Design of biologically active, conformationally constrained GnRH antagonists

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.     

Analogs of pancreatic polypeptide and peptide YY with a locked PP-fold structure are biologically active

Pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY), members of the PP-fold family share a high degree of sequence homology. Nuclear magnetic resonance (NMR) and X-ray crystallography studies have shown these peptides can adopt a tightly organized tertiary structure called the PP-fold, which has long been assumed to be the active structure of this family of peptides. To date, however, no studies have been completed with PYY and PP which confirm if the PP-fold structure is important for their physiological actions. The aim of the study was to test if PYY and PP locked into the PP-fold maintained biological activity. Therefore, we designed and produced analogs of PP and PYY in a cyclic conformation with two cysteine amino acid substitutions at the N-terminus and at position 27. These were oxidized to form a cysteine disulfide bond locking the peptides into the PP-fold structure. Studies demonstrate that the cyclic analogs have both similar in vivo activity to their parent molecules, and affinity for the Y2 and Y4 receptors. Results suggest that the proposed PP and PYY-fold is likely to be their biologically active conformation.     

Synthesis of a bicyclic analog of L-iduronic acid adopting the biologically relevant 2S0 conformation

The synthesis of a bicyclic analogue of the naturally occurring alpha-L-iduronic acid locked in a biologically active (2)S0 skewboat conformation is disclosed. The desired (2)S0 conformation has been obtained by tethering the C-2 and C-5 carbon atoms of the sugar ring with a dimethyloxy bridge and confirmed by NMR and molecular modeling. The new mimic displays the exact hydroxyl pattern of alpha-L-iduronic acid, a major monosaccharide component of glycosaminoglycans and thus represents a closer mimic of the latter, compared to previously reported bicyclic analogs.     

Comparison of conformation and antimicrobial activity of synthetic analogs of gramicidin S: Stereochemical consideration of the role of D-phenylalanine in the antibiotic.

In order to study the role of D -amino acid residues in keeping the stable β-sheet conformation and in the antimicrobial activity of gramicidin S (GS), the four analogs of GS containing D -Ala, L -Ala, Gly, and Aib (α-aminoisobutyric acid) in place of D -Phe were synthesized. D -Ala-and Gly-containing analogs showed antimicrobial activity, while those containing L -Ala and Aib showed no activity. Conformation of these analogs and their derivatives were studied by comparison of ORD and CD spectra and by slective methylation method. It is concluded that the biologically active analogs have β-sheet conformation while inactive analogs have a much different conformation from that of GS. This indicates that D -Ala-Pro and Gly-Pro sequences favor taking a β-bend form but L -Ala-Pro and Aib-Pro sequences do not because the presence of L -side methyl group on the α-carbon atom of L Ala and Aib residues destabilizes the β-bend form. This would explain why the inactive analogs which take a different conformation from that of the active ones result in the loss of activity.     

Empirical solvation models in the context of conformational energy searches: application to bovine pancreatic trypsin inhibitor.

Continuum solvation models that estimate free energies of solvation as a function of solvent accessible surface area are computationally simple enough to be useful for predicting protein conformation. The behavior of three such solvation models has been examined by applying them to the minimization of the conformational energy of bovine pancreatic trypsin inhibitor. The models differ only with regard to how the constants of proportionality between free energy and surface area were derived. Each model was derived by fitting to experimentally measured equilibrium solution properties. For two models, the solution property was free energy of hydration. For the third, the property was NMR coupling constants. The purpose of this study is to determine the effect of applying these solvation models to the nonequilibrium conformations of a protein arising in the course of global searches for conformational energy minima. Two approaches were used: (1) local energy minimization of an ensemble of conformations similar to the equilibrium conformation and (2) global search trajectories using Monte Carlo plus minimization starting from a single conformation similar to the equilibrium conformation. For the two models derived from free energy measurements, it was found that both the global searches and local minimizations yielded conformations more similar to the X-ray crystallographic structures than did searches or local minimizations carried out in the absence of a solvation component of the conformational energy. The model derived from NMR coupling constants behaved similarly to the other models in the context of a global search trajectory. For one of the models derived from measured free energies of hydration, it was found that minimization of an ensemble of near-equilibrium conformations yielded a new ensemble in which the conformation most similar to the X-ray determined structure PTI4 had the lowest total free energy. Despite the simplicity of the continuum solvation models, the final conformation generated in the trajectories for each of the models exhibited some of the characteristics that have been reported for conformations obtained from molecular dynamics simulations in the presence of a bath of explicit water molecules. They have smaller root mean square (rms) deviations from the experimentally determined conformation, fewer incorrect hydrogen bonds, and slightly larger radii of gyration than do conformations derived from search trajectories carried out in the absence of solvent.     

Conformation-activity relationship of peptide T and new pseudocyclic hexapeptide analogs.

Peptide T (ASTTTNYT), a segment corresponding to residues 185-192 of gp120, the coat protein of HIV, has several important biological properties in vitro that have stimulated the search for simpler and possibly more active analogs. We have previously shown that pseudocyclic hexapeptide analogs containing the central residues of peptide T retain considerable chemotactic activity. We have now extended the design of this type of analogs to peptides containing different aromatic residues and/or Ser in lieu of Thr. The complex conformation-activity relationship of these analogs called for a reexamination of the basic conformational tendencies of peptide T itself. Here, we present an exhaustive NMR conformational study of peptide T in different media. Peptide T assumes a gamma-turn in aqueous mixtures of ethylene glycol, a type-IV beta-turn conformation in aqueous mixtures of DMF, and a type-II beta-turn conformation in aqueous mixtures of DMSO. The preferred conformations for the analogs were derived from modeling, starting from the preferred conformations of peptide T. The best models derived from the gamma-turn conformation of peptide T are those of peptides XII (DSNYSR), XIII (ETNYTK) and XVI (ESNYSR). The best models derived from the type-IV beta-turn conformation of peptide T are those of peptides XIV (KTTNYE) and XV (DSSNYR). No low-energy models could be derived starting from the type-II beta-turn conformation of peptide T. The analogs with the most favored conformations are also the most active in the chemotactic test.     

Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). II. Semi-empirical energy calculations.

The molecular conformation of the basic pancreatic trypsin inhibitor (BPTI) is known in considerable detail from both X-ray studies in single crystals and NMR studies in solution. The NMR experiments showed that the aromatic rings of the phenylalanyl and tyrosyl residues can undergo rapid rotational motions about the C beta--Cv bond. The present paper describes a model investigation of the mechanistic aspects of these intramolecular rotational motions. From calculations of the conformational energies for molecular species derived from the X-ray structure by rotations of individual aromatic rings, it was apparent that the rotational motions of the aromatics could only be understood in a flexible structure. Flexibility was simulated by allowing the protein to relax to an energetically favorable conformation for each of the different rotation states of the aromatic rings. It was then of particular interest to investigate how the perturbations caused by different rotation states of the aromatic rings were propagated in the protein structure. It was found that the rotation axes C beta--Cv were only slightly affected (delta X1 approximately less than 20 degrees. The most sizeable perturbations are caused by through space interactions with nearby atoms, which move away from the ring center and thus release the steric hindrance opposing the rotational motions. The values for the energy barriers obtained from the energy minimization are of the same order of magnitude as those measured by NMR.     

The conformation of echinomycin in DMSO solution

The conformation of the antibiotic echinomycin in DMSO solution has been determined from two-dimensional NMR and distance geometry calculation with distance constraints. Five converged conformations were calculated with NOE distance constraints followed by restraint energy minimization.     

Mapping the active site of factor Xa by selective inhibitors: An NMR and MD study

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH₂ and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH₂, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site. Proteins 30:264–279, 1998. © 1998 Wiley-Liss, Inc.     

Assignment of side-chain conformation using adiabatic energy mapping, free energy perturbation, and molecular dynamic simulations

NMR spectroscopic analysis of the C-terminal Kunitz domain fragment (alpha3(VI)) from the human alpha3-chain of type VI collagen has revealed that the side chain of Trp21 exists in two unequally populated conformations. The major conformation (M) is identical to the conformation observed in the X-ray crystallographic structure, while the minor conformation (m) cannot structurally be resolved in detail by NMR due to insufficient NOE data. In the present study, we have applied: (1) rigid and adiabatic mapping, (2) free energy simulations, and (3) molecular dynamic simulations to elucidate the structure of the m conformer and to provide a possible pathway of the Trp21 side chain between the two conformers. Adiabatic energy mapping of conformations of the Trp21 side chain obtained by energy minimization identified two energy minima: One corresponding to the conformation of Trp21 observed in the X-ray crystallographic structure and solution structure of alpha3(VI) (the M conformation) and the second corresponding to the m conformation predicted by NMR spectroscopy. A transition pathway between the M and m conformation is suggested. The free-energy difference between the two conformers obtained by the thermodynamic integration method is calculated to 1.77+/-0.7 kcal/mol in favor of the M form, which is in good agreement with NMR results. Structural and dynamic properties of the major and minor conformers of the alpha3(VI) molecule were investigated by molecular dynamic. Essential dynamics analysis of the two resulting 800 ps trajectories reveals that when going from the M to the m conformation only small, localized changes in the protein structure are induced. However, notable differences are observed in the mobility of the binding loop (residues Thr13-Ile18), which is more flexible in the m conformation than in the M conformation. This suggests that the reorientation of Trp2 might influence the inhibitory activity against trypsin, despite the relative large distance between the binding loop and Trp21.     

Determination of structural elements related to the biological activities of a potent decapeptide agonist of human C5a anaphylatoxin.

The structural features related to the biologic activities of a potent, response-selective decapeptide agonist of human C5a, YSFKPMPLaR (C5a65-74, Y65, F67, P69, P71, D-Ala73), were identified by NMR analysis in H2O, DMSO and TFE. This investigation showed that the KPM residues in H2O and the SFKPM residues in DMSO exhibited an extended backbone conformation, whereas a twisted conformation was found in this region in TFE. In H2O, the C-terminal region (PLaR) adopted a distorted type II beta-turn or a type II/V beta-turn. In the type IIN beta-turn, Leu72 exhibited a conformation typical of a type II beta-turn, whereas D-Ala73 exhibited a conformation characteristic of a type V beta-turn. Furthermore, a gamma-turn involving residues LaR overlapped with the type II/V beta-turn. In DMSO, the C-terminal region had the analogous turn-like motif (type II/V beta-turn overlapping with gamma-turn) found in H2O. In TFE, no beta-turn motifs were formed by the PLaR residues. These turn-like motifs in the C-terminal region of the peptide in both H2O and DMSO were in agreement with the biologically important conformations predicted earlier by a structure-function analysis of a related panel of decapeptide analogs. The motifs determined by the NMR analysis of YSFKPMPLaR in H2O and DMSO may represent structural elements important for C5a agonist activity and thus can be used to design the next generation of C5a agonist, partial agonist and antagonist analogs.     

Theoretical conformational analysis of a encephalitogenic peptide molecule and a study of the structure-activity relationship in a series of its analogs

Semi-empirical energy calculations are used to determine all low-energy conformations of Trp-containing fragment 113-121 of myelin basic protein (experimental allergic encephalomyelitis inducing peptide). The computed conformations are compared with the results of physico-chemical experiments and data on biological testing of the encephalitogenic peptide analogs. The three computed structures are shown to be in a good agreement with the available experimental evidence. However, additional information is required to predict "biologically active" conformation of encephalitogenic peptide.     

Studies of the thyrotropin-releasing factor. II. Conformations of TRF and some analogs

The conformation of the thyrotropin-releasing factor (TRF) and four analogs have been studied by the CNDO/2 molecular orbital method. The N^δ-protonated tautomer of TRF is predicted to be the more stable of the two tautomeric forms; however, it is postulated that the N^ε-tautomer possesses the most active minimum-energy conformation. The peptide backbone for the five compounds remains nearly constant. The conformations predicted from model fragments are very near to those found for the full compound.     

Characterization of a novel type VII beta-turn conformation for a bio-active tetrapeptide rigin A synergy between theoretical and experimental results.

The conformational analysis of an immunomodulating tetrapeptide rigin (H-Gly-Gln-Pro-Arg-OH), shown to possess diverse immunological activity, has been investigated both theoretically and experimentally for its conformational preferences. Unrestrained molecular dynamics simulation studies in implicit dimethylsulfoxide provide strong support for the existence of a significant population of ordered reverse turn structures for the major trans isomer. Of the three different energy minimized families, generated from computer molecular modelling, only one could be complemented by most of the 1D and 2D 1H NMR parameters obtained in dimethylsulfoxide-d6. A variable temperature NMR experiment in dimethylsulfoxide-d6 revealed that the preferred conformation is not stabilized by an intramolecular hydrogen bonding interaction. An analysis of the 2D ROESY experiment provides evidence in favour of an uncommonly observed, rather ill-defined type VII beta-turn structure. A survey of the observed specific inter-and intra-residue NOE connectivities and their comparison with one of the predicted low-energy conformations, demonstrates synergy between the theoretical molecular modelling and experimentally determined NMR spectral data. The primary structure, rather than long-range interactions, appears to be critical in determining the folding behaviour of the bio-active rigin. The present structural attributes may be valuable in peptide drug design and development of the rigin analogs having more effective stimulating activity.     

NMR studies of peptide T, an inhibitor of HIV infectivity, in an aqueous environment.

The synthetic octapeptide peptide T (ASTTTNYT) has been shown to interfere with binding of the HIV-1 envelope glycoprotein gp120 to the chemokine receptor R5, thus preventing viral infection. This study investigated the degree of conformational order of two analogs of peptide T, one biologically active (D-Ala peptide T amide) and one inactive (D-Ala, D-Tyr peptide T amide) using nuclear magnetic resonance (NMR) spectroscopy in an aqueous environment, both in solution and in the frozen solid state. Standard solution NMR techniques such as DQFCOSY, HMQC, ROESY and inversion recovery measurements have been utilized to characterize these peptides. Solid state NMR experiments were likewise employed to study the peptides in a frozen glycerol:water mixture. The NMR results indicate that the monomeric form of both peptide T analogs have considerable conformational heterogeneity. Solid state NMR studies indicate aggregation of D-Ala peptide T, possibly into a beta-sheet structure, at concentrations higher than 10 mM.     

NMR and molecular modeling characterization of RGD containing peptides.

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d6 of the cyclic pentapeptides, [formula: see text], a potent fibrinogen receptor antagonist, and [formula: see text], a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H-1H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH-H alpha coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.     

Inferences about the conformation of somatostatin at a biologic receptor based on NMR studies

Examination of two diastereomeric analogs of somatostatin differing in stereochemistry at the tryptophan residue has revealed a high field resonance in the -Trp isomer which is assigned to the γ-methylene of Lys⁹. The extent of correlation of this shift with biologic activity for a series of analogs of somatostatin is discussed. From comparison of close analogs, it is suggested that the biologically active conformation of somatostatin at the receptor controlling insulin release is not the major conformation of this hormone in solution. It is suggested that the conformation of somatostatin at this receptor resembles more closely the solution conformation of analogs having tryptophan in the -configuration. This latter conformation places the Trp⁸-Lys⁹ side chains in close proximity, thus shifting the γ-methylene protons of Lys⁹ upfield.     

Topography of the active site of the noradrenaline neuronal membrane carrier based on the theoretical conformation analysis of inhibitors of neuronal catecholamine uptake

All equilibrium conformations for nine ligands, essentially different in structure, of the noradrenaline carrier through the synaptosomal membrane of rat hypothalamus were calculated by semi-empirical method. Among these compounds were amphetamine, methylphenidate, tricyclic antidepressants. The conformational energy minimization was performed in the space of torsional and bond angles. Geometrical characteristics of the conformers were determined in the cartesian coordinate system fixed relative to the benzene ring and nitrogen atom of the ligand. The selection of biologically active (productive) conformations was made according to the following criteria: 1) low conformational energy; 2) similarity of the nitrogen atoms and phenyl rings spatial disposition in all ligands; 3) accessibility for intermolecular interactions of the same sides of functional groupings in all ligands. The above criteria enabled the productive conformations for all ligands to be chosen unambiguously. The productive conformation of noradrenaline was found to have the Ph-C-C-N fragment in perpendicular trans-conformation. A topographic model for the carrier active site was suggested, its components being the nucleophilic and two arylophilic groups situated against the most accessible sides of the functional moieties of the productively bound ligands.     

Cooperativity of thymopoietin 32-36 (the active site) and thymopoietin 38-45 in receptor binding

Thymopoietin is a 49 amino acid polypeptide hormone of the thymus whose biological activity is reproduced by the synthetic pentapeptide thymopentin, corresponding to amino acids 32-36. Thymopentin requires the addition of an octapeptide corresponding to thymopoietin 38-45 for full competition with native thymopoietin in a radioreceptor assay with receptor derived from the human T-cell line CEM. Thus thymopoietin appears to bind to its receptor on T-cells by two regions (32-36 and 38-45). Thymopentin alone is biologically active and induces elevations of intracellular cyclic GMP. Whilst occupancy of the adjacent site by thymopoietin 37-45 does not of itself cause an elevation of intracellular cyclic GMP this peptide is not biologically silent as it does enhance the potency of thymopentin.     

Structure-activity relationship and metabolic stability studies of backbone cyclization and N-methylation of melanocortin peptides

Backbone cyclization (BC) and N-methylation have been shown to enhance the activity and/or selectivity of biologically active peptides and improve metabolic stability and intestinal permeability. In this study, we describe the synthesis, structure-activity relationship (SAR) and intestinal metabolic stability of a backbone cyclic peptide library, BL3020, based on the linear alpha-Melanocyte stimulating hormone analog Phe-D-Phe-Arg-Trp-Gly. The drug lead, BL3020-1, selected from the BL3020 library (compound 1) has been shown to inhibit weight gain in mice following oral administration. Another member of the BL3020 library, BL3020-17, showed improved biological activity towards the mMC4R, in comparison to BL3020-1, although neither were selective for MC4R or MC5R. N-methylation, which restrains conformational freedom while increasing metabolic stability beyond that which is imparted by BC, was used to find analogs with increased selectivity. N-methylated backbone cyclic libraries were synthesized based on the BL3020 library. SAR studies showed that all the N-methylated backbone cyclic peptides demonstrated reduced biological activity and selectivity for all the analyzed receptors. N-methylation of active backbone cyclic peptides destabilized the active conformation or stabilized an inactive conformation, rendering the peptides biologically inactive. N-methylation of backbone cyclic peptides maintained stability to degradation by intestinal enzymes.