Targeting Bacterial Nitric Oxide Synthase with Aminoquinoline-Based Inhibitors

Article abstract of DOI:10.1021/acs.biochem.6b00786

Nitric oxide is produced in Gram-positive pathogens Bacillus anthracis and Staphylococcus aureus by the bacterial isoform of nitric oxide synthase (NOS). Inhibition of bacterial nitric oxide synthase (bNOS) has been identified as a promising antibacterial strategy for targeting methicillin-resistant S. aureus [Holden, J. K., et al. (2015) Chem. Biol. 22, 785-779]. One class of NOS inhibitors that demonstrates antimicrobial efficacy utilizes an aminoquinoline scaffold. Here we report on a variety of aminoquinolines that target the bacterial NOS active site, in part, by binding to a hydrophobic patch that is unique to bNOS. Through mutagenesis and crystallographic studies, our findings demonstrate that aminoquinolines are an excellent scaffold for further aiding in the development of bNOS specific inhibitors.

Full text of DOI:10.1021/acs.biochem.6b00786

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Article
Targeting Bacterial Nitric Oxide Synthase
with Aminoquinoline-based Inhibitors
Jeffrey K. Holden, Matthew C. Lewis, Maris A. Cinelli, Ziad Abdullatif,
Anthony V. Pensa, Richard B. Silverman, and Thomas L. Poulos
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00786 • Publication Date (Web): 08 Sep 2016
Downloaded from http://pubs.acs.org on September 16, 2016
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Targeting Bacterial Nitric Oxide Synthase with Aminoquinoline-based Inhibitors
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Jeffrey K. Holden1, Matthew C. Lewis1, Maris A. Cinelli4,5, Ziad Abdullatif1, Anthony V. Pensa4,5,
Richard B. Silverman4,5,6,* and Thomas L. Poulos1,2,3,*
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1Departments of Molecular Biology and Biochemistry, 2Pharmaceutical Sciences, and
3Chemistry, University of California, Irvine, CA 92697ꢀ3900; and 4Departments of Chemistry
and Molecular Biosciences, 5Chemistry of Life Processes Institute, and 6Center for Molecular
Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208ꢀ3113.
Running Title: Inhibition of bNOS with Aminoquinolines
*To whom correspondence should be addressed. T.L.P.: eꢀmail, poulos@uci.edu; phone,
(949) 824ꢀ7020; fax, (949) 824ꢀ3280. R.B.S.: eꢀmail, Agman@chem.northwestern.edu; phone,
(847) 491ꢀ5653; fax, (847) 491ꢀ7713.
†This work was supported by NIH grant GM57353 (TLP) and NIH grant GM49725 (to RBS).
‡Coordinates and structure factors have been deposited in the Protein Data Base under
accession numbers 5G65, 5G66, 5G67, 5G68, 5G69, 5G6A, 5G6B, 5G6C, 5G6D, 5G6E,
5G6F, 5G6G, 5G6H, 5G6I, 5G6J, 5G6K, 5G6L, 5G6M, 5G6N, 5G6O, 5G6P, 5G6Q.
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1Abbreviations
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NOS, nitric oxide synthase; NO, nitric oxide; LꢀArginine, LꢀArg; nNOS, neuronal nitric oxide
synthase; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase;
mNOS, mammalian nitric oxide synthase; bNOS, bacterial nitric oxide synthase; bsNOS,
Bacillus subtilis nitric oxide synthase; saNOS, Staphylococcus aureus nitric oxide synthase;
methicillinꢀresistant Staphylococcus aureus, MRSA.
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Nitric oxide (NO) is produced in Gramꢀpositive pathogens Bacillus anthracis and
Staphylococcus aureus by the bacterial isoform of nitric oxide synthase (NOS). Inhibition of
bacterial nitric oxide synthase (bNOS) has been identified as a promising antibacterial strategy
for targeting methicillinꢀresistant Staphylocoocus aureus1. One class of NOS inhibitors that
demonstrates antimicrobial efficacy utilizes an aminoquinoline scaffold. Here we report on a
variety of aminoquinolines that target the bacterial NOS active site, in part, by binding to a
hydrophobic patch that is unique to bNOS. Through mutagenesis and crystallographic studies,
our findings demonstrate that aminoquinolines are an excellent scaffold to further aid in the
development of bNOSꢀspecific inhibitors.
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Bacterial nitric oxide synthase (bNOS) shares many structural and enzymatic
properties with the mammalian nitric oxide synthases (mNOS), neuronal NOS, inducible NOS,
and endothelial NOS. Both bNOS and mNOS have hemeꢀcontaining oxygenase domains that
produce the free radical gas nitric oxide (NO) through a multiꢀelectron transfer process.
Biosynthesis of NO requires the substrate Lꢀarginine (LꢀArg), NADPHꢀderived electrons, and a
reduced pterin group2. The active sites of all NOS isoforms are very similar and they all bind Lꢀ
Arg directly over the heme, where a conserved LꢀGlu residue holds the LꢀArg in place3.
Despite many similarities between mNOS and bNOS, there are some significant differences.
The mNOS isoforms are multiꢀdomain proteins that transfer electrons from NADPH to the
oxygenase domain via a separate reductase domain. Most bNOS enzymes, however, are
composed of only the oxygenase domain, and thus depend on other redox partners for
electron transfer4. The mNOS isoforms also contain an Nꢀterminal Zn2+ binding motif that helps
stabilize the dimer interface and binding of the pterin cofactor, tetrahydrobiopterin. In sharp
contrast, bNOS lacks the Zn2+ binding motif and therefore has a more open dimer interface5,6
resulting in a larger pterin binding pocket7,8.
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One of the many proposed roles of NO in bacteria is to help protect the bacteria from
host cell or antibioticꢀinduced oxidative stress9,10, and in Staphylococcus aureus NO facilitates
bacterial virulence11.Owing to the important role NO plays in methicillinꢀresistant S. aureus
(MRSA), bNOS has become a promising therapeutic target. Previously, we demonstrated that
NOS inhibitors improve the efficacy of the antimicrobial acriflavine12,13 and hydrogen peroxideꢀ
derived oxidative stress1. From our previous studies on NOS inhibition, we have identified
several key active site differences that can be exploited for the design of bNOS specific
inhibitors. Most notable is the “open” pterinꢀbinding site that is unique to bNOS isoforms
because of the missing Zn2+ binding motif12 found in mNOS isoforms. Another notable
difference is a hydrophobic patch at the distal face of the heme active site1; in bNOS this patch
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is composed of a LꢀIle residue, and in mNOS isoforms the analogous residue is a LꢀVal.
From a chemically diverse library of nNOS inhibitors, aminoquinolineꢀbased inhibitors
were identified for further development of a bNOS specific inhibitor targeting MRSA1. The
aminoquinoline inhibitors were found to bind to the bNOS active site and exploit the
hydrophobic patch contributed by the LꢀIle residue (LꢀVal in mNOS) through van der Waals
interactions. Since the aminoquinoline class of NOS inhibitors presents promising antimicrobial
effects against MRSA, further characterization of aminoquinolines as bNOS inhibitors is
necessary. Here we report on the characterization of 17 aminoquinolineꢀbased bNOS
inhibitors using binding, inhibition, and crystallographic studies. The inhibitors reported herein
are shown in Figure 1.
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Experimental Procedures
Molecular Biology
Bacillus subtilis NOS (bsNOS) DNA was previously cloned into a pET28a (Novagen)
expression plasmid12 with surface entropy reduction mutations E24A/E25A/E316A identified
using the sERP server14. Introduction and expression/purification of bsNOS I218V was
previously described1. Codon optimized DNA for E. coli expression of Homo sapiens inducible
NOS (iNOS) was synthesized by Genewiz (South Plainfield, NJ) and cloned into pET28a
(Novagen) using NdeI and XhoI as the restriction sites. The iNOS heme domain expression
construct encoded residues Arg83 to Arg536. Active site mutation V352I was introduced to the
heme domain expression construct by site directed mutagenesis using PfuTurbo (Agilent). An
Nꢀterminal Hisꢀtag calmodulinꢀexpressing construct was prepared by PCR amplification of the
calmodulin gene from a previous calmodulin expressing construct (a kind gift from Prof. Paul
Ortiz de Montellano, UCSF) and cloned into pET28a (Novagen) using restriction sites NheI
and HindIII, resulting in plasmid pJH114. Expression plasmid pJH114 was then digested with
restriction enzymes XbaI and XhoI. The digested insert containing the calmodulin encoded
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gene was ligated into the XbaI and XhoI restriction sites of pET21a (Novagen) to produce
Calmodulin expression plasmid pJH115.
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Expression and Purification
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Both bsNOS and I218V bsNOS were overexpressed in Escherichia coli BL21(DE3) and
isolated as previously described12,15. Expression of the iNOS heme domain required coꢀ
expression with calmodulin. Hence, the iNOS heme domainꢀexpressing plasmid was coꢀ
transformed with calmodulin expressing plasmid pJH115 into Overexpress C41(DE3)
chemically competent E. coli cells (SigmaꢀAldrich). The following morning an individual colony
was inoculated into 5 mL of LB media supplemented with ampicillin and kanamycin at 50
ng/mL and 35 ng/mL, respectively. The starter culture was then aliquoted to 1 L TB media
supplemented with 500 ꢁM CaCl2, 50 ng/mL ampicillin, and 35 ng/mL kanamycin. Following
inoculation of the media, the culture was shaken overnight at 200 RPM and 30 °C. After this
period, the culture reached OD600 > 2.0 and was induced by addition of 400 ꢁM δꢀ
aminolevulinic acid and 0.5 mM isopropyl βꢀDꢀ1ꢀthiogalactopyranoside. The bacterial cells
were harvested by centrifugation and resuspended in lysis buffer composed of 40 mM BisꢀTris
methane (pH 7.0), 200 mM NaCl, 1 mM CaCl2, 4 mM LꢀArg, 5 ꢁM H4B, 10% glycerol, and 5
mM imidazole. The bacterial cells were lysed using a microfluidics Mꢀ110L microfluidizer, and
cell debris removed by centrifugation prior to loading on to a Ni2+ꢀnitrilotriacetate affinity
column. The column was then washed with 10 CV of lysis buffer supplemented with 15 mM
imidazole, and the targeted proteins were eluted with lysis buffer supplemented with 250 mM
imidazole. The Nꢀterminal His tag was removed by the protease thrombin (MP Biomedicals) at
4 °C overnight. Cleaved protein was resolved from the nonꢀcleaved protein by Ni2+ꢀ
nitrilotriacetate affinity chromatography. The iNOS/calmodulin protein complex was further
purified by Sepharose sizeꢀexclusion chromatography using a buffer composed of 40 mM
HEPES (pH 7.6), 200 mM NaCl, 10% v/v glycerol, 10 ꢁM H4B, 0.5 mM imidazole, and 3 mM
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dithiothreitol.
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Imidazole Displacement
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The sample absorbance was monitored using a Cary 3E UVꢀvisible spectrophotometer as
inhibitors were titrated into a cuvette containing 50 mM Tris (pH 7.4), 1 mM imidazole, 100 ꢁM
dithiothreitol, 10 mM NaCl, and 2 ꢁM of the corresponding NOS. The difference in the
imidazoleꢀbound lowꢀspin state and the inhibitorꢀbound highꢀspin state was calculated as a
function of inhibitor concentration, as previously annotated for bsNOS, bsNOS I218V, iNOS,
and iNOS V352I16. From these data KS,app, was calculated from the concentration of inhibitor
required to transition 50% of the sample from lowꢀspin to highꢀspin and was determined as
previously reported12 using a nonlinear regression analysis available in Sigmaplot version 10.0
(Systat Software, Inc., www.sigmaplot.com). The KS for each ligand was then calculated from
the KS,app as previously described17 using the KD of imidazole to bsNOS, bsNOS I218V, iNOS
and iNOS 352I16.
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IC50 Determination.
Enzyme activity for bsNOS was measured using the previously described bBidomain
construct.15 The bBidomain enzyme is a fusion protein composed of bsNOS at the Nꢀterminus,
flavoprotein YkuN at the Cꢀterminus, and a 30 amino acid peptide linker between the two
proteins. In the presence of reductase YumC and NADPH a catalytic system is established
and electrons are transferred to the bBidomain active site via the flavoproteins. Enzyme
activity was measured by Griess reaction as previously described.15 To calculate the IC50,
nitrite levels were measured at varying concentrations of inhibitor ranging from 0.1 ꢁM to 200
ꢁM for three separate replicates. The average percent inhibition was calculated for each
inhibitor concentration evaluated and a curve was fit to the data in Sigmaplot version 10.0
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(Systat Software, Inc., www.sigmaplot.com). The IC50 was extrapolated from the curve
generated by Sigmaplot version 10.0.
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Crystallization and Sample Preparation
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Crystals of bsNOS and bsNOS I218V were obtained as previously described12,15. Protein
crystals were slowly cryoprotected in the presence of 2 mM H4B and 25% v/v glycerol as
previously reported12. Following cryoprotection, the crystals were soaked in the presence of 5ꢀ
10 mM of the corresponding NOS inhibitor for 3ꢀ5 hours and then flash frozen using N2(l).
X-Ray Data Collection and Processing
Xꢀray diffraction data sets were collected on individual crystals at both the Advanced Light
Source (Berkeley, CA) and the Stanford Synchrotron Radiation Light source (Palo Alto, CA).
Data frames were indexed and integrated using either MOSFLM18 or XDS19. The indexed data
sets were scaled with Aimless20 and some datasets with strong anisotropy were further scaled
using the diffraction anisotropy server21. Structure factors were initially refined using Refmac22
and PHENIX23 was used for later rounds of refinement. The program COOT24 was used to
model inhibitor binding, and PyMOL (Version 1.5.0.4 Schrödinger, LLC.) was used to generate
figures. Data collection and refinement statistics are provided in Table S1.
Chemical Synthesis of 1, 2, and 11
tert-Butyl (3-Hydroxybenzyl)(methyl)carbamate (19). Step 1. Methylamine in THF (4.1 mL,
8.24 mmol) was diluted with CHCl3 (10 mL), and aldehyde 18 (0.500 g, 4.12 mmol) was
added in a solution of CHCl3/MeOH (10:1, 5 mL). Anhydrous sodium sulfate (~2 g) was added,
and the mixture was stirred rapidly under an argon atmosphere at room temp for 90 min.
Glacial AcOH (50 µL) an additional anhydrous sodium sulfate (~1 g) was then added. The
mixture was stirred for a total of 4 h, and the sodium sulfate was filtered from the mixture. The
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filtrate was concentrated, and the residue was diluted with MeOH (20 mL) and cooled to 0 ˚C.
NaBH4 (0.203 g, 5.36 mmol) was added, and the mixture was warmed to room temp and
stirred for 20 min. The resulting mixture was concentrated, and the residue was partitioned
between EtOAc and sat. aq. NaHCO3 (30 mL each). The layers were separated and the
aqueous layer was extracted with EtOAc until no residual amine could be extracted (as
measured by TLC). The organic layer was washed with sat. aq. NaCl (50 mL) and dried.
Concentration afforded the intermediate secondary amine as an oil. Step 2. This amine was
immediately diluted in THF (30 mL) and Boc2O (0.900 g, 4.12 mmol) was added as a solution
in minimal THF. The mixture was stirred at room temp for 3.5 h and then concentrated. The
residue was partitioned between EtOAc and sat. aq. NaHCO3 (30 mL each), the layers were
separated, and the aqueous layer was extracted with EtOAc (3 x 30 mL). The combined
organic layers were washed with H2O and sat. aq. NaCl (50 mL each), dried over anhydrous
sodium sulfate, and evaporated. The residue was purified by flash column chromatography
(SiO2), eluting with a gradient of hexanes to 35% EtOAc in hexanes to yield the product as a
colorless syrup (0.756 g, 77%) that was used without further purification. 1HꢀNMR (500 MHz;
CDCl3): δ 9.28 (br s, 1 H), 7.24ꢀ7.21 (m, 1 H), 7.09 (dd, J = 7.4, 1.6 Hz, 1 H), 6.94 (d, J = 8.1
Hz, 1 H), 6.81 (t, J = 7.4 Hz, 1 H), 4.31 (s, 2 H), 2.88 (s, 3 H), 1.48 (s, 9 H); 13CꢀNMR (126
MHz; CDCl3): δ 156.4, 131.4, 130.1, 122.6, 119.3, 117.7, 81.6, 49.6, 34.1, 28.5.
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7-[(2-((Methylamino)methyl)phenoxy)methyl]quinolin-2-amine Dihydrochloride (11). NaH
(60% suspension in mineral oil, 0.011 g, 0.27 mmol) was diluted with anhydrous DMF (1.5 mL)
and cooled to 0 ˚C under an argon atmosphere. A solution of phenol 19 (0.064 g, 0.27 mmol)
in anhydrous DMF (1.5 mL) was added slowly to the suspension and stirred at 0 ˚C for 20 min,
followed by addition of bromide 20 (see25; 0.075 g, 0.27 mmol) in anhydrous DMF. The
reaction mixture was stirred at 0 ˚C for 1 h and was then quenched by addition of a 1:1 sat. aq.
NaCl/H2O (~15 mL). The mixture was extracted with EtOAc (3 x 20 mL), and the organic
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phase was washed with 5% aq. NaCl (3 x 50 mL) and sat aq. NaCl (50 mL). The organic layer
was dried over anhydrous sodium sulfate, concentrated, and purified by flash column
chromatography (SiO2), eluting with a gradient of 5% EtOAc in CH2Cl2 to 35% EtOAc in
CH2Cl2 to yield acetamide 21 as an offꢀwhite foam (0.099 g, 85%), which was immediately
deprotected. Compound 21 was diluted with anhydrous MeOH (10 mL), and anhydrous K2CO3
(0.063 g, 0.457 mmol) was added. The mixture was heated at reflux for 2.5 h, cooled, and
concentrated. The resulting residue was partitioned between EtOAc and 1:1 H2O/sat. aq. NaCl,
and the aqueous layer was extracted with EtOAc (3 x 20 mL). The organic layers were washed
with sat. aq. NaCl (40 mL), dried over anhydrous sodium sulfate, and concentrated. EtOAc (2
mL) was added to the residue, followed by hexanes (15 mL). The mixture was heated to
boiling, and sonicated vigorously until an offꢀwhite solid precipitate formed, which was
collected by filtration and washed with hexanes. This free aminoquinoline was diluted in 10:1
ether/MeOH, and filtered to remove any particulate matter. To the filtered solution was added
methanolic HCl (3 M, 1 mL), and the mixture was stirred at room temperature overnight. The
resulting precipitate was collected, diluted in hot MeOH (1 mL), and ether (10 mL) was added,
giving 11 as a white flocculent solid (0.063 g, 75%) after washing with ether: mp 284ꢀ286 ˚C.
1HꢀNMR (500 MHz; DMSOꢀd6): δ 14.50 (br s, 1 H), 9.30 (br s, 1 H), 8.99 (br s, 2 H), 8.37 (d, J
= 9.2 Hz, 2 H), 8.30 (br s, 1 H), 7.95 (d, J = 8.2 Hz, 1 H), 7.91 (s, 1 H), 7.60 (d, J = 7.6 Hz, 1 H),
7.49 (dd, J = 7.5, 1.6 Hz, 1 H), 7.43ꢀ7.39 (m, 1 H), 7.17 (d, J = 8.1 Hz, 1 H), 7.09 (d, J = 9.3 Hz,
1 H), 7.05 (td, J = 7.4, 0.7 Hz, 1 H), 5.41 (s, 2 H), 4.21 (t, J = 5.5 Hz, 2 H), 2.62 (t, J = 5.2 Hz, 3
H); 13CꢀNMR (126 MHz; DMSOꢀd6): δ 156.1, 154.4, 142.8, 142.0, 131.4, 130.8, 128.9, 123.6,
121.0, 120.3, 120.2, 115.1, 113.7, 112.5, 68.6, 46.4, 32.6; one of the aminoquinoline carbons
is not visible due to baseline broadening. ESIMS m/z (rel. intensity) 294 (MH+, 100); HRMS
calcd for C18H20N3O+: 294.1601; found, 294.1612. HPLC purity: 100%.
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Quinolin-2-amine Hydrochloride (1). A sealable vial was charged with 2ꢀchloroquinoline (22,
0.244 g, 1.5 mmol), Pd2(dba)3 (13 mg), and DavePhos (13 mg). Anhydrous dioxane (1.1 mL)
and LHMDS (1 M in THF, 3.4 mL, 3.4 mmol) were added, the solution was purged with argon,
and the vial was sealed. The mixture was heated to 100 ˚C for 20 h, and then cooled and
poured into 6 N HCl (20 mL). The solution was washed with EtOAc (20 mL), and the EtOAc
layer was extracted with 6 N HCl (3 x 20 mL). The various aqueous layers were washed with
EtOAc (2 x 30 mL). The EtOAc washes were discarded, and the combined aqueous phase
was basified with NaOH to pH 12. The suspension was extracted with EtOAc (3 x 30 mL), and
the organic layers were washed with 5% aq. NaCl and sat. aq. NaCl (50 mL each), dried over
anhydrous sodium sulfate, and concentrated. The residue was purified by flash column
chromatography (SiO2), eluting with a gradient of EtOAc to 2% MeOH in EtOAc to yield a white
solid after dissolving in EtOAc (5 mL) and precipitating with hexanes (50 mL). The collected
solid was diluted with dry ether (8 mL) and treated with methanolic HCl (3 M, 1.5 mL, 4.5
mmol), upon which a colorless solid precipitated, which was collected by filtration and dried to
afford 1 (0.150 g, 70%): mp 110ꢀ111 ˚C (softens, possibly freeꢀbases); 120ꢀ122 ˚C (melts)
(see26 mp: 222ꢀ223 ˚C (HCl salt); 126ꢀ127 ˚C (freeꢀbase)). 1HꢀNMR (500 MHz; DMSOꢀd6): δ
14.28 (s, 1 H), 9.14 (s, 1 H), 8.38 (d, J = 9.3 Hz, 1 H), 8.21 (br s, 1 H), 7.92 (dd, J = 8.0, 1.0 Hz,
1 H), 7.77 (td, J = 7.7, 1.3 Hz, 1 H), 7.71 (d, J = 8.2 Hz, 1 H), 7.50ꢀ7.47 (m, 1 H), 7.12 (d, J =
9.3 Hz, 1 H); 13CꢀNMR (126 MHz; DMSOꢀd6): δ 154.3, 143.0, 135.7, 132.5, 128.8, 124.9,
120.9, 117.2, 113.8. ESIMS m/z (rel. intensity) 145 (MH+, 100). HPLC purity: 99.6%.
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4-Methylquinolin-2-amine Hydrochloride (2). A mixture of 2ꢀchlorolepidine (23) (0.300 g,
1.69 mmol), K2CO3 (1.17 g, 8.45 mmol), and dry acetamide (8.0 g, 135 mmol) was heated to
240 ˚C for 2 h, during which time a yellow color formed. The solution was then cooled and
poured into H2O (50 mL). The suspension was extracted with EtOAc (3 x 50 mL), and the
organic layer was washed with H2O (3 x 50 mL) and sat. aq. NaCl (50 mL) and then dried over
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anhydrous sodium sulfate. The solution was concentrated and the residue was purified by
flash column chromatography (SiO2), eluting with a gradient of EtOAc to 2% MeOH in EtOAc
to yield a yellow solid after washing with 2% EtOAc in hexanes. The solid was diluted with dry
ether (8 mL) and treated with methanolic HCl (3 M, 1.5 mL, 4.5 mmol), precipitating a colorless
solid. An analytically pure sample was produced by preparative HPLC, using an Agilent Infinity
1200 HPLC pump with MS SQ 6130 detector, with a Phenomenex Gemini 5ꢀ C18 150 x 21.2
mm column, eluting with a gradient of 97% H2O (+ 0.1% formic acid) + 3% MeCN (+ 0.1%
formic acid) to 40% H2O (+ 0.1% formic acid) + 60% MeCN (+ 0.1% formic acid) over 20 min,
at a flow rate of 20 mL/min. The product was obtained as a white solid (87 mg, 33%): mp 199ꢀ
201 ˚C (see27 mp 201ꢀ202 ˚C)); 1HꢀNMR (500 MHz; DMSOꢀd6): δ 14.04 (s, 1 H), 8.94 (br s, 1
H), 8.30 (br s, 1 H), 8.00 (dd, J = 8.2, 1.0 Hz, 1 H), 7.78 (ddd, J = 8.3, 7.1, 1.3 Hz, 1 H), 7.70
(dd, J = 8.3, 0.7 Hz, 1 H), 7.51 (ddd, J = 8.2, 7.1, 1.2 Hz, 1 H), 6.95 (d, J = 1.0 Hz, 1 H), 2.63
(d, J = 1.0 Hz, 3 H); 13CꢀNMR (126 MHz; DMSOꢁd6): δ 153.6, 152.4, 135.5, 132.3, 125.4,
124.8, 121.1, 117.5, 112.6, 19.0; ESIMS m/z (rel. intensity) 159 (MH+, 100). HPLC purity:
100%.
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Results and Discussion
Spectral Binding Analysis. Although the various NOS isoforms have very similar active site
structures, one potentially important difference between mammalian and bacterial NOS
isoforms is that an LꢀIle in bNOS, which is a LꢀVal in mNOS isoforms, interacts with
hydrophobic portions of inhibitors. Previously, we postulated that this subtle difference in
hydrophobicity might contribute to inhibitor selectivity for bNOS1. To further evaluate the role of
the bNOS Ile residue, we performed mutagenesis studies in bsNOS and the reverse mutation
studies in iNOS. From the 17 aminoquinoline inhibitors studied here, Ileꢀ218 was observed to
contribute to the bsNOS inhibitor specificity (Table 1) as noted by an increase in Ks for the
I218V bsNOS mutant. Specifically, for both 1 and 2 we observed a 26ꢀfold and 7ꢀfold decrease
in inhibitor specificity between WT and I218V, respectively. However, this trend was not
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consistently observed for the Ks measured in iNOS WT and V352I iNOS. In fact, the Ks only
decreased for compounds 7, 8, 13, and 14 when V352I was introduced in iNOS and increased
for 10 of the other inhibitors.
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Aminoquinoline Inhibition of bNOS. The two most potent bsNOS inhibitors identified by KS
analysis (1 and 2, Table 1), were also the simplest aminoquinolines studied. To further
evaluate the relationship between Ile218 and the inhibitory potential of aminoquinolines, both 1
and 2 were evaluated using the previously described bBiDomain assay15. The bBidomain is a
construct consisting of the B. subtilis flavodoxin, YkuN, fused to the Cꢀterminal end of bsNOS.
In the presence of the B. subtilis FADꢀcontaining reductase, YumC. and NADPH the
bBidomain oxidizes substrate much more rapidly than the 3 component system consisting of
bsNOS, YkuN, and YumC. 15 This has provided a more facile method for rapidly screening the
ability of inhibitors to block bsNOS activity. 1 Both of these compounds were found to have IC50
values in the low ꢁM range (Table 2). From these studies we also found that introduction of
I218V to bBiDomain resulted in a small increase in the measured IC50 values.
Crystal Structure Analysis. Although 22 crystal structures were solved, we focus here on
those that provide the most important insights into the effect of the I218V mutation. As
expected, in all of these structures the aminoquinoline forms stacking interactions with the
heme group and hydrogen bonds with the conserved active site Glu243, and it directly
contacts Ile218 through a van der Waals interaction (Fig, 2, Fig. S1). For the simplest
inhibitors, 1 and 2, the 8ꢀ27 fold decrease in binding affinity in the I218V bsNOS mutant can be
rationalized based on the additional nonꢀcovalent interactions between Ile218 and the inhibitor
compared to Val218. We estimated the additional stability of the larger Ile vs. Val by
calculating the nonꢀbonded interaction energy between the inhibitor and residue 218 using the
Sander module in Amber. The WT Ile provides an additional ꢀ1.8 kcal/mol greater stability.
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This is in agreement with the ꢀG obtained from the spectral Ks data (≈ꢀ2 kcal/mol).
The addition of a linker to the aminoquinoline with various tail groups results in various
binding orientations of the inhibitor tail, which extends out of the active site. The inhibitor tail
binds better for some inhibitors than others as judged by the electron density. For example,
the electron densities observed for the tail end of inhibitors 6 and 8 are well ordered, while
those for inhibitors 10 and 13 are not (Fig. 2). There is also a relationship between how well
ordered the tail end of the inhibitor is and the effect of the I218V mutant on binding affinity. For
example, both inhibitors 6 and 8 were found to have wellꢀdefined electron density throughout.
The more wellꢀdefined electron density inhibitors, such as 6 and 8, were also minimally
perturbed by the I218V mutation on the basis of differences in KS (Table 1). In contrast,
inhibitors with less defined inhibitor electron density, such as 10 and 13, have KS differences of
8 and 120 fold, respectively, between WT and I218V bsNOS (Table 1). We postulate that for
inhibitors 10 and 13 the tail end of the inhibitor does not contribute much to stability so the
effect of the mutation on binding to the aminoquinoline part of the inhibitor is more pronounced.
Conversely, the effect of the mutant is less pronounced for 6 and 8 since the tail end
contributes more to inhibitor binding. This results in a decreased effect on the mutation on the
aminoquinoline portion of the inhibitor. One reason 6 and 8 have a more ordered tail is due to
the interaction of the secondary amino group with the heme propionates.
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Since the I218V mutation has a large effect on the KS of inhibitor 13, the crystal
structure of the mutant enzyme bound to 13 was also solved (Figure 2). In the mutant, the tail
end of the inhibitor exhibits weak density, and the FoꢀFc maps indicate that the tail of the
inhibitor occupies at least two orientations. An important difference between 13 and some of
the other inhibitors is that the linker in 13 has an ether oxygen rather than a secondary amino
group. As a result, the linker of 13 cannot form a Hꢀbond with the heme propionate, while the
secondary amino group of inhibitors like 8 can form a hydrogen bond. Given the extra
interactions in 8, the I218V mutation has little effect, while for 13, with no linker Hꢀbonding
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possibilities, the effect of the destabilizing I218V mutation is much larger because the
aminoquinoline itself contributes more to overall binding.
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One remaining question is why the I218V mutation has a large effect on KS in bsNOS
but the reverse mutation in iNOS, V352L, has little effect. For example, the I218V mutation
decreases affinity for inhibitor 13 by approximately 120 fold in bsNOS but there is little
difference between WT iNOS and the V352I mutant. There are, however, other differences in
the active site between bsNOS and iNOS. For example, His128 in bsNOS is replaced by Ala
in iNOS. His128 contacts both the central region of the larger inhibitors as well as Ile218
thereby restricting inhibitor mobility within the binding pocket. The bsNOS active site is also
more open than the iNOS active site because, unlike iNOS, bsNOS does not contain an Nꢀ
terminal Zn2+ binding motif. Previously, we have observed that the open pterin site of bsNOS
accommodates the binding of aminopyridineꢀbased inhibitors12 and allows for the binding
modes of thiophenecarboximidamideꢀbased inhibitors that are unique to bsNOS3. Therefore, it
is likely that the Nꢀterminal binding motif in iNOS also influences the binding of the longer
aminoquinoline inhibitors that extend out of the active site in bsNOS.
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Initial structural studies of aminoquinolines binding to bsNOS indicated that they might
bind to the pterin site (data not shown). To determine if aminoquinolines can bind to this site
we prepared crystals of 6 and 16 without H4B present during the cryosoak. Processing of the
crystallographic data provided a twoꢀsite binding model of both 6 and 16 to bsNOS when H4B
is absent (Figures 3B and 3D). In these two site models, one inhibitor is bound at the active
site and the second binds to the pterin site by forming a πꢀπ stacking interaction with Trp329.
In the case of 6, inhibitor binding at the pterin site requires a water molecule to bridge a
hydrogen bond to the heme propionate group. Binding of 6 is further stabilized by a salt bridge
between the secondary linker amine of 6 and R344(C=O) at the dimer interface (Figure 3B).
Unlike 6, a hydrogen bond between inhibitor linker and R344(C=O) is not observed with 16
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because it has an etherꢀlinked tail (Figure 3D). On the basis of 2FoꢀFc maps, the binding of
both 6 and 16 to the pterin site is weak compared to binding of H4B, as H4B can easily outꢀ
compete the aminoquinoline inhibitors evaluated. Inspection of both 6 and 16 bound to the
active site reveals the binding mode to be unchanged regardless of the ligand present at the
pterin site (Figure 3). This confirms our earlier observations that the aminopyridine of
inhibitors prefer the pterin site over aminoquinolines12. Even in the presence of H4B we have
found that aminopyridines can be quite effective at displacing H4B while aminoquinolines
prefer the active site12.
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Conclusions
Aminoquinolineꢀbased inhibitors have been developed to inhibit NOS activity. Since the
aminoquinoline scaffold also has been previously found to function as an antimicrobial agent
against the highly pathogenic organism MRSA1, an improved understanding of aminoquinoline
binding to bNOS was necessary to further drug design efforts and identify new scaffolds for
inhibitor design. Previous evidence also indicates that the potency and specificity afforded by
the aminoquinolineꢀbased inhibitors to be controlled by the subtle differences in hydrophobicity
at the bNOS active site1. Specifically, the hydrophobic patch contributed by Ileꢀ218 (mNOS
equivalent residue is Val) allows for tighter binding of the aminoquinoline group as a result of a
van der Waals interaction between the aminoquinoline group and active site Ileꢀ218 residue.
On the basis of these results one might have anticipated that the reverse mutation in
iNOS, V352I, should improve binding, but there is little difference between WT and mutant.
However, in bsNOS His218 contacts the inhibitor while this equivalent residue is an LꢀAla in
iNOS and a LꢀSer in both H. sapiens endothelial NOS (eNOS) and neuronal NOS (nNOS),
respectively. As a result, the active site of bsNOS provides more contacts with the
aminoquinoline inhibitors. These results, combined with previous studies, indicate at least two
structural and functional differences between bNOS and mNOS that can be exploited in the
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design of bNOSꢀselective inhibitors. First, the larger aminoquinoline compared to the smaller
aminopyridine favors the bNOS active site owing to the more extensive nonbonded contacts
provided by Ile218. Second, aminopyridines favor the pterin site over aminoquinolines.
Targeting the pterin site might be especially useful since pterins bind more weakly to bNOS
than nNOS, which should result in easier displacement of H4B by inhibitors in bNOS. Thus, an
inhibitor with an aminoquinoline in the active site and an aminopyridine in the pterin site has
the potential to be an especially potent and selective bNOS inhibitor.
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Acknowledgements
This work is greatly indebted to the expertise and wisdom provided by Dr. Huiying Li. We are
also very grateful to the beamline staff at both the Advanced Light Source and the Stanford
Synchrotron Radiation Light source (Palo Alto, CA) for help with remote data collection. This
work was supported by National Institute of Health grants GM57353 (to TLP), GM49725 (to
RBS), and F32GM109667 (to MAC).
Supporting Information
A summary of crystallographic and refinement statistics are provided in Table S1 and the
electron density map for various inhibitors in Fig. S1.
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References
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neuronal nitric oxide synthase inhibition. J. Med. Chem. 57, 1513ꢀ1530.
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compounds. US Patent 2892841 A
(27) Kaye, I. A. (1949) 2ꢀLepidyl Substituted Diamines. J. Am. Chem. Soc. 71, 2322ꢀ
2325.
(28) Cinelli, M. A.; Li, H.; Pensa, A. V.; Kang, S.; Roman, L. J.; Martasek, P.; Poulos,
T. L.; Silverman, R. B. (2016) Correction to Phenyl Etherꢀ and AnilineꢀContaining 2ꢀ
Aminoquinolines as Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase. J .Med.
Chem. 59, 1246.
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Table 1. Spectral binding constants for inhibitors against NOS isoforms and mutants. αValue reported
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in,16,β Value reported in.1
bsNOS WT
KS (µM)
bsNOS I218V
KS (µM)
Human iNOS
KS (µM)
Human iNOS
V352I KS (µM)
Ligand
LꢀArg
1
4.8 ± 0.1α
1.25 ± 0.03
0.80 ± 0.08
3.2 ± 0.1
3.2 ± 0.1
1.3 ± 0.1
6.7 ± 0.7β
3.6 ± 0.8β
14.1 ± 0.4
2.0 ± 0.1
19 ± 1
2.0 ± 0.2α
33.3 ± 1.5
6.1 ± 0.3
41 ± 6
16.1 ± 0.7α
4.5 ± 0.5
3.5 ± 0.2
7.1 ± 1.2
91 ± 5
40.9 ± 4.3α
11.1 ± 0.7
14 ± 1
2
3
50 ± 18
76 ± 6
4
97 ± 6
5
31 ± 2
12 ± 1β
40 ± 6
40 ± 3
6
42 ± 8
43 ± 5
7
18 ± 2
70 ± 35
107 ± 39
34 ± 4
50 ± 9
8
11 ± 3
80 ± 19
44 ± 4
9
34 ± 3
10
11
12
13
14
15
16
17
154 ± 12
182 ± 39
266 ± 100
320 ± 140
39 ± 2
14 ± 3
36 ± 11
33 ± 11
56 ± 9
7.5 ± 0.3
3.4 ± 0.1
2.7 ± 0.1
3.0 ± 0.1
2.2 ± 0.1
3.4 ± 0.1
1.3 ± 0.1
18 ± 2
11 ± 1
140 ± 29
39 ± 3
122 ± 16
26 ± 2
184 ± 35
103 ± 22
17 ± 2
61 ± 6
61 ± 7
67 ± 14
2.8 ± 0.3
117 ± 20
8.3 ± 1.5
Table 2. IC50 (ꢁM) of aminoquinoline inhibitors evaluated using bBidomain.
WT
I218V
63.9
1
2
27.3
36.6
53.3
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Figure Legends
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Figure 1. NOS inhibitors reported in this study. Chemical syntheses of inhibitors 1, 2, and 11
are reported here (see Experimental Procedures). Syntheses of 3ꢀ7 are reported in25 and that
of 8 and 9 are reported in http://patents.justia.com/patent/9212144. Syntheses of inhibitors 10
and 12ꢀ17 are reported in28.
Figure 2. Crystal structures of WT and I218V bsNOS complexed with inhibitors shown in
yellow and a FoꢀFc map contoured at 1.0σ. The aminoquinoline group binds to the NOS active
site for all molecules through a hydrogen bond to E243. The tails of inhibitors 6, 8, and 10 are
stabilized by hydrogen bonds to heme propionate. 13 contains an ether group that positions
the inhibitor tail away from the heme propionate group because of electrostatics.
Figure 3. 2FoꢀFc maps contoured at 1 σ for inhibitors 6 and 16 both in the presence and
absence of H4B. Inhibitors are colored yellow and H4B is shown as cyan. When H4B is absent
inhibitor binding is also observe at the pterin site for B) 6 and D) 16.
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Figure 1. NOS inhibitors reported in this study. Chemical syntheses of inhibitors 1, 2, and 11
are reported here (see Experimental Procedures). Syntheses of 3ꢀ7 are reported in25 and that
of 8 and 9 are reported in http://patents.justia.com/patent/9212144. Syntheses of inhibitors 10
and 12ꢀ17 are reported in28.
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Figure 2. Crystal structures of WT and I218V bsNOS complexed with inhibitors shown in
yellow and a FoꢀFc map contoured at 1.0σ. The aminoquinoline group binds to the NOS active
site for all molecules through a hydrogen bond to E243. The tails of inhibitors 6, 8, and 10 are
stabilized by hydrogen bonds to heme propionate. 13 contains an ether group that positions
the inhibitor tail away from the heme propionate group because of electrostatics.
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Figure 3. 2FoꢀFc maps contoured at 1 σ for inhibitors 6 and 16 both in the presence and
absence of H4B. Inhibitors are colored yellow and H4B is shown as cyan. When H4B is absent
inhibitor binding is also observe at the pterin site for B) 6 and D) 16.
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