Natural Product Neopeltolide as a Cytochrome bc1 Complex Inhibitor: Mechanism of Action and Structural Modification

Article abstract of DOI:10.1021/acs.jafc.8b06195

The marine natural product neopeltolide was isolated from a deep-water sponge specimen of the family Neopeltidae. Neopeltolide has been proven to be a new type of inhibitor of the cytochrome bc₁ complex in the mitochondrial respiration chain. However, its detailed inhibition mechanism has remained unknown. In addition, neopeltolide is difficult to synthesize because of its very complex chemical structure. In the present work, the binding mode of neopeltolide was determined for the first time by integrating molecular docking, molecular dynamics simulations, and molecular mechanics Poisson-Boltzmann surface area calculations, which showed that neopeltolide is a Q_o site inhibitor of the bc₁ complex. Then, according to guidance via inhibitor-protein interaction analysis, structural modification was carried out with the aim to simplify the chemical structure of neopeltolide, leading to the synthesis of a series of new neopeltolide derivatives with much simpler chemical structures. The calculated binding energies (ΔG_cal) of the newly synthesized analogues correlated very well (R² = 0.90) with their experimental binding free energies (ΔG_exp), which confirmed that the computational protocol was reliable. Compound 45, bearing a diphenyl ether fragment, was successfully designed and synthesized as the most potent candidate (IC₅₀ = 12 nM) against porcine succinate cytochrome c reductase. The molecular modeling results indicate that compound 45 formed a π-π interaction with Phe274 and two hydrogen bonds with Glu271 and His161. The present work provides a new starting point for future fungicide discovery to overcome the resistance that the existing bc₁ complex inhibitors are facing.

Full text of DOI:10.1021/acs.jafc.8b06195

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Agricultural and Environmental Chemistry
Natural Product Neopeltolide as A Cytochrome bc1 Complex
Inhibitor: Mechanism of Action and Structural Modification
Xiao Lei Zhu, Rui Z'hang, Qiong-You Wu, Yong-Jun Song,
Yu-Xia Wang, Jing-Fang Yang, and Guang-Fu Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06195 • Publication Date (Web): 22 Feb 2019
Downloaded from http://pubs.acs.org on February 23, 2019
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Natural Product Neopeltolide as A Cytochrome bc1 Complex
Inhibitor: Mechanism of Action and Structural Modification
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Xiao-Lei Zhu†, Rui Zhang†, Qiong-You Wu†, Yong-Jun Song†, Yu-Xia Wang†, Jing-Fang Yang†,
Guang-Fu Yang†‡
†Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensor Technology and Health of Ministry of Science and
Technology, Central China Normal University, Wuhan 430079, P.R. China
‡Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300071, P.R.
China.
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Correspondence:
Guang-Fu Yang, Ph.D. & Professor
College of Chemistry
Central China Normal University
Luoyu Road 152, Wuhan 430079
P. R. China
TEL: 86-27-67867800
FAX: 86-27-67867141
E-mail: gfyang@mail.ccnu.edu.cn
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Abstract:
The marine natural product neopeltolide was isolated from a deep-water sponge specimen of the
family Neopeltidae. Neopeltolide has been proven to be a new type of inhibitor of the cytochrome
bc1 complex in the mitochondrial respiration chain. However, its detailed inhibition mechanism
has remained unknown. In addition, neopeltolide is difficult to synthesize because of its very
complex chemical structure. In the present work, the binding mode of neopeltolide was
determined for the first time by integrating molecular docking, molecular dynamics simulations,
and MM/PBSA calculations, which showed that neopeltolide is a Qo-site inhibitor of the bc1
complex. Then, according to guidance via inhibitor-protein interaction analysis, structural
modification was carried out with the aim to simplify the chemical structure of neopeltolide,
leading to the synthesis of a series of new neopeltolide derivatives with much simpler chemical
structures. The calculated binding energies (ΔGcal) of the newly synthesized analogs correlated
very well (R2 = 0.90) with their experimental binding-free energies (ΔGexp), which confirmed that
the computational protocol was reliable. Compound 45, bearing a diphenyl-ether fragment, was
successfully designed and synthesized as the most potent candidate (IC50 = 12 nM) against porcine
succinate cytochrome c reductase (SCR). The molecular modeling results indicate that compound
45 formed a π-π interaction with Phe274 and two H-bonds with Glu271 and His161. The present
work provides a new starting point for future fungicide discovery to overcome the resistance that
the existing bc1 complex inhibitors are facing.
Keywords: neopeltolide, bc1 complex, natural product, molecular design, inhibitor
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1. Introduction
Plant diseases caused by fungal pathogens have been recognized as a worldwide threat to
the agricultural industry. The control of plant fungal infections is very important to the production
of food and has a significant impact on human health. The bc1 complex (also known as complex
III) is one of the most important fungicidal targets.1 The bc1 complex is responsible for the
completion of electron transfer, hydroquinone oxidation and reduction of cytochrome c in the
mitochondrial respiratory chain. If the activity of the bc1 complex is inhibited, the generation of
ATP would be blocked, leading to cell death.2-6 As is known, the development of strobilurin
fungicides targeting the bc1 complex (e.g., azoxystrobin) has been a milestone in the fungicidal
market worldwide. However, according to the Fungicide Resistance Action Committee (FRAC),
an explosive increase of higher resistance has occurred worldwide for strobilurin fungicides,
especially those associated with the G143A mutation in the bc1 complex.7-9 Therefore, an urgent
demand exists to discover new fungicides to overcome the resistance to the existing strobilurin bc1
inhibitors.
Natural products have attracted considerable attention because of their unique molecular
architecture, potent biological activities, and excellent environmental compatibility.10-11 In 2007, a
new marine natural product, neopeltolide (Figure 1), obtained from a deep-water sponge specimen
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of the family Neopeltidae, was reported by Wright et al.
Biological assays revealed that
neopeltolide could inhibit the bovine heart mitochondrial cytochrome bc1 complex with an IC50
value of 2.0 nM.13 Compared with classical inhibitors of the bc1 complex, neopeltolide has a new
chemical scaffold, making it a promising lead compound for the discovery of new fungicides to
overcome the pathogen resistance that existing cytochrome bc1 complex inhibitors are facing.
However, the chemical structure of neopeltolide is too complex to be effectively synthesized, and
determining how to simplify its chemical structure is a challenge. In addition, a structural-activity
relationship (SAR) study showed that the carbamate-containing oxazole moiety was the key
structural feature, whereas its 14-membered macrolactone moiety did not make significant
contribution to its binding.14-19 The fungicide-likeness property also needs to be improved.20
Until now, the inhibition mechanism of neopeltolide was unknown. Because the discovery
of structurally diverse bc1 inhibitors is of interest, herein, we studied the binding mode of
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neopeltolide to the bc1 complex via molecular simulations. Then, structural optimizations of
neopeltolide were performed based on its binding mode. Herein, we describe the binding
mechanism of neopeltolide, compare its binding mode with some other bc1 complex inhibitors,
describe process of the structural optimization of neopeltolide, and describe the inhibition effects
of some newly synthesized compounds against the porcine bc1 complex. In addition, we carried
out molecular docking of these newly synthesized compounds, followed by molecular dynamics
(MD) simulations and the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA)
calculations. Fortunately, some compounds with a simpler chemical structure were successfully
obtained with significantly improved potency compared with the commercial product
azoxystrobin.
2. Materials and Methods
Chemistry.
All chemical reagents and solvents purchased from commercial sources were directly used
without further purification. 1H NMR and 13C NMR spectra were recorded on a Mercury-Plus 600
and or 400 MHz spectrometer (Varian, Palo Alto, CA) with TMS as an internal reference in
DMSO-d6 or CDCl3. An Agilent 6520 Accurate-Mass Q-TOF mass spectrometer (LC/MS)
(Agilent Technologies, Santa Clara, CA) was used to record high solution mass spectra. The
melting points for titled compounds were determined by a B-545 melting point apparatus (Büchi,
Flawil, Switzerland). Silica gel (200-300 mesh) for column chromatography was purchased from
Qingdao Haiyang Chemical Co. Here, our purpose was to obtain the noted compounds quickly,
but the yields of some compounds were not good.
Enzymatic Assays.
The preparation of succinate cytochrome c reductase (SCR, a mixture of complex II and the
bc1 complex) from porcine heart was essentially the same as reported.23 The enzymatic activities
of SCR, complex II, and the bc1 complex were analyzed in respective reaction mixtures as
24-26
reported previously.
The SCR activity was measured in a 1.8 mL reaction mixture that
contained 100 mM PBS (pH 7.4), 0.3 mM EDTA, 20 mM succinate, 10 µM oxidized cytochrome
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c, and an appropriate amount of SCR at 23 °C. The reaction was continuously monitored by
following the absorbance change at 550 mm or 600 mm on a Perkin-Elmer Lambda 45
spectrophotometer equipped with a magnetic stirrer. The extinction coefficients used were 18.5
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mM-1 cm-1 of A550
for the cytochrome c reduction and 21 mM-1 cm-1 of A600
for the
red ox
red ox
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DCIP reduction. To test the IC50 values of all compounds, the reaction was carried out in the
presence of varying concentrations of the inhibitor.
Computational Methods
Molecular Docking. The three-dimensional (3D) structure of the bc1 complex was obtained
from the PDB database (PDB ID: 3CWB).27 The AutoDock 4.2 program28 was used to dock
neopeltolide and its derivatives into the Qo-site and or Qi-site of the bc1 complex. The Gasteiger
charges were used for these inhibitors. To select the best set of docking parameters and to test the
reliability of the docking results, the crocacin derivative (ligand in 3CWB) 27 and antimycin (the
Qi-site inhibitor) were first docked into the Qo and Qi sites, respectively.
In the docking process, the Solis and Wets local search method and the Lamarckian genetic
algorithm (LGA) 28 were selected for the conformational search. The grid size was set to 40 × 50 ×
40, and the grid space was set to 0.375 Å. The others parameters were as set as in our previous
work.23-24 Among a set of 256 candidates of docked complex structures, the best one was selected
according to the interaction energy and was then compared with the conformation of crocacin
derivative/antimycin extracted from the crystal structure. By tuning the docking parameters, the
final inhibitor conformation in the Qi and or Qo sites was obtained based on the smallest
root-mean-square deviation (RMSD). The RMSD between the selected conformation and that
previously noted in the X-ray crystal structure was 0.79 for the Crocacin derivative and 0.85 for
antimycin (data not shown), confirming the reliability of the docking parameters. The same
docking parameters were used to perform molecular docking of other compounds.
Molecular Dynamics and Binding Energy Calculation. All of the complex structures
derived from the molecular docking were used as starting structures for further energy
minimizations using the Sander module of the Amber14 program29 before the final binding
structures were achieved. The Amber ff14 force field was used for amino acids, and the General
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Amber force field was used for ligands.30 For temperature regulation, a Langevin thermostat was
used to maintain a temperature of 300 K. The bcc charges were used as the atomic charges for
ligands. There were five stages for energy minimization. First, the hydrogen atom was minimized
with all heavy atoms fixed. Second, all of the receptor atoms were minimized with the ligand fixed.
Third, minimization was carried out with all the atoms fixed. Fourth, minimization was carried out
with the backbone atoms fixed. The final minimization was performed with both the ligand and
the protein relaxed. In each step, energy minimization was executed by using the steepest descent
method for the first 1000 cycles and the conjugated gradient method for the subsequent 3000
cycles, with a convergence criterion of 0.1 kcal mol−1 Å−1. The atomic coordinates were saved
every picosecond. Then, an additional 30 ns MD simulation was performed for neopeltolide-Qo
and the neopeltolide-Qi complex. To reduce the computational cost, only 20 ps MD simulations
were performed for the newly synthesized analogs.
The binding energy of the protein-ligand complex was calculated using the MM/PBSA
module.31-32. A detailed description of this section is provided in the Supporting Information.
3. Results and Discussion
Binding Mode of Neopeltolide. Neopeltolide was identified as a bc1 complex inhibitor more
than ten years ago; however, its detailed binding mode remained unknown. In our previous
study,24 we developed an integrated computational simulation protocol to assign Ametoctradin as
a Qo site inhibitor of the bc1 complex for the first time, which was later experimentally confirmed
by Fehr and his co-workers.33 Herein, this computational protocol was further used to determine
the detailed binding mechanism of neopeltolide.
First, the chemical structure of neopeltolide was compared with that of existing bc1 inhibitors,
such as pyribencarb and a Crocacin derivative. Interestingly, the key carbamate moiety of
neopeltolide also existed in the commercial fungicide pyribencarb and in the highly potent
compound Crocacin derivative (Figure 1). Therefore, the crystal structure (PDB code: 3CWB)27 of
the chicken bc1 complex bound to the Crocacin derivative was selected herein as the receptor
model.
As is known, the bc1 complex has two binding sites, the Qo-site (a quinol oxidation site) and
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the Qi-site (a quinone reduction site). Then, neopeltolide was docked into the Qo-site and the
Qi-site, producing two neopeltolide-bound complexes (Figure S1), referred to as the
neopeltolide-Qo complex and neopeltolide-Qi complex, respectively. The binding energy obtained
from AutoDock was -9.44 kcal/mol for the neopeltolide-Qo complex and -8.90 kcal/mol for the
neopeltolide-Qi complex, indicating that the neopeltolide-Qo complex was more favorable than the
neopeltolide-Qi complex. To further confirm the binding mode of neopeltolide, we performed 30
ns molecular dynamics simulations for the neopeltolide-Qo and neopeltolide-Qi complexes (Figure
S2), and 200 snapshots obtained from the last 2 ns of each complex were used to calculate the
binding energy using the MM/PBSA module. As shown in Table 1, the binding energies (ΔH)
were -36.77 kcal/mol and -22.05 kcal/mol for the neopeltolide-Qo and neopeltolide-Qi complexes,
respectively. The computational simulation results clearly showed that neopeltolide should bind to
the Qo-site of the bc1 complex.
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The comparison of the MD-simulated neopeltolide-Qo complex with the known crystal
structure of the Qo-site inhibitor-bound bc1 complex was very interesting. As shown in Figure 2,
the complex structures of azoxystrobin (1SQB),34 famoxadone (1L0L),35 crocacin derivative
(3CWB)27 and stigmatellin (1PP9) 36 were superimposed. The common interaction with the bc1
complex was that residues Glu271 and Phe274 (Figure 3A) separately formed H-bonds and a
hydrophobic interaction with the inhibitor, indicating that the two residues played an important
role in the binding of Qo-site inhibitors. In addition, we observed that different types of inhibitors
occupied relatively different spaces inside the Qo pocket, although they shared some common
binding area. For example, as shown in Figure 2, the phenoxypyrimidine moiety in azoxystrobin
took on a nearly parallel conformation with the diphenyl-ether fragment in famoxadone, and their
key pharmacophores (the methoxyacrylate fragment in azoxystrobin and oxazolidinedione
fragment in famoxadone) formed the same H-bond with Glu271. The Crocacin derivative and
stigmatellin showed some translation and then formed an additional new H-bond with His161,
revealing an important binding site for the design of a Qo-site inhibitor.
While considering the chemical structural similarity between neopeltolide and pyribencarb,
we also compared the neopeltolide-Qo complex with the binding model of pyribencarb37-38 (Figure
3B) obtained from AutoDock. Similar to the classical Qo-site inhibitor azoxystrobin, pyribencarb
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also formed an H-bond with Glu271 and a hydrophobic interaction with Phe274. For
neopeltolide-Qo (Figure 3C, Figure S2B), its carbamate moiety inserted into the Qo pocket and
then formed an H-bond with Glu271. Meanwhile, the 14-membered macrolactone moiety of
neopeltolide was located at the entrance of the Qo-site and took on a relative extended
conformation, forming hydrophobic interactions with residues Leu121, Met124, Phe128, Ile146,
Phe150, Leu294, Leu299 and Phe274. The relative position of the neopeltolide in the Qo-site was
very similar to that of the crocacin derivative, but neopeltolide did not form an H-bond with
His161.
Neopeltolide is attractive because its carbamate moiety gives it great potential to overcome
the pathogen resistance associated with the G143A mutation.7 Shimizu and his co-workers37
reported that pyribencarb with a carbamate moiety as a pharmacophore showed a much lower
resistance risk (R/S = 65 for Botrytis cinerea and 16 for Sclerotinia sclerotiorum) than
kresoxim-methyl (R/S = 700 for B. cinerea and 580 for S. sclerotiorum) and azoxystrobin (R/S =
300 for B. cinerea and 1200 for S. sclerotiorum). Our computational simulations revealed that the
steric clash between the inhibitors and residue Ala143 of the mutated bc1 complex reduced the
binding affinity of the inhibitors.24 For the wide-type inhibitor-bound complex (Figure 3B), the
distance between Gly143 and inhibitors ranged from 3.6 Å (e.g., azoxystrobin) to 4.6 Å (e.g.,
pyribencarb). The shorter the distance, the higher the resistance. Therefore, pyribencarb showed
much lower resistance in the presence of the G143A mutation due to the greater distance between
its carbamate moiety and the 143th residue. These results suggest that neopeltolide is a promising
lead for the discovery of new bc1 complex inhibitors to overcome the resistance associated with
the G143A mutation.
Rational Design of Neopeltolide Analogs and Structural Optimization
The above computational simulations showed that His161 is an important binding site for
Qo-site inhibitors. However, neopeltolide did not form an H-bond with His161, possibly due to the
lack of H-bond receptors of neopeltolide, which are marked by the black arrow in Figure 3C.
Therefore, target molecules were designed by introducing an amide bond to replace the carboxylic
ester moiety. Meanwhile, to simplify the chemical structure of neopeltolide, the butylene unit
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between the carboxylic ester moiety and oxazole ring was deleted and the 14-membered
macrolactone moiety of neopeltolide was replaced with a diphenyl-ether fragment. Our previous
study26 showed that the diphenyl-ether was a fragment that forms hydrophobic interactions with
the protein. We have successfully designed a series of diphenyl-ether derivatives, especially those
bearing the 2-Cl-4-CF3 and 2-Cl-4-Cl group, as bc1 complex inhibitors and complex II inhibitors
(Figure 1). In addition, some other groups also reported a series of diphenyl-ether derivatives as
bc1 complex inhibitors.39-40 Therefore, compounds 15~23 were first synthesized, and their activity
against porcine SCR were tested and are shown in Table 2. The commercial fungicide
azoxystrobin was selected as a positive control. Fortunately, compounds 21 (R1 = H; R2 =
2-Cl-4-CF3) and 23 (R1 = 3-Cl-5-Cl; R2 = 2-Cl-4-Cl) showed good potency against porcine SCR,
with IC50 values of 0.17 μM and 0.92 μM, respectively. These results indicated that our design
strategy was reasonable and feasible.
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Then, structural optimization was carried out with compound 21 as a new starting scaffold.
To make the structure optimization more efficient, we first studied the binding mode of compound
21. As shown in Figure 3D, compound 21 could form an H-bond with Glu271 and could interact
hydrophobically with Phe274. However, unfortunately, compound 21 did not form an H-bond
with His161. Superimposing its binding mode with that of pyribencarb, we found that the relative
position of the carbamate pharmacophore of compound 21 was less deep than that of pyribencarb
(Figure 3D) and the Crocacin derivative (Figure S3A). In other words, the carbamate
pharmacophore of compound 21 did not match well with that of the pyribencarb and the Crocacin
derivative. Keeping these differences in mind, we then focused on adjusting the relative position
of the carbamate pharmacophore by inserting a CH2 unit between the NH of the amide and phenyl
ring of the diphenyl-ether or between the oxygen atom and the terminal phenyl ring of the
diphenyl-ether to produce compounds 24~31 and 32~43, respectively. The bioassay indicated that
compounds 24~31 did not show improved potency, whereas six compounds from the second series
displayed significantly improved inhibition activity against porcine SCR compared to compound
21 (IC50 = 0.17 μM). For example, the IC50 values of compounds 36, 37, 38, 41, 42, and 43 were
0.047 μM, 0.039 μM, 0.082 μM, 0.067 μM, 0.044 μM and 0.021 μM, respectively, and had
approximately 2- to 8-times higher potency that compound 21.
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Then, compound 43, which had the highest potency, was selected as a representative
compound to analyze its binding mode. As shown in Figure 3E, except for the H-bond interaction
with Glu271, compound 43 formed an additional H-bond with His161. In addition, its
diphenyl-ester moiety extended toward the entrance of the Qo-site to form hydrophobic
interactions with Phe274 and Phe128. As a consequence, the carbamate pharmacophore of
compound 43 matched very well with that of the pyribencarb and Crocacin derivative (Figure
S3B). The significantly increased potency of compound 43 confirmed that His161 is an important
binding site, which should be taken into account when designing new bc1 complex inhibitors.
Because the diphenyl-ester formed a hydrophobic interaction with its surrounding residues,
introducing a hydrophobic group onto the diphenyl-ester moiety is favorable for the binding
affinity. For example, compounds 36 (3-Cl, IC50 = 0.047 μM), 37 (2-Br, IC50 = 0.039 μM) and 38
(3-Br, IC50 = 0.082 μM) showed higher activity than compound 33 (3-F, IC50 = 0.32 μM).
According to our previous study25, improving the π-π stacking interaction with Phe274 will
benefit the binding affinity. Then, a naphthalene ring was introduced to replace the terminal
phenyl of diphenyl-ester moiety. Meanwhile, the amide bond was also replaced by an ester bond
based on the isostere rule. As expected, the resulting compound, 44, showed high inhibition
activity against the porcine SCR, with an IC50 value of 0.047 μM. Similarly, introducing a bromo
group into the naphthalene ring further improved the potency and produced compounds 45 and 46,
which had IC50 values of 0.012 μM and 0.016 μM, respectively. The binding mode of compound
45 to the bc1 complex is shown in Figure 3F. Compound 45 formed a H-bond not only with
Glu271 but also with His161. In addition, the naphthalene ring of 45 took on a more favorable π-π
interaction with Phe274. The energy calculation results showed that compound 45 had the highest
Van der Waals (VDW) energy (-77.06 kcal/mol) and the highest binding energy (ΔGcal = -48.67
kcal/mol) of all the compounds. Notably, compared with neopeltolide, compound 45 had a slightly
lower activity, but its chemical structure is much simpler and its synthesis is much easier.
Therefore, compound 45 could be regarded as a new lead for the further discovery of bc1 complex
inhibitors.
Synthetic Chemistry
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The titled compounds were synthesized by multiple-step routes, as shown in Scheme 1. In
this synthetic procedure, (Z)-2-(3-((methoxycarbonyl)amino)prop-1-en-1-yl)oxazole-4- carboxylic
acid 6 is a key intermediate for the titled compounds. Here, the cheap and commercially available
2-propynylamine (1a) and methyl chloroformate (1b) were selected as starting materials. The
preparation of 4-((methoxycarbonyl)amino)but-2-ynoic acid 2 was achieved by carboxylation of
prop-2-yn-1-ylcarbamate 1 with butyllithium in THF under a CO2 atmosphere. Reduction of 2
with Lindlar’s catalyst afforded (Z)-4-((methoxycarbonyl)- amino)but-2-enoic acid 3, which
reacted with L-serine methyl ester hydrochloride, N-methylmorpholine and isobutyl chloroformate
to afford methyl(Z)-(4-((methoxycarbonyl)- amino)but-2-enoyl)serinate 4. The intermediate 6
could be obtained by cyclization of 4 with DAST, DBU and BrCCl3 at low temperature, followed
by hydrolysis with lithium hydroxide to provide an excellent yield.
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The substituted 4-diphenyl ether derivatives 8a-i, 10a-f, 12a-l and 14a-c moiety could be
prepared in two routes (A, B) via a nucleophilic substitution reaction. In route A, substituted
fluorobenzenes/phenols were reacted with the corresponding phenols/benzyl bromide to afford
intermediates 7a-i and 9a-f, which were reduced with iron or zinc powder to afford intermediates
8a-i, and 10a-f. In route B, 4-fluorobenzaldehydes were reacted with substituted phenols or
naphthols to afford 11a-l and 13a-c, which were reduced, respectively, with zinc powder or
NaBH4 to afford the key intermediates 12a-l and 14a-c. In the final step, condensation of
4-diphenyl ether derivatives 8a-i, 10a-f, 12a-l and 14a-c with intermediate 6 was performed
to afford the corresponding target compounds 15~46.
Computational Simulations
To understand the structure-activity relationship at the atomic level, we performed the
molecular docking and binding free energy calculations for all the newly synthesized compounds
according to previous methods.41-43 The different kinds of binding energy (ΔGcal, the calculated
binding free energy; ΔEVDW, the VDW energy; ΔEele, the electrostatic energy; ΔEnp, the nonpolar
solvation energy; and ΔEpolar, the polar solvation energy) are shown in Table 4. The highest
binding free energy (ΔGcal) was -48.67 kcal/mol for compound 45, and the lowest binding energy
(ΔGcal) was -40.31 kcal/mol for compound 32. The corresponding experimental binding free
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334
energy (ΔGexp) was -10.84 kcal/mol for compound 45 and -8.47 kcal/mol for compound 32.
Moreover, the ΔGexp showed a qualitatively identical trend with ΔGcal, and their correlation
coefficient of R2 was 0.90 (Figure S4), further confirming the reliability of the computational
models constructed in the present study. In addition, the excellent correlation between ΔGcal and
ΔGexp also further proved that neopeltolide should belong to the Qo-site inhibitor of the bc1
complex.
In summary, the natural product neopeltolide was successfully assigned to be a Qo site
inhibitor by integrating molecular docking, molecular dynamics simulations and MM/PBSA
calculations. Then, a series of neopeltolide derivatives, for use as new potent inhibitors of the bc1
complex, were designed and synthesized, with neopeltolide as the template. Compound 45,
(Z)-4-((7-bromonaphthalen-2-yl)-oxy)benzyl-2-(3-((methoxycarbonyl)amino)prop-1-en-1-yl)-
oxazole-4-carboxylate was identified as the most potent candidate, with an IC50 value of 12 nM
against porcine SCR. Further computational simulations revealed that most of the compounds
inside the Qo-site of bc1 complex formed a H-bond with Glu271 and a π-π interaction with Phe274,
while the most active compound (compound 45) formed an additional H-bond with His161. These
results indicate that Glu271, Phe274 and His161 are the key residues for designing bc1 complex
inhibitors.
Supplementary Data
Supplementary data associated with this article can be found in the online version, at
http://pubs.acs.org/.
AUTHOR INFORMATION
Corresponding Author *E-mail: (GFY) gfyang@mail.ccnu.edu.cn
Funding
The research was supported in part by the National Key Research and Development Program of
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China (2017YFD0200506), the National Natural Science Foundation of China (No. 21837001,
21472065, 21772057), and the Program of Introducing Talents of Discipline to Universities of
China (111 program, B17019).
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
Notes
The authors declare no competing financial interest.
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464
465
466
467
468
469
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471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
44. FIGURE CAPTIONS
Scheme 1. Synthetic route for the target compounds 15~46.
Figure 1. Design protocol of the target compound.
Figure 2. Relative position of known bc1 complex inhibitors in the Qo-site. The PDB structures
(1SQB, 1L0L, 1PP9, and 3CWB) were used. Among them, famoxadone and azoxystribin were
commercial fungicides, and stigmatellin and crocacin-derivative were high potency inhibitors. For
clarity, the long hydrophobic chain of stigmatellin was not displayed.
Figure 3. (A) Binding mode of the Crocacin derivative (yellow stick) with the bc1 complex (PDB
ID 3CWB). (B) The binding mode overlay of pyribencarb (brown stick) with Azoxystrobin (ice
blue stick, 1SQB). The closest distance between the ligand with G143 was measured. (C) The
binding mode of neopeltolide (yellow stick) with the bc1 complex. (D) The binding mode of
compound 21 (yellow stick) overlaid with pyribencarb (brown stick). (E) The binding mode of
compound 43 (yellow stick) overlaid with pyribencarb (brown stick). (F) The binding mode of
compound 45 (yellow stick) overlaid with pyribencarb (brown stick).
Figure 4. Kinetic analysis of inhibition by azoxystrobin (A) and compound 45 (B) against porcine
SCR. (A) Azoxystrobin is noncompetitive with respect to cytochrome c. The reaction mixture
contains 100 mM PBS (pH 7.4), EDTA (0.3 mM), succinate (20 mM), cytochrome c (3-60 μM),
an appropriate amount of SCR, and a series of concentrations of azoxystrobin (0 nM; 70 nM; 200
nM; 500 nM). (B) Compound 45 is noncompetitive with respect to cytochrome c. The reaction
mixture contains 100 mM PBS (pH 7.4), EDTA (0.3 mM), succinate (20 mM), cytochrome c
(3-60 μM), an appropriate amount of SCR, and a series of concentrations of 45 (0 nM; 100 nM;
200 nM; 300 nM).
Table 1. Binding energy of neopeltolide in the Qo and Qi sites (kcal/mol)
Table 2. Percent inhibition and IC50 value of the title compounds against porcine SCR.
Table 3. IC50 value of the title compounds against porcine SCR.
Table 4. Individual energy terms of the calculated binding energies of the title compounds
(kcal/mol)
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O
O
O
O
N
N
O
O
N
H
H
N
Pyribencarb
O
N
Cl
R2
Metominostrobin
O
O
n
I
HN
N
H
N
O
O
O
N
H
O
H
N
R1
m
N
O
O
Crocacin derivation
O
O
O
O
O
n=0,1
m=0,1
Famoxadone
O
H
O
O
H
O
MeO
F2HC
N
O
NH
H3C
N
O
N
H
N
H
O
Neopeltolide
O
N
O
Cl
CF3
Flubenetheram
ref 24
Figure 1. Design protocol of the target compounds
Figure 2. Relative position of known bc1 complex inhibitors in the Qo-site. The PDB structures
(1SQB, 1L0L, 1PP9, and 3CWB) were used. Among them, famoxadone and azoxystribin were
commercial fungicides, and stigmatellin and crocacin-derivative were high potency inhibitors. For
clarity, the long hydrophobic chain of stigmatellin was not displayed. .
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Figure 3. (A) Binding mode of the Crocacin derivative (yellow stick) with the bc1 complex (PDB
ID 3CWB). (B) The binding mode overlay of pyribencarb (brown stick) with Azoxystrobin (ice
blue stick, 1SQB). The closest distance between the ligand with G143 was measured. (C) The
binding mode of neopeltolide (yellow stick) with the bc1 complex. (D) The binding mode of
compound 21 (yellow stick) overlaid with pyribencarb (brown stick). (E) The binding mode of
compound 43 (yellow stick) overlaid with pyribencarb (brown stick). (F) The binding mode of
compound 45 (yellow stick) overlaid with pyribencarb (brown stick).
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Scheme 1. Synthetic route for the target compounds 15-46.
O
O
b
c
O
a
NH
O
O
O
N
N
HO
NH2
HO
O
H
H
O
3
Cl
O
1
2
1a
d
O
1b
N
N
O
O
NH
O
HN
e
f
NH
O
O
O
NH
O
O
O
OH
O
O
HO
O
O
O
5
4
6
Br
Routh A
R1
R2
R1
R1
g
NO2
NH2
NO2
i
OH
Q
Q
O
O
Y
R2
Y: F/ OH
Routh B
7a-i: Q= Phenyl
9a-f: Q= Benzyl
8a-i: Q= Phenyl
10a-f: Q= Benzyl
h
OH
R2
CHO
j / k
Z
g
W
W
CHO
O
O
OH
R2
F
11a-l : W= Phenyl
13a-c: W= Napthyl
12a-l : W= Phenyl; Z=NH2
14a-c: W= Napthyl; Z=OH
g
R1
O
N
Final step
O
N
O
m
O
Z
X
R1
OH
n
l
m
O
R2
+
O
O
O
R2
n
O
N
O
N
H
8a-i: m=0, n=0
10a-f: m=0, n=1
H
15-23: m=0, n=0, X=N
24-31: m=0, n=1, X=N
32-43: m=1, n=0, X=N
44-46: m=1, n=0, X=O
6
12a-l,14a-c: m=1, n=0
Z= NH2, OH
Reagents and conditions: (a) NaHCO3,1,4-dioxane; (b) CO2, n-BuLi, THF, -78°C; (c)H2, Lindlar’s
catalyst, EtOAc; (d) L-serine methyl ester hydrochloride, i-BuOCOCl, N-Me-Morpholine, THF;
(e) (1) DAST,CH2Cl2, -78°C (2) BrCCl3, DBU, -20°C; (f) LiOH,THF/H2O; (g) K2CO3, Acetone,
reflux; (h) K2CO3, DMF, reflux; (i) Fe,NH4Cl, EtOH/H2O, reflux; (j) (1) NH2OH·HCl,NaHCO3,
EtOH, reflux; (2) Zn, HCl, EtOH/H2O, reflux; (k) NaBH4,THF/H2O; (l) EDCI, HOBt, Et3N,
CH3CN/THF. The yield of compound ranges from 8.7% to 91%, and detailed information is
supported in the supporting information.
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Table 1. Binding energy of neopeltolide in the Qo and Qi sites (kcal/mol)
ΔHa
a
a
a
a
AutoDock binding Energy ΔEVDW
ΔEEle
ΔEpolar ΔEnp
neopeltolide-Qo
-9.44
-8.90
-70.79
-50.23
-10.51
-12.74
50.93 -6.40 -36.77
46.54 -5.62 -22.05
neopeltolide-Qi
a Obtained from the 30 ns MD.
Table 2. Percent inhibition and IC50 (μM) value of the title compounds against porcine SCR
R1
R2
O
H
O
N
O
N
O
O
N
H
No.
15
16
17
18
19
20
21
22
23
R1
3-Cl
R2
IC50 or I%
34%
2-Cl-4-CF3
2-Cl-4-CF3
2-Cl-4-CF3
2-Cl-4-CF3
2-Cl-4-CF3
2-Cl-4-CF3
2-Cl-4-CF3
2-Cl-4-Cl
3-Cl-5-Cl
3-Br
2-Me
3-Me
2-OMe
H
30%
49%
55%
42%
59%
0.17±0.016
14%
0.92±0.013
3-Cl
3-Cl-5-Cl
2-Cl-4-Cl
R1
O
H
N
O
R2
O
N
O
O
N
H
24
25
26
27
28
29
30
31
H
H
H
H
H
H
H
H
4-Br
3-Br
H
0.35±0.010
3.71±0.19
11%
4-Me
3-OMe
2-Cl-4-Cl
3-F
16%
<10%
22%
<10%
3-F-5-F
<10%
R1
O
N
O
NH
R2
O
O
O
N
H
32
33
34
35
36
37
38
39
40
41
42
43
H
H
H
H
H
H
H
H
H
H
H
H
-
2-F
3-F
4-F
2-Cl
3-Cl
0.64±0.01
0.32±0.01
0.33±0.01
0.15±0.01
0.047±0.02
0.039±0.01
0.082±0.01
0.11±0.01
0.15±0.01
0.067±0.01
0.044±0.01
0.021±0.01
0.205±0.010
2-Br
3-Br
3-Me
2-Cl-4-Cl
2-Cl-3-F
2-Cl-4-F
2-Cl-4-Me
-
azoxystrobin
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Table 3. IC50 (μM) value of the title compounds against porcine SCR.
O
O
O
O
N
O
R
O
N
H
No.
44
R
H
IC50
No.
45
R
IC50
7-Br 0.012±0.002
0.205±0.010
0.045±0.001
46 6-Br 0.016±0.001 azoxystrobin
-
Table 4. Individual energy terms of the calculated binding energies of the titled compounds
(kcal/mol)
a
Compound
21
ΔEVDW
-68.11
-65.31
-63.66
-61.02
-67.28
-70.63
-65.58
-67.80
-68.53
-66.16
-67.01
-65.57
-66.94
-75.18
-77.06
-73.95
ΔEEle
-15.16
-32.51
-36.08
-29.50
-21.04
-21.86
-27.02
-31.85
-21.57
-23.34
-23.26
-26.66
-26.70
-21.74
-21.74
-21.08
ΔEPolar
45.54
62.47
60.69
54.20
49.54
50.21
49.91
58.75
49.56
50.45
48.60
49.99
51.26
55.23
55.70
51.82
ΔEnp
-4.92
-4.96
-4.89
-4.88
-5.00
-5.11
-5.15
-4.94
-5.16
-5.32
-5.18
-5.24
-5.35
-5.44
-5.58
-5.45
ΔGcal
-42.65
-40.31
-43.94
-41.20
-43.78
-47.40
-47.83
-45.84
-45.69
-44.37
-46.86
-47.49
-47.74
-47.14
-48.67
-48.66
ΔGexp
-9.26
-8.47
-8.88
-8.86
-9.33
-10.02
-10.13
-9.69
-9.52
-9.33
32
33
34
35
36
37
38
39
40
41
42
43
44
45
-9.81
-10.06
-10.50
-10.04
-10.84
-10.65
46
a ΔGexp = -RTLnIC50
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 24
Graphical Abstract
24
ACS Paragon Plus Environment