Bioactive conformations of two seminal delta opioid receptor penta‐peptides inferred from free‐energy profiles

Delta‐opioid (DOP) receptors are members of the G protein‐coupled receptor (GPCR) sub‐family of opioid receptors, and are evolutionarily related, with homology exceeding 70%, to cognate mu‐opioid (MOP), kappa‐opioid (KOP), and nociceptin opioid (NOP) receptors. DOP receptors are considered attractive drug targets for pain management because agonists at these receptors are reported to exhibit strong antinociceptive activity with relatively few side effects. Among the most potent analgesics targeting the DOP receptor are the linear and cyclic enkephalin analogs known as DADLE (Tyr‐D ‐Ala‐Gly‐Phe‐D ‐Leu) and DPDPE (Tyr‐D ‐Pen‐Gly‐Phe‐D ‐Pen), respectively. Several computational and experimental studies have been carried out over the years to characterize the conformational profile of these penta‐peptides with the ultimate goal of designing potent peptidomimetic agonists for the DOP receptor. The computational studies published to date, however, have investigated only a limited range of timescales and used over‐simplified representations of the solvent environment. We provide here a thorough exploration of the conformational space of DADLE and DPDPE in an explicit solvent, using microsecond‐scale molecular dynamics and bias‐exchange metadynamics simulations. Free‐energy profiles derived from these simulations point to a small number of DADLE and DPDPE conformational minima in solution, which are separated by relatively small energy barriers. Candidate bioactive forms of these peptides are selected from identified common spatial arrangements of key pharmacophoric points within all sampled conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 21–27, 2014.     

Biological and conformational studies of [Val4]morphiceptin and [D-Val4]morphiceptin analogs incorporating cis-2-aminocyclopentane carboxylic acid as a peptidomimetic for proline

We report the synthesis, biological activity, and conformational analysis of tetrapeptide analogs related to [Val4]morphiceptin and [D-Val4]morphiceptin in which the proline at the second position has been replaced with cis-2-aminocyclopentane carboxylic acid (cis-2-Ac5c). Since the cis-2-Ac5c residue contains a normal amide, only the trans form has been observed about the amide bond between the first and second residues. The cis-2-Ac5c is a beta amino acid with two chiral centers resulting in two possible configurational isomers, namely (1S, 2R) and 1R, 2S) forms. The analogs containing the (1S, 2R)-Ac5c residue show activity at the mu-receptor but are inactive at the delta-receptor, resulting in a high selectivity for the mu-receptor. The (1R,2S)-Ac5c containing analogs are completely inactive at both the mu- and delta-receptors. The conformational analysis indicates that the separation of the aromatic rings of the tyrosine and phenylalanine residues, as expressed by the center-to-center distance, is 10.1-12.7 A for the preferred conformations of the bioactive analogs containing the (1S,2R)-Ac5c residue while a range of 4.8-7.0 A is observed for the preferred conformations of the inactive analogs with the (IR,2S)-Ac5c residue. A comparison of the findings from the conformational analysis and biological assays establishes the fact that a relatively large separation of the two aromatic side chains is required for the mu-opioid receptor activity of these molecules. Since the tetrapeptide amides studied in this investigation show similar biological profiles to those of the morphiceptin-related analogs, we have compared the preferred conformations estimated for the cis-2-Ac5c containing analogs with those of morphiceptin. One of the low energy conformations calculated for morphiceptin with the cis form about the tyrosine and proline residues has considerable topological similarity with the bioactive analogs containing the (1S,2R)-Ac5c residue, indicating that the cis from about these two residues is required for the biological activity of the morphiceptin-related analogs containing the proline at the second position.     

Single residue modifications of the delta opioid receptor selective peptide, [D-Pen2,D-Pen5]-enkephalin (DPDPE). Correlation of pharmacological effects with structural and conformational features

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [D-Pen2,D-Pen5]enkephalin, Tyr-D-Pen-Gly-Phe-D-PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by 1H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine1-DPDPE ([DAT1]DPDPE) and phenylglycine4-DPDPE ([Pgl4]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced mu receptor binding affinity of the carboxamide terminal DPDPE-NH2 appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the alpha-aminoisobutyric acid substituted analog [Aib3]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the delta opioid receptor.     

Conformational analysis of [Cpp1, Sar7, Arg8] vasopressin by 1H-NMR spectroscopy and molecular mechanics calculations.

A combined 1H-NMR and molecular mechanics study of [Cpp1, Sar7]AVP was performed in order to select the most probable conformations in DMSO solutions. The NMR constraints obtained were employed in the selection of starting conformations of the cyclic moiety of the analog. In particular, the diminished accessibility of the Asn5 NH proton to solvent and the close contact between Cpp1 and Cys6 C alpha H protons suggests a beta-turn conformation at the Phe3-Gln4 residues. Energy minimization was carried out both in the ECEPP/2 (rigid-valence geometry) and in the AMBER (flexible-valence geometry) force fields. Comparison of the experimental and calculated values of NMR characteristics has revealed that conformations containing type I, II, and III beta-turns at the Phe3-Gln4 residues are in reasonable agreement with the experimental data, with a dynamic equilibrium between the beta I (beta III) and beta II type structures of the cyclic part being the most probable. All of these conformations prefer the negative chirality of the disulfide bridge (theta 3 approximately -90 degrees). Five representative conformations were chosen for the acyclic tail: one with a beta I, one with a beta II'-turn at the Sar7-Arg8 residues, two extended-type conformations, and a conformation with a gamma-turn at Sar7. Because only high-energy extended conformations were in agreement with NMR data, it was concluded that the acyclic tail has considerable conformational flexibility in solution. The conformations obtained are discussed in terms of the structure-function relationship of the neurohypophyseal hormone analogs.     

Molecular simulation uncovers the conformational space of the λ Cro dimer in solution

The significant variation among solved structures of the λ Cro dimer suggests its flexibility. However, contacts in the crystal lattice could have stabilized a conformation which is unrepresentative of its dominant solution form. Here we report on the conformational space of the Cro dimer in solution using replica exchange molecular dynamics in explicit solvent. The simulated ensemble shows remarkable correlation with available x-ray structures. Network analysis and a free energy surface reveal the predominance of closed and semi-open dimers, with a modest barrier separating these two states. The fully open conformation lies higher in free energy, indicating that it requires stabilization by DNA or crystal contacts. Most NMR models are found to be unstable conformations in solution. Intersubunit salt bridging between Arg⁴ and Glu⁵³ during simulation stabilizes closed conformations. Because a semi-open state is among the low-energy conformations sampled in simulation, we propose that Cro-DNA binding may not entail a large conformational change relative to the dominant dimer forms in solution.     

Conformational properties of neuromedin NmU-8 and its modified analogs

The comparative study of the spatial organization and conformational properties of NmU-8 neuropeptide and its modified analogs with available experimental data has been carried out. The effect of amino acids point mutation on conformational states of native neuropeptide has been discussed. The low-energy conformations responsible for neuropeptide contractile activity was revealed.     

Detection of nascent polyproline II helices in solution by NMR in synthetic insect kinin neuropeptide mimics containing the X-Pro-Pro-X motif.

The conformations of three synthetic peptide analogs containing the dPro-dPro-dXaa motif (dXaa = dThr, dGlu, dAsn) in aqueous solution were studied by a combination of NMR and molecular modeling simulations. The three compounds were identified from a random D-amino acid tripeptide library on the basis of their ability to either mimic or block the diuretic activity of neuropeptides of the insect kinin family. TOCSY and ROESY correlations, as well as abnormal secondary chemical shifts for protons on the D-proline residues were employed to obtain conformational ensembles consistent with the experimental NMR data for the three analogs using an in vacuo simulated annealing protocol. Similar secondary structures were found for the three molecules after refinement, in agreement with the similarities observed between their NMR spectra. Unrestrained molecular dynamics simulations with explicit water representation indicate that the structural motifs found in vacuo are stable in aqueous solution. The three analogs can be considered initiators of right-handed poly D-proline II helices, mirror images of the poly L-proline II left-handed helical motifs normally found in proline-rich proteins. The role of these secondary folds on binding of the analogs to the kinin receptors is discussed.     

DNA models for A, B, C and D conformations related to fiber X-ray, infrared and NMR measurements

A conformational analysis of the A, B, C and D DNA forms was made in order to establish molecular models presenting a good agreement with experimental data obtained from fiber X-ray, infrared linear dichroism and 31P NMR. The proposed models have been refined and do present good stereochemistry and optimized H-bond distances between bases associated with the Watson-Crick pairing. The DNA conformations proposed are a left handed double helix for the C form and right handed helices for A, B and D. Relations to conformational transitions between these forms are discussed.     

Assessment of the bioactive conformation of the farnesyltransferase protein binding recognition motif by computational methods.

Ras farnesyltransferase catalyzes the carboxyl-terminal farnesylation of Ras as well as other proteins involved in signal transduction processes. Previous studies demonstrated that its inhibition suppresses the activity of Ras transformed phenotypes in cultured cells, causing tumor regression in animal models. This observation led to the consideration of farnesyltransferase as a target for cancer therapy. In the present work we report the results of a computational study aimed at assessing the bioactive conformation of the peptide Cys-Val-Phe-Met, known to be the minimum peptide sequence that inhibits farnesyltransferase. For this purpose the conformational preferences of four analogs of the peptide were assessed by means of thorough searches of their respective conformational spaces, using a simulated annealing protocol as sampling technique. Specifically, two active analogs: Cys-Val-Tic-Met and Cys-Val-psi(CH2NH)Tic-Met and two inactive analogs: Cys-Val-Tic-psi(CH2NH)Met and Cys-Val-Aic-Met were selected for the present study. Low energy conformations of the four analogs were classified according to their structural motifs. The putative bioactive conformation of the minimum farnesyltransferase recognition motif was assessed by cross-comparison of the different classes of conformations obtained for the two active and the two inactive analogs. The putative bioactive conformation is characterized by two structural motifs: i) a C14 pseudo-ring stabilized by a hydrogen bond between the amino group of Cys1 and the carboxylate group of Met4 and a C11 pseudo-ring involving the residues Cys1 and Tic3. In addition, the thiol group of Cys1 side chain of the bioactive conformation points to the carboxylate moiety of Met4.     

Predicting order of conformational changes during protein conformational transitions using an interpolated elastic network model

The decryption of sequence of structural events during protein conformational transitions is essential to a detailed understanding of molecular functions ofvarious biological nanomachines. Coarse‐grained models have proven useful by allowing highly efficient simulations of protein conformational dynamics. By combining two coarse‐grained elastic network models constructed based on the beginning and end conformations of a transition, we have developed an interpolated elastic network model to generate a transition pathway between the two protein conformations. For validation, we have predicted the order of local and global conformational changes during key ATP‐driven transitions in three important biological nanomachines (myosin, F₁ ATPase and chaperonin GroEL). We have found that the local conformational change associated with the closing of active site precedes the global conformational change leading to mechanical motions. Our finding is in good agreement with the distribution of intermediate experimental structures, and it supports the importance of local motions at active site to drive or gate various conformational transitions underlying the workings of a diverse range of biological nanomachines. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The antagonism between 2-methyl-1,25-dihydroxyvitamin D3 and 2-methyl-20-epi-1,25-dihydroxyvitamin D3 in non-genomic pathway-mediated biological responses induced by 1alpha,25-dihydroxyvitamin D3 assessed by NB4 cell differentiation

We synthesized all eight possible A-ring diastereomers of 2-methyl substituted analogs of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and also all eight A-ring diastereomers of 2-methyl-20-epi-1alpha,25(OH)2D3. Their biological activities, especially the antagonistic effect on non-genomic pathway-mediated responses induced by 1alpha,25(OH)2D3 or its 6-s-cis-conformer analog, 1alpha,25(OH)2-lumisterol3, were assessed using an NB4 cell differentiation system. Antagonistic activity was observed for the 1beta-hydroxyl diastereomers, including 2beta-methyl-1beta,25(OH)2D3 and 2beta-methyl-3-epi-1beta,25(OH)2D3. Very interestingly, 2beta-methyl-3-epi-1alpha,25(OH)2D3 also antagonized the non-genomic pathway, despite its 1alpha-hydroxyl group. Other 1alpha-hydroxyl diastereomers did not show antagonistic activity. 20-epimerization diminished the antagonistic effect of all of these analogs on the non-genomic pathway. These findings suggested that the combination of the 2-methyl substitution of the A-ring and 20-epimerization of the side chain could alter the biological activities in terms of antagonism of non-genomic pathway-mediated biological response. Based on a previous report, 2-methyl substitution alters the equilibrium of the A-ring conformation between the alpha- and beta-chair conformers. The 2beta-methyl diastereomers, which exhibited antagonism on non-genomic pathway-mediated response, were considered to prefer the beta-conformer. Further examination to elucidate the relationship between the altered ligand shape and receptors interaction will be important for molecular level understanding of the mechanism of antagonism of the non-genomic pathway.     

Conformational and dynamic considerations in the design of peptide hormone analogs

Efforts to understand the chemical-physical basis for peptide hormone and neurotransmitter action requires integration of conformational parameters and biological properties. Since most peptide hormones are conformationally flexible, the question arises as to which of the manifold of conformations is of biological significance. In molecular terms, it is necessary to carefully distinguish chemical-physical features important to binding (the binding message) from those involved in transduction (the biological activity message). One approach to this involves the design, synthesis, and conformational analysis of semirigid hormone analogs. The distinction between binding and transduction can best be examined by evaluation of full biological profiles of partial agonists, antagonists, and analogs with prolonged biological activity. Using this multidisciplinary approach, we have prepared several semirigid [Pen¹]-oxytocin antagonist analogs and evaluated their conformational properties and biological activities. Specific conformational features can be related to inhibitory activities in several cases. On the basis of structure–activity relationships and conformational considerations, we have designed a series of conformationally restricted cyclic and acyclic analogs of the linear peptide α-melanotropin. Some of these peptides have exceptionally prolonged in vivo activity (weeks), and others exhibit superagonist potency (10,000 times the native hormone). We have evidence that potency and prolonged activity have different structural and conformational requirements. It is suggested that potency is primarily a function of receptor recognition (the binding message), whereas prolonged activity is related to transduction (the biological activity message).     

Disorder and order in unfolded and disordered peptides and proteins: A view derived from tripeptide conformational analysis. I. Tripeptides with long and predominantly hydrophobic side chains

We performed a conformational analysis of the central residues of three tripeptides glycyl‐L ‐isoleucyl‐glycine (GIG), glycyl‐L ‐tyrosyl‐glycine (GYG) and glycyl‐L ‐arginyl‐glycine (GRG) in aqueous solution, based on a global analysis of amide I′ band profiles and NMR J‐coupling constants. The results are compared with recently reported distributions of GVG, GFG and GEG. For GIG and GYG, we found that even though the polyproline II (pPII) fraction is below 0.5, it is still the most populated conformation, whereas GVG and GFG show both a larger β‐strand fraction. For GRG, we observed a clear dominance of pPII over β‐strand, reminiscent of observations for GEG and GKG. This finding indicates that terminal charges on otherwise hydrophobic residue side chains stabilize pPII over β‐strand conformations. For all peptides investigated we found that a variety of compact and turn‐like conformations constitute nearly 20 percent of their conformational distributions. Attempts to analyze our data with a simple two‐state pPII??β model therefore do not yield any satisfactory reproduction of experimental results. A comparison of the obtained GxG ensembles with conformational distributions of GxG segments in truncated coil libraries (helices and sheets omitted) revealed a much larger fraction of type II β_i+2 and type III β like conformations for the latter. Thus, a comparison of conformational distributions of unfolded peptide segments in solution and in coil libraries reveal interesting information on how the interplay between intrinsic propensities of amino acid residues and non‐local interactions in polypeptide chains determine the conformations of loop segments in proteins. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Structural Factors that Distinguish Dopamine D1 and D2 Agonists

To determine the structural features responsible for their selectivity as dopamine D1 agonists, a conformational analysis has been performed on an analog of nomifensine, dihydrexidine, a benzergoline, and an isochroman using the MM2-87 program. The preferred three dimensional structure of the hydroxylated phenyl ring of the nomifensine analog was found to differ from the other compounds with a substantial energy barrier to achieving the planar conformation of the other compounds which may explain its weak potency for D1 receptors. The preferred three dimensional structures of dihydrexidine and the benzergoline were found to differ significantly despite their molecular similarity. These conformational differences were also evident in crystal structures of the compounds or their analogs. The hypothesis that an equatorial ammonium hydrogen (or amine lone pair) is required for D1 agonist selectivity was tested by performing calculations on N-methyl equatorial and N-methyl axial analogs of the compounds. Calculations were also performed on nonselective dopamine agonists (apomorphine and 5,6-diOH- and 6,7-diOH aminotetralin) and dopamine D2-selective agonists ((+)-PHNO and an analog of quinpirole). The energy difference for the N-methyl axial conformations (or their equivalent) were found to be relatively small for the nonselective agonists and more substantial for the D2-selective agonists. This suggests that D2-selectivity may be associated with the relative unfavorability of the N-methyl axial conformation and provides an explanation for the decreased potency of tertiary amine analogs of the D1-selective agonists. In the benzergoline, where the energy difference is computed to be smaller, the addition of the N-methyl group appears to have a smaller deleterious effect on D1 activity. An N-methyl axial conformation has also been observed for the benzergoline in the crystal state suggesting that this conformation is energetically accessible.     

Conformational analysis of receptor selective tachykinin analogs: senktide and septide.

The conformational behavior in solution of two receptor selective tachykinin agonists, senktide (succinyl-D-F-MeF-G-L-M-NH2) and septide (pQ-F-F-P-L-M-NH2) is described. Two dimensional cross relaxation NMR spectroscopy is used together with coupling constant data to obtain interproton distance constraints. These results are used in conjunction with semi-empirical energy computations to indicate favorable conformations. Senktide is found to have a high degree of conformational order which is attributed to rotational restriction associated with the N-methylation of phenylalanine. The lowest energy conformation in accord with the experimental interproton distances contains a beta-turn. Interproton distances indicate that septide exists as a random coil or in an extended chain conformation. Energy computations suggest that septide is primarily an extended chain with internal reorientation restricted by the proline residue. These results may be related to the selectivity of these peptides for different receptors, in that the analogs, with conformations more stable than tachykinins, are more receptor selective.     

Hyperolactone C: determination of its absolute configuration by comparison of experimental and calculated CD spectra

A detailed conformational analysis of hyperolactone C diastereomers and enantiomers ((5R,9R),(5S,9S) and (5S,9R),(5R,9S)) was done with molecular mechanics and density functional theory methods. Time-dependent density functional theory (B3PW91/TZVP) was used to calculate electronic transition energies (UV/vis spectra) and rotational strengths of the respective conformations. The effect of solvation (acetonitrile solution) on excitation energies and electronic circular dichroism was approximated by the polarizable continuum model. By comparison of the simulated CD spectrum with that measured for hyperolactone C isolated from Hypericum lloydii, its absolute configuration can be assigned as (5S,9S).     

Molecular modeling and NMR studies of benzyl substituted mannosyl trisaccharide binding to two mannose‐specific lectins: Allium sativam agglutinin I and Concanavalin A

The interaction of trimannoside, α?benzyl 3, 6‐di‐O‐(α‐D ‐mannopyranosyl)‐α‐D ‐mannopyranoside, 1 with ASAI (Allium sativam agglutinin I, garlic lectin) was studied to reveal the conformational preferences of this ligand in bound‐state and detailed binding mode at atomic level. The binding phenomenon was then compared with another well‐known mannose‐binding lectin, ConA (Concanavalin A). Structural studies of the ligand in free state were done using NMR spectroscopy and Molecular Dynamics simulations. It is found that the substituted‐trimannoside can undergo conformational transitions in solution, with one major and one minor conformation per glycosidic linkage (α 1→3 and α 1→6). On the other hand in the bound‐state only one of the two major conformations was significantly populated. The role of phenyl ring in the binding process was explored. An extended binding site was observed for the trimannoside in ASAI utilizing the aromatic substituent, which is not seen in ConA. Binding data from difference absorption spectroscopy supported this fact that the binding of benzyl‐substituted ligand is tighter with ASAI than ConA. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 952–967, 2010.     

DNA Models for A,B, C and D Conformations Related to Fiber X-ray,Infrared and NMR Measurements

Abstract

A conformational analysis of the A, B, C and D DNA forms was made in order to establish molecular models presenting a good agreement with experimental data obtained from fiber X-ray, infrared linear dichroism and ³¹P NMR. The proposed models have been refined and do present good stereochemistry and optimized H-bond distances between bases associated with the Watson-Crick pairing. The DNA conformations proposed are a left handed double helix for the C form and right handed helices for A, B and D. Relations to conformational transitions between these forms are discussed.     

Comparison of the conformation of poly(dI-dC) with poly(dI-dbr5C) and the B and Z forms of poly(dG-dC). One- and two-dimensional NMR studies

One- and two-dimensional nuclear Overhauser effects (2D NOE) have been used to compare the conformational properties of 60-80 base pair long duplexes of the synthetic DNA polymer poly(dI-dC) with those of poly(dI-dbr5C) and poly(dG-dC) in the B and Z conformations. Cross peaks in the 2D NOE spectra arising from proton-proton dipolar interactions which are more or less independent of the DNA conformation are used to assign the spectra of these molecules. Other cross peaks are sensitive to the conformational details, and these are used to make deductions about the average conformation in solution. The proton-proton interactions that give rise to the cross peaks in the 2D NOE spectrum of poly(dI-dC) are indicative of a B family conformation and rule out the possibility of some alternative conformations, including A, Z, alternating B, and left-handed B-DNA. The spectra are similar to those obtained from B-form poly(dI-dbr5C) and poly(dG-dC) but different from Z-form poly(dG-dC). Taken together, these results indicate that the solution conformation of poly(dI-dC) is not unusual but more closely resembles that of other B-form DNAs.     

Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8

We report the conformational analysis by 1H nmr in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of analogs of the cyclic octapeptide D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys 7]-Thr8-ol (sandostatin, octreotide). The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) contain stereochemical changes in the Thr residues in positions 6 and 8, which allow us to investigate the influence of the stereochemistry within these residues on conformation and binding affinity. The molecular dynamics simulations provide insight into the conformational flexibility of these analogs. The compounds with (S)-configuration at the C(alpha) of residue 6 adopt beta-sheet structures containing a type II' beta-turn with D-Trp in the i+1 position, and these conformations are "folded" about residues 6 and 3. The structures are very similar to those observed for sandostatin, and the disulfide bridge results in a close proximity of the H(alpha) protons of residues 7 and 2, which confirms earlier observations that a disulfide bridge is a good mimic for a cis peptide bond. The compounds with (R)-configuration at the C(alpha) of residue 6 adopt considerably different backbone conformations. The structures observed for these analogs contain either a beta-turn about residue Lys and Xaa6 or a gamma-turn about the Xaa6 residue. These compounds do not exhibit significant binding to the somatostatin receptors, while the compounds with (S) configuration in position 6 bind potently to the sst2, 3, and 5 receptors. The nmr spectra of analogs with (R) or (S) configuration at the C(alpha) of residue 8 are strikingly similar to each other. We have demonstrated that the chemical shifts of protons of residues 3, 4, 5, and 6, which are part of the type II' beta-turn, and especially the effect on the Lys gamma-protons are considerably different in active molecules as compared to inactive analogs. Since the presence of a type II' beta-turn is crucial for the binding to the receptors, the chemical shifts, the amide temperature coefficients of the Thr residue and the medium strength NOE between LysNH and ThrNH can be extremely useful as an initial screening tool to separate the active molecules from inactive analogs.