcutaneous administration. In this paper, we describe the synthesis of two analogs of degarelix incorporating racemic 3-(2-methoxy-5-pyridyl)-alanine (2-OMe-5Pal, 5) at position 3. The two diastereomers were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and the absolute stereochemistry at position 3 in the peptides was determined by enzymatic digestion with proteinase K. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analog 7 with D2-OMe-5Pal was potent in vitro (IC50 = 5.22 nm); however, analog 8 with L2-OMe-5Pal at position 3 in degarelix lost potency as an antagonist of the human GnRH receptor (IC50 = 36.95 nm). Both the analogs were found to be short-acting in vivo.

The three-dimensional NMR structures of six octapeptide agonist analogues of somatostatin (SRIF) in the free form are described. These analogues, with the basic sequence H-DPhe/Phe2-c[Cys3-Xxx7-DTrp8-Lys9-Thr10-Cys14]-Thr-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Ala/Aph, exhibit potent and highly selective binding to human SRIF type 2 (sst2) receptors. The backbone of these sst2-selective analogues have the usual type-II’ β-turn reported in the literature for sst2/3/5-subtype-selective analogues. Correlating biological results and NMR studies led to the identification of the side chains of DPhe2, DTrp8 and Lys9 as the necessary components of the sst2 pharmacophore. This is the first study to show that the aromatic ring at position 7 (Phe7) is not critical for sst2 binding and that it plays an important role in sst3 and sst5 binding. This pharmacophore is therefore different from that proposed by others for sst2/3/5 analogues.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.

Activins, like other members of the transforming growth factor-beta (TGF-beta) superfamily, initiate signaling by assembling a complex of two types of transmembrane serine/threonine receptor kinases classified as type II (ActRII or ActRIIB) and type I (ALK4). A kinase-deleted version of ALK4 can form an inactive complex with activin and ActRII/IIB and thereby acts in a dominant negative manner to block activin signaling. Using the complex structure of bone morphogenetic protein-2 bound to its type I receptor (ALK3) as a guide, we introduced extracellular domain mutations in the context of the truncated ALK4 (ALK4-trunc) construct and assessed the ability of the mutants to inhibit activin function. We have identified five hydrophobic amino acid residues on the ALK4 extracellular domain (Leu40, Ile70, Val73, Leu75, and Pro77) that, when mutated to alanine, have substantial effects on ALK4-trunc dominant negative activity. In addition, eleven mutants partially affected activin binding to ALK4. Together, these residues likely constitute the binding surface for activin on ALK4. Cross-linking studies measuring binding of 125I-activin-A to the ALK4-trunc mutants in the presence of ActRII implicated the same residues. Our results indicate that there is only a partial overlap of the binding sites on ALK4 and ALK3 for activin-A and bone morphogenetic protein-2, respectively. In addition three of the residues required for activin binding to ALK4 are conserved on the type I TGF-beta receptor ALK5, suggesting the corresponding region on ALK5 may be important for TGF-beta binding.

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.

Little is known of the conformation of peptide hormones as they interact with their receptors for a number of reasons: peptide hormones are notoriously flexible in solution, their receptors are particularly complex, and there is strong evidence that receptor-ligand interaction leading to activation is a dynamic process. Insights into the active conformation of the decapeptide gonadotropin releasing hormone (GnRH) have been obtained previously from the solution structures of four constrained GnRH antagonists ?cyclo(1-10)[Ac-Delta(3)-Pro(1),DCpa(2),DTrp(3,6),NMeLeu+ ++(7), betaAla(10)]GnRH (1), cyclo(4-10)[Ac-Delta(3)Pro(1),DFpa(2),DTrp(3), Asp(4),DNal(6),Dpr(10)]GnRH (2), dicyclo(4-10/5-8)[Ac-DNal(1), DCpa(2),DTrp(3),Asp(4),Glu(5),DArg(6),Lys(8),Dpr (10)]GnRH (3), and dicyclo(4-10/5-5'-8)[Ac-DNal(1),DCpa(2),DPal(3), Asp(4),Glu(5)(Gly), DArg(6),Dbu(8),Dpr(10)]GnRH (4)?. However, the precise location of the N-terminal tripeptide in the highly potent (K(i) < 0.4 nM) 2-4 remained unclear due to the lack of constraints in this region. The NMR structure of the newly discovered and potent (K(i) = 0.24 nM) dicyclo(1-1'-5/4-10)[Ac-Glu(1)(Gly),DCpa(2),DTrp(3),As p(4),Dbu(5), DNal(6),Dpr(10)]GnRH (5) now allows the definition of the conformation of this region. A combined computational analysis (consensus forcing) of compounds 2-5, designed to explore the common conformations available to them that are simultaneously consistent with the NMR data corresponding to each compound, leads to a consensus structural model for the GnRH pharmacophore. This model shares some common features with the structure of the nonpeptidic GnRH mimetic T-98475. In the course of that comparative study, two additional contact points to those proposed by the authors are identified, suggesting that this model has predictive value.

We hypothesized that covalent constraints such as side-chain to side-chain lactam rings would stabilize an alpha-helical conformation shown to be important for the recognition and binding of the human corticotropin-releasing factor (hCRF) C-terminal 33 residues to CRF receptors. These studies led to the discovery of cyclo(20-23)[DPhe12,Glu20,Lys23,Nle21,38]hCRF (12-41) and of astressin ¿cyclo(30-33)[DPhe12,Nle21,38,Glu30,Lys33]hCR F(12-41)¿, two potent CRF antagonists, and of cyclo(30-33)[Ac-Leu8,DPhe12,Nle21, Glu30,Lys33,Nle38]hCRF(8-41), the shortest sequence equipotent to CRF reported to date (Rivier et al. J. Med. Chem. 1998, 41, 2614-2620 and references therein). To test the hypothesis that the Glu20-Lys23 and Glu30-Lys33 lactam rings were favoring an alpha-helical conformation rather than a turn, we introduced a D-amino acid at positions 22, 31, and 32 in the respective rings. Whereas the introduction of a D-residue at position 31 was only marginally deleterious to potency (ca. 2-fold decrease in potency), introduction of a D-residue at position 22 and/or 32 was favorable (up to 2-fold increase in potency) in most of the cyclic hCRF, alpha-helical CRF, urotensin, and urocortin agonists and antagonists that were tested and was also favorable in linear agonists but not in linear antagonists; this suggested a unique and stabilizing role for the lactam ring. Introduction of a [DHis32] (6) or acetylation of the N-terminus (7) of astressin had a minor deleterious or a favorable influence, respectively, on duration of action. In the absence of structural data on these analogues, we conducted molecular modeling on an Ac-Ala13-NH2 scaffold in order to quantify the structural influence of specific L- and DAla6 and L- and DAla7 substitutions in [Glu5,Lys8]Ac-Ala13-NH2 in a standard alpha-helical configuration. Models of the general form [Glu5,LAla6 or DAla6,LAla7 or DAla7,Lys8]Ac-Ala13-NH2 were subjected to high-temperature molecular dynamics followed by annealing dynamics and minimization in a conformational search. A gentle restraint was applied to the 0-4, 1-5, and 8-12 O-H hydrogen bond donor-acceptor pairs to maintain alpha-helical features at the N- and C-termini. From these studies we derived a model in which the helical N- and C-termini of hCRF form a helix-turn-helix motif around a turn centered at residue 31. Such a turn brings Gln26 in close enough proximity to Lys36 to suggest introduction of a bridge between them. We synthesized dicyclo(26-36,30-33)[DPhe12,Nle21,Cys26,Glu30 ,Lys33,Cys36, Nle38]Ac-hCRF(9-41) which showed significant alpha-helical content using circular dichroism (CD) and had low, but measurable potency ¿0. 3% that of 6 or ca. 25% that of [DPhe12,Nle21,38]hCRF(12-41)¿. Since the 26-36 disulfide bridge is incompatible with a continuous alpha-helix, the postulate of a turn starting at residue 31 will need to be further documented.

To whom correspondence should be addressedWhile the clinical significance of gonadotrophin-releasing hormone (GnRH)agonists is well recognized, the potential use of GnRH antagonists in humansawaits the availability of potent analogues with no untoward side-effects. Wehave designed, synthesized and tested several hundred linear and cyclic analogues(agonists and antagonists) of GnRH in different rat models; some have highhistamine releasing activity and others have poor solubility in aqueous bufferswith a pH >6.0. Furthermore, we have identified analogues exhibiting short( 72 h) duration of action in the rat(50 |Xg s.c. dose/rat). We have concluded that the basis for such resistance todegradation and elimination must be specific. In order to gain further informationon the optimal nature and sterical requirements of side-chains, preliminaryexperiments were carried out using betidamino acids. Finally, mono- and dicyclicanalogues of GnRH with potencies comparable with that of the most potentlinear analogues were also obtained. Our approach to the development of suchanalogues included the use of nuclear magnetic resonance and computationaltechniques as well as that of state-of-the-art synthetic approaches. We intend touse the information derived from these structure/activity relationship studies todesign conformationally-similar peptido-mimetics.Key words: acyline/azaline B/betidamino acids/drug design/GnRH antagonists

Betidamino acids (a contraction of "beta" position and "amide") are N'-monoacylated (optionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'acyl/alkyl group may mimic naturally occurring amino acid side chains or introduce novel functionalities. Betidamino acids are most conveniently generated on solid supports used for the synthesis of peptides by selective acylation of one of the two amino functions of orthogonally protected aminoglycine(s) to generate the side chain either prior to or after the elongation of the main chain. We have used unresolved Nalpha-tert-butyloxycarbonyl-N'alpha-fluorenylmethoxycarbonyl++ + aminoglycine, and Nalpha-(Nalpha-methyl)-tert-butyloxycarbonyl-N'alpha-fluo renylmethoxycarbonyl aminoglycine as the templates for the introduction of betidamino acids in Acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(A c)-Leu-Ilys-Pro-DAla-NH2, where 2Nal is 2-naphthylalanine, 4Cpa is 4-chlorophenylalanine, 3Pal is 3-pyridylalanine, Aph is 4-aminophenylalanine, and Ilys is Nepsilon-isopropyllysine], a potent gonadotropin-releasing hormone antagonist, in order to test biocompatibility of these derivatives. Diasteremneric peptides could be separated in most cases by reverse-phase HPLC. Biological results indicated small differences in relative potencies (

Through design, synthesis, and biological testing of constrained gonadotropin releasing hormone (GnRH) antagonists, we are studying the structural requirements for biological activity. Here we describe the conformational analysis in solution of a highly potent, dicyclic GnRH antagonist, dicyclo(4-10/5,5‘-8)[Ac-d-2Nal1,d-pClPhe2,d-3Pal3,Asp4,Glu5(Gly),d-Arg6,Dbu8,Dpr10]GnRH (1), using NMR spectroscopy. The dicyclic part of this molecule adopts a preferred conformation containing a type II β turn around residues 5−6, nested with a type I‘ β turn around residues 6−7, and a type II β-turn-like structure involving residue 9 and the side chain of residue 10, which is stabilized by hydrogen bonds between Leu7 NH/Asp4 CO, Dbu8 NHδ/Glu5 CO, and Dpr10 NHγ/Dbu8 CO. This is a novel conformation that had not been observed previously in any constrained GnRH antagonist and is remarkably different from that found for another dicyclic (4-10/5-8) GnRH antagonist with very similar sequence, dicyclo(4-10/5-8)[Ac-d-2Nal1,d-pC...

Predictive methods, physicochemical measurements, and structure activity relationship studies suggest that corticotropin-releasing factor (CRF; corticoliberin), its family members, and competitive antagonists (resulting from N-terminal deletions) usually assume an alpha-helical conformation when interacting with the CRF receptor(s). To test this hypothesis further, we have scanned the whole sequence of the CRF antagonist [D-Phe12,Nle21,38]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, norleucine) with an i-(i + 3) bridge consisting of the Glu-Xaa-Xaa-Lys scaffold. We have found astressin [cyclo(30-33)[D-Phe12,Nle21,38,Glu30,Lys33]r/ hCRF(12-41)] to be approximately 30 times more potent than [D-Phe12,Nle21,38]r/hCRF-(12-41), our present standard, and 300 times more potent than the corresponding linear analog in an in vitro pituitary cell culture assay. Astressin has low affinity for the CRF binding protein and high affinity (Ki = 2 nM) for the cloned pituitary receptor. Radioiodinated [D-125I-Tyr12]astressin was found to be a reliable ligand for binding assays. In vivo, astressin is significantly more potent than any previously tested antagonist in reducing hypophyseal corticotropin (ACTH) secretion in stressed or adrenalectomized rats. The cyclo(30-33)[Ac-Pro4,D-Phe12,Nle21,38,Glu30,Lys33++ +]r/hCRF-(4-41) agonist and its linear analog are nearly equipotent, while the antagonist astressin and its linear form vary greatly in their potencies. This suggests that the lactam cyclization reinstates a structural constraint in the antagonists that is normally induced by the N terminus of the agonist.

In order to investigate conformational preferences of the 21-residue peptide hormone endothelin-1 (ET-1), an extensive conformational search was carried out in vacuo using a combination of high temperature molecular dynamics/annealing and a Monte Carlo/minimization search in torsion angle space. Fully minimized conformations from the search were grouped into families using a clustering technique based on rms fitting over the Cartesian coordinates of the atoms of the peptide backbone of the ring region. A wide range of local energy mining were identified even though two disulfide bridges (Cys1-Cys15 and Cys3-Cys11) constrain the structure of the peptide. Low energy conformers of ET-1 as a nonionized species in vacuo are stabilized by intramolecular interaction of the ring region (residues 1-15) with the tail (residues 16-21). Strained conformations for individual residues are observed. Conformational similarity to protein loops is established by matching to protein crystal structures. In order to assess the influence of aqueous environment on conformational preference, the electrostatic contribution to the solvation energy was calculated for ET-1 as a fully ionized species (Asp8, Lys9, Glu10, Asp18, N- and C-terminus) using a continuum electrostatics model (DelPhi) for each of the conformers generated in vacuo, and the total solvation free energy was estimated by adding a hydrophobic contribution proportional to solvent accessible surface area. Solvation dramatically alters the relative energetics of ET-1 conformers from that calculated in vacuo. Conformers of ET-1 favored by the electrostatic solvation energy in water include conformers with helical secondary structure in the region of residues 9-15. Perhaps of most importance, it was demonstrated that the contribution to solvation by an individual charge depends not only on its solvent accessibility but on the proximity of other charges, i.e., it is a cooperative effect. This was shown by the calculation of electrostatic solvation energy as a function of conformation with individual charges systematically turned "on" and "off". The cooperative effect of multiple charges on solvation demonstrated in this manner calls into question models that relate solvation energy simply to solvent accessibility by atom or residue alone.

The Helmholtz free energy F (rather than the energy) is the correct criterion for stability; therefore, calculation of F is important for peptides and proteins that can populate a large number of metastable states. The local states (LS) method proposed by H. Meirovitch [(1977) Chemical Physics Letters, Vol. 45, p. 389] enables one to obtain upper and lower bounds of the conformational free energy, FB (b, l) and FA (b, l), respectively, from molecular dynamics (MD) or Monte Carlo samples. The correlation parameter b is the number of consecutive dihedral or valence angles along the chain that are taken into account explicitly. The continuum angles are approximated by a discretization parameter l; the larger are b and l, the better the approximations; while FA can be estimated efficiently, it is more difficult to estimate FB. The method is further developed here by applying it to MD trajectories of a relatively large molecule (188 atoms), the potent “Asp4-Dpr10” antagonist [cyclo(4/10)-(Ac-Δ3Pro1-D-pFPhe2-D-Trp3-Asp4-Tyr-5-D-Nal6-Leu7-Arg8-Pro9-Dpr10-NH2)] of gonadotropin releasing hormone (GnRH). The molecule was simulated in vacuo at T = 300 K in two conformational states, previously investigated [J. Rizo et al. Journal of the American Chemical Society, (1992) Vol. 114, p. 2860], which differ by the orientation of the N-terminal tail, above (tail up, TU) and below (tail down. TD) the cyclic heptapeptide ring. As in previous applications of the LS method, we have found the following: (1) While FA is a crude approximation for the correct F, results for the difference, ΔFA = FA(TD) – FA(TU) converge rapidly to 5.6(1) kcal/mole as the approximation is improved (i.e., as b and l are increased), which suggests that this is the correct value for ΔF; therefore TD is more stable than TU. (The corresponding difference in entrophy. TΔSA = 1.3(2) kcal/mole, is equal to the value obtained by the harmonic approximation.) (2) The lowest approximation, which has the minimal number of local states, i.e., based on b = 0 (no correlations) and l = 1 (the angle values are distributed homogeneously), also leads to the correct value of ΔF, within the error bars. This is important since the lowest approximation can be applied even to large proteins. (3) The method enables one to define the entropy of a part of the molecule and thus to measure the flexibility of this part. We have verified that the results for T[SA(TU) – SA(TD)] of the tail alone converged to 2.4(1) kcal/mole, which demonstrates the relatively high flexibility of the tail in the TU state. In order to study the random coil state, the Asp4-Dpr10 analogue and its linear version were simulated by MU at 1000 K. We have been able to calculate a lower bound, ∼ 25 kcal/mole for T[S(linear) – S(cyclic)], which is the reduction in the conformational entropy caused by the ring closure. © 1994 John Wiley & Sons, Inc.

Structural analysis of constrained (monocyclic) analogues of gonadotropin-releasing hormone (GnRH) has led to the development of a model for the receptor-bound conformation of GnRH and to the design of highly potent, dicyclic GnRH antagonists. This is one of the first cases where a dicyclic backbone has been introduced into analogues of a linear peptide hormone with retention of high biological activity. Here we present a conformational analysis of dicyclo(4-10,5-8)[Ac-D-2Nal1-D-pClPhe2-D-Trp3-Asp4+ ++-Glu5-D-Arg6-Leu7-Lys8- Pro9-Dpr10]-NH2 (I), using two-dimensional nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulation. Compound I inhibits ovulation in the rat at a dose of 5-10 micrograms (Rivier et al. In Peptides: Chemistry, Structure ad Biology; Rivier, J. E., Marshall, G. R., Eds.; ESCOM: Leiden, The Netherlands, 1990; pp 33-37). The backbone conformation of the 4-10 cycle in this dicyclic compound is very similar to that found previously for a parent monocyclic (4-10) GnRH antagonist (Rizo et al. J. Am. Chem. Soc. 1992, 114, 2852-2859; ibid. 2860-2871), which gives strong support to the hypothesis that GnRH adopts a similar conformation upon binding to its receptor. In this conformation, residues 5-8 form a "beta-hairpin-like" structure that includes two transannular hydrogen bonds and a Type II' beta turn around residues D-Arg6-Leu7. The "tail" of the molecule formed by residues 1-3 is somewhat structured but does not populate a single major conformation. However, the orientation of the tail on the same side of the 4-10 cycle as the 5-8 bridge favors interactions between this bridge and the tail residues. These observations correlate with results obtained previously for the parent monocyclic (4-10) antagonist, and have led to the design of a series of new dicyclic GnRH antagonists with bridges between the tail residues and residues 5 or 8.

We illustrate a strategy for identifying the possible structures which can be assigned to a series of fragment ions. The strategy uses the computer program amass to generate a list of all possible fragment structures for each ion and discards those structures that were not common within each list. We illustrate this strategy by comparing several “d” fragment ions present in the plasma desorption (PD) mass spectrum of peptide 1 (H-Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Ala-Ala-OH). From this comparison only three fragment structures were consistently observed; the “x/z” internal sequence fragment, the “z/a” internal sequence fragment and the “d” backbone, and sidechain fragment. On the basis of the measurement of an analog of peptide 1, both the “x/z” and “z/a” internal sequence fragment structures could be excluded. This strategy was used to help assign a series of unassigned fragment ions observed for peptide 1. The unassigned fragment ions were each observed about 14Da above the “d” series of fragment ions. Comparison of the possible fragment structures indicated that an internal sequence fragment could not explain the series of fragment ions. Only two fragment structures were consistently observed for all of the fragment ions. These fragment structures involve “a” type backbone cleavage with side chain cleavage between the γ—γ atoms or “b” type backbone cleavage with side chain cleavage between α—β atoms.

A nomenclature scheme for exhaustively labeling peptide fragment ions is proposed. The scheme is based on IUPAC nomenclature and the previously proposed Roepstorff nomenclature scheme used to label fragment ions in linear peptides. The descriptor used is specifically defined in order to increase the number of peptide and side chain linkages to which the nomenclature scheme can be applied compared with the Roepstorff scheme. The proposed descriptor can be used unambiguously to assign all possible fragments from linear, cyclized, branched, extended (i.e. beta-amino acids) and retro inverso peptides. A significant advantage of the proposed scheme is its simple interface with the currently accepted Roepstorff scheme. This nomenclature scheme is able to label all theoretical fragments generated by the computer program 'AMASS'. AMASS is proposed as a means of systematically calculating the mass of all possible fragment ions from known precursor structures. The program can help determine whether a peptide fragment was derived from an internal sequence fragment or a combination of side chain and backbone cleavages. The program AMASS and the proposed nomenclature scheme are used to illustrate a procedure for identifying fragment ions in the metastable product ion spectrum of somatostatin-14. We envisage that this procedure will be useful for identifying fragment ions which are characteristic of particular structural arrangements in dicyclic and polycyclic peptides.

Truncated, branched, and/or cyclic neuropeptide Y (NPY) analogues were tested for their ability to bind to the neuroblastoma cells, SK-N-MC (Y1 receptor) and SK-N-BE(2) (Y2 receptor). The design of such analogues was inspired by models of NPY based on the crystal structure of avian pancreatic polypeptide. The minimum length of the backbone was investigated using the following truncated analogues [binding affinity (nM) for Y1 and Y2 receptor subtypes respectively are given in parentheses]: des-AA10-17[D-Ala9]NPY (100, 0.9), des-AA7-23[D-Ala6]NPY (> 1000, 1.2), des-AA4-26[D-Ala3]NPY (> 1000, 120), cyclo(7,20)-des-AA10-17[Glu7,D- Ala9,D-Dpr20]NPY (100, nd), cyclo(2,27)-des-AA7-23[Glu2,D-Ala6,D-Dpr27]NPY (> 1000, 3.6), cyclo(2,30)- des-AA7-23[Glu2,D-Ala6,-D-Dpr30]NPY (> 1000, nd), cyclo(1,30)-des-AA4-26[Glu1,D-Ala3,D-Dpr30]NPY (> 1000, > 1000). A new family of branched NPY analogues corresponding to the partial deletion of the polyproline helix with conservation of the N-terminus was also examined: des-AA7-23[(Ac-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 2.1), des-AA7-23[(Ac-NPY7-22)-epsilon-D-Lys6]NPY (> 1000, 5.1), des-AA7-23-[(Ac-LEALEG-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 4.8). Finally, the role played by the flexible tail (residues 32-36) was studied with the following cyclic analogues: cyclo(30,34)-[Lys30,Glu34]NPY18-36 (> 1000, 360), cyclo(30,34)-[Orn30,Gly34]NPY18-36 (> 1000, 950), cyclo(30,34)-[Dpr30,Glu34]NPY18-36 (> 1000, 590), cyclo(33,36)-[Lys33,Glu36]NPY (> 1000, > 1000), cyclo(33,36)-[Lys33,Glu36]NPY18-36 (> 1000, > 1000). These results suggest that the Y1 receptor is highly discriminatory since deletion of residues 10-17, shown to have little effect on Y2 binding affinity, reduces Y1 affinity 50-fold. Bridging sites and constructs have been identified that may serve as useful leads in the design of more potent and selective analogues. We have identified two positions (9 and 6) where the introduction of a D amino acid is not detrimental to binding affinity. Whether this modification leads to the stabilization of a yet unidentified turn compatible with high Y2 receptor affinity will have to be determined by spectroscopic methods. Finally, stabilizing a putative alpha-helical conformation of the C-terminal heptapeptide of NPY18-36 has a deleterious effect on the Y1 and Y2 receptors.

A brief review of 2 lines of research and development of potent analogs of gonadotropin releasing hormone (GnRH) cyclic analogs and linear analogs intended to be safe long acting and hydrophilic is provided. Cyclic analogs of the linear peptide are highly conformationally constrained and hopefully resistant to enzymatic destruction therefore potentially orally active. Work with linear analogs has focused on substituting for the highly basic arginine residues to lower the pKa and thereby to prevent local histamine release. An example is to substitute cyano derivatives with a pKa of 0.4 for guanidino functional group with a pKa of 14.5. Other antagonists have contained aryl guanidino groups on homoarginine arginine or cyanoguanidino peptides or amino triazolyl groups on the distal amino function of lysine ornithine or p-amino-phenylalanine. Using the rat antiovulatory assay a pituitary cell culture assay a pituitary cell membrane binding assay and a histamine release assay the authors group proposed 2 new derivatives Azaline and Azaline B. Azaline B appears to be long acting in castrated male rats resistant to degradation in the blood stream and relatively soluble in aqueous solutions. Azaline B has been selected for further development in human clinical trials as it is more hydrophilic than Antide or "Nal-Glu" and less of a histamine releaser than "Nal-Glu" ganirelix or cetrorelix.

Conformational analysis of a cyclic decapeptide analogue of gonadotropin-releasing hormone (GnRH) led to the design of a series of highly potent GnRH antagonists constrained by a bridge between residues 4 and 10 (Struthers, R. S.; et al. Proteins 1990, 8, 295). We have now used nuclear magnetic resonance (NMR) to study the conformational behavior of one of the most potent of these GnRH antagonists: Ac-Δ 3 -Prol-D-pFPhe2-D-Trp3-c(Asp4-Tyr5-D-2Nal6-Leu7-Arg8-Pro9-Dpr10)-NH 2 . Our data indicates that a β-hairpin conformation within residues 5-8, stabilized by two transannular hydrogen bonds, is the best defined feature of the molecule

Molecular dynamics simulations in vacuo and in solvent have been used, in combination with the NMR data praented in the preceding paper in this tissue, to analyze the conformational behavior of a highly potent antagonist of gonadotropin-releasing hormone (GnRH). An initial conformational search in vacuo yielded two fundamentally different classes of structures that differ in the location of the tail formed by residues 1-3, above or below the cyclic part of the molecule. NMR restraints applied progressively on both families of structures, leading to a consistent conformational model that confirms and refines the interpretation of the experimental data

Strategies for the design of peptide agonists and antagonists of gonadotropin releasing hormone (GnRH, one ligand and one receptor) and of receptor-selective agonists and antagonists of somatostatin (SRIF, three ligands and five receptors) and corticotropin releasing factor (CRF, three ligands and two receptors) are described. These strategies include the use of unusual amino acids, a versatile scaffold based on aminoglycine (betidamino acids) and side chain as well as backbone conformational constraints.

CRF [1] is involved in a wide spectrum of central nervous system (CNS)-mediated effects, suggesting that this peptide plays an important role within the brain, especially in response to stressful stimuli [2]. Systematic SAR investigations have resulted in the development of CRF antagonists such as [3], members of the (standard) family [4] and conformationally restricted analogs [5] that are effective in the CNS. Those results, predictive methods and physicochemical measurements have suggested that CRF and its family members (urotensins and sauvagine) assume an conformation when interacting with the CRF receptors. To further test this hypothesis, we have scanned the whole rat/human sequence with an i-(i + 3) bridge consisting of the Glu-Xaa-Xbb-Lys scaffold which we and others had shown to be compatible with maintenance or enhancement of structure in at least some unpredictable cases.

The three-dimensional NMR structures of six analogues of somatostatin (SRIF) are described. These analogues with the amino acid 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at position 9 exhibit potent and highly selective binding to human SRIF subtype 1 receptors (sst(1)). The conformations reveal that the backbones of these analogues have a hairpin-like structure similar to the sst(2)-subtype-selective analogues. This structure serves as a scaffold for retaining a unique arrangement of the side chains of d-Trp(8), IAmp(9), Phe(7), and Phe(11) or m-I-Tyr(11) (m-I-Tyr = mono-iodo-tyrosine). The conformational preferences and results from biological analyses of these analogues(1,2) allow a detailed study of the structure-activity relationship of SRIF. The proposed consensus pharmacophore of the sst(1)-selective analogues requires a unique set of distances between an indole/2-naphthyl ring, an IAmp side chain, and two aromatic rings. This motif is necessary and sufficient to explain the binding affinities of all of the analogues studied and is distinct from the existing models suggested for sst(4) as well as sst(2)/sst(5) selectivity.

The three-dimensional NMR structures of eight cyclic octapeptide analogues of somatostatin (SRIF) are described. These analogues, with the basic sequence H-c[Cys(3)-Phe(6)-Xxx(7)-Yyy(8)-Lys(9)-Thr(10)-Zzz(11)-Cys(14)]-OH (the numbering refers to the position in native SRIF), with Xxx(7) being Phe/Ala/Tyr, Yyy(8) being Trp/DTrp/D-threo-beta-Me2Nal/L-threo-beta-Me2Nal, and Zzz(11) being Phe/Ala, exhibit potent and highly selective binding to human SRIF type 4 (sst(4)) receptors. The conformations reveal that the backbones of these analogues do not have the usual type-II' beta-turn reported in the literature for sst(2)-subtype-selective analogues. Instead, the structures contain a unique arrangement of side chains of Yyy(8), Lys(9), and Phe(6) or Phe(11). The conformational preferences and results from biological analyses of these analogues (parts 1-3 of this series, Rivier et al., Erchegyi et al., and Erchegyi et al., J. Med. Chem. 2003, preceding papers in this issue) allow a detailed study of the structure-activity relationship of SRIF. The proposed consensus structural motif at the binding pocket for the sst(4)-selective analogues requires a unique set of distances between an indole/2-naphthyl ring, a lysine side chain, and another aromatic ring. This motif is necessary and sufficient to explain the binding affinities of all of the analogues studied and is distinct from the existing model suggested for sst(2)/sst(5) selectivity.

The heptahelical receptors for corticotropin-releasing factor (CRF), CRFR1 and CRFR2, display different specificities for CRF family ligands: CRF and urocortin I bind to CRFR1 with high affinity, whereas urocortin II and III bind to this receptor with very low affinities. In contrast, all the urocortins bind with high affinities, and CRF binds with lower affinity to CRFR2. The first extracellular domain (ECD1) of CRFR1 is important for ligand recognition. Here, we characterize a bacterially expressed soluble protein, ECD1-CRFR2beta, corresponding to the ECD1 of mouse CRFR2beta. The K(i) values for binding to ECD1-CRFR2beta are: astressin = 10.7 (5.4-21.1) nm, urocortin I = 6.4 (4.7-8.7) nm, urocortin II = 6.9 (5.8-8.3) nm, CRF = 97 (22-430) nm, urocortin III = sauvagine200 nm. These affinities are similar to those for binding to a chimeric receptor in which the ECD1 of CRFR2beta replaces the ECD of the type 1B activin receptor (ALK4). The ECD1-CRFR2beta possesses a disulfide arrangement identical to that of the ECD1 of CRFR1, namely Cys(45)-Cys(70), Cys(60)-Cys(103), and Cys(84)-Cys(118). As determined by circular dichroism, ECD1-CRFR2beta undergoes conformational changes upon binding astressin. These data reinforce the importance of the ECD1 of CRF receptors for ligand recognition and raise the interesting possibility that different ligands having similar affinity for the full-length receptor may, nevertheless, have different affinities for microdomains of the receptor.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.

A series of analogs of gonadotropin-releasing hormone (GnRH) containing the conformationally restrictive residue tetrahydroisoquinoline carboxylic acid (Tic) or its non-restricted parent phenylalanine were synthesized and evaluated for anti-ovulatory activity in the rat. The series, based on the potent linear parent compound Ac-DNal1-DCpa2-DPal3- Ser4-Tyr5-DPal6-Leu7-Arg8-Pro9-DAla10-NH2, included L-Tic in positions 1, 2, 3, 7 and 9, D-Tic in positions 1, 2, 3, 6 and 9, or D-Phe in positions 2 and 3 for comparison. The most potent analog in this series, with D-Tic in position 6, fully inhibited ovulation at 2.5 micrograms compared to near complete inhibition at 0.5 microgram for the parent compound. A theoretical analysis of the conformational restrictions imposed on mainchain and sidechain torsional angles by the incorporation of Tic was conducted in vacuo using molecular mechanics techniques. Using cyclo(4-10)-[Ac-delta 3Pro1,DCpa2,DTrp3,Asp4,DNal6,Dpr10]- GnRH as a template conformer for which NMR conformational data is available, it was found that the potency of the different analogs correlated with the strain energy required to deform the mainchain and backbone angles of residues to values which would be expected if Tic were present and if the analog assumed the same solution structure. In particular, the effect of DTic at position 6 is to maintain the type II' beta-turn involving residues 5-8 found in active GnRH analogs.

The introduction of conformational constraints into a flexible peptide hormone can be exploited to develop models for the conformation required for receptor binding and activity. In this review, we illustrate this approach to analog design using our work on antagonists of gonadotropin-releasing hormone (GnRH). Design of a conformationally constrained, competitive antagonist of GnRH, cyclo[delta 3,4 Pro-D4ClPhe-DTrp-Ser-Tyr-DTrp-NMeLeu-Arg-Pro-bet a Ala] led to the prediction of its bioactive conformation. Template forcing experiments show that this conformation is accessible to other active GnRH analogs. Two-dimensional NMR studies verified the predicted conformation in solution. The predicted binding conformation has recently been used to design two new analogs incorporating side chain-side chain linkages suggested by the conformational model: Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-DTrp-Leu-Arg-Asp-Gly- NH2 and Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-D2Nal-Leu-Arg-Pro-Asp -NH2. These analogs were synthesized and the one predicted to be most similar to the parent conformation had equivalent potency while the second, designed to refine the conformational hypothesis, was found to exhibit enhanced potency, thus confirming the original binding conformation hypothesis. These compounds and their derivatives now provide a new class of GnRH antagonists possessing both high biological potency and limited conformational flexibility, thus making them ideal for both biophysical and structure-activity studies.

In order to determine the bioactive conformation of GnRH we decided to synthesize fully rigid and biologically active analogs which could be studied ultimately by NMR spectroscopy or X-ray crystallography. Because we had found that bridging GnRH sequences at positions 1 and 10 or 4 and 10 in fact lead to partial agonists and antagonists resp., and because we have more latitude in the selection of amino acid substitutions in the design of antagonists, we designed, synthesized and tested cyclic GnRH antagonists. These antagonists are characterized by specific substitutions at positions 1, 2 and 3. Here, we show that some antagonists of GnRH which had been bridged at positions 4 and 10 and had been found to be equipotent to the most potent linear analogs, could be further constrained by the introduction of a second bridge between residues 5 and 8 with retention of high potency.

These rapid-scanning stopped-flow kinetic studies of the equine liver alcohol dehydrogenase-catalyzed reduction of p-nitrobenzaldehyde by NADH and (4R)-4-deuterio NADH (NADD) under single turnover conditions establish : (1) The reaction is biphasic using NADD as coenzyme, k1 approximately 200 sec-1, k2 = 0.5 sec-1 and the amplitude ratio (A1)/(A1 + A2) approximately equal to 0.5. (2) Each phase of the reaction involves the oxidation of enzyme-bound reduced coenzyme. (3) The recycling of sites in the presence of 20 mM pyrazole is negligible. (4) The rates of E(NAD-pyrazole) complex formation at all sites and oxidation of reduced coenzyme in the slow phase are limited by the same process. We conclude that the biphasicity arises from nonequivalent but interconvertible forms of the enzyme, and that interconversion of the two forms is mediated by ligand-dependent protomer-protomer interactions.

The compounds 3-hydroxy-4-nitrobenzaldehyde and 3-hydroxy-4-nitrobenzyl alcohol are introduced as new chromophoric substrates for probing the catalytic mechanism of horse liver alcohol dehydrogenase (LADH). Ionization of the phenolic hydroxyl group shifts the spectrum of the aldehyde from 360 to 433 nm (pKa = 6.0), whereas the spectrum of the alcohol shifts from 350 to 417 nm (pKa = 6.9). Rapid-scanning, stopped-flow (RSSF) studies at alkaline pH show that the LADH-catalyzed interconversion of these compounds occurs via the formation of an enzyme-bound intermediate with a blue-shifted spectrum. When reaction is limited to a single turnover of enzyme sites, the formation and decay of the intermediate when aldehyde reacts with enzyme-bound reduced nicotinamide adenine dinucleotide E(NADH) are characterized by two relaxations (lambda f approximately equal to 3 lambda s). Detailed stopped-flow kinetic studies were carried out to investigate the disappearance of aldehyde and NADH, the formation and decay of the intermediate, the displacement of Auramine O by substrate, and 2H kinetic isotope effects. It was found that NADH oxidation takes place at the rate of the slower relaxation (lambda s); when NADD is substituted for NADH, lambda s is subject to a small primary isotope effect (lambda Hs/lambda Ds = 2.0); and the events that occur in lambda s precede lambda f. These findings identify the intermediate as a ternary complex containing bound oxidized nicotinamide adenine dinucleotide (NAD+) and some form of 3-hydroxy-4-nitrobenzyl alcohol. The blue-shifted spectrum of the intermediate strongly implies a structure wherein the phenolic hydroxyl is neutral. When constrained to a mechanism that assumes only the neutral phenolic form of the substrate binds and reacts and that the intermediate is an E(NAD+, product) complex, computer simulations yield RSSF and single-wavelength time courses that are qualitatively and semiquantitatively consistent with the experimental data. We conclude that the LADH substrate site can be divided into two subsites: a highly polar, electropositive subsite in the vicinity of the active-site zinc and, just a few angstroms away, a rather nonpolar region. The polar subsite promotes formation of the two interconverting reactive ternary complexes. The nonpolar region is the binding site for the hydrocarbon-like side chains of substrates and in the case of 3-hydroxy-4-nitrobenzaldehyde conveys specificity for the neutral form of the phenolic group.

The Z isomer of 4-trans-(N,N-dimethylamino)-cinnamaldoxime, (Z)-DMOX (lambda maxH2O 354 nm), forms a ternary complex with NAD+ and equine liver alcohol dehydrogenase. The 3-acetyl (3-acetyl-PdAD+), 3-thiocarboxamide (3-thio-NAD+), 3-iodo (io3PdAD+) and nicotinamide mononucleotide (NMN+) analogues of NAD+ also form ternary complexes with enzyme and (Z)-DMOX. These complexes are characterized by large red-shifts in the UV-visible spectrum of bound (Z)-DMOX (lambda max 428 nm for the NAD+ complex) and new spectral bands in the 280-340-nm region associated with the pyridine moieties of NAD+ and the NAD+ analogues. The ternary enzyme-NAD+-(Z)-DMOX complex is weakly fluorescent (lambda ex 430 nm; lambda em max 505 nm) and strongly quenches the residual tryptophan fluorescence of the enzyme-NAD+ binary complex. (Z)-DMOX binds with high affinity to the enzyme-NAD+ complex (Kd less than or equal to 4 X 10(-9) M at pH 8.75 and 25 degrees C), and similarly high affinities were found for the 3-acetyl-PdAD+, 3-thio-NAD+, and io3PdAD+ complexes. Binding is much weaker to the enzyme-NMN+ complex. The active site specifically substituted Co(II), Ni(II), Cu(II), and Cd(II) enzyme derivatives and the enzyme species lacking any metal ion at the active site (apoenzyme) also form ternary complexes with (Z)-DMOX in which the DMOX UV-visible spectrum is red-shifted (ranging from 43 to 83.5 nm). The complexes formed with the Zn(II) and Co(II) enzymes are characterized by relatively high affinities for (Z)-DMOX and by spectra that are independent of pH over the range 6-10. The affinity of the apoenzyme-NAD+ complex for (Z)-DMOX is much lower, and the spectrum of the complex is pH dependent with lambda max = 430 nm at pH 7 and lambda max = 397 nm at pH 10. The rate of (Z)-DMOX dissociation from the apoenzyme complex was found to be approximately 10(3)-fold greater than the rates observed for the metal ion substituted enzymes. The 280-340-nm spectral bands appear to result from the dihydropyridine moieties of covalent adducts formed between (Z)-DMOX and NAD+ and the NAD+ analogues. The large red-shifts of the (Z)-DMOX spectrum result from the bonding of the oxime nitrogen to a strong electrophilic center (either the active site zinc ion or the nicotinamide ring of NAD+.)(ABSTRACT TRUNCATED AT 400 WORDS)

An improved procedure is described for the isolation of the flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida as well as methods for the separation of its subunits in native form and their recombination to reconstitute the original flavocytochrome. Under appropriate conditions, the reconstitution is stoichiometric and results in complete recovery of the catalytic activity of the flavocytochrome. The separated flavoprotein subunit shows only 2% of the catalytic activity of the original enzyme on p-cresol and is characterized by converging lines in bisubstrate kinetic analysis, while the intact and reconstituted enzymes show parallel line kinetics in steady-state experiments. van't Hoff plots of the dependence of the dissociation constant of the subunits of PCMH on temperature show a break near 15 degrees C. Above this temperature, KD is characterized by a positive delta H value of 12.6 kcal mol-1; below 15 degrees C, the dissociation is essentially temperature independent. The subunit dissociation is strongly dependent on ionic strength in the oxidized form of PCMH but not in the reduced form of the enzyme. Reduction also lowers the KD significantly, while substrates and nonoxidizable competitive inhibitors lower the dissociation constant even further, suggesting a conformation change. Combination of the subunits to form PCMH entails a small but measurable change in the absorption spectra of the component proteins.