(η6-Cp*Rh-Tyr1)-Leu-enkephalin - ACS Publications - American

Aug 7, 2015 - ABSTRACT: Recently reported studies by Kobilka et al. (Nature 2012, 485, 321, 400) and Stevens et al. (Nature 2012,. 485, 327) have ...
1 downloads 0 Views 5MB Size
Download PDF
Article pubs.acs.org/Organometallics

Quantum Chemical and Molecular Docking Studies of [(η6‑Cp*RhTyr1)‑Leu-enkephalin]2+ to G‑Protein-Coupled μ‑, ∂‑, and κ‑Opioid Receptors and Comparisons to the Neuropeptide [Tyr1]‑Leuenkephalin: Conformations, Noncovalent Amino Acid Binding Sites, Binding Energies, Electronic Factors, and Receptor Distortion Forces Irena Efremenko*,† and Richard H. Fish*,‡ †

Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, United States

‡

S Supporting Information *

ABSTRACT: Recently reported studies by Kobilka et al. (Nature 2012, 485, 321, 400) and Stevens et al. (Nature 2012, 485, 327) have characterized the structures of the G-proteincoupled μ-, ∂-, and κ-opioid receptors (GPCORs) via X-ray crystallography, including the use of guest, morphinan antagonist, drug analogues. These GPCORs have been shown to control the physiological functions of pain and, therefore, have been designated as a prime target for new, nonaddictive, pain drug discoveries. Moreover, Fish et al. (J. Am. Chem. Soc. 2012, 134, 10321) have recently reported on a chemoselective reaction of GPCR tyrosine-containing peptides with [Cp*Rh(H2O3)](OTf)2 to provide [(η6-Cp*Rh-Tyr#)GPCR-peptide](OTf)2 complexes. For example, the agonist, endogenous neuropeptide [Tyr1]-Leu-enkephalin, 1 (Tyr1-Gly-GlyPhe-Leu), upon reaction with the Cp*Rh tris aqua complex, at pH 5−6, gave the [(η6-Cp*Rh-Tyr1)-Leu-enkephalin](OTf)2 complex 2, also an agonist, which was found to bind to individual and coexpressed μ- and ∂-opioid receptor cells. Therefore, we present, in this contribution, the first comprehensive quantum chemical and molecular docking studies of an organometallic− neuropeptide complex, 2, to structurally characterized μ-, ∂-, and κ-GPCORs. We found that the docked conformations of dication 2 at the three opioid receptors were in similar receptor locations to the natural neuropeptide 1, as well as the morphinan drug derivatives, all antagonists, used in the X-ray structures of the μ-, ∂-, and κ-opioid receptors, but, importantly, had distinctly different noncovalent H-bonding, π−π, and CH−π interactions with the nearby transmembrane receptor amino acids compared to 1, with only H-bonding interactions. Therefore, quantum chemical calculations showed this was due to four critical factors: (a) Dication 2 was found to be a non-zwitterion versus 1 being a zwitterion; (b) significant differences in the electron density and hydrophobic effects of the (η6-Cp*Rh-Tyr1)2+ versus the (Tyr1) moieties on the message paradigm for receptor molecular recognition; (c) binding energies of 2 in comparison to 1, for the opioid receptors; and (d) receptor distortion forces that could possibly hinder binding regimes of 1 and 2, especially to the κ-opioid receptor. Furthermore, we have attempted to understand how these factors might possibly be related to the previously reported EC50 receptor binding values (nM) of agonists 1 and 2 at the μ-, ∂-, and κ-opioid receptors.

■

al. providing the μ- and ∂-opioid structures with their antagonist morphinan guest derivatives, while the κ-opioid receptor structure, also with a antagonist morphanin guest, was solved by Stevens et al. (Scheme 1).2a−c The X-ray data showed a common seven-transmembrane structure (Scheme 1, left, ∂-OR as an example) for the μ-, ∂-, and κ-opioid receptors, with conserved areas (Scheme 1, center), including conserved βstrand folds (Scheme 1, right).2b Moreover, B. Kobilka and his mentor, R. Lefkowitz, won the 2012 Nobel Prize in Chemistry

INTRODUCTION G-Protein-coupled receptors (GPCRs) have been studied extensively, since they have been shown to influence the physiological responses to hormones, neurotransmitters, and environmental stimulants, and, thus, have great importance and focus for new drug discoveries for a wide spectrum of diseases.1 The μ-, ∂-, and κ-opioid receptors have been some of the most extensively studied GPCRs, but what was missing was their uniquivocal X-ray crystal structures, in order to better design nonaddictive drug candidates for pain and addiction therapy. Recently, the X-ray structures of the μ-, ∂-, and κ-opioid receptors (OR) were published by two groups, with Kobilka et © XXXX American Chemical Society

Received: June 23, 2015

A

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics

Scheme 1. ∂-Opioid Receptor (OR) Showing the Typical Seven-Transmembrane Structure (Left); Usual Conserved Fold Structures for ∂-, μ-, and κ-ORs (Center, Color Coded); and the Usual Conserved β-Strand Folds, with an Expansive, Open Binding Pocket (Right)2b

for their seminal studies of GPCRs, which definitively showed the significance of this exciting field at the interface of chemistry and biology.3 The bioorganometallic chemistry discipline has clearly shown that organometallic chemistry and biology are also compatible at their respective interfaces.4 Furthermore, the bioconjugation of peptides with organometallic complexes has been studied to a great extent, in order to discover new metal-based drugs.5 However, the methods that have been utilized to derivitize peptides with organometallic reagents have focused mainly on the terminal amino and carboxylic acid groups, in mostly organic solvents, but this may impinge on the molecular recognition process at the designated receptor sites and be deliterious for bioactivity. For example, in the μ-, ∂-, and κopioid receptors, these terminal groups were found to be totally necessary for molecular recognition and biological activity.1 More recently, Fish et al. have shown, for the first time, that the dication, Cp*Rh tris aqua complex [Cp*Rh(H2O)3](OTf)2 reacted with tyrosine-containing GPCR peptides, in water at pH 5−6 and RT, to chemoselectively provide the [(η6-Cp*RhTyr#)-GPCR-peptide](OTf)2 dicationic complexes (eq 1).6 For

Figure 1. DFT-calculated lowest energy forms of [Tyr1]-Leuenkephalin, as a zwitterion, 1, and dication [(η6-Cp*Rh-Tyr1)-Leuenkephalin]2+, a non-zwitterion, 2 (vide inf ra).

opioid receptors. This included zwitterion versus nonzwitterion calculations for 1 and 2 and the role of the terminal [η6-Cp*Rh-Tyr1]2+ residue, the message (signal transduction) aspect of the message−address paradigm for molecular recognition, as to the lowest energy conformations to accommodate the receptor amino acid noncovalent binding regimes, receptor binding energies, receptor distortion forces, and the critical electronic effects of the pharmacophore, dicationic [η6-Cp*Rh-Tyr1]2+ moiety on the noncovalent phenol O−H, amino N−H, and amide CO binding with receptor amino acids. Furthermore, we also included the phenylalanine residue (message sequence) and the terminal leucine residue (address sequence, which provides selectivity for an opioid receptor) in these molecular docking experiments. Finally, we will attempt to show how these factors may possibly be related to the previously found EC50 receptor binding values (nM) of agonists 1 and 2 at the μ-, ∂-, and κ-opioid receptors.6 Moreover, a much less sophisticated molecular docking approach with zwitterion [Tyr1]-Leu-enkephalin and [(η6Cp*Rh-Tyr1)-leu-enkephalin], as a non-dication zwitterion, to the μ- and ∂-opioid receptors, was recently reported7 as we were preparing our own manuscript, and many neglected, critical factors not addressed in that preliminary account, such as zwitterion/non-zwitterion energy calculations, important electronic effects of the [η6-Cp*Rh-Tyr1]2+ dicationic moiety on noncovalent binding of complex 2 to the μ- and ∂-opioid receptors, binding energies, receptor distortion forces that could possibly affect binding regimes, and using optimized conformations with the lowest energy forms of [Tyr1]-Leuenkephalin, 1, and complex 2, at the μ- and ∂-opioid receptors, will be, as stated, the focus of this contribution.

example, reaction of the neuropeptide [Tyr1]-Leu-enkephalin as a zwitterion, 1, with [Cp*Rh(H2O)3](OTf)2, at pH 5−6 and RT, gave a quantitative yield of the [(η6-Cp*Rh-Tyr1)-Leuenkephalin](OTf)2 complex and the computationally calculated non-zwitterion (vide inf ra) 2, an agonist, which was found to also bind (EC50 values, nM) to the individual and coexpressed μ- and ∂-opioid receptor cells, as does the natural neuropeptide 1 (Figure 1).6 Thus, in this contribution, we present a comprehensive quantum chemical and molecular docking study on the noncovalent interactions for 1 and 2, at the μ-, ∂-, and κB

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics

■

RESULTS AND DISCUSSION Zwitterions versus Non-zwitterions for [Tyr1]-Leuenkephalin, 1, and [(η6-Cp*Rh-Tyr1)-Leu-enkephalin]2+, 2. It has been universally accepted that amino acids readily undergo self-ionization with formation of zwitterions. In several cases, amino acids in the form of zwitterions were found in the solid state or even in the gas phase, but were readily found as zwitterions in aqueous solution, between pH 2.2 and 9.4. Computational results indicated that for stabilization of zwitterions direct solvation by two to five water molecules, with stabilization energies of up to 35 kcal mol−1, was needed.8 In small molecules, where the positive and negative charges were close to each other, these explicit water molecules were interconnected by cyclic hydrogen-bonding networks, terminating at the charged carboxyl and ammonium groups. Thus, we first calculated the relative energies of the two forms of 1 and 2 in water, i.e., non-zwitterion and zwitterion forms, 1a, 1b and 2a, 2b, respectively. Energetic results, at the PBE0/D3BJ/ PC-1/COSMO level of theory, demonstrated that, in water media, the [Tyr1]-Leu-enkephalin zwitterion, 1b, was more stable than the neutral isomer, 1a, by 4.7 kcal/mol at room temperature. No explicit water molecules were needed in this case, because of the large spatial separation of the charged carboxyl and amino groups of 15.2 Å. More importantly, although in the zwitterion form of complex 2b, the distance between the charged carboxyl and amino groups was ∼15.0 Å, the dicationic charge of 2b made this zwitterion form less stable than the non-zwitterion form, 2a, by 10.9 kcal/mol, in water, at room temperature. Moreover, the monocationic form of complex 2a (NH2−...−COO−) was also found to be even less stable, i.e., 23.8 kcal/mol higher in energy than the nonzwitterion dicationic form. These results indicated that zwitterion 1b and dicationic, non-zwitterion 2a represented the most stable forms of the free molecules in water. The optimized, DFT-calculated geometries of both zwitterion 1b and dicationic non-zwitterion 2a are shown in Figure 2; the atomic xyz coordinates can be found in the Supporting Information.

nature of 2a, as depicted in Figure 2. Clearly, there was an ∼180° rotation turn of the [η6-Cp*Rh-Tyr1]2+ residue in 2a, which was now trans to the phenyl group of the phenylalanine residue, in comparison to 1b, and defined the role of the η6Cp*Rh moiety in the conformational change of the phenol group of [Tyr1]-Leu-enkephalin 1b. Since the X-ray structures of the μ-, ∂-, and κ-opioid receptors were recently published in 2012,2a−c we decided to evaluate the molecular recognition sequences, the message− address paradigm, that have been previously designated for neuropeptides, such as 1b, at GPCORs and compare them to those found for complex 2a via molecular docking calculations.1 Furthermore, [Tyr1]-Leu-enkephalin 1b, an endogenous neuropeptide agonist, has been shown to bind to μ- and ∂-opioid receptor cells, but negligible/weak binding was found for the κopioid receptor.9 However, we wanted to compare all three, μ-, ∂-, and κ-opioid receptors, in noncovalent binding with neuropeptide 1b and complex 2a, to ascertain the differences in conformations and binding to all opioid receptor amino acid residues that were structurally characterized. Mulliken Charges for the (Tyr1-) and [(η6-Cp*Rh-Tyr12+ )] Moieties of Neuropeptide Leu-enkephalin 1b and Complex [(η6-Cp*Rh-Tyr1)-Leu-enkephalin]2+ 2a. Since the η6-Cp*Rh moiety will have a profound effect on the atomic charges of the phenol, O−H, terminal amino, N−H, and amide CO of the tyrosine residue (message sequence), we analyzed this aspect for its possible role in the molecular recognition process (Figure 3). What the Mulliken charge

Figure 3. Calculated Mulliken charges on the [Tyr1] residue of 1b (left) and on the [η6-Cp*Rh-Tyr1]2+ residue of complex 2a (right).

analysis demonstrated was that this interaction caused significant depletion of electron density on the phenol ring; it was found to be virtually neutral in 1b, for comparison, but in complex 2a, its Mulliken charge was +0.32. This resulted in the redistribution of electron density in the tyrosine moiety (message paradigm). Thus, the phenolic O atom has lost most of its nucleophilicity; the total Mulliken charge had changed from −0.16 to −0.05 upon η6 coordination to the Cp*Rh moiety. Similarly, the terminal NH3+ group of 1b was found to be electrophilic, +0.68, in comparison to the terminal amino group of 2a, −0.04. It should also be noted that a more detailed analysis showed that the O−H bond in the phenol O− H group, and the N−H bond in the terminal amino group, became more polar in complex 2a; the exact numbers in the phenol O atom changed from −0.40 to −0.34 and the H atom from +0.23 to +0.28, while in the amino group the effect was not significant. The N atom changed from −0.41 to −0.43, and similarly, the H atoms were also not significant, with values changing from +0.18 and +0.16 to +0.19 and +0.20. More importantly, the electron density on the carbonyl oxygen of the

Figure 2. Optimzed structures of [Tyr1]-Leu-enkephalin, as a zwitterion, 1b, and dication, [(η6-Cp*Rh-Tyr1)-Leu-enkephalin]2+, a non-zwitterion, 2a.

The DFT-calculated optimized structures of 1b and 2a, shown in Figure 2, demonstrated the pronounced effect on the conformation of the neuropeptide 1b when an [η6-Cp*RhTyr1)]2+ complex was formed; interestingly, the DFTcalculated, lowest energy structure of 2a appeared somewhat different from that of the 2D NMR structure recently reported,7 which failed to represent the non-zwitterion and dicationic C

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics tyrosine amide group was slightly higher than that in 1b (−0.57 in comparison to −0.51), such that this atom becomes important as a potential H-bond acceptor in complex 2a. More significantly, Figure 3 demonstrated that the NH3+ group of 1b is now a H-donor, while the NH2 group in 2a is a Hacceptor. Moreover, the phenol O−H group for 1b is both a potential H-acceptor and H donor, while the now electrondepleted phenol O−H for 2a is a better H-donor than Hacceptor. Furthermore, the terminal amide carbonyl of 2a is now a potentially better H-acceptor, due to the increased electron density on the amide carbonyl oxygen atom, in comparison to the amide carbonyl oxygen of 1b. These dramatic electron density consequences for 2a, in comparison to 1b, will be shown to have a pronounced effect on the noncovalent molecular recognition regimes of amino acid residues in the μ-, ∂-, and κ-opioid receptors for both guest molecules. Molecular Recognition Sequences of Peptide 1b and Complex 2a at the μ-, ∂-, and κ-Opioid Receptors. The important molecular recognition components of the opioid neuropeptide [Tyr1]-Leu-enkephalin 1b (Tyr1-Gly-Gly-PheLeu) and consequently complex 2a were the terminal tyrosine residue with the phenol, terminal amino, and amide carbonyl groups and the phenylalanine residue, which have been designated as prominent recognition points at the μ-, ∂-, and κ-opioid receptors and labeled the message sequence (signal transduction) of the message−address paradigm for binding to the opioid receptors.1 Moreover, the terminal leucine for 1b, and now for 2a, provided selectivity and/or reduced affinity for another opioid receptor and has been designated the address sequence, while the spacers are the glycine, glycine amino acids between the message and address sequences (Figure 4).1

Thus, the message−address paradigm was validated for the pharmacological aspects with these opioid receptor X-ray structural results, in that any single drug provided both message and address recognition aspects, which were contained in an individual drug molecule that formed noncovalent H-bonds with receptor amino acid residues and had conformational flexibility.2a−c However, it should be clearly pointed out that all three morphanin derivatives used in the X-ray structural determinations of the three opioid receptors were antagonists that do not elicit a biological response and, therefore, are not in biologically active conformations. This factor should allow us to verify with agonists 1b and 2a the differences in noncovalent binding to the opioid receptor amino acids, in comparison to the biologically inactive morphanin derivatives used for the Xray structural analysis and possibly use this information to better understand the noncovalent binding criteria that elicit bioactivity. Comparison of the Conformations inside the Receptors, Noncovalent Binding Regimes of Peptide 1b and Complex 2a at the μ-, ∂-, and κ-Opioid Receptors within 5 and 3.5 Å, and H-Bond, π−π, and CH−π Noncovalent Interactions. The neuropeptide 1b, docked at the μ-, ∂-, and κ-opioid receptors (Figure 5), showed the conformation inside the opioid receptors (left, center left), the message sequences, the tyrosine residue, and the phenylalanine carbonyl, along with the address sequence, the leucine residue terminal carboxyl, as the major sites of noncovalent molecular recognition (right). Furthermore, in the reported X-ray structure of the μ-opioid receptor, the morphinan derivative, the β-FNA guest ligand, was found to have reacted with the Lys 233 amino acid residue, via a Michael addition, to provide a covalent bond to the receptor; this would have invalidated our quest for noncovalent binding sites, and in this case, the covalent ligand−receptor bond was removed prior to building the μ-opioid binding site for docking 1b and 2a. Since both guest ligand and binding site torsion angles were set to be flexible during our docking process, the binding site was thought to have been relaxed to its original structure (see the Experimental Section). Figure 5 also shows (center right) that there are more than 15 amino acid residues within 5 Å (back side not shown) of docked neuropeptide 1b at the μ-opioid receptor, more than 15 residues at the κ-opioid receptor, and more than 16 residues at the ∂-opioid receptor. The number of amino acid residue sites with noncovalent binding to neuropeptide 1b, within 3.5 Å (right), for the μ-opioid receptor had four prominent Hbonding sites that included the message tyrosine phenol O and H atoms with the Ala 304 N−H atom, 2.84 Å, and the Val 300 carboxyl O atom, 2.01 Å; the spacer Gly carbonyl O atom with the N−H atom of Lys 233, 2.72 Å; and the address terminal Leu carboxyl O atom with the phenol O and H atoms of Tyr 148, 2.90 Å. The docked conformation of 1b in the μ-opioid receptor is shown on the left to demonstrate the position in the receptors (center left). The κ-opioid receptor with docked 1b had 10 prominent Hbonding sites within 3.5 Å (Figure 5), which included the message tyrosine phenol O and H atoms with the Cys 210 carboxyl O atom, 2.35 Å, and a H2O molecule H atom, 2.67 Å; the message NH3+ H atom to both the Thr 111 COO− and O− H atoms, 2.96, 2.78 Å; the spacer Gly carbonyl O atom with the phenol O−H of Tyr 312, 2.35 Å, and the Gly N−H with the phenol O atom, Tyr 312, 3.05 Å; the message phenylalanine N−H bond with the amide carbonyl O atom of Asp 138, 3.20

Figure 4. Message, spacer, and address sequences for molecular recognition at the μ-, ∂-, and κ-opioid GPCRs of complex 2a: the message−address paradigm.

Therefore, from the Kobilka/Stevens et al. X-ray studies of the μ-, ∂-, and κ- opioid receptors, it was found that the binding sites were separated into two regions, where the lower part of the binding pocket (message; signal transduction) was greatly conserved among all opioid receptors (Scheme 1), while the less conserved upper part of the binding pocket provided opioid receptor selectivity (address paradigm).2a−c D

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics

Figure 5. Conformation of 1b in the designated opioid receptor (left, center left); noncovalent binding regimes of [Tyr1]-Leu-enkephalin 1b to the μ-, κ-, and ∂-opioid receptors: the amino acid residues of the binding sites within 5 Å of docked peptide 1b (center right) and hydrogen bonds responsible for the noncovalent receptor interactions. H-bond lengths indicate the distance between the heavy atoms (not hydrogen) within 3.5 Å (right). “Top” refers to the binding pocket receptor opening.

Å; and the address terminal Leu COO− atoms with the NH3+ H atom of Tyr 320, 2.58 Å, and the N−H of Gly 319, 2.97 Å, with the Leu carboxyl carbonyl. The ∂-opioid receptor with docked 1b had 11 prominent Hbonding sites within 3.5 Å that included the message tyrosine phenol O−H atoms with the amide carbonyl of Asp 128, 2.42 Å, the message phenol H to O Tyr 308, 2.98 Å, and the Asp 128 carboxyl H with the message Tyr phenol O−H, 3.40 Å; the message NH3+ H atom to both the carboxyl of Ile 277, 2.99 Å, and N ring atom of His 278, 3.12 Å; the message phenylalanine amide carbonyl with the O−H of Thr 285, 3.05 Å, and the N− H of Lys 214, 3.28 Å; the address Leu carboxyl carbonyl with the ring N−H, Trp 284, 2.64 Å, and carboxyl O atom with the N−H of Lys 214, 2.87 Å, and the O−H of Thr 285, 2.43 Å. Interestingly, Figure 5 also demonstrated the differences in binding modes of the μ- and ∂-opioid receptors with 1b. For example, the message tyrosine phenol of 1b in the μ-opioid receptor was at the top binding, less conserved sites, while the message phenyl of phenylalanine and address leucine residue were at the inner conserved areas of the receptor. The ∂-opioid receptor had the reverse situation with guest 1b, with the message tyrosine residue (signal transduction) in the more conserved inner areas, while the address (selectivity) leucine residue was in the less conserved top of the receptor. It was clear that the ∂-opioid receptor binding site of amino acids with guest 1b had the most critical message−address noncovalent Hbonding interactions (11 for the ∂-OR versus four for the μ-

OR) with neuropeptide 1b. This was also reflected in the EC50 binding constants we found for 1b in our previous study, with values for the μ-opioid receptor of 14.3 nM versus the ∂-opioid receptor of 4.4 nM (vide inf ra), the ∂-opioid receptor having a potency/selectivity factor of 3.25 times that of the μ-opioid receptor for 1b.6 While the κ-opioid receptor also had many noncovalent Hbonds (10 interactions) to the message−address structural aspects of 1b, it was stated in the literature that 1b had a weak binding value (EC50 > 1000 nM) to the κ-opioid receptor,9 in contrast to the potency of the μ- and ∂-opioid receptors, which seems to indicate that selectivity to the κ-opioid receptor and its biological activity (EC50 value) are compatible; however, other factors not related to the noncovalent binding regimes must be responsible for the weak interactions of 1b and 2a to the κ-opioid receptor. Moreover, Asp 138 has been shown by Stevens et al. to be important for selectivity to the κ-opioid receptor with the morphanin analogue JDTic as the guest.2c Furthermore, we see an Asp 138 carboxyl carbonyl H-bond to the phenylalanine amide N−H of 1b, but this may not be enough stabilization to facilitate any selectivity to the κ-opioid receptor or be important for biological activity, since JDTic is an antagonist, which does not elicit a biological response, while being conformationally stable. We have shown, in Figure 3, that the electronic effect of the [η6-Cp*Rh-Tyr1] moiety of 2a dramatically reduced the nucleophilicity of the phenol O atom while making the O−H E

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics

Figure 6. Lowest energy conformation of 2a inside the μ-, κ-, and ∂-opioid receptors (left, center left); the structural basis for noncovalent binding regimes of dication complex 2a with amino acid residues at the binding sites within 5 Å of the docked ligand (center right); hydrogen bond, π−π, and CH−π noncovalent interactions responsible for the noncovalent receptor interactions. The H-bond lengths indicate the distance between the heavy atoms (not hydrogen) within 3.5 Å (right).

more acidic and a H-donor. Further, the −NH2 group was converted into a H-acceptor, as well as the amide CO. Thus, the Mulliken charge differences between 1b and 2a were dramatic and have changed the noncovalent bonding modes in all three opioid receptors in comparison to neuropeptide 1b. Moreover, the Cp*Rh moiety became a site for π−π and CH−π molecular recognition, while the phenyl group of the phenylalanine residue was also a site for π−π interactions; we found none of these π−π or CH−π interactions within 3.5 Å for 1b. Thus, the important noncovalent H-bond, π−π, and CH−π interactions (within 3.5 Å) with selective amino acids in the conserved (bottom of the binding pocket) or less conserved (top of binding pocket) areas of the μ-, ∂-, and κ-opioid

receptors for 2a are as follows. Figure 6 shows the lowest energy conformation of 2a inside the μ-opioid receptor (left, center left), while more than 17 amino acid residues (back side not shown) for the μ-opioid receptor are within 5 Å (center right, “Top” refers to the receptor binding pocket opening) and eight H-bond, π−π, and CH−π noncovalent interactions (right) are within 3.5 Å. Importantly, the [η6-Cp*Rh-Tyr1]2+ residue of complex 2a was found to be in the more inner conserved area for the μ- and ∂-opioid receptors and similar to the ∂-opioid receptor result with 1b, but opposite the μ- and κopioid receptor with 1b. Moreover, the conformation of 2a in the κ-opioid receptor was found to have the [η6-Cp*Rh-Tyr1]2+ residue in the somewhat less conserved area, which might F

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics reflect its weak binding value (>1000 nM)9 that was found for 1b and also theoretically for this trend with 2a, since we found that 2a was less potent in binding to the μ- and ∂-opioid by a factor of ∼2, in comparison to 1b. The H-bond interactions for 2a in the μ-opioid receptor included a C−H bond of a Cp*CH3− to the N of His 297, 2.9 Å; a tyrosine amide carbonyl with the H of H2O, 2.96 Å; O−H of Tyr 148, 2.19 Å, and NH3+ of Leu 219, 2.74 Å, with both Gly amide carbonyls; NH3+ of Lys 233 with the phenylalanine amide carbonyl, 2.00 Å; and the NH3+ of Lys 233 with the Leu carboxyl carbonyl, 1.95 Å. No π−π interactions were found within 3.5 Å, but His 297 was ∼4 Å from the Cp*Rh, as a hydrophobic amino acid residue, and Tyr 326 was ∼4 Å from [η6-Cp*Rh-Tyr1]2+, and CH−π interacted with the Cp*CH3− residue in 2a with Trp 293 at 1.9 Å (phenyl), with a possible weaker interaction at ∼4 Å (indole). Tyr 326 also appeared to prevent H-bonding to the phenol O−H and could be one reason the EC50 value was high at 93 nM in comparison to the 1b value of 14.3 nM.6 The interaction of 2a with the κ-opioid receptor shown in Figure 6 provided 22 amino acid residues within 5 Å, six Hbonds, and one π−π interaction within 3.5 Å. Again, we see the important Asp 138 residue of the κ-opioid receptor, this time H-bonding to the [η6-Cp*Rh-Tyr1]2+ phenol O−H, carboxyl H, 2.23 Å, and O, 2.85 Å; Ser 211 terminal O−H bonding to the Tyr carbonyl, 2.61 Å; Tyr 139 O−H bonding to the Gly CO; Asp 138 carboxyl O−H bonding to the NH of phenylalanine, 3.00 Å; H2O molecule H-bonding to the phenylalanine carbonyl, 2.86 Å; and a phenyl group of phenylalanine π−π interaction with the Tyr 312 phenyl group, 2.3 Å. Interestingly, no π−π interactions with aromatic amino acid residues were observed with the Cp*Rh group of 2a at the less conserved areas of the κ-opioid receptor, but even with the phenol OH Hbonding regimes, as noted above, the extrapolated EC50 value from 1b was speculated to be >1000 nM, indicating very weak binding to the κ-opioid receptor. The interaction of 2a with the ∂-opioid receptor shown in Figure 6 provided more than 20 amino acid residues within 5 Å, with four H-bonds and two π−π interactions within 3.5 Å. The message portion, for signal transduction, of 2a with a [η6Cp*Rh-Tyr1]2+ group clearly shows the Cp* group’s π−π interaction with the Trp 284 residue indole ring, 3.2 Å. This is an important find, since Trp amino acid residues are highly hydrophobic/lipophilic; therefore, the hydrophobic Cp* group would stabilize its conformation with this π−π interaction in the inner part of the ∂-opioid receptor (Figure 6). Furthermore, the other message portion of 2a, the phenyl group of the phenylalanine residue, revealed another critical π−π interaction with the Tyr 308 phenol, 3.8 Å. Moreover, H-bond, noncovalent interactions were also important for conformational stability of 2a inside the ∂-opioid receptor. For example, the terminal Tyr NH2 group of 2a, an H-bond acceptor, had a H-bond to the Tyr 109 phenol O−H, 3.00 Å, while the Lys 214 NH2 H-bonds with the 2a Tyr phenol O−H, 3.06 Å, both part of the message paradigm. The Lys 214 NH2 also H-bonds with a Gly amide carbonyl, 2.57 Å. Interestingly, no other amino acid residues from the μ- and κ-opioid receptors H-bond to the terminal Tyr NH2 group of 2a. This was also reflected in the EC50 value of 2a at the ∂-opioid (15.6 nM) versus the μ-opioid receptor (93 nM), with a ∂/μ ratio of 0.17, the ∂-opioid receptor being more selective to 2a, without having any Hbonds to the address portion, the protonated, terminal Leu carboxylic acid.

When we compared 1b to 2a in each intra and interseries of μ-, κ-, and ∂-opioid receptors, we saw that no similarities of their respective noncovalent binding regimes, within the message−address paradigm, were apparent (Figures 5 and 6). This clearly demonstrates the varied binding regimes for 1b to 2a in each opioid receptor, as epitomized in the lowest energy conformations (Figures 5 and 6), including the message and address aspects, and the degree of H-bond, π−π, and CH−π interactions, all of which dictate signal transduction (message region) and opioid receptor selectivity (address region) and, therefore, their overall EC50 binding values. Thus, 1b binds to both the μ- and the ∂-opioid receptors, with selectivity to the ∂-opioid by a factor, ∂/μ, of 0.31.6 The anomaly was the κ-opioid receptor, which has many noncovalent interactions with the message tyrosine residue and the address terminal Leu carboxylic acid (Figure 5); however, its EC50 binding value was found to be >1000 nM,9 a weak binding to 1b, and we postulate a similar situation for 2a from the published results of the μ- and ∂-opioid receptors, where 1b was more potent in comparison to 2a by factors of 0.15 (1b /2a, μ-OR) and 0.28 (1b /2a, ∂-OR). Thus, 1b was more selective to the ∂-OR in comparison to 2a by a factor, ∂/μ, of 1.86.6 The significant receptor binding differences revolved around the total lack of π−π or CH−π noncovalent interactions at the μ-, κ-, and ∂-opioid receptors for 1b in comparison to 2a. We presume that when you bind a [(η6-Cp*Rh-Tyr1)]2+ moiety to 2a, you create a more lipophilic/hydrophobic molecule, 2a, which when docked to an opioid receptor will seek receptor aromatic amino acid residues for π−π and CH−π noncovalent interactions that are weak; however, multiple weak, noncovalent interactions provide an overall fundamental stabilization in binding guest compounds to all GPCRs. Furthermore, a detailed analysis of the conformations of 1b and 2a inside the binding sites of the opioid receptors showed that their weak binding to the κ-opioid receptor might be due to the high internal energy of the guests, 1b and 2a. Indeed, torsional rotation was considered in the docking calculations as being free, while the distortion effect of the scoring function accounts only for the entropy effects, due to the constraints in conformations of both 1b and 2a and the receptor amino acid residues.10 However, accurate calculations showed that conformations of sufficiently large organic molecules, such as biological and pharmacological compounds, could differ in energy by 10 kcal/mol and were more dependent on the presence of internal hydrogen bonds and dipole and quadrupole contacts of the Ar···O and Ar···N variety.11 Our DFT calculations showed that the 1b conformation inside the κ-OR binding pocket was 18.5 and 12.7 kcal/mol higher in energy than the corresponding conformations inside the μ- and δopioid receptors, respectively. For complex 2a, the corresponding differences were 5.9 and 9.6 kcal/mol. By comparing these values along with the estimated docking energies found in Figure 7 (vide inf ra), this demonstrated that bonding in the κopioid receptor pocket was found to be energetically much less favorable in comparison with the μ- and ∂-opioid receptors, especially for 1b, and could be a major factor in 1b and 2a having weak binding values in the κ-opioid receptor. Binding Energies (ΔG) for Neuropeptide 1b and Complex 2a at the μ-, κ-, and ∂-Opioid Receptors. The binding energies (ΔG = −energy released on binding) were calculated for both [Tyr1]-Leu-enkephalin 1b and the dication [(η6-Cp*Rh-Tyr1)-Leu-enkephalin]2+ complex, 2a, when docking to the μ-, κ-, and ∂-opioid receptors (see Figure 7). These G

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics

Figure 7. Docking of the lowest energy binding modes (ΔG = −energy released on binding) of neuropeptide 1b (top) and complex 2a (bottom) in binding sites of the μ-, κ-, and ∂-opioid receptors, together with their corresponding calculated docking energies. The docked guest is rendered as CPK models.

Figure 8. Superposition of the lowest energy modes of neuropeptide 1b (in ball-and-stick representation, top) and complex 2a (in ball-andstick representation, bottom) docked in the binding sites of the opioid receptors, with the corresponding X-rayed ligands (highlighted in yellow) presented in the initial PDB structures, i.e., μ- (β-FNA), κ(JDTic), and ∂-opioid receptor (Naltrindole).

binding values are estimations, especially in the case of 2a, since the parameters applied in the docking calculations are relevant for a neutral peptide molecule, but do not account for the total positive charge of [2a]2+. Therefore, the relative binding energies for both 1b and 2a in individual receptors should be accurate, but intercomparisons to each other may be considered tenuous. However, we could compare the binding energies of 1b or 2a to those we calculated for antagonists JDTic and Naltrindole in the κ- and ∂-opioid receptors. Thus, complex 2a was on average providing ΔΔG values that are about −3 to −5 kcal/mol more stable than the corresponding binding energies of −10.7 and −12.4 kcal/mol for JDTic docking at the κ-opioid receptor and Naltrindole docking (structures, vide inf ra) at the ∂-opioid receptor, respectively (see Figure 7 for 1b and 2a binding energies, for comparison). Similar results with 1b provided ΔΔG values that are lower by about −0 to −2 kcal/ mol than the corresponding binding energies for JDTic and Naltrindole at the κ- and ∂-opioid receptors. The dicationic and hydrophobic nature of 2a, we surmised, was a plausible reason for the more stable binding energies, including the H-bond, π−π, and CH−π noncovalent interactions we observed with 2a in all the opioid receptors. Superposition of Both Agonists Neuorpeptide 1b and Complex 2a with Antagonists μ- (β-FNA), κ- (JDTic), and ∂- (Naltrindole) Opioid Receptor Morphanin Guests. We were interested in understanding the binding/conformational differences between 1b and 2a with the morphanin derivatives μ- (β-FNA); κ- (JDTic), and ∂- (Naltrindole) antagonists used in the X-ray structures of the designated opioid receptors.2 Figure 8 shows the superposition of 1b and 2a in molecular docking to the μ-, κ-, and ∂-opioid receptors and the clear differences in conformation flexibility demonstrated by agonists 1b and 2a in comparison to antagonists β-FNA (covalently bound to Lys 233), JDTic, and Naltrindole, respectively. Structures are shown below.2 Importantly, there were many differences in binding regimes between those antagonist compounds utilized in the X-ray structures and the agonists 1b and 2a, which were reflected in both message and address areas of all three opioid receptors. To reiterate, these differences are apparently due to the conformational flexability shown by the agonists and possibly

their biological function that elicits a biological response, i.e., a cascade of events leading to the release of Ca2+ ions, in contrast to the antagonists that do not elicit a biological response. Furthermore, the antagonists used in the X-ray structures of the μ-, κ-, and ∂-opioid receptors were in stable and biologically inactive conformations, while 1b and 2a were in their lowest energy and, presumably, mostly in biologically active conformations, reflecting the conformational differences in the message−address areas, and provided evidence of being able to discern what critical H-bond, π−π, and CH−π noncovalent interactions, seen in Figures 5 and 6, could possibly be associated with biological activity or lack thereof. Lastly, when we compared the binding regime results in the Kobilka et al. paper2b for the highly selective ∂-opioid receptor with agonists 1b and 2a and the antagonist Naltrindole, we observed no apparent similarities, which may be a factor in any drug being classified as an agonist versus being an antagonist.

■

CONCLUSIONS The overall analysis of the quantum chemical and molecular docking calculations showed a significant understanding of the differences between noncovalent binding regimes of 1b and 2a to the μ-, κ-, and ∂-opioid receptors (message−address paradigm) and their biological receptor binding values (EC50), the κ-opioid receptor being the anomaly, i.e., many noncovalent binding sites in the message−address areas with 1b and 2a (Figures 5 and 6), but a EC50 value of >1000 nM for both, showing very weak binding, which could be explained by H

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics a smaller total internal energy, −3.7 for 1b and −3.47 kcal/mol for 2a (see Table S2 in the Supporting Information), in comparison to the values for the μ- and ∂-opioid receptors, −5.58 and −4.77 kcal/mol for 1b and −7.43 and −5.09 kcal/ mol for 2a. As stated, calculations showed that the 1b conformation inside the κ-opioid receptor binding pocket was 18.5 and 12.7 kcal/mol higher in energy than the corresponding conformations inside the μ- and δ-opioid receptors, respectively. For complex 2a, the corresponding differences are 5.9 and 9.6 kcal/mol. By comparing these values along with the estimated docking energies found in Figure 7 (vide inf ra), this demonstrated that bonding in the κ-opioid receptor pocket was found to be energetically much less favorable in comparison with the μ- and ∂-opioid receptors, especially for 1b, and could be a major factor in 1b and 2a having weak binding values (>1000 nM) in the κ-opioid receptor. Moreover, we have also shown very clearly by the intracomparison of opioid receptors, as well as the intercomparison, that none of the noncovalent binding regimes were similar for both 1b and 2a, simply because of the conformational differences of both 1b and 2a inside the respective opioid receptors. We also have shown a systematic approach to understanding the various quantum chemical parameters that affect the molecular docking results. First, one needed to know the energy differences between a zwitterion and a non-zwitterion peptide or Cp*Rh dicationic complex, [Tyr1]-Leu-enkephalin 1b being a zwitterion, but in this case, the dication [(η6-Cp*Rh-Tyr1)-Leu-enkephalin]2+ complex, 2a, was found to be a non-zwitterion. This led to an entirely different electronic effect on the message region of 1b in comparison to 2a, including the η6-Cp*Rh moiety, having a dramatic effect on the charges of the phenol, O−H, terminal amino, N−H, and amide CO, of the tyrosine residue (Figure 3). The Mulliken charge analysis demonstrated, for example, that this interaction caused a significant depletion of electron density on the phenol ring, being virtually neutral in 1b, but in complex 2a, the electron charge was +0.32. Furthermore, the Cp*Rh moiety creates a hydrophobic environment that provided an entirely different binding regime to the μ-, κ-, and ∂-opioid receptors; interestingly, we found C−H, π−π, and CH−π noncovalent interactions (message paradigm) for 2a in docking to the three opioid receptors, and no π−π or CH−π noncovalent interactions for 1b in docking to the three opioid receptors. We also found it surprising that the docking of both 1b and 2a at the ∂-opioid receptor’s more conserved areas provided aromatic amino acid noncovalent interactions on opposite sides (Figures 5 and 6, respectively). For example, with 1b, the message phenol H-bonds to O Tyr 308, 2.98 Å, while the address Leu carboxyl carbonyl H-bonds with the ring N−H, Trp 284, 2.64 Å. This was opposite of what occurred with 2a at the ∂-opioid receptor; for example, with the message [η6Cp*Rh-Tyr1]2+ group, the Cp* moiety π−π interacted with the Trp 284 residue indole ring, 3.2 Å, while the message phenyl of phenylalanine π−π interacts with the phenol of Tyr 308, 2.3 Å; no Leu carboxylic H-bonds for 2a were found. Thus, even with these dramatic noncovalent binding differences, the EC50 binding values for both 1b and 2a showed excellent biological activity and selectivity for the ∂-opioid receptor; EC50 = 4.4 nM for 1b and 15.6 nM for 2a. The latter result also demonstrated that greater flexibility in binding conformations, as epitomized in Figures 5 and 6 for 1b and 2a, respectively, still provides

meaningful EC50 receptor binding values and, concomitantly, also showed that the opioid receptors were also flexible in binding a variety of guest conformations, which then maintained biological activity. We believe that this study is an important beginning in understanding the role of metal complexes of GPCR peptides, as to a variety of conformations, zwitterion/non-zwitterion energy calculations, electron density charge effects at the message−address regions, distortion forces, guest ΔG binding energies to opioid receptors, and the effect of these parameters on the opioid receptor EC50 binding values. A more pertinent finding was that there were very dramatic differences in the binding conformations of antagonist guests at the three opioid receptors used for the X-ray analysis, in comparison to agonists 1b and 2a (Figure 8). This point is critical, since the designation of a drug/inhibitor as agonist/antagonist occurs only from biological assays, where it either elicits a biological response or has a better receptor binding value, but no biological response. These types of quantum chemical and molecular docking calculations, especially for organometallic complexes of GPCR peptides, could conceivably provide more information on these designations, and we are pursuing this line of research in future studies with a variety of Cp*Rh-GPCR peptide complexes.

■

EXPERIMENTAL SECTION

Computational Methods. The optimized geometry of complex 2a was obtained from the density functional theory (DFT) calculations,12 using the Weizmann Institute modified version of the Gaussian 09 package.8 Full geometry optimization has been performed using the PBE1PBE hybrid density functional,13 also known as PBE0, combined with the basis set of double-ζ plus polarization quality.14 This basis set, denoted PC1, combined Jensen’s polarization consistent pc-1 basis set on the main group elements and the Stuttgart−Dresden basis set-RECP sdd15 on Rh with an added f-type polarization exponent taken as the geometric average of the two f-exponents given in the appendix of ref 15.16 Previously, it was shown that the [η6Cp*Rh-hydroxytamoxifen] 2+ complex was a dication in polar solutions; thus, the OTf counterions were omitted in the calculations, and the total charge 2+ was defined for the complex.17 Molecular docking calculations were performed using ArgusLab 4.0.18 The ArgusDock engine was applied in conjunction with the program’s default settings for parameters of calculations. Initial geometries of the binding sites on the opioid receptors were taken from the reported X-ray structures provided by the Protein Database (PDB).19 We have applied the μ-opioid receptor binding of the morphinan antagonist (entry 4DKL),2a the κ-opioid receptor binding of JDTic (entry 4DJH),2b and the ∂-opioid receptor bound to a morphine analogue Naltrindole (entry 4EJ4).2c We have also docked [Tyr1]-Leu-enkephalin 1b, the natural neuropeptide, and the [(η6Cp*Rh-Tyr1)-Leu-enkephalin]2+ complex 2a to the μ-, κ-, and ∂-opioid receptors. The docking accuracy of such computational techniques was expected to be close to that of rigorous commercial programs.20,21 The binding sites of the κ- and ∂-opioid receptors were defined based on the X-ray structures of the corresponding ligands. The reported structure of the μ-opioid receptor contained the morphinan ligand β-FNA, which was covalently bound to the receptor and could cause distortions at the binding site. In this case, the covalent ligand−pocket bond was removed prior to constructing the binding site. Since both ligand and binding site geometries were set as being flexible during the docking process, the binding site was made to relax to its original form. I

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics

■

Organometallics. In Bioorganometallics; Jaouen, G., Ed.; Wiley-VCH: Weinheim, 2005; pp 125−179. (c) Barragán, F.; López-Senín, P.; Salassa, L.; Betanzos-Lara, S.; Habtemariam, A.; Moreno, V.; Sadler, P. J.; Marchán, V. J. Am. Chem. Soc. 2011, 133, 14098. (d) Lemke, J.; Metzler-Nolte, N. J. Organomet. Chem. 2011, 696, 1018. (e) Patra, M.; Metzler-Nolte, N. Chem. Commun. 2011, 47, 11444. (f) Zagermann, J.; Molon, M.; Metzler-Nolte, N. Dalton Trans. 2011, 40, 1011. (g) Raszeja, L.; Maghnouj, A.; Hahn, S.; Metzler-Nolte, N. ChemBioChem 2011, 12, 371. (6) Albada, H. B.; Wieberneit, F.; Dijkgraaf, I.; Harvey, J. H.; Whistler, J. L.; Stoll, R.; Metzler-Nolte, N.; Fish, R. H. J. Am. Chem. Soc. 2012, 134, 10321. (7) Wieberneit, F.; Korste, A.; Albada, H. B.; Metzler-Nolte, N.; Stoll, R. Dalton Trans. 2013, 42, 9799. (8) (a) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T. Montgomery, T. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D..; Farkas, O.; Foresman, J. B.; Ortiz, J. V; Cioslowski, J. W.; Fox, D. J. Gaussian 09, Revision C.01wis4; Gaussian, Inc.: Wallingford, CT, 2009; J. Mol. Struct. Theochem. 2003, 626, 127. (b) Sung, D. D.; Koo, I. S.; Yang, K.; Lee, I. Chem. Phys. Lett. 2006, 426, 280. (c) Yang, Z. W.; Wu, X. M.; Zhou, L. J.; Yang, G. Int. J. Mol. Sci. 2009, 10, 3918. (9) Raynor, K.; Kong, H.; Chen, Y.; Yasuda, K.; Yu, L.; Bell, G. I.; Reisine, T. J. Pharmacol. Exp. Ther. 1993, 45, 330. (10) For further development and validation of empirical scoring functions for structure-based binding affinity predictions, see: Wang, R.; Lai, L.; Wang, S. J. J. Comput.-Aided Mol. Des. 2002, 16, 11. (11) Fogueri, U. R.; Kozuch, S.; Karton, A.; Martin, J. M. L. J. Phys. Chem. A 2013, 117, 2269. (12) (a) Kohn, W.; Sham, L. J. Phys. Rev. 1965, 140, 1133. (b) Parr, R. G.; Yang, W. Density Functional Theory of Atoms and Molecules; Oxford University Press: New York, 1970; p 230. (13) Adamo, C.; Cossi, M.; Barone, V. J. Mol. Struct.: THEOCHEM 1999, 493, 145. (14) Jensen, F. J. Chem. Phys. 2002, 116, 7372. (15) Dolg, M. In Modern Methods and Algorithms of Quantum Chemistry; Grotendorst, J., Ed.; John von Neumann Institute for Computing: Jülich, 2000; Vol. 1, pp 479−508. (16) Martin, J. M. L.; Sundermann, A. J. Chem. Phys. 2001, 114, 3408. (17) Efremenko, I.; Top, S.; Martin, J. M. L.; Fish, R. H. Dalton Trans. 2009, 4334. (18) Thompson, M. A. ArgusLab 4.0.1; Planaria Software LLC: Seattle, WA, http://www.arguslab.com. (19) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235. (20) Oda, A.; Okayasu, M.; Kamiyama, Y.; Yoshida, T.; Takahashi, O.; Matsuzaki, H. Bull. Chem. Soc. Jpn. 2007, 80, 1920. (21) Top, S.; Efremenko, I.; Rager, M.-N.; Vessières, A.; Yaswen, P.; Jaouen, G.; Fish, R. H. Inorg. Chem. 2011, 50, 271.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.5b00542. Atomic xyz coordinates for 1b.xyz XYZ) Atomic xyz coordinates for 2a.xyz (XYZ) Table S2 with added QC/MD data (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail for R. H. F.: rhfi[email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS I.E. gratefully acknowledges the financial support at the Weizmann Institute of Science, by the Helen and Martin Kimmel Center for Molecular Design, the Israel Science Foundation (grant 709/05), the Minerva Foundation, and the Lise Meitner-Minerva Center for Computational Quantum Chemistry. R.H.F. thanks Dr. H. Bauke Albada, formerly of the Department of Bioinorganic Chemistry, Ruhr University, Bochum, and now of the Department of Organic Chemistry, Hebrew University, Jerusalem, for collaboration on the synthesis and purification of peptide 1 and complex 2 (ref 6). We also thank Dr. Jennifer Whistler, Department of Neurology, University of California, San Francisco, for critical discussions on the biological aspects. R.H.F. also gratefully acknowledges support by the Department of Energy under Contract No. DE AC02-05CH11231.

■

REFERENCES

(1) GPCR reviews and book chapters: (a) Rosenbaum, D. M.; Rasmussen, S. G. F.; Kobilka, B. Nature 2009, 459, 356. (b) Gentilucci, L. Curr. Top. Med. Chem. 2004, 4, 19. (c) Lappano, R.; Maggiolini, M. Nat. Rev. Drug Discovery 2011, 10, 47. (d) Baselga, J. Science 2006, 312, 1175. (e) Klabunde, T.; Hessler, G. ChemBioChem 2002, 3, 928. (f) Biochemistry of Signal Transduction and Regulation, 3rd ed.; Krauss, G., Ed.; Wiley-VCH: Weinheim, Germany, 2003; Chapters 8 and 11. (2) (a) The X-ray crystal structure of the μ-opioid receptor bound to a morphinan antagonist, β-FNA: Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F. S.; Mathiesen, J. M.; Sunahara, R. K.; Pardo, L.; Weis, W. I.; Kobilka, B. K.; Granier, S. Nature 2012, 485, 321. (b) Structure of the ∂-opioid receptor bound to naltrindole: Granier, S.; Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F. S.; Weis, W. I.; Kobilka, B. K. Nature 2012, 485, 400. (c) Structure of the human κ-opioid receptor with JDTic: Wu, H.; Wacker, D.; Mileni, M.; Katritch, V.; Han, G. W.; Vardy, E.; Liu, W.; Thompson, A. A.; Huang, X.-P.; Carroll, F. I.; Mascarella, S. W.; Westkaemper, R. B.; Mosier, P. D.; Roth, B. L.; Cherezov, V.; Stevens, R. C. Nature 2012, 485, 327. (3) Chem. Eng. News 2012, October 15, 6. (4) (a) Fish, R. H.; Jaouen, G. Organometallics 2003, 22, 2166 and references therein. (b) Fish, R. H. Aust. J. Chem. 2010, 63, 1505 and references therein. (c) Bioorganometallics: Biomolecules, Labeling, Medicine; Jaouen, G., Ed.; Wiley-VCH, 2005. (d) Fish, R. H. J. Organomet. Chem. 2015, 782, 3 and references therein (ISBOMC’14 Award paper). (5) (a) Metzler-Nolte, N. Biomedical Applications of OrganometalPeptide Conjugates. In Topics in Organometallic Chemistry: Medicinal Organometallic Chemistry; Jaouen, G., Metzler-Nolte, N., Eds.; Springer-Verlag: Berlin, Heidelberg, 2010; Vol 32, pp 195−217. (b) Metzler-Nolte, N. Labelling of Peptides and PNA with J

DOI: 10.1021/acs.organomet.5b00542 Organometallics XXXX, XXX, XXX−XXX