Bioactive conformation of linear peptides in solution: An elusive goal?

Bioactive peptides of natural origin have, in general, short linear sequences, and are characterized by a large conformational flexibility. It is very difficult to study their conformation in solution since they exist, almost invariably, as a complex mixture of numerous conformers, most of which are extended. The so-called bioactive conformation may be one of them, although the solvents used in solution studies often have properties drastically different from those of the biological system in which the peptide acts. There is, however, no simple way of identifying the bioactive conformation amid the many existing conformers. It is possible to approach a solution to this problem using two distinct strategies: (a) Limiting the conformational freedom of the peptide, e.g., by increasing the viscosity of the solution and decreasing the temperature, in the assumption that the bioactive conformation is, even slightly, more stable than the others. (b) Trying to mimic in solution the physicochemical features of the more reliable receptor models. These two approaches will be illustrated with examples taken mainly from opioid peptides.     

The nociceptin pharmacophore site for opioid receptor binding derived from the NMR structure and bioactivity relationships

Nociceptin, a 17 amino acid opioid-like peptide that has an inhibitory effect on synaptic transmission in the nervous system, is involved in learning, memory, attention, and emotion and is also implicated in the perception of pain and visual, auditory, and olfactory functions. In this study, we investigated the NMR solution structure of nociceptin in membrane-like environments (trifluoroethanol and SDS micelles) and found it to have a relatively stable helix conformation from residues 4-17 with functionally important N-terminal residues being folded aperidoically on top of the helix. In functional assays for receptor binding and calcium flux, alanine-scanning variants of nociceptin indicated that functionally important residues generally followed helix periodicity, consistent with the NMR structural model. Structure-activity relationships allowed identification of pharmacophore sites that were used in small molecule data base searches, affording hits with demonstrated nociceptin receptor binding affinities.     

Conformational restrictions of biologically active peptides via amino acid side chain groups

Determining the relationships between conformation and biological activity in peptide hormones and neurotransmitters is an important goal of contemporary biology. A major difficulty in these studies is the conformational flexibility of most peptides and the high dependence of the conformations on environment. The question arises whether conformations determined in solution are relevant to those important to the peptide at the membrane receptor(s). One recent approach to overcome these difficulties has been the use of conformational constraints by covalent bonding of side chain groups of residues in the peptide. In this manner linear peptides are rendered cyclic, and cyclic peptides are further conformationally constrained either by ring contractions or by other conformational constraints. Biologically active peptides specifically designed by this approach have been found to possess several useful properties including: 1) greater conformational integrity; 2) increased agonist or antagonist potency; 3) prolonged biological activity; 4) increased enzymatic stability; and 5) increased specificity for a particular receptor. Careful applications of this approach have provided important new designs features for peptide structure-function studies, and new insights into peptide conformation-activity relationships for oxytocin, somatostatin, enkephalin, bradykinin, vasopressin, and other biologically active peptides.     

Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion

Peptides containing the tripeptide sequence Arg-Gly-Asp can duplicate or inhibit the cell attachment-promoting effects of fibronectin and vitronectin. Peptides analogous to a prototype peptide, Gly-Arg-Gly-Asp-Ser-Pro-Cys, the sequence of which was taken from the cell attachment site of fibronectin, were assayed for their relative abilities to inhibit the attachment of cells to a fibronectin or vitronectin substrate. A peptide having the L-Arg residue replaced with D-Arg showed no difference in this capacity, whereas substituting Gly with D-Ala or L-Asp with D-Asp resulted in completely inactive peptides. Replacement of L-Ser with D-Ser drastically reduced the influence that the resulting peptide had on the vitronectin interaction, but this peptide showed little difference in its effect on the binding of cells to fibronectin when compared with the prototype peptide. Furthermore, substitution of the Ser with L-Asn resulted in a peptide that had an apparent increased preference for the fibronectin receptor and decreased preference for the vitronectin receptor. Conversely, threonine in this position gave a peptide with increased preference for the vitronectin receptor, whereas L-Pro in this position gave a completely inactive peptide. Finally, by cyclicizing the prototype peptide to restrict its conformational flexibility, a peptide was obtained that was a much improved inhibitor of attachment of cells to vitronectin and yet nearly inactive with respect to the interactions of cells with fibronectin substrates. These studies lend support to the hypothesis that different Arg-Gly-Asp-directed adhesion receptors can recognize differences in the conformation and environment of the Arg-Gly-Asp tripeptide, and they establish the feasibility of obtaining synthetic probes that are more selective for individual receptors than are the peptides modeled after the natural sequences of adhesive extracellular matrix molecules.     

Modeling of a C‐end rule peptide adsorbed onto gold nanoparticles

The RPAR peptide, a prototype C‐end Rule (CendR) sequence that binds to neuropilin‐1 (NRP‐1), has potential therapeutic uses as internalization trigger in anticancer nanodevices. Recently, the functionalization of gold nanoparticles with CendR peptides has been proved to be a successful strategy to target the NRP‐1 receptor in prostate cancer cells. In this work, we investigate the influence of two gold surface facets, (100) and (111), on the conformational preferences of RPAR using molecular dynamics simulations. Both clustering and conformational analyses revealed that the peptide backbone becomes very rigid upon adsorption onto gold, which is a very fast and favored process, the only flexibility being attributed to the side chains of the two Arg residues. Thus, the different components of RPAR tend to adopt an elongated shape, which is characterized by the pseudo‐extended conformation of both the backbone and the Arg side chains. This conformation is very different from the already known bioactive conformation, indicating that RPAR is drastically affected by the substrate. Interestingly, the preferred conformations of the peptide adsorbed onto gold facets are not stabilized by salt bridges and/or specific intramolecular hydrogen bonds, which represent an important difference with respect to the conformations found in other environments (e.g. the peptide in solution and interacting with NRP‐1 receptor). However, the conformational changes induced by the substrate are not detrimental for the use of gold nanoparticles as appropriate vehicles for the transport and targeted delivery of the RPAR. Thus, once their high affinity for the NRP‐1 receptor induces the targeted delivery of the elongated peptide molecules from the gold nanoparticles, the lack of intramolecular interactions facilitates their evolution towards the bioactive conformation, increasing the therapeutic efficacy of the peptide.     

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.     

Conformation-activity relationships of opioid peptides with selective activities at opioid receptors

The discovery of endogenous opioid peptides 25 years ago opened up a new chapter in efforts to understand the origins and control of pain, its relationships to other biological functions, including inflammatory and other immune responses, and the relationships of opioid peptides and their receptors to a variety of undesirable or toxic side effects often associated with the nonpeptide opiates such as morphine including addiction, constipation, a variety of neural toxicities, tolerance, and respiratory depression. For these investigations the need for potent and highly receptor selective agonists and antagonists has been crucial since they in principle allow one to distinguish unequivocally the roles of the different opioid receptors (mu, delta, and kappa) in the various biological and pathological roles of the opioid peptides and their receptors. Conformational and topographical constraint of the linear natural endogenous opioid peptides has played a major role in developing peptide ligands with high selectivity for mu, delta, and kappa receptors, and in understanding the conformational, topographical, and stereoelectronic structural requirements of the opioid peptides for their interactions with opioid receptors. In turn, this had led to insights into the three-dimensional pharmacophore for opioid receptors. In this article we review and discuss some of the developments that have led to potent, selective, and stable peptide and peptidomimetic ligands that are highly potent and selective, and that have delta agonist, mu antagonist, and kappa agonist biological activities (other authors in this issue will discuss the development of other types of activities and selectivities). These have led to ligands that provide unique insight into opioid pharmacophores and the critical roles opioid ligands and receptor scan play in pain, addiction, and other human maladies.     

Structure-function of the TNF receptor-like cysteine-rich domain of osteoprotegerin

Osteoprotegerin (OPG) is a soluble decoy receptor that inhibits osteoclastogenesis and is closely associated with bone resorption processes. We have designed and determined the solution structures of potent OPG analogue peptides, derived from sequences of the cysteine-rich domain of OPG. The inhibitory effects of the peptides on osteoclastogenesis are dose-dependent (10(-6) M-10(-4) M), and the activity of the linear peptide at 10(-4) M is ten-fold higher than that of the cyclic OPG peptide. Both linear and cyclic peptides have a beta-turn-like conformation and the cyclic peptide has a rigid conformation, suggesting that structural flexibility is an important factor for receptor binding. Based on structural and biochemical information about RANKL and the OPG peptides, we suggest that complex formation between the peptide and RANKL is mediated by both hydrophobic and hydrogen bonding interactions. These results provide structural insights that should aid in the design of peptidyl-mimetic inhibitors for treating metabolic bone diseases caused by abnormal osteoclast recruitment.     

Structure of the R3/I5 chimeric relaxin peptide, a selective GPCR135 and GPCR142 agonist

The human relaxin family comprises seven peptide hormones with various biological functions mediated through interactions with G-protein-coupled receptors. Interestingly, among the hitherto characterized receptors there is no absolute selectivity toward their primary ligand. The most striking example of this is the relaxin family ancestor, relaxin-3, which is an agonist for three of the four currently known relaxin receptors: GPCR135, GPCR142, and LGR7. Relaxin-3 and its endogenous receptor GPCR135 are both expressed predominantly in the brain and have been linked to regulation of stress and feeding. However, to fully understand the role of relaxin-3 in neurological signaling, the development of selective GPCR135 agonists and antagonists for in vivo studies is crucial. Recent reports have demonstrated that such selective ligands can be achieved by making chimeric peptides comprising the relaxin-3 B-chain combined with the INSL5 A-chain. To obtain structural insights into the consequences of combining A- and B-chains from different relaxins we have determined the NMR solution structure of a human relaxin-3/INSL5 chimeric peptide. The structure reveals that the INSL5 A-chain adopts a conformation similar to the relaxin-3 A-chain, and thus has the ability to structurally support a native-like conformation of the relaxin-3 B-chain. These findings suggest that the decrease in activity at the LGR7 receptor seen for this peptide is a result of the removal of a secondary LGR7 binding site present in the relaxin-3 A-chain, rather than conformational changes in the primary B-chain receptor binding site.     

Solution conformational study of nociceptin an(d its 1-13 and 1-11 fragments using circular dichroism and two-dimensional NMR in conjunction with theoretical conformational analysis.

Conformational studies of nociceptin (NC-NH2), its fully active fragment, NC(1-13)-NH2, and two significantly less potent fragments, NC(1-13)-OH and NC(1-11)-OH, were conducted in water and TFE solutions by the employment of circular dichroism, and in DMSO-d6 by 2DNMR spectroscopy in conjunction with theoretical conformational analysis. The conformations of all thepeptides studied were calculated taking two approaches. The first assumes multiconformational equilibrium of the peptide studied, which is characterized by a set of conformations (and their statistical weight values)obtained from a global conformational analysis using three methods: the electrostatically driven Monte-Carlo (EDMC) with the ECEPP/3 force field, the simulated annealing (SA) protocols in the AMBER and CHARMM force fields. The second approach incorporates the interproton distance and dihedral angle constraints into the starting conformation. Calculations were performed using the distance geometry and SA protocol in the CHARMM force field implemented in the X-PLOR program. The CD experiments indicated that for the active peptides, hydrophobic solvents induced a significantly higher (compared with those remaining)content order, probably a helical structure. Unfortunately, as a result of the conformational flexibility of thepeptides, the analysis of conformations obtained with both approaches and different force fields did not alllow the selection of any structural elements of the NC peptides that might be connected with their bioactivity. The only common element found in most conformations of the active peptides was a helical character of fragment 8-13, which allowed the side chains of basic amino acid residues to be exposed to the outside of the molecule and probably to interact with the ORL1 receptor.     

Conformation and environment of channel-forming peptides: a simulation study

Ion channel-forming peptides enable us to study the conformational dynamics of a transmembrane helix as a function of sequence and environment. Molecular dynamics simulations are used to study the conformation and dynamics of three 22-residue peptides derived from the second transmembrane domain of the glycine receptor (NK4-M2GlyR-p22). Simulations are performed on the peptide in four different environments: trifluoroethanol/water; SDS micelles; DPC micelles; and a DMPC bilayer. A hierarchy of alpha-helix stabilization between the different environments is observed such that TFE/water < micelles < bilayers. Local clustering of trifluoroethanol molecules around the peptide appears to help stabilize an alpha-helical conformation. Single (S22W) and double (S22W,T19R) substitutions at the C-terminus of NK4-M2GlyR-p22 help to stabilize a helical conformation in the micelle and bilayer environments. This correlates with the ability of the W22 and R19 side chains to form H-bonds with the headgroups of lipid or detergent molecules. This study provides a first atomic resolution comparison of the structure and dynamics of NK4-M2GlyR-p22 peptides in membrane and membrane-mimetic environments, paralleling NMR and functional studies of these peptides.     

Conformations of neurotensin in solution and in membrane environments studied by 2-D NMR spectroscopy

Two-dimensional HOHAHA and ROESY nuclear magnetic resonance techniques are used to obtain complete proton resonance assignments and to perform a conformational investigation of the neuropeptide neurotensin (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) in aqueous solution, methanol, and membrane-mimetic [deuterated sodium dodecylsulfate (SDS)] environments. Results suggest the absence of discernible elements of secondary structure in water and methanol. ROESY spectra confirm that Lys-Pro and Arg-Pro peptide bonds are all-trans, but that a significant population of cis Arg-Pro bonds arises in aqueous solution, which increases in the environment of SDS micelles. The conformational ensemble of the peptide is observed to narrow as it becomes bound through its cationic mid-region to SDS micelles, with the accompanying advent of local extended structure. The overall results indicate the inherent conformational flexibility of neurotensin, and emphasize the environmental dependence of conformation in peptides of medium length.     

New insights on mu/delta selectivity of opioid peptides: conformational analysis of deltorphin analogues

The message domain of dermorphin (Tyr-D-Ala-Phe), a natural mu-opioid heptapeptide, has long been considered the main cause of the high mu selectivity of this peptide and of its analogues. The recent discovery, in the skin of Phyllomedusa sauvagei (i.e., the same natural source of dermorphin) and of Phyllomedusa bicolor of deltorphins, challenges this belief. Deltorphins, in fact, are three heptapeptides characterized by a message domain typical of mu-selective peptides, but endowed of an extremely high delta selectivity, the highest of all natural opioid peptides. A conformational analysis of dermorphin and deltorphins, based on nmr studies in DMSO and cryoprotective mixtures and internal energy calculations, showed that the enormous differences in receptor selectivity can be interpreted on the basis of receptor models for mu and delta opioids that recognize the same beta-turn in the N-terminal part, but discriminate for the conformation and polarity of the C-terminal part. Here we present the synthesis, biological activity, and conformational analysis in solution of three deltorphin analogues with very similar constitution, but with different net charge, different location of negative residues, or even without negative residues, which confirm these hypotheses and show that His4 can play a specific structural role.     

Opioid receptor selectivity of peptide models of beta-endorphin

Two peptides, designed to contain structural models of the proposed hydrophilic linker domain (residues 6-12) and amphiphilic alpha-helical domain (residues 13-29) in beta-endorphin, have been tested for their abilities to mimic the opioid receptor selectivity profile of the natural hormone. In competitive binding assays employing guinea-pig brain membranes, both peptides displayed a much higher affinity for mu- and delta-opioid receptors than for kappa opioid receptors. Relative to beta-endorphin, the peptide models were 2-3 times more potent in the mu and kappa receptor binding assays, and about equipotent in the delta receptor binding assay. In guinea-pig ileum assays, one peptide was equipotent to beta-endorphin and the other was twice as potent. Like beta-endorphin, their actions on this tissue were highly sensitive to naloxone antagonism, indicating that they were mediated by mu receptors and not kappa receptors. In view of the design of the two peptide models, and their minimal homology to the natural hormone, these results provide additional evidence in support to our proposal for the functional conformation of beta-endorphin.     

Structure determination and dynamics of peptides overlapping the catalytic hairpin of the Ras-specific GEF Cdc25(Mm)

Ras proteins are small G proteins playing a major role in eukaryotic signal transduction. Guanine nucleotide exchange factors (GEF) stimulate GDP/GTP exchange, resulting in the formation of the active Ras-GTP complex. In mammalian cells, two major Ras-specific GEF exist: Sos-like and Cdc25-like. To date, structural data are available only for Cdc25(Mm). We designed and synthesized Cdc25(Mm)-derived peptides spanning residues corresponding to the hSos1 HI helical hairpin that has been implicated in the GEF catalytic mechanism. NMR experiments on a chemically synthesized Cdc25(Mm)(1178-1222) peptide proved that helix I readily reaches a conformation very similar to the corresponding helix in hSos1, while residues corresponding to helix H in hSos1 show higher conformational flexibility. Molecular dynamics studies with the appropriate solvent model showed that different conformational spaces are available for the peptide. Since helix H is making several contacts with Ras and a Cdc25(Mm)(1178-1222) peptide is able to bind nucleotide-free Ras in a BIAcore assay, the peptide must be able to obtain the proper Ras-interacting conformation, at least transiently. These results indicate that rational design and improvement of the Ras-interacting peptides should take into account conformational and flexibility features to obtain molecules with the appropriate biochemical properties.     

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.     

New features of the delta opioid receptor: conformational properties of deltorphin I analogues

Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.     

Nociceptin/orphanin FQ in spinal nociceptive mechanisms under normal and pathological conditions

Nociceptin and its receptor are present in dorsal spinal cord, indicating a possible role for this peptide in pain transmission. The majority of functional studies using behavioral and electrophysiological studies have shown that nociceptin applied at spinal level produces antinociception through pre- and post-synaptic mechanisms. The spinal inhibitory effect of nociceptin is not sensitive to antagonists of opioid receptors such as naloxone. Thus, nociceptin-induced antinociception is mediated by a novel mechanism independent of activation of classic opioid receptors. This has raised the possibility that agonists of the nociceptin receptor may represent a novel class of analgesics. Supporting this hypothesis, several groups have shown that intrathecal nociceptin alleviated hyperalgesic and allodynic responses in rats after inflammation or partial peripheral nerve injury. Electrophysiological studies have also indicated that the antinociceptive potency of spinal nociceptin is maintained or enhanced after nerve injury. It is concluded that the predominant action of nociceptin in the spinal cord appears to be inhibitory. The physiological role of nociceptin in spinal nociceptive mechanisms remains to be defined. Moreover, further evaluation of nociceptin as a new analgesic calls the development of non-peptide brain penetrating agents.     

Non-opioid actions of opioid peptides

Beside the well known actions of opioid peptides on mu-, delta- and kappa-opioid receptors, increasing amount of pharmacological and biochemical evidence has recently been published about non-opioid actions of various opioid peptides. These effects are not abolished by naloxone treatments. Such non-opioid effects are observed both in nervous tissues and in the cellular elements of the immune system. Peptides exhibiting non-opioid effects include beta-endorphin, dynorphin A, nociceptin/OFQ, endomorphins, hemorphins and a number of Proenkephalin A derived peptides, such as Met-enkephalin, Met-enkephalin-Arg-Phe (MERF) and bovine adrenal medullary peptide (BAM22). Non-opioid actions are exerted through different neuronal receptors, e.g., dynorphin hyperalgesia through NMDA receptor, Met-enkephalin induced regulation of cell growth through zeta receptors, pain modulation by nociceptin through ORL-1 or NOP receptors, while BAM22 acts through sensory neuron specific G protein-coupled receptors (SNSR). We have investigated Met-enkephalin-Arg-Phe (MERF) and its analogues by the means of direct and indirect radioligand binding assays. It has been found that in addition to kappa(2) and delta-opioid receptors, MERF can act also through sigma(2)- or probably via FMRF-NH(2) receptors in rat cerebellum. A role of functionally assembling heterodimer receptors in mediating the non-conventional actions of these peptide ligands can not be excluded as well.     

Role of solution conformation and flexibility of short peptide ligands that bind to the p56(lck) SH2 domain

A general approach in drug design is making ligands more rigid in order to avoid loss in conformational entropy (deltaS(conf)) upon receptor binding. We hypothesized that in the high affinity binding of pYEEI peptide ligands to the p56(lck) SH2 domain this loss in deltaS(conf) might be diminished due to preorganization of the fourfold negatively charged pYEEI peptide in the bound, extended, conformation. A thermodynamic analysis was performed on the peptides Ac-pYEEI-NH(2), Ac-pYAAI-NH(2) and Ac-pYGGI-NH(2) using surface plasmon resonance (SPR) competition experiments to assay affinity constants at different temperatures. To study the effect of solution conformation and flexibility a computational conformation analysis was performed from which low energy conformations in solution were calculated, and S(conf) estimated. It was found that the calculated low energy conformations for especially the pYE moiety in solution resemble that in the bound state. In the calculated minimum energy conformation in solution isoleucine is bent towards the pY aromatic ring, the occurrence of such conformation is experimentally confirmed by NMR. The estimated values for S(conf) of the EE- and AA-peptide were similar, suggesting no predominant role of preorganization of the solution conformation due to electrostatic repulsion. Apparently the thermodynamics obey the same entropy-enthalpy compensation relationship, which also was found to hold for other peptides and peptidomimetics binding to p60(src) family SH2 domains. The implications of the results for drug design are discussed.