Modulation of the Interaction between a Peptide Ligand and a G Protein-Coupled Receptor by Halogen Atoms

Article abstract of DOI:10.1021/acsmedchemlett.5b00126

Systematic halogenation of two native opioid peptides has shown that halogen atoms can modulate peptide-receptor interactions in different manners. First, halogens may produce a steric hindrance that reduces the binding of the peptide to the receptor. Second, chlorine, bromine, or iodine may improve peptide binding if their positive σ-hole forms a halogen bond interaction with negatively charged atoms of the protein. Lastly, the negative electrostatic potential of fluorine can interact with positively charged atoms of the protein to improve peptide binding.

Full text of DOI:10.1021/acsmedchemlett.5b00126

Letter
pubs.acs.org/acsmedchemlett
Modulation of the Interaction between a Peptide Ligand and a
G Protein-Coupled Receptor by Halogen Atoms
‡,§
‡,§
Monica Rosa,†,∇ Gianluigi Caltabiano,#,∇ Katy Barreto-Valer,
Veronica Gonzalez-Nunez,
́
̀
Jose C. Gomez-Tamayo,# Ana Arda,∥,⊥ Jesus Jimenez-Barbero,∥,⊥ Leonardo Pardo,*,#
́
́
́
́
́
‡,§
Raquel E. Rodríguez,
Gemma Arsequell,† and Gregorio Valencia*,†
†Institut de Química Avanca
̧
da de Catalunya (I.Q.A.C.-C.S.I.C.), E-08034 Barcelona, Spain
‡Department of Biochemistry and Molecular Biology, Faculty of Medicine, Instituto de Neurociencias de Castilla y Leon
́
(INCyL),
University of Salamanca, 37008 Salamanca, Spain
§Institute of Biomedical Research of Salamanca (IBSAL), E-37007 Salamanca, Spain
∥CIC bioGUNE, Bizkaia Technological Park, 48160 Derio, Spain
⊥Ikerbasque, Basque Foundation for Science, E-48013 Bilbao, Spain
#Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Auton
Bellaterra, Barcelona, Spain
̀
oma de Barcelona, E-08193
S
* Supporting Information
ABSTRACT: Systematic halogenation of two native opioid
peptides has shown that halogen atoms can modulate
peptide−receptor interactions in different manners. First,
halogens may produce a steric hindrance that reduces the
binding of the peptide to the receptor. Second, chlorine,
bromine, or iodine may improve peptide binding if their
positive σ-hole forms a halogen bond interaction with
negatively charged atoms of the protein. Lastly, the negative
electrostatic potential of fluorine can interact with positively
charged atoms of the protein to improve peptide binding.
KEYWORDS: Halogen bond, drug design, G protein-coupled receptors, opioid receptors, halogenated peptides, endomorphin-1,
Leu-enkephalin, neuropeptides
alogenation has traditionally been used in medicinal
halogen bonds are the prevalent interactions between
Hchemistry to modulate both potency and ADME halogenated ligands and target proteins.2,6,8−10 These halogen
properties of drugs.1,2 For many years halogens were only
bonds are preferentially formed between the halogen atom of
the ligand and the carbonyl oxygen of the protein backbone,
which is the most abundant Lewis base in proteins.7 In these
cases the halogen atom shares its donor oxygen with the
backbone hydrogen of the amide bond of the protein (the
backbone CO···H−N hydrogen bond forms the secondary
structure of proteins) in such a manner that the halogen bond
tends to be geometrically perpendicular to the shared hydrogen
bond.8
The evolution and structural integration of fluorine into
drugs introduced over the past 50 years have been recently
evaluated.11 We have extended this analysis to the other
halogen atoms (Figure 1) by surveying their presence in
compounds deposited in DrugBank. Out of the 1584 FDA-
considered for their hydrophobic properties and as Lewis bases
due to their electronegativities. However, theoretical studies
have shown that, on the van der Waals surface of the halogen
atom, fluorine (F) remains entirely electronegative, whereas
chlorine (Cl), bromine (Br), and iodine (I) show a small
positively charged surface (named σ-hole) on the opposite side
of the CX axis (X is Cl, Br, I).3,4 The size and intensity of the
σ-hole increases with the charge of the σ-hole (I > Br > Cl).
Thus, fluorines are most likely to act as hydrogen bond
acceptors (F···HD, D is O, N, S, C).5 In contrast, the other
three halogens can act as electron acceptors (CX···B, B is O,
N, S), via the σ-hole, forming the so-called halogen bond, a
Lewis acid−base interaction in which halogens act as Lewis
acids.6 The strength of the halogen bond depends on the
halogen atom and the Lewis base involved in the interaction,
but it is comparable in magnitude to hydrogen bond
interactions.7 A survey of protein crystal structures shows that
Received: March 24, 2015
Accepted: July 16, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00126
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ACS Medicinal Chemistry Letters
Letter
determined in the presence of 10 μM of the nonspecific
antagonist naloxone. Results are shown in Table 1 (see also SI).
Table 1. Summary of the Ki Values (Mean SEM) Found
for the Halogenated Series of Endo-1 and Leu-ENK when
a
Tested on the μ and δ Opioid Receptors
Figure 1. Presence of halogens in ligands deposited in the DrugBank
(FDA-approved), ZINC (commercially available), and IUPHAR/BPS
(Class A GPCR ligands) databases.
approved drugs, 10.4% of them contain fluorine, 14.6%
chlorine, 1.5% bromine, and 1.4% iodine. The largest database
of commercially available compounds, the ZINC database,
shows similar trends. Out of the almost 30 million compounds
deposited in ZINC, 16.5% contain fluorine, 14% chlorine, 5.2%
bromine, and 0.3% iodine. Similar percentages (F, 12.3%; Cl,
16.5%; Br, 1.7%; I, 2.1%) are found in the analysis of the
IUPHAR/BPS database of ligands for the G protein-coupled
receptor (GPCR) superfamily. Clearly, halogens have a key role
in drug development.
Therefore, in this study we aimed to test the effect of halogen
substitution in endogenous peptides of opioid receptors, which
belong to the GPCR superfamily.12 Like other GPCRs,13 the
structures of the μ-, δ-, and κ-opioid receptors14−16 are formed
by seven membrane-spanning α-helical segments (TMs 1−7),
connected by extracellular (ECL 1−3) and intracellular loops.
It is known that chlorination of aromatic residues (i.e., 4-Cl-
Phe4) of certain opioid peptides ([D-Pen2, D-Pen5]enkephalin
(DPDPE); biphalin) increases their lipophilicity and hence cell
permeability and blood−brain barrier passage.17,18 Other
peptides like DPDPE, metkephamid, and dermorphin/
deltorphin analogues when halogenated at Phe4 or Phe3
increased their affinity for δ-opioid receptors.19−21 More
recently, chlorination or fluorination of Phe4 residues on a
series of Dmt-substituted enkephalin-like tetrapeptides have led
to the development of nonselective potent opioid agonists for
μ- and δ-opioid receptors.22 Due to the absence of systematic
halogenation of Phe residues to produce homologous series (F,
Cl, Br, I), in the present work we wanted to examine the
possibility to induce stabilizing ligand−receptor interactions by
the systematic halogenation of two native opioid ligands,
endomorphin-1 and Leu-enkephalin.
a
#
Values taken from previous works: , ref 25; *, ref 26.
Halogenation of the selective Endo-1 peptide reduces affinity
for μ-OR in a linear manner along the series (Table 1), which
suggests an increasing steric hindrance in the ligand−receptor
interaction proportional to the bulkiness of the halogen atom.
In contrast, this effect along the -Cl, -Br, and -I derivatives is
positive for δ-ORs (more significant in δ1a than in δ1b, Table
1). An interesting result of the combination of these opposite
effects of enhanced binding for δ-ORs and less binding for μ-
OR is a modification of peptide selectivity. The selectivity of
Endo-1, between μ-OR and δ1a-OR gradually decreases from
258 to 5 times (Table 1). In the second and third series we
constructed ortho-Phe4- and para-Phe4-halogenated Leu-ENK
peptides with different effects (Table 1).
The binding pattern of the [2-X-Phe4]-Leu-ENK series to
both types of δ receptors indicates the formation of a halogen
bond interaction. There is a linear (R2 of 0.94 and 0.97 for δ1a
and δ1b, respectively) decrease of Ki values with an increase of
the halogen volume, as measured by the effective van der Waals
radii for phenyl substitution (H, 1.20 Å; Cl, 1.77 Å; Br, 1.92 Å;
I, 2.06 Å).27 In the [4-X-Phe4]-Leu-ENK series, the -Cl, -Br,
and -I substitutions reduce affinity for δ-OR, compared to F-
substitution, which markedly improves binding affinity for both
δ-ORs, leading to the most potent peptide tested in this work.
In order to understand the nature of the repulsive interaction
between the halogen atom of [4-X-Phe4]Endo-1 and the μ-OR
(steric hindrance), and the attractive interaction between the
-Cl, -Br, and -I atom of [2-X-Phe4]-Leu-ENK and δ-ORs
(halogen bond donor) and the -F atom of [4-F-Phe4]-Leu-ENK
and δ-ORs (hydrogen bond acceptor), we performed a
combined NMR and modeling study.28 Indeed, in their free
state in water solution, and independently of the substitution,
no medium-range or long-range NOEs were detected. These
data indicate that these molecules behave similarly to other
endogenous enkephalin peptides and analogues, with a
remarkable flexibility and major extended conformations.29−32
The halogen substitution does not modify the natural
presentation of these peptides, and therefore, the observed
different binding affinities are likely to be due to intermolecular
interactions with the key receptors.
Three homologous series of halogenated analogues have
been prepared. The first series corresponds to ortho-Phe4
halogenated endomorphin-1 ([4-X-Phe4]Endo-1), while the
other two are based on ortho-Phe4 ([2-X-Phe4]Leu-ENK) and
para-Phe4 ([4-X-Phe4]Leu-ENK) halogenated Leu-ENK. Bind-
ing affinities of these three series of halogenated peptides were
determined by competition experiments on the μ-opioid
receptor (μ-OR) from zebrafish23 and on two duplicate δ-
opioid receptors (δ-ORs), dre-δ1a24 and dre-δ1b,25 using [3H]-
diprenorphine as radioligand. All tested ligands displaced [3H]-
diprenorphine binding, and the experimental data fitted better
to one-site displacement model. Nonspecific binding was
B
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Figure 2. Computational models of the complexes between halogenated Endo-1 and Leu-ENK peptides in complex with μ- and δ1b-opioid
receptors. (A) [4-I-Phe4]Endo-1 (in cyan) in complex with μ-OR. Phe4 is placed in a small cavity formed by Thr2.56 and Val3.28, making
halogenation (iodine is shown) at the para position repulsive (steric hindrance). (B) [4-F-Phe4]Leu-ENK (in magenta) in complex with δ1b-OR.
The negative electrostatic potential of fluorine forms a F···H−C hydrogen bond with the methyl group of Thr2.56. (C) [2-I-Phe4]-Leu-ENK-1 (in
light blue) in complex with δ1b-OR. The positive σ-hole of iodine forms a halogen bond with the backbone carbonyl oxygen of Phe2.59. (D) The
orientation of Phe4 in Endo-1 (cyan sticks), [4-F-Phe4]Leu-ENK (magenta), and [2-I-Phe4]Leu-ENK (light blue) inside the small cavity located
between TMs 2 and 7. The negatively charged area (red) created by Asp3.32 is shown. The color code of the helices is TMs 1 in white, 2 in yellow, 3
in red, 4 in gray, 5 in green, 6 in blue, and 7 in brown.
Endo-1 and Leu-ENK peptides were docked in extended
conformations, as suggested by the NMR experiments, into
homology models of μ- and δ1b-opioid receptors, respectively,
in such a manner that the observed binding modes in crystal
structures of nonpeptidic ligands to these receptors11 are
mimicked (see Experimental Procedures). In the modeled
positively charged σ-hole and an electronegative crown,3 as
depicted in electrostatic potential surfaces (Figure S3)
calculated at the B3LYP level of theory (see Methods),
impedes the proper modeling of halogen atoms by the standard
single point charge. Thus, halogen atoms (with the exception of
the entirely electronegative F) are modeled using two point
charges following the scheme proposed by Ibrahim36 (see
Experimental Procedures and SI). Halogenation at the para
position of Phe4 in Endo-1 (steric hindrance) and Leu-ENK
(fluorine acts as hydrogen bond acceptor) has different effects
(see above) despite Phe4 is located at the same hydrophobic
pocket. The MD simulations reproduce slightly different
orientations of Phe4 in Endo-1 (the preceding amino acid
before the C-terminus) and Leu-ENK (two amino acids before
the C-terminus) in the cavity (Figures 2 and S1). The
formation of the interactions between the amidated C-terminus
Endo-1 and Gln2.60 and Asn2.63 (Figures 2A and S1)
profoundly restrains Phe4 to adapt inside this small hydro-
phobic cavity, formed by the β-branched Thr2.56 and Val3.28
residues. Thus, halogenation of Endo-1 ([4-X-Phe4]Endo-1) is
repulsive due to a steric hindrance (Figure 2A). In contrast,
Phe4 in Leu-ENK is positioned two amino acids before the
amidated C-terminus, which confer significant flexibility due to
a less restrictive interaction of Leu5, the following amino acid,
with Ile202 in ECL 2 (Figure S1). Thus, Leu-ENK benefits
from fluorine substitution in para ([4-F-Phe4]-Leu-ENK),
yielding a peptide with high affinity for δ-opioid receptors
(Table 1). para-Fluorinated phenylalanine is characterized by a
slightly negative electrostatic potential that is distributed as a
hollow ring around the tip of the opposite side of the C−F axis,
whereas this trend is the opposite (positive σ-hole) in the other
three halogen atoms (Figure S3). Thus, the facts that fluorine
substitution benefits peptide binding whereas chlorine,
bromine, and iodine substitution is detrimental for peptide
binding (Table 1) suggest that the halogen atom at the para
position acts as hydrogen bond acceptor. MD simulations of [4-
F-Phe4]-Leu-ENK in complex with the δ-opioid receptor show
that the slightly negative electrostatic potential of fluorine
interacts with the slightly positive charge of the methyl group of
Thr2.56, forming a F···H−C hydrogen bond (Figure 2B).
Noteworthy, interactions with hydrophobic residues account
complexes, the message part33 of Endo-1 and Leu-ENK (NH3 -
+
Tyr1) interact with μ- and δ-opioid receptors, respectively, in a
similar manner. The protonated N-terminus amine of the
peptide forms an ionic interaction with Asp3.32, whereas Tyr1
interacts with His6.52 via a conserved water molecule, present
in all released crystal structures of opioid receptors (Figure S1).
In contrast, the address part (Pro2Trp3Phe4 in Endo-1 and
Gly2Gly3Phe4Leu5 in Leu-ENK), responsible for selectivity,
forms slightly different interactions. In the complex between
Endo-1 and μ-OR, Pro2 induces a tight turn of the backbone to
facilitate an aromatic−aromatic interaction between Trp3 and
Tyr2.64, and Phe4 is positioned in a hydrophobic pocket
formed by Thr2.56, Phe2.59, Val3.38, and a conserved Trp127
aromatic residue (the WxFG motif in ECL 1,13 and the
−CONH2 C-terminus of the peptide hydrogen bonds Gln2.60
and Asn2.63) (Figure S1). In the complex between Leu-ENK
and δ-ORs, the flexible Gly2Gly3 spacer mimics the tight turn of
Pro2 in Endo-1, positioning Phe4 in the same hydrophobic
pocket (but with slightly different orientation, see below)
shaped by Thr2.56, Phe2.59, Val3.38, and Trp118 in ECL 1,
Leu5 forms hydrophobic interactions with Ile202 in ECL 2, and
the -CONH2 C-terminus of the peptide hydrogen bonds with
the exposed backbone of ECL2. These binding modes explain
the observed selectivity of Endo-1 for μ-OR and Leu-ENK for
δ-ORs. Asn2.63 in μ-OR (Lys2.63 in δ-ORs) provides
selectivity of Endo-1 for μ-OR, whereas Ile 202 in ECL 2 of
δ-ORs (Asp 213 in μ-OR) provides selectivity of Leu-ENK for
δ-ORs (Figure S2). These are the only nonconserved/
nonsimilar residues in the binding pocket of both receptors.
This is in agreement with previous work suggesting that the
amino acid at position 2.63 is responsible for the selectivity of
the enkephaline DAMGO between rat μ- and δ-opioid
receptors.34,35
Molecular dynamics (MD) simulations were used to study
the observed effects of halogen substitution in the peptide−
receptor interaction. Importantly, the presence of both a
C
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1
2,2,3,3-d4 acid (TSP) was used as a chemical shift reference in the H
NMR experiments [δ(TSP) = 0 ppm] (see SI for spectra).
for almost 40% of the observed complexes of fluorinated
ligands in a recent survey of the PDB database.9
The orientation of Phe4, in MD simulations of the complex
between [2-I-Phe4]-Leu-ENK with the δ-opioid receptor
(Figure 2C), shows that halogen substitution at the ortho
position forms a halogen bond interaction with the oxygen
atom of the backbone carbonyl of F2.59. In accordance,
fluorine substitution at this ortho position is repulsive while
chlorine, bromine, and iodine substitution increases peptide
binding linearly with the charge of the σ-hole (Table 1). In our
computer simulations the halogen bond remains stable and
almost geometrically perpendicular to the backbone carbonyl
group (Figure S4). Notably, the chlorinated ligand bound to
OX2 orexin receptor, whose structure has been released
recently,37 forms halogen bond with the same carbonyl oxygen
as postulated for [2-I-Phe4]-Leu-ENK, in a similar position
(Figure S4). Figure 2D shows the different orientation of Phe4
in [Phe4]-Leu-ENK, [2-I-Phe4]-Leu-ENK, and [4-F-Phe4]-Leu-
ENK, illustrating the flexibility of Phe4 inside the hydrophobic
cavity.
Here, we have shown that halogen atoms can modulate
peptide−receptor interactions in different manners. First,
substitution of a hydrogen atom by bulkier fluorine, chlorine,
bromine, or iodine atoms, in small constraint cavities, might
produce steric hindrances that reduce the binding of the
peptide to the receptor (proportional to the bulkiness of the
halogen atom) as shown in the [4-X-Phe4]Endo-1 series. In
contrast, substitution of a hydrogen atom by chlorine, bromine,
or iodine might be beneficial (proportional to the charge of the
σ-hole) if the positive σ-hole of the halogen forms a halogen
bond interaction with negatively charged atoms of the protein
(i.e., backbone carbonyls) as shown in the [2-X-Phe4]-Leu-
ENK series. We believe this interaction is purely σ-hole halogen
bonding and does not involve lipophilicity. Substitution of
hydrogen by fluorine stabilizes the ligand−receptor complex if
the negative electrostatic potential of fluorine interacts with
positively charged atoms of the proteins (i.e., hydrophobic CH
groups) as shown in the [2-X-Phe4]-Leu-ENK series. In
conclusion, our data show how halogen atoms can be used to
fine-tune ligand−protein interactions in different protein
environments. Each halogen atom can sensibly affect ligand
binding as well as ligand selectivity.
Molecular Models of Peptide−Receptor Complexes. Modeler
was used to model zebrafish μ- and δ1b-opioid receptors using the
structure of mouse μ- (PDB 4DKL)14 and δ- (PDB 4N6H)15 opioid
receptors, respectively, as templates. Endo-1 and Leu-ENK, in the
extended conformation as determined by NMR, were docked into the
homology models using the Autodock Vina tool. All docking solutions
were visually inspected, and the poses in which the N-terminus amine
of the peptide mimicked the mode of interaction observed in crystal
structures of opioid receptors in complex with nonpeptidic ligands
were selected for energy minimization and MD simulations in a
explicit lipid bilayer (see SI for details) with the GROMACS software
using the protocol previously described.38 Cl, Br, and I were modeled
by two point charges (a negative charge centered on the halogen atom
to reproduce the electronegative crown,3 and a positive charge
centered on a dummy atom on the opposite side of the CX axis in
order to reproduce the positively charged σ-hole).
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00126.
Experimental details for the synthesis and character-
ization of individual halogenated endomorphin-1 and
Leu-enkephalin analogues, additional figures, competi-
tion binding studies, details for computational studies,
and 2D NMR spectra of Leu-enkephalin analogues.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: leonardo.pardo@uab.es.
*E-mail: gregorio.valencia@iqac.csic.es.
Author Contributions
∇These authors (M.R. and G.C.) contributed equally to this
work.
Funding
The work was supported by a grant from the Fundacio
de TV3 (Pain, 070430-31-32-33), MINECO (SAF2010-18597;
SAF2013-48271-C2-2-R) and Junta de Castilla y Leon (B1039/
́ ́
Marato
́
SA25/10). L.P. participates in the European COST Action
CM1207 (GLISTEN). M.R. acknowledges a fellowship of
EXPERIMENTAL PROCEDURES
■
́
Formacion del Profesorado Universitario (AP2009-2534) from
MINECO.
Chemistry. Peptides have been prepared by standard stepwise solid
phase peptide synthesis techniques using Nα-Fmoc chemistry. Halogen
substitutions have been introduced on the peptides from appropriate
commercially available Fmoc-Phe building blocks. Crude peptides
were purified by RP-HPLC to afford >98% pure compounds.
Characterization of compounds was effected by ESI-UPLC methods
for the determination of the peptide exact mass (see SI for full
description).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
Endo-1, endomorphin-1; dre, Danio rerio zebrafish; Dmt, 2,6-
dimethyltyrosine; Leu-ENK, Leu-enkephalin; GPCR, G-protein
coupled receptor; SEM, standard error of the mean; OR, opioid
receptor; TM, transmembrane domain
Binding Studies to Opioid Receptors. Stably transfected
HEK293 cells expressing the μ, δ1a, or δ1b receptor from zebrafish
were grown to 80% confluence, harvested, and collected by
centrifugation. Membrane preparation, radioligand binding, and data
analysis were performed as previously described26 (SI for details).
Naloxone was purchased from Sigma-Aldrich (Madrid, Spain) and
[3H]-diprenorphine (50 Ci/mmol) from PerkinElmer (Boston, MA).
Conformation in Solution of Peptides by NMR. Samples were
prepared in H2O/D2O (90:10), and spectra were recorded on a Bruker
Avance 600 MHz instrument equipped with a triple-channel cryoprobe
and at 278 K. NMR assignments were reached using standard 2D
TOCSY experiments at 20 and 60 ms mixing times and 2D NOESY
experiments at 300 ms. The resonance of 3-(trimethylsilyl) propionic-
REFERENCES
■
(1) Hernandes, M. Z.; Cavalcanti, S. M.; Moreira, D. R.; de Azevedo,
W. F., Jr.; Leite, A. C. Halogen atoms in the modern medicinal
chemistry: hints for the drug design. Curr. Drug Targets 2010, 11,
303−314.
(2) Wilcken, R.; Zimmermann, M. O.; Lange, A.; Zahn, S.; Boeckler,
F. M. Using halogen bonds to address the protein backbone: a
systematic evaluation. J. Comput.-Aided Mol. Des. 2012, 26, 935−945.
D
DOI: 10.1021/acsmedchemlett.5b00126
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(3) Auffinger, P.; Hays, F. A.; Westhof, E.; Ho, P. S. Halogen bonds
in biological molecules. Proc. Natl. Acad. Sci. U. S. A. 2004, 1, 16789−
16794.
(4) Clark, T.; Hennemann, M.; Murray, J. S.; Politzer, P. Halogen
bonding: the σ-hole. J. Mol. Model. 2007, 13, 291−296.
(5) Dalvit, C.; Invernizzi, C.; Vulpetti, A. Fluorine as a Hydrogen-
Bond Acceptor: Experimental Evidence and Computational Calcu-
lations. Chem. - Eur. J. 2014, 20, 11058−11068.
(6) Xu, Z.; Yang, Z.; Liu, Y.; Lu, Y.; Chen, K.; Zhu, W. Halogen
bond: its role beyond drug-target binding affinity for drug discovery
and development. J. Chem. Inf. Model. 2014, 54, 69−78.
(7) Xu, Z.; Liu, Z.; Chen, T.; Chen, T.; Wang, Z.; Tian, G.; Shi, J.;
Wang, X.; Lu, Y.; Yan, X.; Wang, G.; Jiang, H.; Chen, K.; Wang, S.; Xu,
Y.; Shen, J.; Zhu, W. Utilization of halogen bond in lead optimization:
a case study of rational design of potent phosphodiesterase type 5
(PDE5) inhibitors. J. Med. Chem. 2011, 54, 5607−5611.
(8) Voth, A. R.; Khuu, P.; Oishi, K.; Ho, P. S. Halogen bonds as
orthogonal molecular interactions to hydrogen bonds. Nat. Chem.
2009, 1, 74−79.
(9) Parisini, E.; Metrangolo, P.; Pilati, T.; Resnati, G.; Terraneo, G.
Halogen bonding in halocarbon-protein complexes: a structural survey.
Chem. Soc. Rev. 2011, 40, 2267−2678.
(10) Sirimulla, S.; Bailey, J. B.; Vegesna, R.; Narayan, M. Halogen
interactions in protein-ligand complexes: implications of halogen
bonding for rational drug design. J. Chem. Inf. Model. 2013, 53, 2781−
2791.
(11) Ilardi, E. A.; Vitaku, E.; Njardarson, J. T. Data-mining for sulfur
and fluorine: an evaluation of pharmaceuticals to reveal opportunities
for drug design and discovery. J. Med. Chem. 2014, 57, 2832−2842.
(12) Filizola, M.; Devi, L. A. Grand opening of structure-guided
design for novel opioids. Trends Pharmacol. Sci. 2013, 34, 6−12.
(13) Gonzalez, A.; Cordomí, A.; Caltabiano, G.; Pardo, L. Impact of
Helix Irregularities on Sequence Alignment and Homology Modeling
of G Protein-Coupled Receptors. ChemBioChem 2012, 13, 1393−
1399.
(14) 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. Crystal structure of the μ-opioid receptor bound to a
morphinan antagonist. Nature 2012, 485, 321−326.
(15) Granier, S.; Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F.
S.; Weis, W. I.; Kobilka, B. K. Structure of the delta-opioid receptor
bound to naltrindole. Nature 2012, 485, 400−404.
(16) 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. Structure of the human kappa-opioid
receptor in complex with JDTic. Nature 2012, 485, 327−332.
(17) Weber, S. J.; Greene, D. L.; Sharma, S. D.; Yamamura, H. I.;
Kramer, T. H.; Burks, T. F.; Hruby, V. J.; Hersh, L. B.; Davis, T. P.
Distribution and analgesia of [3H][D-Pen2, D-Pen5]enkephalin and
two halogenated analogs after intravenous administration. J. Pharmacol.
Exp. Ther. 1991, 259, 1109−1117.
(18) Abbruscato, T. J.; Williams, S. A.; Misicka, A.; Lipkowski, A. W.;
Hruby, V. J.; Davis, T. P. Blood-to-central nervous system entry and
stability of biphalin, a unique double-enkephalin analog, and its
halogenated derivatives. J. Pharmacol. Exp. Ther. 1996, 276, 1049−
1057.
(19) Vaughn, L. K.; Knapp, R. J.; Toth, G.; Wan, Y. P.; Hruby, V. J.;
Yamamura, H. I. A high affinity, highly selective ligand for the delta
opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]encephalin. Life Sci.
1989, 45, 1001−1008.
(20) Gesellchen, P. D.; Shuman, R. T. Substitution on the Phe3
aromatic ring in cyclic.delta. opioid receptor-selective dermorphin/
deltorphin tetrapeptide analogs: electronic and lipophilic requirements
for receptor affinity. Pharmacologically active peptides. US Patent
US4265808, 1981.
tetrapeptide analogs: electronic and lipophilic requirements for
receptor affinity. J. Med. Chem. 1992, 35, 1535−1541.
(22) Lee, Y. S.; Kulkarani, V.; Cowell, S. M.; Ma, S. W.; Davis, P.;
Hanlon, K. E.; Vanderah, T. W.; Lai, J.; Porreca, F.; Vardanyan, R.;
Hruby, V. J. Development of potent μ and δ opioid agonists with high
lipophilicity. J. Med. Chem. 2011, 54, 382−386.
(23) Barrallo, A.; Gonzalez-Sarmiento, R.; Alvar, F.; Rodriguez, R. E.
ZFOR2, a new opioid receptor-like gene from the teleost zebrafish
(Danio rerio). Mol. Brain Res. 2000, 84, 1−6.
(24) Rodriguez, R. E.; Barrallo, A.; García-Malvar, F.; Mcfadyen, I. J.;
Gonzalez-Sarmiento, R.; Traynor, J. R. Characterization of ZFOR1, a
putative delta-opioid receptor from the teleost zebrafish (Danio rerio).
Neurosci. Lett. 2000, 288, 207−210.
(25) Pinal-Seoane, N.; Martin, I. R.; Gonzalez-Nunez, V.; de Velasco,
E. M.; Alvarez, F. A.; Sarmiento, R. G.; Rodriguez, R. E.
Characterization of a new duplicate delta-opioid receptor from
zebrafish. J. Mol. Endocrinol. 2006, 37, 391−403.
́
(26) Gonzalez-Nunez, V.; Jimenez-Gonzalez, A.; Barreto-Valer, K.;
Rodriguez, R. E. In vivo regulation of the mu opioid receptor: role of
the endogenous opioid agents. Mol. Med. 2013, 19, 7−17.
(27) Bondi, A. van der Waals volumes and radii. J. Phys. Chem. 1964,
68, 441−451.
(28) Arsequell, G.; Rosa, M.; Mayato, C.; Dorta, R. L.; Gonzalez-
Nunez, V.; Barreto-Valer, K.; Marcelo, F.; Calle, L. P.; Vaz
́
quez, J. T.;
Rodríguez, R. E.; Jimenez-Barbero, J.; Valencia, G. Synthesis, biological
́
evaluation and structural characterization of novel glycopeptide
analogues of nociceptin N/OFQ. Org. Biomol. Chem. 2011, 9,
6133−6142.
(29) Wieberneit, F.; Korste, A.; Albada, H. B.; Metzler-Nolte, N.;
Stoll, R. Structural and biological implications of the binding of Leu-
enkephalin and its metal derivatives to opioid receptors. Dalton Trans.
2013, 42, 9799−9802.
(30) Naito, A.; Nishimura, K. Conformational analysis of opioid
peptides in the solid states and the membrane environments by NMR
spectroscopy. Curr. Top. Med. Chem. 2004, 4, 135−145.
(31) Hu, X. G.; Thomas, D. S.; Griffith, R.; Hunter, L. Stereoselective
fluorination alters the geometry of a cyclic peptide: exploration of
backbone-fluorinated analogues of unguisin A. Angew. Chem., Int. Ed.
2014, 53, 6176−6179.
(32) Wang, Y. C.; Wu, Y. C.; Yeh, C. C.; Hwang, C. C. Structure-
activity relationships of Leu-Enkephalin analog with (4-Carboxamido)-
phenylalanine substituted for tyrosine: a molecular dynamics study.
Biopolymers 2007, 86, 231−239.
(33) Portoghese, P. S.; Sultana, M.; Takemori, A. E. Design of
peptidomimetic delta opioid receptor antagonists using the message-
address concept. J. Med. Chem. 1990, 33, 1714−1720.
(34) Fukuda, K.; Terasako, K.; Kato, S.; Mori, K. Identification of the
Amino Acid Residues Involved in Selective Agonist Binding in the
First Extracellular Loop of the delta- -and mu-Opioid Receptors. FEBS
Lett. 1995, 373, 177−181.
(35) Minami, M.; Nakagawa, T.; Seki, T.; Onogi, T.; Aoki, Y.; Katao,
Y.; Katsumata, S.; Satoh, M. A single residue, Lys108, of the delta-
opioid receptor prevents the mu-opioid-selective ligand [D-Ala2,N-
MePhe4,Gly-ol5]enkephalin from binding to the delta-opioid receptor.
Mol. Pharmacol. 1996, 50, 1413−1422.
(36) Ibrahim, M. A. A. Molecular mechanical study of halogen
bonding in drug discovery. J. Comput. Chem. 2011, 32, 2564−2574.
(37) Yin, J.; Mobarec, J. C.; Kolb, P.; Rosenbaum, D. M. Crystal
structure of the human OX2 orexin receptor bound to the insomnia
drug suvorexant. Nature 2015, 519, 247−250.
(38) Cordomí, A.; Caltabiano, G.; Pardo, L. Membrane Protein
Simulations Using AMBER Force Field and Berger Lipid Parameters.
J. Chem. Theory Comput. 2012, 8, 948−958.
(21) Heyl, D. L.; Mosberg, H. I. Substitution on the Phe3 aromatic
ring in cyclic.delta. opioid receptor-selective dermorphin/deltorphin
E
DOI: 10.1021/acsmedchemlett.5b00126
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