Selective monocationic inhibitors of neuronal nitric oxide synthase. Binding mode insights from molecular dynamics simulations

Article abstract of DOI:10.1021/ja302269r

The reduction of pathophysiologic levels of nitric oxide through inhibition of neuronal nitric oxide synthase (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. We have developed a series of pyrrolidine-based nNOS inhibitors that exhibit excellent potencies and isoform selectivities (J. Am. Chem. Soc.2010, 132, 5437). However, there are still important challenges, such as how to decrease the multiple positive charges derived from basic amino groups, which contribute to poor bioavailability, without losing potency and/or selectivity. Here we present an interdisciplinary study combining molecular docking, crystallography, molecular dynamics simulations, synthesis, and enzymology to explore potential pharmacophoric features of nNOS inhibitors and to design potent and selective monocationic nNOS inhibitors. The simulation results indicate that different hydrogen bond patterns, electrostatic interactions, hydrophobic interactions, and a water molecule bridge are key factors for stabilizing ligands and controlling ligand orientation. We find that a heteroatom in the aromatic head or linker chain of the ligand provides additional stability and blocks the substrate binding pocket. Finally, the computational insights are experimentally validated with double-headed pyridine analogues. The compounds reported here are among the most potent and selective monocationic pyrrolidine-based nNOS inhibitors reported to date, and 10 shows improved membrane permeability.

Full text of DOI:10.1021/ja302269r

Article
pubs.acs.org/JACS
Selective Monocationic Inhibitors of Neuronal Nitric Oxide Synthase.
Binding Mode Insights from Molecular Dynamics Simulations
He Huang,† Haitao Ji,†,‡ Huiying Li,§ Qing Jing,† Kristin Jansen Labby,† Pavel Martasek,∥,⊥
́
Linda J. Roman,∥ Thomas L. Poulos,*,§ and Richard B. Silverman*,†
†Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, amd Center for Molecular
Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States
§Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine,
California 92697-3900, United States
∥Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78384-7760, United States
⊥Department of Pediatrics, First School of Medicine, Charles University, Prague, Czech Republic
S
* Supporting Information
ABSTRACT: The reduction of pathophysiologic levels of
nitric oxide through inhibition of neuronal nitric oxide
synthase (nNOS) has the potential to be therapeutically
beneficial in various neurodegenerative diseases. We have
developed a series of pyrrolidine-based nNOS inhibitors that
exhibit excellent potencies and isoform selectivities (J. Am.
Chem. Soc. 2010, 132, 5437). However, there are still
important challenges, such as how to decrease the multiple
positive charges derived from basic amino groups, which
contribute to poor bioavailability, without losing potency and/or selectivity. Here we present an interdisciplinary study
combining molecular docking, crystallography, molecular dynamics simulations, synthesis, and enzymology to explore potential
pharmacophoric features of nNOS inhibitors and to design potent and selective monocationic nNOS inhibitors. The simulation
results indicate that different hydrogen bond patterns, electrostatic interactions, hydrophobic interactions, and a water molecule
bridge are key factors for stabilizing ligands and controlling ligand orientation. We find that a heteroatom in the aromatic head or
linker chain of the ligand provides additional stability and blocks the substrate binding pocket. Finally, the computational insights
are experimentally validated with double-headed pyridine analogues. The compounds reported here are among the most potent
and selective monocationic pyrrolidine-based nNOS inhibitors reported to date, and 10 shows improved membrane permeability.
is difficult because of the challenge to achieve highly selective
inhibition of the specific isoforms.13 Each one of the three
isozymes is associated with different functions: nNOS is
devoted to neuronal signaling, iNOS to the immune response,
and eNOS to smooth muscle relaxation and blood pressure
regulation.14 Therefore, selective inhibition of nNOS over the
other isozymes is highly desirable for the treatment of
neurodegenerative diseases to avoid undesirable effects related
to iNOS and eNOS inhibition.15−19
In our continuous efforts to develop nNOS-selective
inhibitors, we established a new approach for fragment-based
de novo design and discovered a series of highly potent and
nNOS-selective small-molecule inhibitors with a 2-amino-
pyridinomethyl pyrrolidine scaffold, including 1a, 1b, 2a, and
2b (Figure 1).20−24 There are two chiral centers (the 3′ and 4′
carbons) in each structure. In vitro enzyme assays of the
enantiopure compounds indicate that (3′R,4′R)-enantiomers 1b
and 2b are much more potent and selective for nNOS than
INTRODUCTION
■
The key physiologic mediator, nitric oxide (NO), which plays
an important role in the regulation of various biological
processes, is produced in mammalian cells by three distinct
nitric oxide synthases (NOSs): neuronal NOS (nNOS),
endothelial NOS (eNOS), and inducible NOS (iNOS).1,2
The three isoforms share significant sequence homology
(∼50%) and catalyze the oxidation of L-arginine to NO and
citrulline with NADPH and O2 as co-substrates.3,4 Each isoform
consists of an N-terminal oxygenase domain that binds heme,
substrate, and tetrahydrobiopterin (H4B); a central linker
region that binds calmodulin; and a C-terminal reductase
domain with binding sites for FAD, FMN, and NADPH.5,6
NO overproduction by nNOS has been associated with
chronic neurodegenerative pathologies including Alzheimer’s,
Parkinson’s, and Huntington’s diseases as well as neuronal
damage resulting from stroke, cerebral palsy, and migraine
headaches.1,7−10 The reduction in pathophysiologic levels of
NO through inhibition of nNOS has the potential to be
beneficial as an approach to develop new therapeutics for these
diseases.11,12 However, the therapeutic control of NO synthesis
Received: March 7, 2012
Published: June 25, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of 1−3.
Figure 2. Crystallographic binding conformation of 2a (A, PDB ID 3NLK) and 2b (B, PDB ID 3NLM) in rat nNOS.
(3′S,4′S)-analogues 1a and 2a.20,21 These pyrrolidine-based
Table 1. Inhibition of NOS Isozymes by the
inhibitors were designed from structure−activity studies based
on a series of dipeptide inhibitors.25 The 2-aminopyridine
moiety mimics the guanidine group in the dipeptide inhibitors
for potential hydrogen-bonding interactions with the NOS
active-site Glu592, which is conserved in all mammalian NOS
isoforms. The pyrrolidine nitrogen mimics the α-amino group
of dipeptide inhibitors and provides a positive charge next to
the Glu592 in nNOS and close to another negatively charged
residue, Asp597, which is Asn in eNOS. Therefore, nNOS has
two negative charges in the active site compared to one in
eNOS, and, as a result, nNOS provides better electrostatic
stabilization to the positively charged inhibitors and is the
primary reason why these inhibitors are selective for nNOS. An
additional complication with the pyrrolidine inhibitors is that
the binding orientation depends on the stereochemistry at the
3′- and 4′-positions of the pyrrolidine.20,21 The (3′S,4′S)
inhibitors behave just like the dipeptide inhibitors, hydrogen-
bonding with their 2-aminopyridine to the NOS active-site Glu
residue, while the pyrrolidine nitrogen also interacts with this
same Glu, as does the α-amino group of dipeptide inhibitors.
We refer to this binding mode as the “normal” binding mode.
However, the (3′R,4′R) inhibitors bind in a 180° flipped over
mode, so that the 2-aminopyridine makes bifurcated hydrogen
bonds with heme propionate D, and the hydrophobic tail binds
above the heme next to the active-site Glu. We refer to this
binding mode as the “flipped” binding mode. In the flipped
binding mode there also is a new π-stacking interaction of the
2-aminopyridine with Tyr706. These two different binding
modes are illustrated in Figure 2 for 2a and 2b bound to nNOS.
Although the (3′R,4′R) inhibitors show great potency (Ki <
10 nM) and excellent selectivity for nNOS over eNOS (>2500-
fold) and iNOS (>700-fold) (Table 1), the positive charges
derived from the side-chain basic groups dramatically impair
Enantiomerically Pure Isomers of 1−3
a
a
Ki (μM)
selectivity
compd
nNOS
eNOS
iNOS
n/e
n/i
1a
0.052
0.005
0.116
0.007
0.080
26.4
20.3
26.2
19.2
62.0
3.8
3.9
506
3830
226
74
743
65
1b
2a
7.5
2b
5.8
2667
780
806
650
( )-3
52.0
a
n/e and n/i are the selectivity ratios of Ki for eNOS or iNOS to Ki for
nNOS.
the ability of these inhibitors to penetrate the blood−brain
barrier (BBB).26 To improve the bioavailability of these
inhibitors, two fluorines were further introduced to the benzylic
position of the m-fluorophenylethyl tail of 2b to lower the side-
chain NH pKa (to about 5.5). The nNOS cell-based assay
indicated that compound 3 (Figure 1, IC50 = 19 μM in cell-
based assay) crossed the cell membrane 2.5-fold better than
racemic 1.27,28 Computational studies indicate that the flipped
mode binds more tightly as a result of better electrostatic
interactions between the protein and inhibitor, because in the
“normal” binding mode the aminopyridine is only partially
protonated as a result of unfavorable electrostatic interactions
between the aminopyridine and pyrrolidine N atoms.20
Removal of the side-chain NH group would be the most
direct strategy to circumvent the side-chain positive charge and
further improve BBB permeability. Here, we report the results
of a series of monocationic pyrrolidine-based analogues as
nNOS inhibitors (Figure 3A). In addition, on the basis of the
crystal structures of these inhibitors with nNOS, we carried out
steered molecular dynamics (SMD) simulations, which provide
valuable information about the ligand−enzyme interactions and
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Figure 3. Target molecules synthesized and tested in this study.
the pharmacophoric requirements needed for the design of
potent inhibitors.29−31 Finally, the derivatives with doubly
substituted pyridine heads (Figure 3B) were made to validate
the predictions of our simulations.
(3′R,4′R) Inhibitors 4b and 5b exhibit much better nNOS
inhibitory potency and selectivity over the other two NOS
isozymes than the corresponding (3′S,4′S)-enantiomers 4a and
5a, respectively. Introduction of a trans double bond into
(3′S,4′S)-4a (compound 5a) results in a reduction in activity,
whereas the potency increases a little by introduction of a trans
double bond into (3′R,4′R)-4b (compound 5b). Compared to
the 3-fluorophenyl analogue 4b, the double halo-substituted
phenyl compound 6 exhibits 4-fold better inhibitory potency
and selectivity for nNOS over eNOS. Structural optimization of
6 leads to the generation of 3-fluoro-5-chlorophenyl ether 7,
which exhibits 2-fold better inhibitory potency but 4-fold lower
selectivity for nNOS over eNOS than 6.
Crystallographic Studies of 4−7. To better understand
the structure−activity relationships for 4−7, the crystal
structures of nNOS complexed with these inhibitors were
determined (Figure 4). Consistent with the binding preference
of parent compound 2a, the (3′S,4′S)-isomers 4a and 5a are
found to bind in the normal binding mode with their
aminopyridine moiety hydrogen-bonded to the side chain of
Glu592 in nNOS (Figure 4A,C), and the pyrrolidine nitrogen
also forms a hydrogen bond with Glu592. A water molecule
provides a hydrogen-bonding bridge for the pyrrolidine N atom
and the side chains of both Tyr588 and Asp597. The side chain
beyond the ether O atom and the fluorophenyl end is
disordered and only visible at low electron density contour
levels. Surprisingly, (3′R,4′R)-isomers 4b and 5b break the rule
of our previous pyrrolidine-based inhibitors. Despite their
(3′R,4′R) stereochemistry, they exhibit the unexpected normal
binding mode, similar to the conformation of the (3′S,4′S)
enantiomers. The 2-aminopyridine moieties interact with
Glu592, while the 3-fluorophenyl group extends out of the
substrate catalytic site and points toward the entrance of the
binding pocket (Figure 4B,D). The locations of the pyrrolidine
rings of 4b and 5b are similar to that of 4a. Although the
pyrrolidine nitrogen can no longer directly hydrogen-bond to
Glu592, it can still form a hydrogen bond with the same water
molecule found in the 4a-nNOS structure (Figure 4A) that
bridges to a hydrogen-bond network with both Tyr588 and
Asp597. However, the rest of both inhibitor molecules beyond
RESULTS AND DISCUSSION
■
Chemistry. Scheme 1 shows the synthetic route for target
compounds 4−9. The synthesis of 12a and 12b was described
previously.21,23 Single enantiomer 12a or 12b was treated with
NaH, and the resulting anion was allowed to react with (Z)-
(((4-bromobut-2-en-1-yl)oxy)methyl)benzene. Catalytic hy-
drogenation of the crude product then removed the benzyl
protecting group and also reduced the double bond, giving high
yields of purified alcohol 13a or 13b. The oxidation of 13a and
13b gave aldehydes 14a and 14b, respectively. Next, a Wittig
reaction of the aldehydes with the corresponding phosphorus
ylides allowed the isolation of intermediates 19a, 19b, and 20−
22 in moderate yields. Finally, reduction of 19a, 19b, and 20−
22 was performed to reduce the double bonds, followed by Boc
deprotection to form the corresponding target products 4a, 4b,
6, 8, and 9.
The bromination of 13b, followed by nucleophilic sub-
stitution with 3-chloro-5-fluorophenol, provided intermediate
27. The Boc protecting groups of 19a, 19b, and 27 were
removed to generate inhibitors 5a, 5b, and 7.
The synthetic route for 10 and 11 is shown in Scheme 2.
Boc-protected 2-aminopyridine analogues 28 and 29 were
treated with n-butyllithium and allowed to react with (Z)-((4-
bromobut-2-en-1-yl)oxy)(tert-butyl)dimethylsilane. The amino
groups of the products were further protected with benzyl
groups. The TBS protecting group was then removed, followed
by bromination to generate intermediates 30 and 31.
Subsequently, nucleophilic substitution of alcohol 12b with
the allyl bromides gave the corresponding ethers. Debenzyla-
tion of the ethers with hydrogen gave 32 and 33, in which the
double bond was also reduced. Finally, removal of the Boc
protecting group proceeded smoothly, giving high yields of the
target products 10 and 11.
In Vitro NOS Inhibition by 4−7. Table 2 shows the results
of inhibition assays using purified NOS enzymes with 4−7.
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a
Scheme 1. Synthesis of 4−9
a
Reagents and conditions: (a) (i) NaH, DMF, 0 °C, 3 h, (ii) 20 wt % Pd(OH)2/C, H2, EtOH, 60 °C, 36 h, 91−94%; (b) Dess−Martin periodinane,
CH2Cl2, rt, 18 h, 89−92%; (c) LHMDS, −78 °C to rt, THF, 8 h; (d) 10% Pd/C, H2, MeOH, rt, 12 h; (e) 3 N HCl/MeOH, rt, 24 h, 47−69% for
three steps; (f) 3 N HCl/MeOH, rt, 24 h, 90−93%; (g) (i) PPh3, CBr4, CH2Cl2, 0 °C, 3 h, (ii) 3-chloro-5-fluorophenol, K2CO3, acetone, reflux, 12 h,
quantitative; (h) 3 N HCl/MeOH, rt, 24 h, quantitative.
the ether oxygen is poorly defined and can only be modeled on
the basis of partial electron density at low contour levels. In
chain B the position of the fluorophenyl tail of 4b is closer to
heme propionate D, with the Tyr706 side chain adopting the
rotamer associated with the flipped binding mode (see
Supporting Information (SI) Figure S1A). More interestingly,
5b shows alternate binding conformations in chain B and binds
mostly (∼70%) in the normal mode, but with a small
population (∼30%) in the flipped mode (Figure S1B). The
crystallographic results indicate that both binding modes are
almost isoenergetic, which means that small perturbations can
apparently change the ligand binding conformation without
significantly changing stability. Similar observations have been
made for inhibitor binding to elastase.32
5). The aminopyridine makes bifurcated hydrogen bonds with
heme propionate D. The pyrrolidine N atom hydrogen-bonds
to both heme propionate A and H4B. The flexible linker
extending from the pyrrolidine brings the 3-fluoro-5-chlor-
ophenyl ring to the substrate catalytic site to form π-stacking
interactions with the heme. However, the larger size of the 3-
fluoro-5-chlorophenyl group forces Glu592 into an alternate
rotamer position. This alternate Glu side-chain position also
was observed in the eNOS-2-bromo-7-nitroindazole complex.33
Overall, one extra ether oxygen in 7 does not change its binding
conformation much from that seen for 6, which is consistent
with the similar potency shared by the two inhibitors (Table 2).
Docking and Steered Molecular Dynamics Simula-
tions. The normal binding mode of (3′R,4′R)-isomers 4b and
5b was completely unexpected because it breaks the precedent
that had been established by many crystal structures,
demonstrating that the (3′R,4′R)-pyrrolidine analogues adopt
Despite their high structural similarity to 4b, both of the 3-
fluoro-5-chlorophenyl analogues 6 and 7 adopt the flipped
binding mode as expected from their (3′R,4′R) chirality (Figure
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a
Scheme 2. Synthesis of 10 and 11
a
Reagents and conditions: (a) (i) n-BuLi, THF, −78 °C to rt, 2 h, (ii) NaH, DMF, 0 °C, 3 h, (iii) TBAF, DMF/THF, 0 °C, 2 h, (iv) PPh3, CBr4,
CH2Cl2, 0 °C, 3 h, 26−43%; (b) (i) NaH, DMF, 0 °C, 3 h, (ii) 20 wt % Pd(OH)2/C, H2, EtOH, 60 °C, 36 h, 84−89%; (c) 3 N HCl/MeOH, rt, 24
h, 89−94%.
aminopyridine ring. The pyrrolidine N atom in 4b-F makes
favorable hydrogen bonds with propionate A, while the
hydrogen bond with the O4 atom of H4B is broken.
Table 2. Inhibition of NOS Isozymes by Synthetic
Compounds 4−7
a
b
Ki (μM)
selectivity
The crystallographically flipped pose of 2b, crystallo-
graphically normal pose of 4b, and flipped docking pose 4b-F
were subsequently submitted to 5 ns equilibrium molecular
dynamics (MD) simulations. The root-mean-square deviation
(rmsd) values of all atoms and the Cα atoms of nNOS as a
function of time for the 5 ns MD simulation are shown in SI
Figure S2, suggesting the whole structure has been equilibrated
after about 1.5 ns.
compd
nNOS
eNOS
iNOS
n/e
n/i
4a
4b
5a
5b
6
9.057
0.922
15.402
0.637
0.230
0.117
34.0
33.8
69.0
77.0
35.6
4.4
468.5
83.8
4
37
52
91
133.8
35.1
4
9
121
155
37
55
106.0
12.4
461
106
7
a
The rmsd values of ligands 2b, 4b, and 4b-F versus
simulation time are shown in Figure 7A. These results indicate
that both 4b and 4b-F are more flexible than 2b. The structure
of 2b appears to have been stabilized, with the rmsd value of ∼1
Å after ∼500 ps of simulation, while the structures of 4b and
4b-F are stabilized after ∼1500 ps of simulation, with rmsd
values of ∼2 and ∼3 Å, respectively. To further probe the
differences among the moieties located in the substrate catalytic
pocket above the heme, center−center distances between the
Fe atom of the heme co-factor and the aromatic group of the
ligands were monitored during the MD simulation, and the
results are shown in Figure 7B. During the simulation, the
aromatic heads (aminopyridine) of 2b and 4b vibrate slightly in
the catalytic pocket, their distances to heme-Fe being relatively
stable at ∼3.5 Å. The profile of the center−center distance
between the heme-Fe and the aromatic tail (fluorophenyl) of
4b-F forms the first platform from 0 to 380 ps at ∼3.3 Å. The
distance then sharply increases to ∼4.5 Å and forms a short
platform from 380 to 1100 ps, followed by a long platform after
1000 ps at ∼5 Å.
To understand the interactions between nNOS and the
ligands, the direct hydrogen bonds were analyzed. Compound
2b can potentially form more hydrogen bonds with the enzyme
than 4b and 4b-F. Figure 7C shows the statistics of hydrogen
bonds formed every 4 ps. Statistical analysis shows that the
hydrogen bonds of 2b and 4b-F with the highest occurrence
frequency involve heme propionate A, heme propionate D, and
H4B. For the 4b-nNOS system, the dominant hydrogen bonds
were formed by Glu592. In our simulation, Glu592 is a flexible
residue, which can swing toward the side-chain N atom of 2b,
forming a salt bridge or hydrogen bond, and stabilize 2b
(Figure 8A). Both the hydrogen bond numbers and the
hydrophobic interaction numbers for 2b and 4b are relatively
The apparent Ki values are represented as the mean of two or more
independent experiments preformed in duplicate with five or six data
points each. n/e and n/i are the selectivity ratios of Ki for eNOS or
iNOS to Ki for nNOS.
b
the flipped binding mode. Only one bioisosteric change
(replacing the side-chain NH by CH2) from parent compound
2b both dramatically affects inhibitory potency and radically
changes the binding mode. Clearly, the chemical nature of the
side chain and the tail end affects the inhibitor binding mode,
but how? What kind of inhibitor−enzyme interactions, other
than chirality, determine the binding mode? To study the
molecular interactions involved in formation of the ligand−
enzyme complex and to answer these questions, we carried out
computational studies combining docking, equilibrium, and
SMD simulations.
The two most populated clusters of docking conformations
were obtained by using different nNOS receptors. One, in
which 2a is bound, exhibits the normal binding mode, and the
other, with 2b bound, exhibits the flipped binding mode. After
2a and 2b were removed, the receptors were used to dock 4b,
which gives 4b-nNOS complexes corresponding to the normal
and flipped poses, respectively (Figure 6). Hereafter, the
normal and flipped docking poses are referred to as 4b-N and
4b-F, respectively. Consistent with the binding preference of 4b
in the crystal structure, the aminopyridine moiety of docking
pose 4b-N establishes hydrogen-bonding interactions with
Glu592. Its pyrrolidine nitrogen also interacts with Glu592 and
is part of a water-mediated hydrogen-bonding network
involving Tyr588 and Asp597. In contrast, the aminopyridine
group in 4b-F makes bifurcated hydrogen bonds with the
propionate of pyrrole ring D. Tyr706 rotates farther out,
without making an optimized π-stacking interaction with the
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Figure 4. Crystallographic binding conformations of 4a (A), 4b (B), 5a (C), and 5b (D) with rat nNOS. The omit Fo − Fc electron density maps for
the inhibitors are shown at 2.5σ contour level. Major hydrogen bonds are depicted with dashed lines. Atom color scheme: oxygen, red; nitrogen,
blue; sulfur, yellow; fluorine, light cyan; chlorine, green.
Figure 5. Crystallographic binding conformation of 6 (A) and 7 (B) with rat nNOS. The omit Fo − Fc electron density maps for inhibitors are
shown at 2.5σ contour level. Major hydrogen bonds are depicted with dashed lines. The atom color scheme from Figure 4 is used.
stable during the simulation, while those for 4b-F decrease
gradually from 4 and 35 to 2 and 20 before 1000 ps,
respectively, and then stabilize at 3−4 and ∼20 after 1100 ps.
This is in good agreement with the variation of the center−
center distance between heme-Fe and the aromatic fluoro-
phenyl tail of 4b-F.
Figure 8 displays snapshots of the structures of ligand−
nNOS complexes at 5000 ps. Both the 2b-nNOS X-ray crystal
structure and the dynamics simulation demonstrate the
importance of the presence of water molecules in ligand
binding to nNOS. A water molecule bridging the side-chain
amine nitrogen in 2b with heme propionate D was seen during
the entire simulation (Figure 8A). The water molecule and the
atoms between the O atom and N atom in the side chain of 2b
form a rigid five-membered ring structure, which greatly
stabilizes the flipped binding conformation of 2b. In contrast to
2b, no water bridge between the O atom of 4b-F (lack of the
side-chain N atom) and heme propionate D was detected in the
simulation.
To investigate the molecular mechanism, at the atomic level,
of a ligand leaving the binding pocket of nNOS, we performed
SMD simulations, which can reveal features characteristic of the
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Figure 6. Docking conformation of 4b with different rat nNOS crystal structures: (A) 4b-N, in which 2a-nNOS complex (PDB ID 3NLK) was used
as the receptor, and (B) 4b-F, in which 2b-nNOS complex (PDB ID 3NLM) was used as the receptor.
Figure 7. (A) Time dependence of the rmsd. (B) Distance between the heme-Fe and the center of the aromatic head. (C) Numbers for the direct
hydrogen bonds. (D) Numbers for hydrophobic interactions in the equilibrium MD simulations. All curves were obtained over 4 ps intervals.
Figure 8. Snapshots of 2b (A), 4b (B), and 4b-F (C) isolated from the equilibrium MD at 5000 ps. The dashed lines represent hydrogen bonds and
a water bridge. The water molecule in (A) is shown as a red sphere. Residues around the ligand within 8 Å were selected to show the surface.
reverse process of binding. The profiles of the force exerted on
the system to encourage the unbinding of the ligands along a
carefully predefined reaction coordinate are shown in Figure 9A
(see also the Experimental Section and SI Figure S3 for further
details). During the unbinding of 2b, the concerted rupture of
the anchoring interactions (mainly electrostatic interactions,
hydrogen bonds, and hydrophobic interactions) between
nNOS and 2b defines the primary event with an unfolding
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Figure 9. Plot of the rupture force (A), number of direct hydrogen bonds (B), and number of hydrophobic interactions (HI) (C) versus time in the
equilibrium MD simulations. All curves were obtained over 4 ps intervals.
Figure 10. Snapshots of 2b (A), 4b (B), and 4b-F (C) isolated from the SMD before and after dissociation. The dashed lines represent hydrogen
bonds and the water bridge. The water molecule in (A) is shown as a red ball. Residues around the ligand within 8 Å were selected to show the
surface.
force of ∼77 kJ/(mol·Å) (Figure 9A), significantly higher than
the 58.3 and 50.1 kJ/(mol·Å) measured for 4b and 4b-F,
respectively, at the same pulling force constant. In particular,
the unbinding is controlled by a breakup in the charge-
reinforced hydrogen-bonding network between the ligand
pyrrolidine N atom and heme propionate A. The pulling
force profile of 4b is similar to that of 2b; the forces increase in
the beginning of ligand unbinding and reach a maximum
around 2500 ps, followed by a return to zero. Notably, both the
responses of 2b and 4b to the pulling force evolve in two
distinct stages (Figure 9A): (i) from 0 to 2600 ps (4b) or 3200
ps (2b), a buildup of force during which hydrogen bonds
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between the ligands with heme propionate A, heme propionate
D, and Glu592 are ruptured; (ii) after those times, the ligands
are pulled out of the binding pocket.
Interestingly, 4b-F suffers a three-state pathway for forced
unbinding: an intermediate state is maintained from 2150 to
2350 ps between the maximum rupture force and breakdown of
the primary unbinding barrier (Figure 9A). From 2140 to 2260
ps, the hydrophobic interactions between 4b-F and nNOS are
broken, while the hydrogen-bonding number remains relatively
constant.
Ligands in nNOS are shown in their bound states before and
after dissociation in Figure 10. A key observation during this
time is that 4b-F curls up within the binding pocket. This
curling, clearly discernible in Figure 10C, is the result of the
rupture of hydrophobic interactions between the fluorobenzene
moiety and the substrate binding pocket, while most hydrogen
bonds between 4b-F and heme propionate D remain intact
(Figure 9B,C). The curling then orients the fluorophenyl end
of 4b-F into a position that permits it to easily dissociate from
the binding pocket. These results clearly indicate the instability
of the fluorophenyl end located in the catalytic pocket and favor
4b in a normal binding mode.
What are the determining factors that result in a normal
binding pose for the (3′R,4′R)-pyrrolidine analogue 4b? The
SMD simulation calculations provide some clues. The structural
features of 4b have the potential to make it bind to nNOS with
either the normal or flipped pose. We thought the hydrogen
bonds from the aminopyridine and pyrrolidine to the
surrounding protein would be the key factors that determine
which way the inhibitors bind.
However, the chemical nature of the side chain and the
aromatic end group also matters. SMD simulation results
shown in Figure 10 indicate that 2b in its flipped binding mode
utilizes its side-chain nitrogen to interact with a water molecule
that bridges in between 2b and heme propionate D. This N
atom also interacts with the Glu592 side chain for another
hydrogen bond. The side chain and the fluorophenyl end of 4b-
F are extremely unstable because of the lack of this side-chain N
atom, resulting in a curling of the tail prior to its much easier
dissociation from the substrate catalytic pocket. Therefore, 4b
prefers to adopt the normal binding mode. Comparisons of the
force profiles for 4b and 2b (Figure 9A) show that the binding
affinity of 4b is considerably weaker than that for 2b. This is
understandable for the following reasons: (i) in the normal
binding mode 4b has fewer hydrogen bonds, hydrophobic
interactions, and electrostatic interactions than 2b in a flipped
mode; (ii) the side chain without the amino N atom causes the
fluorophenyl moiety of 4b to vibrate more vigorously in the
pocket surrounded by Met336, Leu337, and Tyr706 because of
no hydrogen-bonding possibilities; (iii) the orientation of the
pyrrolidine ring of 4b in the normal mode makes it impossible
to form a hydrogen bond to Glu592.
These results show that SMD simulations can be used to
guide the development of better nNOS inhibitors. As shown in
Figure 11, the substrate binding pocket (region A) should be
occupied by an aromatic ring, which not only stacks with the
heme but should also interact with Glu592 for additional
stability. This implies that introducing one or more
heteroatoms embedded in the aromatic ring or linker chain
should be favorable. In addition, an anchoring part that can
interact with heme propionate A or Asp597 should be located
in region B. This part functions to anchor the ligand in the
substrate catalytic pocket and to position the tail aromatic ring
Figure 11. Pharmacophoric requirements needed for the design of
potent nNOS inhibitors.
to region A appropriately. Moreover, an aminopyridine
fragment in region C is favorable to enhance the affinity
through the bifurcated hydrogen bonds with heme propionate
D and the π-stacking interaction with Tyr706.
One final question to address is why 6 and 7 bind in the
flipped mode, even though both 6 and 7 are close analogues of
4b, which binds in the normal orientation. The only difference
between 4b and 6 is the additional Cl atom added to the
fluorophenyl group of 4a. The bulkiness of the doubly-
substituted phenyl group forces the Glu592 side chain to adopt
a different rotamer, resulting in less steric clashes between the
chlorofluorophenyl group and Glu592. In addition, the second
halogen atom increases the hydrophobic character of the
protein−ligand interface within the active-site pocket.34,35 It
also reduces the electron density of the aromatic ring, allowing
more favorable parallel π-stacking interactions between the
chlorofluorophenyl tail and the heme.36,37 Both increased
interactions enhance the binding affinity of the tail phenyl
group to region A, thus tipping the balance in favor of the
flipped orientation.
New Analogue Design, NOS Inhibition, and Crystallo-
graphic Analysis of 8−11. As mentioned above, the
simulation results imply that one or more heteroatoms
embedded in the aromatic ring or linker chain should favorably
stabilize the ligand by providing an additional hydrogen bond
with the protein. A pyridine ring is a good candidate to replace
the fluorophenyl moiety because it has the potential to form
hydrogen-bonding interactions with Glu592 and importantly, it
would not be protonated under physiological conditions.
Both the activities and the crystal structures of double-
headed pyridine analogues 8−11 correlate well with the
insights gained from the above MD simulations. The
corresponding Ki values and nNOS selectivity ratios of 8−11
are shown in Table 3. The enzyme assay results indicate that
the tested compounds are potent and selective inhibitors
toward nNOS, with Ki values in the low nanomolar range. With
the aim of investigating the effect of 4-methyl and 6-amino
groups on the pyridine head, we synthesized compounds 9−11.
Each of the substitutions increases the potency of the nNOS
inhibitor relative to that of parent compound 8. Compounds 9
and 10 have the greatest combined potency for nNOS and
selectivity over eNOS and iNOS of any of the monocationic
pyrrolidine-based nNOS inhibitors reported to date. Com-
pound 11, with both 4-methyl and 6-amino groups on the
pyridine head, maintains an nNOS inhibitory activity
comparable to those of 9 and 10, but its selectivities over
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strongly stabilizes inhibitor binding, which confirms the
predictions from our MD simulations.
Table 3. Inhibition of NOS Isozymes by Synthetic
Compounds 8−11
Addition of the 6-amino group to the pyridine ring in 10 and
11 further enforces the hydrogen-bonding interaction between
the inhibitor and Glu592, as shown in Figure 12C,D, although
it does not necessarily improve the potency (Table 3). These
two compounds are double-headed aminopyridine inhibitors;
however, they bind to the other two isoforms more tightly as
well, thus lowering the selectivity.
The two best inhibitors, 9 and 10, were further tested for
their potency in a cell-based nNOS assay, which can provide
information about their membrane permeability.38 As shown in
Figure 13, the IC50 value for 9 is 20 μM, very similar to that of
the difluorinated analogue 3 (19 μM). Remarkably, compound
10 is 2.7 times more potent than 3, which demonstrates a
successful improvement in membrane permeability for the
pyrrolidine-based nNOS inhibitors and validates our approach
toward eliminating the positively charged amines in the
scaffold.
Compared to their in vitro activities, the potencies of these
pyrrolidine-based analogues in the cell-based assay are weaker,
which may relate to both their membrane permeability and
pharmacokinetic properties. Besides their potential affinity for
one of the active efflux transporters (e.g., p-glycoprotein),
metabolic properties also play important roles.39 Metabolism
may make the inhibitors ineffective or decompose them, thus
weakening their cellular activities. The metabolic stability of 10
a
b
Ki (μM)
selectivity
compd
nNOS
eNOS
iNOS
n/e
n/i
8
0.074
0.031
0.030
0.038
148.9
45.2
33.5
26.1
9.8
17.3
18.6
6.5
2012
1459
1117
687
132
558
619
172
9
10
11
a
The apparent Ki values are represented as the mean of two or more
independent experiments preformed in duplicate with five or six data
points each. n/e and n/i are the selectivity ratios of Ki for eNOS or
b
iNOS to Ki for nNOS.
eNOS (687-fold) and iNOS (172-fold) are decreased relative
to those of 8−10.
Crystal structures of nNOS complexed with 8 and 9 show
that these inhibitors adopt the flipped binding mode again
(Figure 12A,B). Their pyridine heads interact with active-site
Glu592, which stabilizes binding. The extra methyl group in the
pyridine ring of 9 further helps to tightly position the ring by
providing additional hydrophobic contacts with the protein.
The positively charged N atoms in the pyrrolidine rings interact
with heme propionate A, and the aminopyridine heads extend
out of the active site, where they form bifurcated salt bridges
with heme propionate D. The presence of the pyridine N atom
Figure 12. Crystallographic binding conformation of 8 (A), 9 (B), 10 (C), and 11 (D) with rat nNOS. The omit Fo − Fc electron density maps for
inhibitors are shown at 2.5σ contour level. Major hydrogen bonds are depicted with dashed lines. The atom color scheme shown in Figure 4 is used.
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Figure 13. Dose−response curves and IC50 values for 9 (A) and 10 (B) in a cell-based nNOS assay. Curves represent the average of three separate
experiments.
docking grid, and the active site was defined using AutoGrid. The grid
size was set to 60×60×60 points with grid spacing of 0.375 Å. The grid
box was centered on the ligand from the corresponding crystal
structure complexes. The Lamarckian genetic algorithm was used for
docking with the following settings: a maximum number of 2,500,000
energy evaluations, an initial population of 150 randomly placed
individuals, a maximum number of 27 000 generations, a mutation rate
of 0.02, a crossover rate of 0.80, and an elitism value (number of top
individuals that automatically survive) of 1. The structure of 4b was
built and geometry optimized using the molecular modeling suite of
programs Sybyl-X 1.2 (Tripos International, St. Louis, MO). The
ligand was fully optimized inside the binding site during the docking
simulations. Finally, the conformation with the lowest predicted
binding free energy of the most frequently occurring binding modes in
the nNOS active pocket was selected.
MD Simulation. The models used in the present study were based
on the X-ray crystal structures of the corresponding nNOS−ligand
complexes. The coordinates of the missing residues (339−349 in chain
A and 339−347 in chain B) were added according to the intact X-ray
crystal structures of nNOS (PDB entry code 1ZVL) using the
molecular modeling software Sybyl-X 1.2. The repaired residues were
subjected to energy minimization in Sybyl-X 1.2 using the steepest
descent method up to a gradient tolerance of 0.05 kcal/(mol·Å) to
relieve possible steric clashes and overlaps of side chains, with the rest
of the complex fixed. The ionization states of the residues were
determined by the Web-based program H++ (http://biophysics.cs.vt.
edu/H++).42,43 All of the simulations were carried out using the
GROMACS 4.5.3 package with an identical protocol.44,45 The ligands
were extracted from the corresponding complex crystal structures or
docking results. The secondary amines were modeled as charged.
Topology files and parameters for the ligands were generated using the
AmberTools 1.5 package (http://ambermd.org/). A modification of
the AMBER03 force field and the generalized AMBER force field were
applied for the proteins and ligands, respectively.46,47 The reported
AMBER force field parameters for the heme and Cys-Fe bond were
adopted.47 TIP3P water molecules were added in cuboid periodic
boxes, which were 120 Å × 110 Å × 120 Å.48 To ensure overall
neutrality of the system, appropriate Na+ and Cl− were added at
physiological concentration in the box.
can be compared to that of (R,R)-3. The in vitro metabolic rate
was 0.05 (nmol/min/mg protein) for (R,R)-3 and 0.02 (nmol/
(min·mg) protein) for 10; 69% of (R,R)-3 and 85% of 10
remain after 60 min in the presence of NADPH. It is apparent
that 10 has improved metabolic stability over (R,R)-3.
CONCLUSIONS
■
In summary, we have described an interdisciplinary study of a
series of selective monocationic inhibitors of nNOS. Three
stages of investigation allowed for the following conclusions:
(i) The potencies and selectivities of a series of monocationic
nNOS inhibitors decreased sharply, even with the small
modification of removing the side-chain amino nitrogen
compared to the potent and selective parent compound. The
crystal structures revealed that they adopt an unexpected
normal binding mode.
(ii) To investigate the underlying binding characteristics and
reveal the important potential pharmacophoric features of the
nNOS inhibitors, we carried out computational studies
combining molecular docking, equilibrium, and SMD simu-
lations. These computational results suggest that one or more
heteroatoms embedded in the aromatic head or linker chain
should be favorable to stabilize the ligand and allow it to tightly
occupy the substrate binding pocket.
(iii) The enzyme inhibition results of four double-headed
pyridine analogues, in conjunction with their crystal structures,
confirm the computational insights very well. Remarkably, 9
and 10 are the most combined potent and selective
monocationic pyrrolidine-based nNOS inhibitors reported to
date. Moreover, 10 has improved membrane permeability and
serves as a lead molecule for further development.
(iv) Both computational and experimental insights from this
study can be utilized to design even more effective nNOS
inhibitors.
EXPERIMENTAL SECTION
All covalent bonds to H atoms were constrained using the linear
constraint solver method. Electrostatic interactions were calculated
using the particle mesh Ewald algorithm.49,50 Periodic boundary
conditions were applied to avoid edge effects in all calculations. Before
the MD run, the energy of these complexes was minimized to remove
conflicting contacts. The systems were then heated gradually from 0 to
310 K. After a 50 ps equilibration of water, two 250 ps equilibrations of
the system were carried out with the restriction of main chain and
carbon α-helix, respectively. Finally, 5 ns simulations were carried out,
with coordinates saved every 2 ps during the entire process. Afterward,
selective structures from the end of these trajectories were used as
starting configurations for single-molecule pulling simulations. Ligand
in the active site of chain A was pulled out along the previously defined
axis that extends from heme-CAB atom in chain A to the center of
mass (COM) of atomic group of Trp711-CE2 in chain A and Trp306-
■
Organic Synthesis. All reagents were purchased from Sigma-
Aldrich, Alfa Aesar, or TCI and were used without further purification.
Analytical thin-layer chromatography was visualized by ultraviolet,
ninhydrin, or phosphomolybdic acid (PMA). Flash column
1
chromatography was carried out under a positive pressure of air. H
NMR and 13C NMR spectra were recorded on 500 and 125 MHz
spectrometers, respectively. High-resolution mass spectra were
measured by liquid chromatography/time-of-flight mass spectrometry
(LC-TOF). For synthetic details, see the SI.
Molecular Docking. AutoDock 4.2 was used to predict the
potential binding between 4b and nNOS.40,41 The crystal structures of
rat nNOS oxygenase domain in complex with 2a and 2b (PDB entries
3NLK and 3NLM) were used as the receptors. The ligand and solvent
molecules were removed from the crystal structures to obtain the
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Table 4. Crystallographic Data Collection and Refinement Statistics
nNOS-4a
nNOS-4b
nNOS-5a
nNOS-5b
nNOS-6
nNOS-7
3UFT
nNOS-8
nNOS-9
nNOS-10
nNOS-11
Data Collection
3UFS
PDB code
3UFO
3UFP
3UFQ
3UFR
3UFU
P212121
3UFV
P212121
3UFW
4EUX
space group
P212121
P212121
P212121
P212121
P212121
P212121
P212121
P212121
cell dimens a, b, c 52.2, 111.6, 52.0, 110.9, 52.1, 111.0, 51.9, 111.3, 51.8, 111.3,
51.8,111.2,164.1
52.1, 110.8, 51.7, 110.2, 51.9, 111.2, 52.0, 111.2,
164.4 164.3 164.1 164.3
2.18 (2.22− 2.10 (2.21− 2.05 (2.09− 2.10 (2.14− 1.97 (2.00− 2.08 (2.12−2.08) 1.89 (1.92− 2.08 (2.12− 2.00 (2.03− 2.14 (2.18−
2.18) 2.10) 2.05) 2.10) 1.97) 1.89) 2.08) 2.00) 2.14)
0.079/0.570 0.095/0.489 0.067/0.536 0.070/0.656 0.053/0.460 0.084/0.656 0.056/0.546 0.055/0.662 0.077/0.654 0.096/0.630
(Å)
165.0
164.5
164.5
164.5
164.2
resolution (Å)
Rsym/Rmerge
)
b
I/σI
22.5(2.0)
5.4(1.6)
56 388
24.5(1.9)
59 092
24.1(2.1)
55 153
31.9(2.4)
67 604
29.8(2.1)
57 805
22.7(1.9)
75 429
24.3(2.2)
55 597
24.5(1.6)
64 761
20.4(1.8)
52 877
no. unique reflns 50 641
completeness
(%)
97.8 (94.6) 99.7 (100.0) 98.4 (94.5) 97.2 (100.0) 98.6 (83.0)
99.6 (99.7)
97.2 (96.8) 96.7 (96.2) 99.4 (99.9) 99.3 (100.0)
redundancy
4.1 (4.1)
3.9 (3.9)
3.7 (3.7)
4.1 (4.1)
4.0 (3.7)
Refinement
1.97
4.8 (3.9)
4.1 (4.2)
3.9 (3.9)
4.0 (3.9)
3.9 (4.0)
resolution (Å)
2.18
2.10
2.06
2.10
2.08
1.89
2.08
2.00
2.14
no. reflns used
47 872
53 442
55 921
52 229
64 012
54 727
71 429
52 658
61 504
50 181
a
Rwork/Rfree
0.198/0.254 0.208/0.262 0.190/0.231 0.199/0.249 0.193/0.233 0.206/0.260
0.175/0.209 0.175/0.218 0.196/0.244 0.180/0.228
no. of atoms
protein
6678
196
6660
185
6660
185
6669
209
6671
187
6660
187
6681
183
6690
185
6665
185
6671
185
ligand/ion
water
195
259
270
220
284
167
367
325
252
268
rms deviations
bond lengths (Å) 0.016
0.015
1.565
0.015
1.317
0.016
1.640
0.015
1.485
0.019
1.865
0.014
1.421
0.015
1.485
0.015
1.513
0.011
1.981
bond angles
(deg)
1.589
a
Rfree was calculated with the 5% of reflections set aside throughout the refinement. The set of reflections for the Rfree calculation were kept the same
b
for all data sets according to those used in the data of the starting model (1OM4). This data set was processed with MOSFLM. All others were
processed with HKL2000.
CE2 in chain B. Chain A was used as an immobile reference for the
COM pulling simulation. It should be emphasized that the pulling
velocity is an important parameter in the COM pulling simu-
lations.51,52 The simulations at higher pulling velocity may lead to
substantial nonequilibrium effects. The low-velocity pulling simu-
lations that were carried out on a millisecond time scale can overcome
these disadvantages; however, the corresponding computational cost
will be very expensive. To find an appropriate simulation velocity,
several pulling simulations were carried out at different pulling
velocities. The simulation results are listed in SI Figures S2 and S3.
Finally, the SMD simulations with a pulling rate of 0.01 Å/ps and a
force constant of 2.5 kJ/(mol·Å2) were selected. LIGPLOT 5.0.4 was
used to analyze the hydrogen bonds and hydrophobic interactions
between nNOS and the ligands in the MD simulation trajectories.53
Figures 2, 4−6, 8, and 10−12 were prepared with PyMol (http://
www.pymol.org).
Enzyme Assays. The three isozymes, rat nNOS, murine
macrophage iNOS, and bovine eNOS, were recombinant enzymes,
overexpressed (in E. coli) and isolated as reported.54−56 IC50 values for
inhibitors 4−11 were measured for the three different isoforms of
NOS using L-arginine as a substrate. The formation of NO was
measured using the hemoglobin capture assay described previously.57
All NOS isozymes were assayed using a Synergy H1 Hybrid Multi-
Mode Microplate Reader (BioTek Instruments, Inc.) at 37 °C in a 100
mM Hepes buffer (pH 7.4) containing 10 μM L-arginine, 0.83 mM
CaCl2, 320 units/mL calmodulin, 100 μM NADPH, 10 μM H4B, and
3.0 μM oxyhemoglobin (for iNOS assays, no Ca2+ and calmodulin was
added). The assay was initiated by the addition of enzyme, and the
initial rates of the enzymatic reactions were determined by monitoring
the formation of methemoglobin complex at 401 nm for 60 s. The
apparent Ki values were obtained by measuring the percent enzyme
inhibition in the presence of 10 μM L-arginine with at least five
concentrations of inhibitor. The parameters of the following inhibition
equation were fitted to the initial velocity data: % inhibition = 100[I]/
{[I] + Ki(1 + [S]/Km)}. Km values for L-arginine were 1.3 (nNOS), 8.2
(iNOS), and 1.7 μM (eNOS). The selectivity of an inhibitor was
defined as the ratio of the respective Ki values.
Cell-Based nNOS Inhibition Assay. HEK293t cells stably
transfected with rat nNOS were cultured as previously described.38
The nNOS inhibition assay was performed as previously reported,38
with the following modifications: assays were performed in 96-well
plates with a total volume of 100 μL, and 10 μM A23187 (Sigma-
Aldrich, St Louis, MO) (added as 100× stock in 50% DMSO) was
used in place of 5 μM. Eight concentrations of each inhibitor were
tested, in at least triplicate wells. All inhibitors reported here were
assayed within the same experiment to ensure consistencies in cell
concentration and passage number. The entire assay was repeated at
least three times for each inhibitor, and the IC50 values were averaged.
Cells were plated in the 96-well plates 24 h before activation at a
concentration of approximately 8.5 × 106 cells/mL, which resulted in
80−90% confluency. After 6 h of A23187 activation (in the presence
or absence of inhibitor, which was added 30 min before activation), 50
μL aliquots of the media were removed from the wells. Nitrite
production was quantified using Griess reagent and measuring
absorbance at 540 nm.
Pharmacokinetic Data. All pharmacokinetic results were obtained
by LC/MS/MS analysis in the DMPK group, Sai Advantium Pharma
Ltd., Pune, India.
Inhibitor Complex Crystal Preparation. The nNOS heme
domain proteins used for crystallographic studies were produced by
limited trypsin digest from the corresponding full length enzymes and
further purified through a Superdex 200 gel filtration column (GE
Healthcare) as described previously.58 The enzyme−inhibitor complex
crystals were obtained by soaking, rather than co-crystallization, as
reported in the earlier structural work.59 The nNOS heme domain at
7−9 mg/mL containing 20 mM histidine was used for the sitting drop
vapor diffusion crystallization setup under the conditions reported
before.58 Fresh crystals (1−2 days old) were first passed stepwise
through cryo-protectant solutions as described58 and then soaked with
10 mM inhibitor for 4−6 h at 4 °C before being mounted on nylon
loops and flash-cooled by plunging into liquid nitrogen.
X-ray Diffraction Data Collection, Processing, and Structure
Refinement. The cryogenic (100 K) X-ray diffraction data were
collected remotely at various beamlines at Stanford Synchrotron
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Radiation Lightsource or Advanced Light Source through the data
collection control software Blu-Ice60 and a crystal mounting robot.
Raw data frames were indexed, integrated, and scaled using
HKL2000.61 The binding of inhibitors was detected by the initial
difference Fourier maps calculated with REFMAC.62 The inhibitor
molecules were then modeled in COOT63 and refined using
REFMAC. Water molecules were added in REFMAC and checked
by COOT. The TLS64 protocol was implemented in the final stage of
refinements with each subunit as one TLS group. The omit Fo − Fc
density maps were calculated by repeating the last round of TLS
refinement with inhibitor coordinates removed from the input PDB
file to generate the coefficients DELFWT and SIGDELFWT. The
refined structures were validated in COOT before deposition in the
RCSB protein data bank. The crystallographic data collection and
structure refinement statistics are summarized in Table 4 with PDB
accession codes included.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Details of chemical synthesis, alternate crystallographic binding
conformation of 4b and 5b in chain B of rat nNOS, rupture
force versus time in the process of 2b leaving the nNOS
binding gorge at different pulling force constant and pulling
rates, time dependence of the rmsd of the ligand−nNOS
complex for the all atoms and Cα in the equilibrium MD
simulation, and complete spectroscopic data of compounds 4−
11. This material is available free of charge via the Internet at
http://pubs.acs.org.
(21) Ji, H. T.; Delker, S. L.; Li, H. Y.; Martasek, P.; Roman, L. J.;
Poulos, T. L.; Silverman, R. B. J. Med. Chem. 2010, 53, 7804−7824.
(22) Ji, H.; Stanton, B. Z.; Igarashi, J.; Li, H.; Martasek, P.; Roman, L.
J.; Poulos, T. L.; Silverman, R. B. J. Am. Chem. Soc. 2008, 130, 3900−
3914.
(23) Ji, H.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. L.;
Silverman, R. B. J. Med. Chem. 2009, 52, 779−797.
́
(24) Ji, H.; Tan, S.; Igarashi, J.; Li, H.; Derrick, M.; Martasek, P.;
Roman, L. J.; Vas
Neurol. 2009, 65, 209−217.
́
quez-Vivar, J.; Poulos, T. L.; Silverman, R. B. Ann.
AUTHOR INFORMATION
■
(25) Flinspach, M. L.; Li, H. Y.; Jamal, J.; Yang, W. P.; Huang, H.;
Hah, J. M.; Gomez-Vidal, J. A.; Litzinger, E. A.; Silverman, R. B.;
Poulos, T. L. Nat. Struct. Mol. Biol. 2004, 11, 54−59.
(26) Lawton, G. R.; Ranaivo, H. R.; Chico, L. K.; Ji, H. T.; Xue, F. T.;
Martasek, P.; Roman, L. J.; Watterson, D. M.; Silverman, R. B. Bioorg.
Med. Chem. 2009, 17, 2371−2380.
Corresponding Author
poulos@uci.edu; Agman@chem.northwestern.edu
Present Address
‡Department of Chemistry, University of Utah, Salt Lake City,
UT 84112-0850
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful for financial support from the National
Institutes of Health (GM049725 to RBS and GM057353 to
TLP). We thank Dr. Bettie Sue Siler Masters (NIH grant
GM52419, with whose laboratory P.M. and L.J.R. are affiliated).
B.S.S.M. also acknowledges the Welch Foundation for a Robert
A. Welch Distinguished Professorship in Chemistry (AQ0012).
P.M. is supported by grant 0021620849 from MSMT of the
Czech Republic. We also thank the beamline staff at SSRL and
ALS for their assistance during the remote X-ray diffraction
data collections.
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