Elucidation of Mechanism for Ligand Efficacy at Leukotriene B4Receptor 2 (BLT2)

Article abstract of DOI:10.1021/acsmedchemlett.0c00065

G protein-coupled receptors (GPCRs) have always been important drug targets in the pharmaceutical industry. One major question for the current GPCR drug discovery is how drugs have distinct efficacies at the same GPCR target. Related to this question, we studied how different ligands can have disparate efficacies at Leukotriene B4 receptor (BLT2). By using molecular modeling studies, we predicted that Tyr2716.51 located at TM6 of BLT2 performs as a key trigger for its activation and verified the prediction by site-directed mutagenesis, chemotactic motility studies, which included a chemical derivative of agonist CAY10583. We further identified Asn2756.55 located at TM6 as a weak activation trigger in BLT2 and performed double mutation studies to confirm our computational results. Our results provide strong evidence for the exact mechanism of ligand efficacy at BLT2.

Full text of DOI:10.1021/acsmedchemlett.0c00065

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Letter
Elucidation of Mechanism for Ligand Efficacy at Leukotriene B4
Receptor 2 (BLT2)
Minsup Kim,∥ Jun-Dong Wei,∥ Dipesh S. Harmalkar,∥ Ja-il Goo, Kyeong Lee, Yongseok Choi,*
Jae-Hong Kim,* and Art E. Cho*
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ABSTRACT: G protein-coupled receptors (GPCRs) have always
been important drug targets in the pharmaceutical industry. One
major question for the current GPCR drug discovery is how drugs
have distinct efficacies at the same GPCR target. Related to this
question, we studied how different ligands can have disparate
efficacies at Leukotriene B4 receptor (BLT2). By using molecular
modeling studies, we predicted that Tyr2716.51 located at TM6 of
BLT2 performs as a key trigger for its activation and verified the
prediction by site-directed mutagenesis, chemotactic motility
studies, which included a chemical derivative of agonist
CAY10583. We further identified Asn2756.55 located at TM6 as a
weak activation trigger in BLT2 and performed double mutation
studies to confirm our computational results. Our results provide
strong evidence for the exact mechanism of ligand efficacy at BLT2.
KEYWORDS: Leukotriene B4 receptor 2, ligand efficacy, molecular modeling, mutagenesis study, chemical study
treatment of several pulmonary inflammatory diseases,
protein−coupled receptors (GPCRs) are the first
1
Gtransmitters for cells to accept signals from the outside. including asthma, acute respiratory distress syndrome
(ARDS), acute lung injury (ALI), and chronic obstructive
pulmonary disease (COPD).10,11 Because of its association
with various diseases, BLT2 has multiple feasibilities as a drug
target.
These receptors recognize various external stimuli such as
extracellular chemical, sensory, and mechanical stimuli.2 Drugs
binding to GPCRs can promote or block physiological
responses by modulating intracellular signal pathways.3 The
term ligand efficacy refers to the capability of a molecule to
induce a specific physiological response of receptor activation.4
The efficacy of a full agonist is by definition 100%, whereas
that of a full antagonist is 0%. The efficacy of a partial agonist
ranges from 0 to 100% while an inverse agonist has a negative
efficacy.5 Drugs that act on the same receptor can be used for
different purposes based on their efficacies. Thus, the
elucidation of efficacies of ligands at GPCRs has recently
become a critical research subject in the GPCR drug discovery
field.6
In the pharmaceutical industry, GPCRs are among the most
extensively investigated drug targets.7 Among them, leuko-
triene B4 receptor 2 (BLT2) is a receptor for leukotriene B4
(LTB4), which is a pro-inflammatory lipid mediator, and is a
recent promising GPCR drug target. It has been mainly
recognized as a drug target for the management of
inflammatory diseases8 as well as cancer treatment.9 BLT2 is
minimally expressed in the homeostatic normal state and
specifically overexpressed in response to stress environment-
inflammation in asthma and cancer.8,9 Recent studies suggest
that blocking BLT2 is a potentially useful strategy for the
However, due to the immune system-related role of BLT2,
only ligands that exhibit antagonist efficacy are eligible for new
drug candidates for BLT2. In this context, understanding the
mechanism of ligand efficacy at the molecular level is necessary
for the selective design of a compound. Based on recently
solved GPCR structures,12 it has been shown that the
difference between agonists and antagonists can be explained
by distinct interactions with a few binding site residues. In the
case of the β2-adrenergic receptor, agonist BI-167107 forms a
distinct hydrogen bond with S2075.46 (the superscript indicates
Ballesteros−Weinstein numbering.) The serine undergoes
conformational changes in the agonist-bound active state and
stabilizes the active state by forming a hydrogen bond with the
agonist. However, the crystal structure has not been solved for
Received: February 10, 2020
Accepted: July 14, 2020
Published: July 14, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00065
© XXXX American Chemical Society
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BLT2 and the molecular mechanism responsible for ligand-
dependent efficacy for BLT2 remains poorly understood.
In this study, we aim to elucidate the mechanism of ligand
efficacies at BLT2 using a series of molecular modeling
techniques and experimental verification. First, we predicted
the three-dimensional (3D) structures of BLT2 in apo and
agonist- or antagonist-bound forms at an atomic scale using
molecular modeling. Based on the predicted structures, we
identified residues that could determine ligand efficacy.
Subsequently, we verified our predictions through site-directed
mutagenesis and cell motility experiments. By chemically
modifying a known agonist, we further confirmed our
proposed ligand efficacy mechanism thus enabling the design
of partial agonists. Additionally, we discuss in detail our
molecular modeling procedures of class A GPCR structure
prediction and GPCR ligand docking used to identify the
mechanism for ligand efficacy at BLT2.
according to their efficacies.16 Our predicted structure of BLT2
is based on an antagonist-bound structure of BLT1; therefore,
to accommodate the possibility of conformational changes
when an agonist binds BLT2, we introduced a structure
optimization step for protein−ligand complex after the
docking. It is well-known that the overall conformational
changes of GPCRs in active and inactive forms are difficult to
simulate.17 On the other hand, the structure optimization was
performed only on the binding site residues within 5 Å of the
ligand using the Prime18 module of the Schrodinger suite.
̈
We first predicted a binding pose of a native agonist
leukotriene B4 (LTB4). In the binding pose, LTB4 adopts a U-
shaped conformation in the binding site of BLT2 (Figure 1).
First, we predicted the 3D structure of human BLT2 (Figure
S1) using the GPCR homology modeling method we have
developed (Supporting Information). The crystal structure of
guinea pig BLT1 (PBD ID: 5X33) recently revealed was used
as the template for homology modeling. BLT2, like BLT1,
belongs to the leukotriene receptor family and recognizes the
same native ligands including LTB4. Human BLT2 and BLT1
exhibit 39.5% sequence identity and have similar amino acid
compositions in the ligand binding sites (Figure S2).
Seven transmembrane (TM) helices of GPCRs show
considerable variations in the helix shape because of kink
structures.13 Because conformations of TMs determine the
shape and volume of the ligand-binding site, accurately
predicting the helix conformation is crucial. Therefore, we
added two additional modeling steps. First, we ensured that the
position of proline or glycine that can form a kink in the helix
structure is consistent between template-BLT1 and target-
BLT2 (Figure S2). Second, we performed molecular dynamics
(MD) simulation to optimize the predicted structure of BLT2
under explicit membrane and solvent environments. MD
results showed that the predicted BLT2 structure rapidly
entered the equilibrium state at 200 ns and maintained stable
conformations to 1 μs (Figure S3). For comparison, we
performed another MD calculation on the crystal structure of
BLT1. At the equilibrium state, average heavy atom RMSD
values of the crystal structure of BLT1 and the predicted
structure of BLT2 were 4.84 and 4.86 Å, respectively.
Considering the thermal fluctuation of the BLT1 crystal
structure, one can conclude that the predicted BLT2 structure
did not undergo significant structural changes during the MD
simulation. This result also implies that the initial predicted
BLT2 structure is as stable as the crystal structure of BLT1.
The only notable structural difference between the predicted-
BLT2 and template-BLT1 is the conformation of extra-cellular
loop 2 (ECL2). Among the six loop structures in GPCRs,
ECL2 was found to be in direct interactions with the bound
ligand.14 The predicted BLT2 structure has a distinct parallel β
sheet in ECL2 like other members of γ-branch GPCRs
including BLT1 and opioid receptors.15 However, the ECL2 of
BLT2 is four residues shorter and located further inside the
ligand binding site than that of BLT1 (Figure S1).
Figure 1. Structural features of agonist LTB4 binding in BLT2 (green
surface: hydrophobic, cyan surface: polar uncharged).
The position 1 carboxyl group located in the binding site
entrance participates in various interactions with residues in
ELC2; the position 4 hydroxyl group forms hydrogen bonds
with Gln2987.33; and the position 12 hydroxyl group forms a
hydrogen bond with residue Tyr2716.51, located deep in the
binding site pocket. Long hydrophobic chain position 13−20
forms van der Waals contacts with the hydrophobic region in
the inner binding site. The predicted binding pose of LTB4 is
similar to that obtained from the nuclear magnetic resonance
(NMR) experiment.19 This agreement strongly suggests that
our predicted BLT2 structure and modeling methods are
adequate for studying BLT2 and ligand interactions.
We conducted additional docking simulations using the
agonists 12(S)-HETE, 12-HHT, and CAY1058320 to find
distinct interactions of agonists. 12(S)-HETE and 12-HHT are
native BLT2 agonists derived from arachidonic acids such as
LTB4, whereas CAY10583 is a synthetic agonist. Prior to the
interaction analysis, we calculated binding free energies of the
four agonists based on the docking results using the molecular
mechanics/generalized Born surface area (MM/GBSA)
method, and compared the calculation results with the
experimentally measured binding affinities to ensure the
reliability of the docking results. Experimentally, 12-HHT
exhibited the highest binding affinity for BLT2, followed by
LTB4 and 12(S)-HETE, respectively.21 Our calculated binding
energies were in the order consistent with the experimental
results (Table S1). The two native BLT2 agonists commonly
adopt a U-shaped conformation similar to LTB4 and
CAY10583 was bound in the transverse form of the binding
Following the structure prediction of BLT2, we performed a
docking simulation on the BLT2 structure to predict the
binding pose of ligands. GPCRs have various conformational
ensembles between active and inactive states, and the ligands
form complexes with their respective states of receptors
B
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site. Analysis of the interactions revealed that all agonists
commonly formed hydrogen bonds with residue Tyr2716.51 in
helix 6 (Figure 2). As the TM6 participates in the activation
mechanism of GPCR,22 we hypothesized that Tyr2716.51 serves
as an activation trigger for BLT2.
Figure 3. (A) Migrating cells were fixed and stained with
hematoxylin/eosin. (B) Agonists-induced chemotactic motility was
determined in wild-type and mutant BLT2 (Y271A) expressed CHO
cells. (C) Agonists-induced chemotactic motility was determined in
wild-type and mutant BLT2 (Y271A/N275A) expressed CHO cells.
Figure 2. Binding poses of four agonists. (A) LTB4, (B) 12(S)-HETE,
(C) 12-HHT, (D) CAY10583 (white ribbon: BLT2, gray carbon
tube: binding site residues of BLT2, green carbon tube: agonists,
dotted-cyan line: hydrogen bond, dotted-green line: π−π stacking
interaction).
To test the above hypothesis, we performed further docking
simulations using antagonists SC-41930,23 CGS-25019C,24
LY255283,25 and CP-195543.26 The docking results showed
that all four antagonists do not form hydrogen bonds with
Tyr2716.51 (Figure S4), which confirms our hypothesis that
agonists selectively form hydrogen bonds with Tyr271; thus,
Tyr271 acts as an activation trigger for BLT2.
The simulation results were verified using a site-directed
mutagenesis approach. Recent studies have shown that
activation of BLT2 regulates cell motility functions, such as
chemotaxis.27 Measuring the biological activities with chemo-
taxis logically validates how the agonists trigger the activity. We
performed a competitive binding experiment of CAY10583
with LTB4 to confirm that it does bind in the same pocket
(Figure 4). To check if Tyr2716.51 triggers BLT2 activation, we
produced a BLT2 Y271A mutant and examined its ability to
elicit chemotaxis in transiently transfected CHO-K1 cells. In
accordance with our prediction, the potency of the four
agonists of BLT2 to induce chemotaxis decreased in the BLT2
Y271A mutant when compared with the wild-type BLT2
(Figure 3B).
Figure 4. Competitive binding for CAY10583 against LTB4.
bonds with residues of TM6. In our modeling results, all
agonists form hydrogen bonds with Tyr271 of TM6 and van
der Waals contacts with binding site residues of TM2 and
TM3, as shown in Figure 2. In our mutagenesis experiments,
however, LTB4 and 12(S)-HETE did not lose their activity
completely at BLT2 Y271A mutant, as shown in Figure 3. This
phenomenon can be attributed to the observation that the two
agonists have longer hydrophobic chains than 12-HTT, which
maintain van der Waals contacts with TM2 and TM3 (Figure
2) and form additional hydrogen bonds with Asn2756.55
located at a higher position than Tyr2716.51 in TM6. Therefore,
we formulated an additional hypothesis that for LTB4 and
12(S)-HETE, there exists a second agonist-specific interaction,
namely the hydrogen bond with Asn275, which leads to partial
agonism.
To test this additional hypothesis, we examined the ability of
each ligand to elicit chemotaxis from the BLT2 Y271A/N275A
double mutant. As shown in Figure 3C, now LTB4-elicited
chemotaxis was completely blocked in cells transfected with
the BLT2 Y271A/N275A double mutant along with all the
other ones elicited by other agonists. Our experiments verified
that Tyr2716.51 is the preferential residue that leads to BLT2
activation and Asn2756.55 plays an assisting role.
However, the chemotactic motility of the cells transfected
with the Y271A mutant was not completely blocked in
response to LTB4 or 12(S)-HETE. According to previous
GRCR activation mechanistic studies,4,28 GPCR undergoes
helical rearrangement of TM5, TM6, and TM7 while
transforming from the inactivation form to the activation
form. The bound agonist stabilizes the active state by
interacting with the key residues. A crystal structure of the
A
2A complexed with agonist NECA offers structural features of
agonist binding.12 In this case, NECA has hydrophobic
contacts with residues of TM2 and TM3 and hydrogen
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Our prediction for the role of Tyr2716.51 was also verified
through chemical variation. We synthesized a new compound,
dubbed AC-1657, in which a methyl group replaces the
carboxyl group of CAY10583 (Figure 5). As the carboxyl group
Figure 6. Chemotactic motility decreased with increasing distance of
hydrogen bond between Tyr2716.51 and carboxyl group. (A) Distance
measurements between Tyr271 and carbon atoms of terminal
aromatic ring. (B) Chemotactic motility results. (C) Chemical
structures of LMT-1886-L and LMT-1887-L.
Figure 5. Synthetic compound AC-1657 could not induce chemo-
tactic motility.
of CAY10583 formed a hydrogen bond with Tyr2716.51, we
could verify the role of Tyr2716.51 through a chemotactic
motility test using LMT-2657-L. The cells transfected with
wild-type BLT2 exhibited significant chemotactic motility in
response to CAY10583 but not to LMT-2657-L as shown in
Figure 5. Furthermore, we tested the criticality of the hydrogen
bond between Tyr2716.51 and the carboxyl group of CAY10583
via binding affinity measurement. By measuring the ability of
LMT-2657-L to compete for 3H-LTB4 binding to the
membranes of cells expressing BLT2, we observed that the
AC-1657 compound has no ligand binding affinity. Tyr2716.51
plays important roles not only as an activation trigger for
agonists but also as an important residue in the ligand binding
of them. The major interaction force between Tyr2716.51 and
the agonists is revealed to be the hydrogen bond.
ionic interaction network with Arg301 of TM7, Gln278 of
TM6, and His203 of ECl2. AC-1074 not only acted as an
antagonist in cell-based chemotaxis test but also displayed IC50
value of 132 nM from competitive ligand binding affinity
measurement using radiolabeled LTB4 (Figure 7), which is
Our simulation results showed that the agonists form
hydrogen bonds with Tyr2716.51 and this interaction stabilizes
the active state of the receptor. Thus, we hypothesized that a
compound that has a weak hydrogen bond with Tyr2716.51
could act as a partial agonist. To test this hypothesis, we
synthesized two compounds called LMT-1886-L and LMT-
1887-L in which the carboxyl group at the ortho- position of
CAY10583 was shifted to the meta- and para-positions,
respectively (Figure 6). The chemical modifications increased
the distances of the hydrogen bond between the carboxyl
group and Tyr2716.51, and the strength of the two hydrogen
bonds would be weakened. Indeed, the chemotactic motilities
of the cells transfected with the wild-type BLT2 partially
decreased in response to LMT-1886-L and LMT-1887-L. Our
results strongly suggest that the hydrogen bond between
Tyr2716.51 and the agonist is critical in stabilizing the active
state of the receptor.
We also designed selective BLT2 antagonists based on the
revealed activation mechanism of BLT2 thereby proving our
hypotheses even further. Using the LMT compound as a
template scaffold, we designed a series of derivatives and
predicted their ligand efficacies and binding energies using the
molecular modeling approaches. LMT-2074-L, the full
structure of which we cannot reveal in this paper, emerged
in the end. LMT-2074-L does not form any hydrogen bond
with Tyr2716.51 and Asn2756.55 but a hydrogen bond and an
Figure 7. Competitive binding for LMT-2074-L against LTB4.
lower than that of a known BLT2 antagonist LY255283 (150
nM). Our calculation of the binding affinities indeed conforms
to this result giving −6.15 kcal/mol for LMT-2074-L and
−5.985 kcal/mol for LY255283.
In conclusion, we identified the binding site residues, which
determine ligand efficacy at BLT2. In GPCRs, ligand efficacy is
related to the structural rearrangement of the receptor;
however, gaining structural insights by exclusively using
experimental methods is difficult. In this work, we overcame
such difficulty by employing molecular modeling techniques.
We constructed a 3D structure of BLT2 and predicted binding
poses of agonists and antagonists using our own GPCR-specific
structure modeling protocol and docking method. Utilizing
molecular modeling, we predicted that Tyr2716.51 and
Asn2756.55 act as activation triggers of BLT2. Then we verified
the modeling results through chemotactic motility tests using
BLT2 mutants. The chemical variation test further confirmed
that the hydrogen bond between the agonist and Tyr2716.51 is
an important interaction for agonist binding and BLT2 activity.
Finally, we designed two partial agonists based on the BLT2
activation mechanism we constructed herein.
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Several site-directed mutagenesis experiments have stated
that residues at position 6.51 are associated with GPCR
activation.29 However, to the best of our knowledge, this is the
first study that identifies residues related to the activation of
leukotriene receptor BLT2. Furthermore, because of the
variety of residues expressed at the 6.51 position of GPCRs,
investigating the distinct interactions between the 6.51 residue
and agonists using only site-directed mutagenesis experiments
is difficult. However, we have successfully shown that the
hydrogen bond between Tyr2716.51 and agonists is an
important interaction to stabilize the active state of BLT2
using docking and LMT-2657-L chemical variation studies.
The discovery of activation triggers would help develop
selective BLT2 agonists and antagonists. We were able to
design an antagonist with significant binding affinity to BLT2,
but as is evident in comparison of its binding with that of a
known agonist (Figures 4 and 7), missing the interaction to the
trigger residues weakens the binding. Therefore, to design an
effective antagonist, how to strengthen the binding without
interacting with the two trigger residues must be investigated.
We are continuing our research effort along this line to design
more potent antagonists.
Kyeong Lee − College of Pharmacy, Dongguk University,
Goyang-si 10326, Republic of Korea; orcid.org/0000-0002-
5455-9956
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00065
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Author Contributions
∥M.K., J.-D.W., and D.S.H. contributed equally.
Funding
This work was partly supported by National Research
Foundation of Korea (NRF) [2020R1A2C1011059,
2020R1A2B5B01002046, 2019R1F1A1059339,
2018R1D1A1B07046939, 2017M3A9D8063317, and
2018R1A5A2023127], Technology Development Program of
MSS [S2832693], and Korea University Grant [K1903051].
Notes
The authors declare no competing financial interest.
Additionally, our strategy of combining computational
prediction with site-directed mutagenesis can be used to
elucidate different biases of other agonists by highlighting
involved residues. Overall, the cooperation of computational
and experimental approaches reported herein provides a
promising strategy for investigating ligand efficacy at GPCRs.
ACKNOWLEDGMENTS
■
̈
We thank Schrodinger, Inc. (New York, NY, USA) for the
generous supply of the software used in this research.
ABBREVIATIONS
■
GPCRs, G protein-coupled receptors; BLT2, leukotriene B4
receptor 2; LTB4, leukotriene B4; MD simulation, molecular
dynamics simulation; ECL2, extra cellular loop 2; QM/MM,
quantum mechanics/molecular mechanics; ARDS, acute
respiratory distress syndrome; ALI, acute lung injury; COPD,
chronic obstructive pulmonary disease; HA, hemeagglutinin;
MM/GBSA, molecular mechanics/generalized Born surface
area; PCR, polymerase chain reaction; BAC, bacterial artificial
chromosome
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00065.
Experimental details for molecular modeling, cell
chemotaxis assay and compound synthesis. NMR data
for newly synthesized compounds. Molecular modeling
figures. (PDF)
REFERENCES
■
AUTHOR INFORMATION
Corresponding Authors
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