Unexpected binding modes of nitric oxide synthase inhibitors effective in the prevention of a cerebral palsy phenotype in an animal model

Article abstract of DOI:10.1021/ja910228a

Selective inhibition of the neuronal isoform of nitric oxide synthase NOS (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. However, given the high active site conservation among all three NOS isoforms, the design of selective inhibitors is an extremely challenging problem. Here we present the structural basis for why novel and potent nNOS inhibitors exhibit the highest level of selectivity over eNOS reported so far (～3,800-fold). By using a combination of crystallography, computational methods, and site-directed mutagenesis, we found that inhibitor chirality and an unanticipated structural change of the target enzyme control both the orientation and selectivity of these novel nNOS inhibitors. A new hot spot generated as a result of enzyme elasticity provides important information for the future fragment-based design of selective NOS inhibitors.

Full text of DOI:10.1021/ja910228a

Published on Web 03/25/2010
Unexpected Binding Modes of Nitric Oxide Synthase
Inhibitors Effective in the Prevention of a Cerebral Palsy
Phenotype in an Animal Model
Silvia L Delker,† Haitao Ji,‡ Huiying Li,† Joumana Jamal,† Jianguo Fang,‡
Fengtian Xue,‡ Richard B. Silverman,*,‡ and Thomas L. Poulos*,†
Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry,
UniVersity of California, IrVine, California 92697-3900, and Department of Chemistry,
Department of Biochemistry, Molecular Biology, and Cell Biology, Center for Molecular
InnoVation and Drug DiscoVery, and Chemistry of Life Processes Institute, Northwestern
UniVersity, EVanston, Illinois 60208-3113
Received December 9, 2009; E-mail: poulos@uci.edu; agman@chem.northwestern.edu
Abstract: Selective inhibition of the neuronal isoform of nitric oxide synthase NOS (nNOS) has been shown
to prevent brain injury and is important for the treatment of various neurodegenerative disorders. However,
given the high active site conservation among all three NOS isoforms, the design of selective inhibitors is
an extremely challenging problem. Here we present the structural basis for why novel and potent nNOS
inhibitors exhibit the highest level of selectivity over eNOS reported so far (∼3,800-fold). By using a
combination of crystallography, computational methods, and site-directed mutagenesis, we found that
inhibitor chirality and an unanticipated structural change of the target enzyme control both the orientation
and selectivity of these novel nNOS inhibitors. A new hot spot generated as a result of enzyme elasticity
provides important information for the future fragment-based design of selective NOS inhibitors.
peutic benefit.10 However, humans also have two other NOS
isoforms, one of which, endothelial NOS (eNOS), is essential
Introduction
Nitric oxide (NO),1 an essential signaling molecule involved
in various physiological functions in humans,2-4 is synthesized
by a family of enzymes called nitric oxide synthase (NOS, EC
1.14.13.39).5 NOS is active as a homodimer with each monomer
containing a C-terminal reductase domain (with binding sites
for NADPH, FAD, and FMN) and a N-terminal oxygenase
domain containing the heme prosthetic group.6 Both the
substrate L-arginine and a redox cofactor, (6R)-5,6,7,8-tetra-
hydro-L-biopterin (H4B), bind near the heme center in the
oxygenase domain.7,8 The over and under production of NO is
responsible for a number of pathological conditions. The
biosynthesis of NO by brain neuronal NOS (nNOS) is associated
with stroke and chronic neurodegenerative diseases, such as
Alzheimer’s, Parkinson’s, and Huntington’s diseases.9 As a
result, drugs targeting nNOS should be of considerable thera-
for maintaining proper blood pressure.11-13 Inhibition of eNOS
results in hypertension and is an undesirable and even dangerous
side effect of nonselective inhibitors targeted to nNOS. Isoform-
selective drugs are essential if nNOS is to be a viable therapeutic
target.14 Herein lies the challenge, because the crystal structures
of the catalytic domain of all three NOS isoforms show that
the active sites are nearly identical,7,8,15 making structure-based
isoform-selective drug design a difficult and challenging
problem.16-19 Here we describe the synthesis, inhibitory
constants, and crystal structures of a series of novel NOS
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D. A.; Stella, A. M. Nat. ReV. 2007, 8, 766–775.
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† University of California, Irvine.
‡ Northwestern University.
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2002, 41, 13868–13875.
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10.1021/ja910228a  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 5437–5442 5437

A R T I C L E S
Delker et al.
Table 1. Inhibition of NOS Isozymes by 1, 2, and Four
Enantiomerically Pure Isomers of 2
Ki (nM)
selectivity
compound
nNOS
iNOS
eNOS
n/i
n/e
1
130
14
52.2
5.3
18.9
171.0
25 000
4 060
3 850
3 940
16 100
26 600
20 000
28 000
26 400
20 300
57 100
34 500
192
290
73.7
743
852
155
1538
2000
505
3830
3020
202
(()-2
(3′S,4′S)-2
(3′R,4′R)-2
(3′R,4′S)-2
(3′S,4′R)-2
contains Asp597 in nNOS and Asn368 in eNOS. This pocket
is only occupied with water molecules when dipeptide inhibitors
bind. The flexible dipeptide inhibitors can adopt a curled
conformation that allows the free R-amino group to interact with
both the active site Glu592 and Asp597 in nNOS (Figure 1).
Since eNOS has Asn368 at this position rather than Asp, these
dipeptide inhibitors are electrostatically far less stable in the
eNOS active site. As expected, the potency of these inhibitors
can be dramatically increased in eNOS by replacing Asn368
with Asp, and Ki rises substantially in nNOS if Asp597 is
replaced by Asn.21
Inhibitor Design and Ki Measurements. Recently, we de-
scribed a new strategy for the de noVo design of nNOS-selective
inhibitors called fragment hopping.27 Using this novel approach
together with what we learned from the dipeptide inhibitors
described in the previous section, a series of compounds with
a pyrrolidine methylaminopyridine scaffold (Figure 2) were
designed and synthesized, which showed nanomolar nNOS
inhibitory potency and more than 1000-fold nNOS selectivity.
These inhibitors were designed with the idea that the
aminopyridine ring mimics the guanidinium group of L-arginine
and anchors the compound in the active site by interacting with
the active site glutamate. The rigid pyrrolidine ring is placed
between the same conserved Glu and the selective residue nNOS
Asp597/eNOS Asn368, which results in similar interactions
observed by the R-amino group of dipeptide inhibitors bound
to nNOS. Very recently, we showed that the racemic mixture
of 2, (()-2, decreases NO levels and NOS activity in the brains
of newborn rabbit kits, is nontoxic, and has no effect on the
cardiovascular function of rabbit dams (indicating no impairment
in eNOS function). Most importantly, (()-2 is very effective
at protecting the rabbit fetuses from experimentally induced
ischemic brain damage and preventing severe cerebral palsy
symptoms in the newborn kits.28 This is consistent with previous
studies that have shown that nNOS knockout mice experience
less neuronal damage as a result of experimentally induced
ischemia.29
Figure 1. Inhibitor binding pocket in NOS (A) and the active site structure
of nNOS (C) and eNOS (D) showing the different binding modes of the
dipeptide inhibitor 1 (B) that exhibits about 1500-fold selectivity for nNOS
over eNOS (Table 1).
inhibitors complexed to both nNOS and eNOS leading to the
identification of unexpected binding modes as well as the
identification of a flexible region of the NOS active site that
can be exploited for structure-based drug design.
Results and Discussion
Structural Basis for Selectivity. Previous structure-activity
relationship studies in our laboratories on a series of Nω-nitro-
L-arginine containing dipeptide inhibitors (1 in Figure 1) have
enabled us to identify a family of compounds that have high
potency and selectivity for inhibition of nNOS over eNOS and
iNOS.20-26 The key structural features in the active site that
are responsible for the lower Ki of these inhibitors to nNOS
over eNOS have been identified by crystallographic and
computational simulations. Most importantly, a single amino
acid difference, Asp597 in nNOS and Asn368 in eNOS, has
been identified as the major structural determinant for why these
dipeptide inhibitors bind more tightly to nNOS than eNOS.21
As shown in Figure 1, all NOS isoforms have a conserved Glu
(Glu592 in nNOS and Glu363 in eNOS) in the active site pocket
that helps to anchor the natural substrate, L-arginine, in place.
The CR end of the substrate is anchored in a second pocket that
There are two chiral centers (3′ and 4′ carbons) in the structure
of 2 (Figure 2). Our initial studies were carried out with the
cis-racemic mixtures. Recognizing that chirality is critically
important in drug design and that the enantiomers should
eventually improve in ViVo efficacy, we synthesized four
enantiomerically pure isomers of 2 (see Supporting Information).
The in Vitro enzyme assay shows dramatic and unexpected
(20) Silverman, R. B. Acc. Chem. Res. 2009, 42, 439–451.
(21) Flinspach, M.; Li, H.; Jamal, J.; Yang, W.; 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.
(22) Huang, H.; Martasek, P.; Roman, L. J.; Masters, B. S.; Silverman,
R. B. J. Med. 1999, 42, 3147–3153.
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L. J.; Poulos, T. L.; Silverman, R. B. J. Am. Chem. Soc. 2008, 130
(12), 3900–3914. (b) Ji, H.; Li, H.; Martasek, P.; Roman, L. J.; Poulos,
T. L.; Silverman, R. B. J. Med. Chem. 2009, 52, 779–797.
(28) Ji, H.; Tan, S.; Igarashi, J.; Li, H.; Derrick, M.; Martasek, P.; Roman,
L. J.; Vasquez-Vivar, J.; Poulos, T. L.; Silverman, R. B. Ann. Neurol.
2009, 65, 209–217.
(23) Seo, J.; Igarashi, J.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. L.;
Silverman, R. B. J. Med. Chem. 2007, 50, 2089–2099.
(24) Ji, H.; Gomez-Vidal, J. A.; Martasek, P.; Roman, L. J.; Silverman,
R. B. J. Med. Chem. 2006, 49, 6254–6263.
(25) Gomez-Vidal, J. A.; Martasek, P.; Roman, L. J.; Silverman, R. B.
J. Med. Chem. 2004, 703–710.
(26) Hah, J. M.; Roman, L. J.; Martasek, P.; Silverman, R. B. J. Med.
Chem. 2001, 44, 2667–2670.
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Binding Modes of Nitric Oxide Synthase Inhibitors
A R T I C L E S
Figure 3. (3′R,4′R)-2 (yellow) modeled in the same orientation as
(3′S,4′S)-2 (cyan) with an emphasis placed on minimizing steric clashes. It
is not possible to model (3′R,4′R)-2 in the (3′S,4′S)-2 orientation and still
maintain optimal inhibitor-protein H-bonds and minimal steric clashes.
this compound. Crystallographic analysis of (3′S,4′R)-2 bound
to nNOS (Figure 2B) is more ambiguous owing to the poorer
quality of the electron density. Nonetheless, it is clear that this
compound cannot be positioned to enable both the pyrrolidine
and aminopyridine moieties to interact with the active site Glu.
The (3′R,4′S)-inhibitor, therefore, forms better electrostatic
interactions with neighboring protein groups (Figure 2A), which
is the basis for the observed lower Ki of the (3′R,4′S)-inhibitor
over the (3′S,4′R)-inhibitor. In addition, the 3-fluorophenyl ring
is stabilized in a hydrophobic pocket defined by Met 336,
Leu337, and Trp306 from the second monomer of nNOS. iNOS
does not have this hydrophobic pocket. The residue correspond-
ing to Leu337 of nNOS is Thr121 in human iNOS or Asn115
in murine iNOS. That is why the (3′R,4′S)-isomer exhibits more
than 800-fold nNOS selectivity over iNOS.
As shown in Figure 2, the (3′S,4′S)-isomer binds to nNOS
as expected, with the aminopyridine group interacting with
active site Glu592, which is very similar to the binding mode
of the trans (3′R,4′S)- and (3′S,4′R)-compounds. The (3′R,4′R)-
isomer, however, exhibits a totally different and unexpected
binding mode; it flips 180° from the expected orientation. The
fluorophenyl group is positioned over the heme while the
aminopyridine extends out of the active site where it forms a
bifurcated salt bridge with heme propionate D. For this to occur,
Tyr706 must swing out of the way to make room for the
aminopyridine. The pyrrolidine N atom is placed right in the
middle between heme propionate A (2.5 Å) and the carbonyl
group of H4B (2.8 Å) (Figure 2). Strong H-bonding and
charge-charge interactions are expected between these groups.
In an attempt to understand why (3′R,4′R)-2 flips relative to
(3′S,4′S)-2, we modeled (3′R,4′R)-2 in the normal orientation
(Figure 3). Optimization of H-bonds between the inhibitor and
protein would result in severe steric clashes. In the modeled
orientation shown in Figure 3 the inhibitor would lose the
H-bond from its pyrrolidine nitrogen and only form two H-bonds
between the aminopyridine and Glu592. Indeed, it is not possible
for the inhibitor to adopt a conformation that enables H-bonding
interactions between the pyrrolidine N atom and protein. There
is the possibility for a third H-bond with a heme propionate,
but this would require movement of the aminopyridine, thereby
Figure 2. A series of 2Fo - Fc electron density maps contoured at 1.0σ
for four various aminopyridine inhibitors bound to nNOS. Shown also their
chemical formula and Ki values. Detailed refinement statistics are provided
in Table S2 in Supporting Information. Briefly the resolution and Rwork
/
Rfree values for the 4 structures shown are (A) 1.93 Å, 0.18/0.21; (B) 1.98
Å, 0.18/0.21; (C) 1.95 Å, 0.19/0.22; and (D) 2.01 Å, 0.19/0.23.
results. The (3′R,4′R)-2, rather than (3′S,4′S)-2, is more potent
and more selective for nNOS (Table 1). Its Ki is 5.3 nM, and
the selectivities for nNOS over eNOS and over iNOS are more
than 3,800-fold and 700-fold, respectively. With a Ki of ∼5
nM and ∼3,800-fold selectivity, this compound is, to the best
of our knowledge, the most potent and dual-selective nNOS
inhibitor reported to date. Trans (3′R,4′S)-2 also is a very potent
and selective inhibitor of nNOS with a Ki value of 19 nM for
nNOS, and the nNOS selectivities over eNOS and iNOS are
about 3,000-fold and 800-fold, respectively (Table 1).
Crystal Structures. Crystal structures of the trans (3′R,4′S)-
isomer and the trans (3′S,4′R)-isomer show that these inhibitors
bind as expected with the aminopyridine moiety interacting with
active site Glu592 and the fluorophenyl group extending out of
the substrate-binding site (Figure 2A,B). The reason why the
(3′S,4′R)-isomer exhibits nNOS inhibitory activity lower than
that of the (3′R,4′S)-isomer is readily understood on the basis
of the crystal structures (Figure 2). In the structure with the
(3′R,4′S)-isomer bound (Figure 2A), the pyrrolidine five-
membered ring is positioned to H-bond with the active site Glu
in nNOS. The ordered water bridges the pyrrolidine N atom
and Asp597 in the (3′R,4′S) isomer-bound structure (Asn368
in eNOS, Figure S1 in Supporting Information). The positively
charged nitrogen atom in the pyrrolidine ring interacts favorably
with the side chain carboxylic group of Asp597 of nNOS (but
not with the amide of Asn368, the corresponding residue in
eNOS), which results in high nNOS selectivity over eNOS with
9
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A R T I C L E S
Delker et al.
Table 2. Inhibition of NOS Isozymes and Mutants by (3′S,4′S)-2
and (3′R,4′R)-2
disrupting the stacking interaction with the heme and H-bonds
with Glu592. Thus, it is not possible to maintain both optimal
H-bonding and minimal steric clashes in this modeled orienta-
tion. The flipped orientation in the crystal structure provides
better H-bonding opportunities with no steric problems.
A critical factor in controlling the flipped orientation of
(3′R,4′R)-2 is the ability of Tyr706 to swing out of the way
and form π-π interactions with the aminopyridine of the
inhibitor. This is the first time that movement of this tyrosine
residue has been observed in the NOS enzymes, and this
generates a new hot spot for fragment-based inhibitor design
with appropriate inducement.
Ki (µM)
enzyme
(3′S,4′S)-2
(3′R,4′R)-2
nNOS wild type
nNOS D597N
0.0522
1.19
0.0053
0.29
nNOS D597N/M336V
nNOS D597N/M336V/Y706A
eNOS wild type
1.19
2.18
26.4
0.18
1.29
20.3
eNOS Y477A
46.0
35.2
eNOS N368D
2.29
0.50
eNOS N368D/V106M
1.41
0.23
Given that (3′R,4′R)-2 has 3,800-fold lower Ki with nNOS
than eNOS, we might anticipate yet another binding mode in
eNOS, as in the case of 1. However, the 2.0 Å structure of eNOS
complexed to (3′R,4′R)-2 and related inhibitors (Figure S1 in
Supporting Information) show exactly the same binding mode
and structural changes found in nNOS.
protonated in the (3′R,4′R)-2 orientation. As shown in Figure
S2 in Supporting Information, the calculated free energies agree
much better with the experimental free energies if we assume
that the aminopyridine is only partially charged in the (3′S,4′S)-2
orientation. It thus appears that the reason (3′R,4′R)-2 is a better
inhibitor than (3′S,4′S)-2 is because of stronger electrostatic
interactions between the inhibitor and the protein, including the
heme.
Basis for Ki Differences. There are two central questions to
be addressed. First, why does (3′R,4′R)-2 have 7- to 8-fold lower
Ki with nNOS than (3′S,4′S)-2, and second, why is (3′R,4′R)-2
more selective for nNOS over eNOS than is (3′S,4′S)-2? The
answer to the first question centers on the different binding
modes of the pyrrolidine moieties in these two inhibitors, and
the distance-dependent electrostatic interaction is probably the
key factor that determines the difference in Ki. The pKa of the
2-amino-4, 6-dimethylpyridine moiety is about 7.1,30 and it
would be expected that the aminopyridine moiety of (3′S,4′S)-2
is fully protonated when bound to NOS owing to direct contact
with the conserved active site Glu592. However, the curled
conformation of (3′S,4′S)-2 places the pyrrolidine N atom about
4.1 Å from the aminopyridine. Since the protonated pyrrolidine
N atom has a much higher pKa, it should be fully protonated at
neutral pH, and its close proximity to the aminopyridine will
prevent full protonation of that moiety to avoid electrostatic
repulsion. Moreover, the pyrrolidine N atom directly contacts
the active site Glu, and therefore, charge neutrality is achieved
by having only the pyrrolidine carry a positive charge and not
the aminopyridine.
In sharp contrast, the aminopyridine and the pyrrolidine
moieties of (3′R,4′R)-2 adopt an extended conformation with
the pyrrolidine N atom 4.9 Å from the aminopyridine. As a
result of the extended conformation, the aminopyridine carries
a full positive charge with little electrostatic repulsion from the
positive charge on the pyrrolidine. Since the aminopyridine
interacts with the heme D-ring propionate and the pyrrolidine
with the heme A-ring propionate, charge neutrality is achieved
by having each group in the inhibitor carry a positive charge.
Furthermore, the coplanarity of the positively charged pyrroli-
dine N atom of (3′R,4′R)-2 with heme propionate A leads to
strong charge-charge interactions, while the pyrrolidine N atom
of (3′S,4′S)-2 is not at the center of the area of influence from
Glu592, which results in relatively weak charge-charge
interactions.
The 700-fold nNOS selectivity of (3′R,4′R)-2 over iNOS is
primarily from the 4-methyl group of the 2-aminopyridine
moiety, which can be stabilized in a hydrophobic pocket defined
by Met336, Leu337, and Trp306 from the second monomer of
nNOS. The therapeutically more interesting and more challeng-
ing question, however, is how to explain why the flipped
orientation of (3′R,4′R)-2 leads to greater selectivity for nNOS
over eNOS despite the fact that the crystal structures show
identical binding modes in both eNOS and nNOS. In previous
studies we have shown that Asp597 (Asn in eNOS) and to a
lesser extent Met336 (Val in eNOS) are largely responsible for
the higher affinity of dipeptide inhibitors to nNOS.21 Met336
provides more extensive interactions with the aminopyridine
that extends out of the substrate-binding site pocket in nNOS,
whereas Asp597 might provide greater electrostatic stabilization.
As shown in Table 2, the D597N/M336V double nNOS
mutant exhibits a Ki for (3′R,4′R)-2 of 0.18 µM compared to
5.3 nM for wild type nNOS, and 0.23 µM for the eNOS double
mutant compared to 20.3 µM for wild type eNOS. While the
Asp/Asn and Met/Val differences contribute to selectivity, there
clearly must be other factors involved. The one additional subtle
difference we have consistently noted is that the interaction
between Tyr706 (Tyr477 in eNOS) and the aminopyridine is
more extensive in nNOS than in eNOS (Figure S1 in Suporting
Information). This could provide better nonbonded contacts as
well as better desolvation of the inhibitor in nNOS. We therefore
made a triple nNOS mutant, D597N/M336V/Y706A, and
compared the Ki values to that of the single Y477A eNOS
mutant. The D597N/M336V/Y706A triple nNOS mutant ex-
hibits a Ki with (3′R,4′R)-2 of 1.29 µM compared to 5.3 nM
for wild type nNOS and 20.3 µM for wild type eNOS.
The single Y477A eNOS mutant exhibits a Ki with (3′R,4′R)-2
of 35.2 µM (see Table 2). Tyr477 contributes little to no binding
in eNOS but does contribute substantially in nNOS. Therefore,
the reason (3′R,4′R)-2 in the flipped orientation is more selective
for nNOS than other inhibitors is a combination of better
electrostatic interactions between the inhibitor and the active
site in nNOS and the more favorable nonbonded contacts formed
between Tyr706 and the inhibitor bound to nNOS.
To test this hypothesis we have used the MM-PBSA
computational approach to compute the free energy of binding
of a series of (3′S,4′S)-2 and (3′R,4′R)-2-like inhibitors for which
we have crystal structures. In the first series of calculations we
assume that the aminopyridine is fully protonated in all
complexes while in the second we assume that the aminopyri-
dine is half protonated in the (3′S,4′S)-2 orientation but fully
In summary, we have designed, prepared, and solved crystal
structures of a series of very potent and highly selective nNOS
inhibitors, which we have previously demonstrated exhibit
(30) Paudler, W. W.; Blewitt, H. L. J. Org. Chem. 1966, 31, 1295–1298.
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Binding Modes of Nitric Oxide Synthase Inhibitors
A R T I C L E S
remarkable protection of newborn rabbit kits against the
phenotype of cerebral palsy experimentally induced by hypoxia-
ischemia. By using a combination of crystallography, compu-
tational biochemistry, and site-directed mutagenesis, we found
that inhibitor chirality and the unexpected structural elasticity
of NOS cause the inhibitor to adopt a novel binding mode and
generate a new hot spot for ligand binding. These findings now
can be utilized to design even more selective and potent drug-
like NOS inhibitors.
∆Gbind ) (Gcomplex - Greceptor - Ginhibitor
)
As others have done the solute entropy is ignored.39 Given that
the inhibitors used for these calculations are structurally very similar
with a similar number of rotatable bonds, ignoring inhibitor entropy
introduces little error in comparing relative calculated and experi-
mental free energies but does, of course, preclude the calculation
of absolute free energies.
Inhibitor parameters and charges are assigned using the GAFF
force field40 and AM1-BCC charge scheme41,42 as implemented
in the Antechamber module in Amber 9.0. Heme parameters
developed for cytochrome P450 were provided by Dr. Dan Harris.43
It is necessary to carefully check the Antechamber output to make
sure the correct atom types have been assigned. For some inhibitors
it has been necessary to increase the force constant on improper
torsion angles from 1.1 to 10.1 kcal/Å in order to maintain planarity
of the aminopyridine groups. To prepare the models for energetic
calculations all crystallographic waters are removed and TIP3 waters
added back within 30 Å of the inhibitor. The resulting solvated
structure is first energy minimized using the steepest descent method
for 1,000 cycles with the inhibitor and heme heavy atoms restrained
to the starting crystallographic positions. The restraints are relaxed
to 10.0 kcal/Å2 for the inhibitor and heme followed by another
1,000 cycles of refinement. In the last step the restraints for the
heme and inhibitor were relaxed to 1.0 kcal/Å2 followed by 1,000
cycles of minimization.
Enzymes, Assays, and Ki. All of the NOS isozymes used were
recombinant enzymes overexpressed in E. coli. The murine mac-
rophage iNOS was expressed and isolated according to the
procedure of Hevel et al.44 The constitutive full-length isozymes
nNOS and eNOS were isolated as described previously,21,31 with
the exception that buffer containing 15 mM NADP+ was used to
elute wild type eNOS and eNOS N368D mutant from the 2′,5′-
ADP sepharose column (GE Healthcare). Nitric oxide formation
from NOS was monitored by the hemoglobin capture assay as
described with some modifications.45 The Hb assay mixture
contained L-arginine (10 µM), NADPH (0.1 mM), tetrahydrobiop-
terin (10 µM), dithiothreitol (100 µM), Hb (0.1 mg/mL), CaM (10
µg/mL), CaCl2 (0.1 mM), and different amounts of inhibitors. The
final volume was adjusted to 600 µL with 100 mM Hepes buffer,
pH 7.4. The enzymatic reaction was initiated by addition of enzyme,
and the rate of NO production was monitored by the change of
absorbance at 401 nm in the initial 60 s on a Perkin-Elmer Lambda
10 UV/vis spectrophotometer. The IC50 values were obtained from
the dose-dependent inhibition curves. The inhibition constant (Ki)
was calculated on the basis of the following equation:46 Ki ) IC50/
(1 +[substrate]/Km), where the Km values of WT enzymes were as
reported47 and the Km values of the mutant enzymes were
experimentally determined by the Hb assay (Supplementary Table
2). All assays were performed at room temperature. The selectivity
of an inhibitor was defined as the ratio of the respective Ki values.
Experimental Section
Crystallography. For crystallization the heme domain of isozymes
nNOS and eNOS were isolated as described previously.15,21,31
Cocrystallization of nNOS or eNOS crystals with inhibitors was
abandoned due to the disturbance by inhibitors to the growth conditions.
Instead, 10 mM histidine or 2 mM imidazole was added to the nNOS
or eNOS samples, respectively, to occupy the heme active site before
crystallization setup. Crystals grew within 24-48 h at 4 °C (eNos,
20 mg/mL or nNOS, 7-9 mg/mL) using the sitting drop vapor
diffusion method as described.15,21,31 Crystals were passed stepwise
through a series of cryoprotectant solutions15,21,31 before soaking
with 10 mM inhibitors at 4 °C for 4-6 h and then were flash cooled
with liquid nitrogen.
Both isoforms crystallized in space group P212121 with typical
unit cell dimensions a ) 52.0, b ) 112.4, c ) 164.6 Å for nNOS
and a ) 58.6, b ) 107.1, c ) 157.7 Å for eNOS. The X-ray
diffraction data were collected under a liquid nitrogen stream (100K)
with CCD detectors either at Advanced Light Source (ALS,
Berkeley, CA) or Stanford Synchrotron Radiation Lightsource
(SSRL, Menlo Park, CA). Raw data were processed with
HKL2000.32 The binding of inhibitor was detected by difference
Fourier synthesis. The inhibitor was modeled in using O33 and
refined with CNS34 and then with REFMAC35 to include the TLS
protocol.36 Water molecules were added automatically and inspected
visually in COOT.37 The refined structures were validated before
deposition to the PDB. The data collection and refinement statistics
are summarized in Table S1 in Supporting Information.
Computational Methods. The MM-PBSA method as imple-
mented in Amber 9.0 was used to compute binding free energy.38
In this method the total free energy of the NOS-inhibitor complex
is taken as the sum of the following energy terms:
G ) EMM + Gsolv + Gnp - TSsolute
where EMM ) the total molecular mechanics energy computed with
the Sander module in Amber 9.0, Gsolv is the solvation free energy
estimated from the Poisson-Boltzmann equation, Gnp is the
nonpolar solvation energy estimated from the solvent accessible
surface area, and TSsolute is the solute entropy. From a single energy
minimized structure the free energy is computed for the NOS-
inhibitor complex, NOS alone with the inhibitor removed, and the
inhibitor alone. The overall free energy of binding is computed
from the following equation:
(38) Massova, I.; Kollman, P. A. J. Am. Chem. Soc. 1999, 8133–8143.
(39) Brown, S. P.; Muchmore, S. W. J. Chem. Inf. Model 2006, 46, 999–
1005.
(31) Li, H.; Flinspach, M. L.; Igarashi, J.; Jamal, J.; Yang, W.; Gomez-
Vidal, J. A.; Litzinger, E. A.; Huang, H.; Erdal, E. P.; Silverman,
R. B.; Poulos, T. L. Biochemistry 2005, 44, 15222–15229.
(32) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307–326.
(33) Jones, T. A.; Zou, J.-Y.; Cowan, S. W.; Kjeldgaarrd, M. Acta
Crystallogr. 1991, A47, 110–119.
(40) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D.
J. Am. Chem. Soc. 2004, 25, 1157–1174.
(41) Jakalian, A.; Bush, B. L.; Jack, D. B.; Bayly, C. I. J. Comput. Chem.
2000, 21, 132–146.
(42) Jakalian, A.; Jack, D. B.; Bayly, C. I. J. Comput. Chem. 2002, 23,
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(34) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.;
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(36) Winn, M. D.; Murshudov, G. N.; Papiz, M. Z. Methods Enzymol. 2003,
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(46) Segel, I. H. Enzyme Kinetics; John Wiley and Sons: New York, 1975.
(47) Hah, J. M.; Martasek, P.; Roman, L. J.; Silverman, R. B. J. Med.
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J. AM. CHEM. SOC. VOL. 132, NO. 15, 2010 5441

A R T I C L E S
Delker et al.
Chemical Synthesis. The synthetic route, experimental details,
and 1H NMR and 13C NMR spectra for the final products are
provided in Supporting Information.
Supporting Information Available: Details of chemical
synthesis, a table of crystallographic statistics, and a description
of 11 additional NOS-inhibitor complexes. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. The authors are grateful for financial support
from NIH with grants GM57353 (T.L.P) and GM49725 (R.B.S.).
We thank the beamline staff at ALS and SSRL for their excellent
support during the data collections.
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