Research Article
ZLc002, a putative small-molecule inhibitor
of nNOS interaction with NOS1AP,
suppresses inflammatory nociception and
chemotherapy-induced neuropathic pain
and synergizes with paclitaxel to reduce
tumor cell viability
Wan-Hung Lee1, Lawrence M Carey2,3, Li-Li Li4,5, Zhili Xu3,
Yvonne Y Lai3,6, Michael J Courtney4,5,7 and
Andrea G Hohmann1,2,3,8
Abstract
Elevated N-methyl-D-aspartate receptor activity contributes to central sensitization. Our laboratories and others recently
reported that disrupting protein–protein interactions downstream of N-methyl-D-aspartate receptors suppresses pain.
Specifically, disrupting binding between the enzyme neuronal nitric oxide synthase and either its upstream (postsynaptic
density 95 kDa, PSD95) or downstream (e.g. nitric oxide synthase 1 adaptor protein, NOS1AP) protein partners suppressed
inflammatory and/or neuropathic pain. However, the lack of a small-molecule neuronal nitric oxide synthase-NOS1AP
inhibitor has hindered efforts to validate the therapeutic utility of disrupting the neuronal nitric oxide synthase-NOS1AP
interface as an analgesic strategy. We, therefore, evaluated the ability of a putative small-molecule neuronal nitric oxide
synthase-NOS1AP inhibitor ZLc002 to disrupt binding between neuronal nitric oxide synthase and NOS1AP using ex vivo, in
vitro, and purified recombinant systems and asked whether ZLc002 would suppress inflammatory and neuropathic pain in
vivo. In vitro, ZLc002 reduced co-immunoprecipitation of full-length NOS1AP and neuronal nitric oxide synthase in cultured
neurons and in HEK293T cells co-expressing full-length neuronal nitric oxide synthase and NOS1AP. However, using a cell-
free biochemical binding assay, ZLc002 failed to disrupt the in vitro binding between His-neuronal nitric oxide synthase1-299
and glutathione S-transferase-NOS1AP400-506, protein sequences containing the required binding domains for this protein–
protein interaction, suggesting an indirect mode of action in intact cells. ZLc002 (4–10mg/kg i.p.) suppressed formalin-
evoked inflammatory pain in rats and reduced Fos protein-like immunoreactivity in the lumbar spinal dorsal horn. ZLc002
also suppressed mechanical and cold allodynia in a mouse model of paclitaxel-induced neuropathic pain. Anti-allodynic
efficacy was sustained for at least four days of once daily repeated dosing. ZLc002 also synergized with paclitaxel when
administered in combination to reduce breast (4T1) or ovarian (HeyA8) tumor cell line viability but did not alter tumor cell
viability without paclitaxel. Our results verify that ZLc002 disrupts neuronal nitric oxide synthase-NOS1AP interaction in
intact cells and demonstrate, for the first time, that systemic administration of a putative small-molecule inhibitor of
neuronal nitric oxide synthase-NOS1AP suppresses inflammatory and neuropathic pain.
1Biochemistry Interdisciplinary Graduate Program, Molecular and Cellular
Biochemistry Department, Indiana University, Bloomington, IN, USA
2Program in Neuroscience, Indiana University, Bloomington, IN, USA
3Department of Psychological and Brain Sciences, Indiana University,
Bloomington, IN, USA
4Neuronal Signalling Lab, Turku Centre for Biotechnology, University of
Turku; A˚bo Academy University, Turku, Finland
5Turku Centre for Biotechnology and Institute of Biomedicine, Screening
Unit, University of Turku, Turku, Finland
6Anagin, Inc., Indianapolis, IN, USA
7Turku Brain and Mind Center, Turku, Finland
8Gill Center for Biomolecular Science, Bloomington, IN, USA
Corresponding Author:
Andrea G Hohmann, Department of Psychological and Brain Sciences,
Indiana University, 1101 E, 10th Street, Bloomington, IN 47405-7007, USA.
Email: hohmanna@indiana.edu
Molecular Pain
Volume 14: 1–17
! The Author(s) 2018
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DOI: 10.1177/1744806918801224
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Keywords
N-methyl-D-aspartate, central sensitization, neuronal nitric oxide synthase, NOS1AP, postsynaptic density 95 kDa (PSD95)
Date Received: 25 May 2018; revised: 16 July 2018; accepted: 14 August 2018
Introduction
Elevated N-methyl-D-aspartate receptor (NMDAR)
activity is one of the key mechanisms contributing to
central sensitization.1,2 However, NMDAR antagonists
have limited therapeutic applications due to unwanted
side effects (e.g. motor impairment, memory deficits,
cognitive dysfunction, dissociation from reality, and
abuse liability).3–5 Strategies that counteract or prevent
aberrant elevated NMDAR activity without altering the
basal activity of NMDAR would, therefore, be advan-
tageous. Our laboratories and others have previously
demonstrated that disrupting protein–protein interac-
tions downstream of NMDAR (i.e. NR2B-PSD95,
PSD95-nNOS, and nNOS–NOS1AP) produces antinoci-
ceptive efficacy without unwanted side effects associated
with NMDAR antagonists.6–10 We recently proposed
that the nNOS–NOS1AP protein–protein interface was
a previously unrecognized target for analgesic drug
development.11 We showed that TAT-GESV, a peptide
inhibitor of the nNOS–NOS1AP interface, disrupted
binding between nNOS and NOS1AP in vitro, sup-
pressed glutamate-induced cell death in cultured cortical
neurons, and produced antinociceptive efficacy in mech-
anistically distinct models of neuropathic pain.11
TAT-GESV, administered intrathecally (i.t.), suppressed
mechanical and cold allodynia induced by either toxic
challenge with the chemotherapeutic agent paclitaxel or
traumatic nerve injury produced by a partial sciatic
nerve ligation.11 These effects were not observed follow-
ing the administration of a control peptide (e.g.
TAT-GESVD1, which lacks the terminal valine residue
of TAT-GESV) which also failed to disrupt nNOS–
NOS1AP interactions.11 However, peptides are not ideal
therapeutics due to limited bioavailability and poor phar-
macokinetics.12,13 Moreover, the impact of nNOS–
NOS1AP disruption on inflammatory pain is unknown.
Systemically active small-molecule nNOS–NOS1AP
inhibitors are needed to better evaluate the therapeutic
potential of disrupting the nNOS–NOS1AP interface.
A putative small-molecule inhibitor of nNOS–
NOS1AP, ZLc002 (Figure 1) was recently shown to
exhibit anxiolytic-like efficacy in a mouse model of
chronic mild stress without altering appetite, general
activity, or locomotor activity or interfering with the
resting potential of neurons.14 ZLc002 also inhibited
co-immunoprecipitation of NOS1AP with nNOS in
hippocampal cells.14 However, despite the promising
preclinical therapeutic and side effect profile of
ZLc002, it is unclear whether these effects occur through
direct disruption of the nNOS–NOS1AP complex. This
evaluation is important because the ability of ZLc002 to
uncouple NOS1AP from nNOS in hippocampal cells ex
vivo could occur through either direct or indirect mech-
anisms. Moreover, whether ZLc002 produces antinoci-
ceptive efficacy or suppresses neurochemical markers of
pain-evoked neuronal activation is unknown.
We evaluated the ability of ZLc002 to suppress
inflammatory and neuropathic pain in vivo and further
characterized its mechanism of action in vitro. We ver-
ified that ZLc002 disrupts nNOS–NOS1AP interaction
in primary cultures of cortical neurons. We documented
that, in a cell-free binding assay, ZLc002 did not directly
disrupt binding between purified nNOS and NOS1AP
containing the known interacting sites.15,16 We also
established that, in intact HEK293T cells transfected
with the full-length tagged proteins, ZLc002 disrupted
the co-immunoprecipitation of nNOS with NOS1AP.
We demonstrated that ZLc002 suppressed formalin-
evoked pain behavior and neuronal activation in
lumbar spinal dorsal horn. We established that ZLc002
suppressed the maintenance of chemotherapy-induced
neuropathic pain induced by paclitaxel, which is used
clinically to treat breast, ovarian, and lung cancers but
produces dose-limiting toxic neuropathy in humans.17
Finally, we showed that ZLc002 acted synergistically
with paclitaxel to enhance ovarian and breast cancer
tumor cell line cytotoxicity in vitro.
Materials and methods
Drugs and chemicals
Peptides were purchased from GeneCust (Dudelange,
Luxembourg) or GenicBio (Shanghai, China) with at
Figure 1. Chemical structure of ZLc002, putative small-molecule
inhibitor of nNOS–NOS1AP interaction.
2 Molecular Pain
least 95% of purity: L-TAT-GESV (GRKKRRQ
RRRYAGQWGESV); L-TAT-GESVD1 (GRKKRR
QRRRYAGQWGES): lacking the last C-terminal
valine residue. Peptides were dissolved in phosphate-
buffered saline (PBS) for AlphaScreen. ZLc002 was syn-
thesized by RTI international (Research Triangle Park,
NC) for Anagin Inc. (Indianapolis, IN) and provided to
the investigators. MK-801 and ZLc002 were dissolved in
dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis,
MO, USA) at 20 mM for AlphaScreen biochemical
binding assays and in a vehicle composed of 3%
DMSO, 1:1:18 of emulphor (Alkamuls EL 620L;
Solvay), 95% ethanol (Sigma Aldrich), 0.9% NaCl
(Aquilite System; Hospira, Inc, Lake Forest, IL) for in
vivo administration. All drugs were delivered via intra-
peritoneal (i.p.) injection in a volume of 5 ml/kg for
in vivo studies performed in mice and in a volume of
1ml/kg for in vivo studies performed in rats. In the rat
formalin study, ZLc002 and MK-801 were dissolved in a
vehicle containing 20% DMSO (Sigma Aldrich), and the
remaining 80% consisting of 95% ethanol, emulphor,
and 0.9% saline at a ratio of 1:1:8 (final ratio of 5: 2:
2: 16). MK-801 and formaldehyde (37% in H2O) were
purchased from Sigma Aldrich. Formalin was diluted to
2.5% in saline from formaldehyde stock (100% forma-
lin) and administered via local intraplantar (i.pl.) injec-
tion in a volume of 50 ml.
Protein purification
Purification of glutathione S-transferase (GST), His-
tagged nNOS, and NOS1AP was performed as previous-
ly described.9,18 nNOS1-299 containing the core PSD-95,
Dlg (discs large homolog), and ZO-1 (zona occludens-1)
(PDZ) domain that binds NOS1AP and the b-finger that
binds to PDZ2 of PSD95, but lacking the catalytic
domain, was expressed as an N-terminal His-tagged
fusion protein. GST-NOS1AP400-506, containing the
internal ExF motif and the C-terminal tail that is recog-
nized by the core PDZ domain of nNOS, was expressed
as an N-terminal GST-tagged fusion protein.
AlphaScreen assay
AlphaScreen assays were set up and performed as previ-
ously described.9 Briefly, binding between nNOS and
NOS1AP was set up using His-nNOS1-299 and GST-
NOS1AP400-506 proteins. AlphaScreen nickel chelate
acceptor beads (PerkinElmer, Waltham, MA) and
AlphaScreen glutathione donor beads (PerkinElmer)
were sequentially added and incubated at room temper-
ature for 1 h with each addition. The reaction was car-
ried out in a 40 ll final volume using 96-well 1=2 area
plates in 1X PBS containing bovine serum albumin
(1mg/mL) and Tween-20 (0.1%). An EnSpireVR
Multimode Plate Reader (PerkinElmer, Waltham, MA)
equipped with AlphaScreen optical detection module
was used to read plates. Titration was performed to
determine 50% binding between His-nNOS1-299 and
GST-NOS1AP400-506 (0–100 nM each). To test the dis-
ruption of the protein–protein binding by inhibitors, the
reaction was carried out using concentrations of His-
nNOS1-299 and GST-NOS1AP400-506 which lead to
50% of maximum binding. Inhibitors or vehicle (PBS
or DMSO) were added to the protein pairs at the begin-
ning of the experiments. All the peptides used in this
experiment were dissolved in PBS. ZLc002 was dissolved
in DMSO. Peptides and ZLc002 were prepared as 20
mM stocks, and subsequent dilutions were made from
this stock for use in each assay. The concentrations of
peptides and ZLc002 ranging from 0 to 100 mM were
used to determine IC50. Each data point represents the
mean % AlphaScreen signal count derived from at least
four determinations (i.e. duplicate determinations
obtained in two independent assays performed on sepa-
rate days).
Cell culture and transfection
HEK293T cells were cultured in Dulbecco’s minimal
essential medium (with 10% fetal bovine serum,
19.4mM supplementary glucose, 2mM glutamine,
50 lg/ml streptomycin sulfate, and 50 U/ml penicillin)
at 37
C under a 5% CO2 humidified atmosphere. The
cells were transfected by the calcium phosphate method
as previously described19 with full-length plasmid DNA
pEGFP-nNOSa (human sequence fused to enhanced
green fluorescent protein) and full-length pLuc-
NOS1AP (human, transcript variant 1, fused to firefly
luciferase) or pLuc-PSD95-PDZ2 (encoding aa 159–249
of human Dlg4 transcript variant4, fused to firefly lucif-
erase) as indicated, or empty luciferase vector pLuc-C1
as negative control. These constructs have been previ-
ously described in the studies by Li et al.15,18
Inhibitor treatment and co-immunoprecipitation assay
using HEK293T cells
Twenty-two hours after transfection, HEK293T cells
were treated in 0.5 mM probenecid-supplemented con-
ditioned cell culture medium with or without 10 lM
ZLc002 for 90 min. The probenecid was added because
cell lines, in contrast to neurons, have a high rate of
extrusion of esters like calcium dyes20 and, therefore,
most likely ZLc002 also. Cells were then lysed in low
stringency buffer21 supplemented with protease inhibi-
tors, 1 mM DTT, and 0.5% Igepal CA-630 and pre-
cleared at 4
C by centrifugation at 20,000g. Cell lysates
were precipitated by agarose-coupled single-chain anti-
green fluorescent protein (GFP) camelid antibody-based
Lee et al. 3
protein (“GFP-Trap,” Chromotek) for 1 h at 4
C. The
co-precipitated luciferase-fused proteins were
measured using a tube luminometer and %
co-immunoprecipitation determined by normalization
to lysate levels of luciferase-fusion. Equal immunopre-
cipitation of the EGFP-nNOS in all samples was deter-
mined by Western blotting with anti-GFP antibody
(mouse monoclonal clone JL8, RRID:AB_2313808,
used at 0.1 lg/ml; Clontech).
Cortical neuronal culture
Cortical neuron cultures were prepared from P0 rats of
either sex (mixed) as described previously.22 The isola-
tion of cells and tissues from animals was performed in
accordance with the corresponding local, national, and
European Union regulations. The neurons were cultured
in Neurobasal-A/B-27 medium (Thermofisher), and one
third of medium was changed every three days.
Inhibitor treatment and co-immunoprecipitation assay
using cortical neurons
At eight days in vitro, neuronal medium was replaced by
minimum essential medium (MEM cat. # 11700077,
Thermofisher) and neurons were then treated with or
without 10 mM ZLc002 for 90 min. Following pretreat-
ments for 90 min, neurons were stimulated with 50 mM
NMDA for 10 min, followed by immediate lysis in low
stringency buffer (LSB), supplemented with protease
inhibitors, 1 mM DTT, and 0.5% Igepal CA-630 and
precleared at 4
C by centrifugation at 20,000g.21
Immunoprecipitating (IP) antibody, nNOS (mouse
monoclonal clone A-11, RRID: AB_626757, Santa
Cruz Biotechnology, 2.5 lg/ml) was added to the
lysate. Samples were rotated for 2 h at 4
C, after
which 5 ll of protein-A resin (GenScript) was added,
and rotation continued for 1 h. The resin was then
washed three times with the LSB, and protein was
eluted from drained resin by boiling at 95
 for 10 min
in SDS-PAGE sample loading buffer and analyzed by
Western blotting.18,23 Immunoprecipitated nNOS and
co-immunoprecipitated NOS1AP in all samples were
determined by Western blotting with anti-nNOS anti-
body (A-11) and anti-NOS1AP antibody (rabbit poly-
clonal IgG, R-300, RRID: AB_2251417, Santa Cruz
Biotechnology), respectively.
Tumor cell viability assay
4T1 mouse breast cancer cells were a gift from Dr
Harikrishna Nashatri (IUPUI) and were maintained in
RPMI-1640 supplemented with 10% fetal bovine serum
and 1% penicillin–streptomycin. HeyA8 human ovarian
cancer cells were a gift from Dr Kenneth Nephew (IU
Bloomington) and were maintained in DMEM
supplemented with 10% fetal bovine serum and 1% pen-
icillin–streptomycin. All the cells were kept in a 37
C
incubator equipped with 5% CO2. Tumor cell viability
was measured with the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay accord-
ing to the manufacturer’s instructions (Roche,
Indianapolis, IN), as described in our previously pub-
lished work.24 Briefly, cells were seeded at a density of
3000 cells/well in a 96-well plate and cultured overnight.
The following day, 4T1 andHeyA8 cells were treated with
an increasing concentration of ZLc002 (0 50 mM), pac-
litaxel (0 500 nM), or the combination of both and incu-
bated for a further 72 h. At the end of the incubation, 10
lL MTT solution (5 mg/mL) was added to each of the
wells. After 4 h of incubation, 100 lL solubilization solu-
tion was added. The solubilized crystals were measured at
optical density 570 nm. The effect of the drugs on cells
was expressed as a percentage of viability compared to
untreated cells. Data were derived from multiple experi-
ments (n¼ 7 for 4T1 cell line and n¼ 3 for HeyA8), all
performed on separate days; all data sets were normalized
and subjected to nonlinear regression analysis to generate
IC50. The combination response (additivity, synergy, or
antagonism) was analyzed using Combenefit (Cancer
Research UK Cambridge Institute; Cambridge, UK), a
software tool that enables the visualization, analysis, and
quantification of drug combination effects.25 The data
from the combination treatments were processed using
three synergy reference models: the Bliss independence
model, the Loewe additivity model, and the highest
single agent (HSA) model using Combenefit
(‘Combination Benefit’).25
Subjects
Adult C57BL/6J male mice, weighing 23–33 g (Jackson
Laboratory, Bar Harbor,ME), were used in the studies of
chemotherapy-induced peripheral neuropathic pain pro-
duced by paclitaxel. Adult male Sprague Dawley rats,
weighing 285–446 g (Envigo, Indianapolis, IN, USA),
were used in the formalin study. The positive control
(MK-801) and vehicle-treated groups that appear in the
rat formalin study described here (vehicle and MK801
data points in Figure 5 only6) were published previously
by our group as comparators in a separate evaluation of
structurally distinct PSD95-nNOS protein–protein inter-
action disruptors (i.e. IC87201 and ZL006) and are
included here with permission from the publisher.6 The
vehicle- and MK-801-treated groups were tested, per-
fused, and processed concurrently, under blinded condi-
tions, with the ZLc002-treated groups described for the
first time in the present report, in accordance with our
obligations to comply with the guidelines from the
Association for Assessment and Accreditation of
Laboratory Animal Care to reduce unnecessary animal
4 Molecular Pain
use.6 Tissue from all subjects was processed concurrently
for Fos immunohistochemistry. Animals were housed in
a temperature-controlled facility (73 2
F), 45% humid-
ity under a 12-h light/dark cycle with standard rodent
chow and water ad libitum. All experimental procedures
were approved by the Bloomington Institutional Animal
Care and Use Committee of Indiana University and fol-
lowed guidelines of the International Association for the
Study of Pain.
Formalin test
Rats received a single i.p. injection of ZLc002 (4 or 10
mg/kg), MK-801 (0.1 mg/kg) or vehicle 30 min before i.
pl. formalin injection. Animals were placed on an elevat-
ed clear glass table in Plexiglass observation chambers
immediately following i.p. injection and were allowed to
habituate to the testing apparatus for 30 min. Next, rats
received a unilateral i.pl. injection of 2.5% formalin
(50ml) into the superficial plantar surface of the hind
paw. Behavior was videotaped for 60 min immediately
following i.pl. formalin and nociceptive behaviors were
quantified by a single experimenter (LMC) blinded to
the experimental conditions. Composite pain scores
(CPS) were calculated for every 5 min time bin as
described in our previous work.6,26 No pain behavior
was scored as 0, lifting of the paw was scored as 1, and
shaking/biting/flinching was scored as 2. The area under
the curve (AUC) for formalin-evoked pain was calculated
for the early phase of pain behavior (phase 1, 0–10 min)
and the late phase (phase 2, 10–60 min) for each subject.
Tissue preparation for immunohistochemistry
Immunohistochemical experiments were conducted on
tissues from the same subjects that are used to evaluate
the impact of ZLc002 (4 or 10 mg/kg i.p.), MK-801
(0.1mg/kg i.p.) and vehicle on formalin-evoked pain
behavior. Immediately after concluding behavioral pro-
cedures, rats were anesthetized with 25% urethane and
immediately perfused transcardially with 0.1% heparin-
ized 0.1 M PBS followed by 4% paraformaldehyde (i.e.
1 h post i.pl. formalin). Lumbar spinal cord tissue was
dissected and kept in the same fixative for 24 h and then
cryoprotected in 30% sucrose for three days prior
to sectioning.
Immunohistochemistry
Immunohistochemical experiments were conducted as
described previously.6,27–30 Briefly, transverse sections
(30 lm) of the L4-L5 lumbar spinal cord were cut on a
cryostat and kept in an antifreeze solution (50% sucrose
in ethylene glycol and 0.1 M PBS) prior to staining.
Every fourth section was processed for immunostaining
to avoid counting the same cell twice in adjacent
sections. Sections were washed in 0.1 M PBS, then
endogenous peroxidases were quenched in 0.3% H2O2
for 30min. Tissue was then incubated for 1 h in a block-
ing solution consisting of 5% normal goat serum and
0.3% Triton X-100 in 0.1 M PBS. Tissue was incubated
with rabbit polyclonal Fos protein antibody (1:1500,
Santa Cruz Biotechnology, Dallas, TX, USA) for 24
h at 4
C. Fos protein expression was visualized using
the avidin-biotin peroxidase method using diaminoben-
zidine as the chromagen. Three sections per animal
which displayed the greatest numbers of Fos-like immu-
noreactive (FLI) cells, based upon qualitative evalua-
tion, were quantified by an experimenter blinded to
treatment conditions. Images were obtained using a
Retiga 1300 digital camera mounted on a Leica
DMLB microscope. The number of FLI cells were
counted manually using ImageJ software in spinal sub-
divisions as first described by Presley et al.31 The spinal
subdivisions subjected to quantification were the super-
ficial dorsal horn (laminae I and II), the nucleus proprius
(lamina III and IV), the neck region of the dorsal horn
(laminae V and VI), and the ventral horn (laminae
VII-X). Statistical analyses were conducted on the
number of FLI cells per subdivision averaged across the
three sections quantified per animal to generate a single
mean for each subdivision per animal. FLI cells were
largely absent in rats receiving an i.pl. injection of saline
in lieu of formalin (data not shown and Carey et al.6).
Paclitaxel-induced neuropathic pain
Paclitaxel (Tecoland Corporation, Irvine, CA) was dis-
solved in a vehicle consisting of a 1:1:4 ratio of cremo-
phor EL (Sigma Aldrich), ethanol (Sigma Aldrich) and
saline (Aquilite System; Hospira, Inc, Lake Forest, IL).
Mice were injected with either the cremophor-vehicle or
paclitaxel (4 mg/kg, i.p.) on days 0, 2, 4, and 6 following
initiation of paclitaxel dosing (16 mg/kg i.p. cumulative
dose). Responsiveness to mechanical and cold stimula-
tion was assessed before initiation of paclitaxel or
cremophor-vehicle dosing (i.e. baseline, day 0) and
during development and maintenance phases of
paclitaxel-induced hypersensitivity on days 4, 7, 11,
and 15 as previously described.
Time course of anti-allodynic effects of ZLc002 in the
paclitaxel model of neuropathic pain
Paclitaxel-treated mice were randomly divided into two
groups and injected systemically with either vehicle or
ZLc002 (10 mg/kg, i.p.) on day 16 following initiation of
paclitaxel dosing. Responsiveness to mechanical and
cold stimulation was assessed starting at 30 min after
drug injection and reevaluated at 60, 90, and 150 min
postinjection.
Lee et al. 5
Effects of repeated systemic dosing with ZLc002 in
the paclitaxel model of neuropathic pain
The same mice used in the time course evaluation were
repeatedly injected with vehicle or ZLc002 (10 mg/kg, i.
p.) once daily for another seven consecutive days.
Repeated dosing was initiated on day 16 following initi-
ation of paclitaxel dosing. Responsiveness to mechanical
stimulation was assessed at 30 min posttreatment on
days 1, 4, and 8 of chronic injection.
Assessment of mechanical allodynia
Withdrawal thresholds (g) to mechanical stimulation
were measured in duplicate for each paw using an elec-
tronic von Frey anesthesiometer supplied with a 90-g
semi-flexible probe (IITC Life Science, Woodland
Hills, CA) as described previously.32,33
Assessment of cold allodynia
Cold allodynia was assessed by applying one drop
(5–6 ml) of acetone (Sigma Aldrich) to the plantar sur-
face of the hind paw. Time spent reacting to acetone
stimulation was measured in triplicate for each paw.33,34
Statistical analysis
Data were analyzed using GraphPad Prism for Windows
5 (Graphpad Software, San Diego, CA USA). IC50
values in AlphaScreen were calculated by nonlinear
regression analysis using the equation of a sigmoid con-
centration–response curve using GraphPad Prism.
Co-immunoprecipitation data were analyzed by one-
way analysis of variance (ANOVA) followed by post
hoc Bonferroni test. In vivo data were analyzed by
two-way repeated measures ANOVA and one-way
ANOVA, as appropriate. Post hoc comparisons were
performed using Bonferroni’s post hoc tests or, in the
case of comparisons to control, Bonferroni’s multiple
comparison test. P< 0.05 was considered statistical-
ly significant.
Results
ZLc002 reduced co-immunoprecipitation of
NOS1AP with nNOS immunoprecipitated from
primary cultured cortical neurons
We verified that ZLc002 can disrupt nNOS–NOS1AP
interactions in primary neuronal cultures as previously
suggested14 using methodology published previously by
our group.18 Our results confirm that NMDA (50 mM)
challenge elevated nNOS–NOS1AP interaction relative
to control conditions in primary cortical neurons as
determined with co-immunoprecipitation (Figure 2).
Moreover, pretreatment with ZLc002 (10 mM) reduced
NMDA-induced nNOS–NOS1AP interaction (F2,11=
21.26, p< 0.001, Figure 2). NMDA-treated cells
displayed higher levels of co-immunoprecipitated
nNOS–NOS1AP than either control cells not treated
with NMDA (p< 0.001; Bonferroni’s post hoc test) or
NMDA-treated cells pretreated with ZLc002 (p< 0.01;
Bonferroni’s post hoc test) (Figure 2).
ZLc002 failed to disrupt nNOS–NOS1AP
protein–protein interactions in the AlphaScreen
in vitro binding assay
To investigate whether ZLc002 disrupts the binding
between nNOS and NOS1AP through a direct mecha-
nism, we set up AlphaScreen assays for cell-free
(a) (b)
Figure 2. ZLc002 reduces co-immunoprecipitation of NOS1AP with nNOS in primary cortical neurons challenged with NMDA (50 mM).
(a) Immunoblots show that nNOS–NOS1AP association was increased after exposure of cells to NMDA and this effect was reduced by
ZLc002 (10 mM) pretreatment. (b) Quantification of mean S.E.M from n¼ 4 experiments (from two separate primary neuronal cultures)
as shown in (a). **p< 0.01; ***p< 0.001 vs. NMDA-treated controls (one-way ANOVA followed by Bonferroni’s post hoc test).
NMDA: N-methyl-D-aspartate; nNOS: neuronal nitric oxide synthase.
6 Molecular Pain
measurement of direct binding between purified His-
nNOS1-299 (containing the extended PDZ domain of
nNOS) and GST-NOS1AP400-506 (containing the ExF
motif and C-terminal tail). ZLc002 failed to disrupt
this interaction event at the highest concentration
tested (100 lM) in this reductionist Alphascreen binding
assay, whereas the consensus peptide inhibitor of
nNOS–NOS1AP, TAT-GESV, reliably disrupted the
interaction with an IC50 of 4.9 lM (Figure 3) under
analogous conditions. Moreover, consistent with our
previous findings, the inactive peptide TAT-GESVD1,
had no effect on binding between nNOS and NOS1AP
(Figure 3). These results are consistent with ZLc002
acting in cells as a prodrug as previously suggested14
and therefore showing no activity in a cell-free assay.
ZLc002 reduced co-immunoprecipitation of full-length
NOS1AP but not of PSD95-PDZ2 from HEK293T
cells co-expressing full-length nNOS
Although ZLc002 disrupts NMDA-evoked interaction
of nNOS with NOS1AP (Figure 2), this effect need not
be direct and could, instead, result from an indirect
effect on NMDA receptor signaling. For example,
nNOS inhibitors also reduce NMDA-evoked
nNOS–NOS1AP interaction in neurons.18 To determine
whether ZLc002 directly disrupts the targeted protein–
protein interaction14 in intact cells as intended, we
evaluated the effect of ZLc002 exposure on the
co-immunoprecipitation of NOS1AP preassembled
with over-expressed nNOS in HEK293T cells, cells that
would not be expected to express endogenous nNOS,
PSD95, or NMDAR subunits. Full-length GFP-tagged
nNOS was immunoprecipitated from transfected 293T
cells co-expressing full-length NOS1AP (tagged with
luciferase tag for quantification, see methods) with or
without a 90-min preexposure to 10 mM ZLc002.
Immunoblotting demonstrated comparable immunopre-
cipitation of GFP-nNOS in all samples in each replicate
(n¼ 3), and quantification of co-immunoprecipitated
NOS1AP showed that ZLc002 reduced the
nNOS–NOS1AP interaction by 40% (F2,8¼ 495.5,
p< 0.0001; Figure 4(a)). To evaluate the selectivity of
this inhibition for the nNOS interaction with
NOS1AP, the experiments were repeated using nNOS
and PSD95-PDZ2. While higher levels of binding of
nNOS to PSD95-PDZ2 were observed in control relative
to empty vector samples (F2,8¼ 859.4, p< 0.0001;
p< 0.0001; Figure 4(b)), ZLc002 had no effect on
co-immunoprecipitation of PSD95-PDZ2 with nNOS.
These observations suggest that ZLc002 is selective for
a specific function of the nNOS-PDZ domain, i.e. the
recruitment of NOS1AP.
ZLc002 reduced formalin-evoked nociceptive behavior
and Fos-like immunoreactivity in the spinal dorsal horn
The i.pl. injection of formalin increased CPS in a biphas-
ic manner (F12,18¼ 19.22, p< 0.0001; Figure 5(a)).
ZLc002, administered 30 min prior to i.pl. formalin
injection, reduced formalin-evoked CPS (F3,18¼ 9.964,
p< 0.001, Figure 5(a)), and the interaction between
time and drug treatment was significant (F38,18¼ 4.187,
p< 0.0001, Figure 5(a)). Post hoc analyses revealed that
both the high (10 mg/kg i.p.) and low dose of ZLc002
(4mg/kg i.p.) reduced formalin-evoked CPS from 30–50
min following formalin (i.pl.) injection relative to vehicle
(p< 0.05 for each comparison; Bonferroni’s multiple
comparison test). MK-801 similarly reduced formalin-
evoked CPS from 25 to 50 min postformalin relative to
vehicle (p< 0.05 for each comparison, Bonferroni’s mul-
tiple comparison test (Figure 5(a)).
The i.pl. formalin increased the AUC of formalin-
induced pain behavior in a phase-dependent manner
(F1,18¼ 40.49, p< 0.0001; Figure 5(b)). ZLc002 treat-
ment decreased the AUC (F3,18¼ 40.49, p< 0.0001;
Figure 5(b)), and the interaction between phase and
treatment was significant (F3,18¼ 8.205, p< 0.01;
Figure 5(b)). None of the treatment groups altered
phase 1 of formalin-evoked pain behavior (p> 0.5;
Figure 5(b)). The NMDAR antagonist MK-801 (0.1
mg/kg i.p.) (p< 0.001), used here as a positive control,
and both the high (10 mg/kg i.p.) (p< 0.001) and low
(4mg/kg) (p< 0.001) dose of ZLc002, all reduced the
AUC of phase 2 of formalin-evoked pain behavior rela-
tive to vehicle treatment (Figure 5(b)).
ZLc002 reduced formalin-evoked Fos protein-like
immunoreactivity (F5,24¼ 85.86, p< 0.0001; Figure 5(c)
-10 -8 -6 -4
0
50
100
PBS
DMSO
TAT-GESV
TAT-GESV1
ZLc002
%
A
lp
ha
Sc
re
en
Si
gn
al
C
ou
nt
s
Log [M]
Figure 3. ZLc002 failed to disrupt the interaction between His-
nNOS1-299 and GST-NOS1AP400-506 in AlphaScreen. The peptide
nNOS–NOS1AP disruptor TAT-GESV disrupted this interaction
with an IC50 of 4.9 lM, whereas the inactive peptide TAT-GESVD1
failed to do so (n¼ 4 replicates derived from two separate assays
performed on separate days). Data are mean S.E.M.
Lee et al. 7
and (d)) in a lamina-dependent manner (F3,24¼ 21.43,
p< 0.0001; Figure 6(a) and (b)), and the interaction
between drug treatment and spinal cord laminar expres-
sion of Fos-protein like immunoreactivity was signifi-
cant (F15,24¼ 8.7, p< 0.0001; Figure 6(a)). ZLc002, at
doses of both 4 and 10 mg/kg i.p., reduced formalin-
evoked Fos-like immunoreactivity in the superficial
dorsal horn (p< 0.001; Bonferroni’s post hoc test) and
the neck region of the dorsal horn (p< 0.001;
Bonferroni’s post hoc test) but not in the nucleus pro-
prius or the ventral horn (p> 0.05 for each comparison;
Bonferroni’s post hoc test) relative to vehicle treatment.
Control ZLc002
0
5
10
15
20
25 *** ***
NOS1AP
full length
Empty vector
C
o-
Im
m
un
op
re
ci
pi
ta
tio
n
(%
of
In
pu
t)
Control ZLc002
0
1
2
3
PSD95-PDZ2
Empty vector
C
o-
Im
m
un
op
re
ci
pi
ta
tio
n
(%
of
In
pu
t)
***
(a) (b)
GFP
EGFP-nNOS full length
250 kDa
130 kDa
250 kDa
130 kDa
GFP
EGFP-nNOS full length
Figure 4. ZLc002 reduces co-immunoprecipitation of NOS1AP with nNOS but not PSD95-PDZ2 in HEK293T cells co-expressing the
full-length proteins. ZLc002 (10 mM) treatment disrupts co-immunoprecipitation with full-length EGFP-nNOS of (a) full-length pLuc-
NOS1AP but not of (b) pLuc-PSD95-PDZ2 from HEK293T cell lysates. Data are mean S.E.M. (n¼ 3) ***p< 0.001 (one-way ANOVA
followed by Bonferroni’s post hoc test). Immunoblots under each bar chart demonstrate equal levels of nNOS among compared samples.
GFP: green fluorescent protein.
0 10 20 30 40 50 60
0.0
0.2
0.4
0.6
0.8
1.0
Vehicle ZLc002 4 mg/kg
ZLc002 10 mg/kgMK-801 0.1 mg/kg
*** ***
***
***
###
**
*****
*
***
* *
**
*
Time (min)
C
PS
Phase 1 Phase 2
0
5
10
15
20
25
Vehicle
ZLc002 10 mg/kg
ZLc002 4 mg/kg
MK-801 0.1 mg/kg
***
*** ***A
UC
(b)(a)
Figure 5. ZLc002 reduces formalin-evoked pain behavior and Fos-like immunoreactivity in spinal dorsal horn. (a) ZLc002 (4 and 10mg/kg,
i.p.) reduced composite pain scores (CPS) from 30 to 50 min postformalin relative to vehicle. MK-801 (0.1 mg/kg) reduced CPS from 25 to
50 min postformalin relative to vehicle. (b) ZLc002 (4 and 10 mg/kg i.p.) (p< 0.001) and MK-801 (0.1 mg/kg i.p.) reduced formalin-evoked
AUC of pain behavior scores relative to vehicle in phase 2 but not in phase 1 of formalin-evoked pain behavior. Data are mean S.E.M.
(n¼ 5–6 per group) ***p< 0.001; **p< 0.01; *p< 0.05 vs. vehicle; ###p< 0.001 vs. all other groups, (two-way ANOVA followed by
Bonferroni’s multiple comparison test). MK-801 and vehicle groups were published previously but run and processed concurrently with
ZLc002-treated groups shown here (see methods and Carey et al.6).
CPS: composite pain score; AUC: area under the curve.
8 Molecular Pain
By contrast, MK-801 (0.1 mg/kg i.p.) reduced formalin-
evoked Fos-like immunoreactivity in laminae I-IV
(p< 0.001) (Figure 6(a) and (b)) and in the ventral
horn (p< 0.01) relative to vehicle (Figure 6(a) and (b)).
Effects of ZLc002 (4 and 10 mg//kg i.p.) did not
differ from each other in any spinal cord region
(P> 0.05). Example photomicrographs depicting the
impact of vehicle, ZLc002, and MK-801 on formalin-
evoked Fos protein expression are shown in Figure 6
(c) and (f).
ZLc002 attenuates mechanical and cold allodynia
evoked by paclitaxel in mice
Paclitaxel treatment decreased mechanical paw with-
drawal thresholds, mechanical paw withdrawal thresh-
olds differed across test days, and the interaction
between treatment and test day was significant
(F1,22¼ 33.7, p< 0.0001 (treatment); F4,88¼ 20.81,
p< 0.0001 (day); F4,88¼ 17.49, p< 0.0001 (interaction);
Figure 7(a)). Similarly, paclitaxel increased the duration
Vehicle ZLc002 4 mg/kg
ZLc002 10 mg/kg MK-801
(d)(c)
(f)(e)
I-II III-IV V-VI Ventral
0
20
40
60
80
Vehicle
MK-801 0.1 mg/kg ZLc002 10 mg/kg
ZLc002 4 mg/kg
**
*** ***
***
Lamina
N
um
be
ro
fc
el
ls
Ventral Horn
V, VI
III, IV
I, II
(b)(a)
Figure 6. (a) ZLc002 (4 and 10 mg/kg i.p.) reduced formalin-evoked Fos-like immunoreactivity in laminae I-II (p< 0.001) and laminae V-VI
(p< 0.001) relative to vehicle. MK-801 reduced formalin-evoked Fos-like immunoreactivity in laminae I-II (p< 0.001), laminae III-IV
(p< 0.001), V-VI (p< 0.001), and the ventral horn (p< 0.01) relative to rats treated with vehicle. (b) Schematic adapted from the rat brain
atlas of Paxinos and Watson35 showing spinal cord laminae quantified for Fos-like immunoreactivity. Data are mean S.E.M. (n¼ 5–6 per
group) ***p< 0.001; **p< 0.01; *p< 0.05 vs. vehicle (two-way ANOVA followed by Bonferroni’s multiple comparison test). MK-801 and
vehicle groups were published previously but run and processed concurrently with ZLc002-treated groups shown here (see methods and
Carey et al.6). Example photomicrographs taken at 10x magnification showing formalin-evoked Fos-like immunoreactivity in lumbar dorsal
horn of rats treated with vehicle (c), ZLc002 (4 mg/kg i.p.) (d), ZLc002 (10 mg/kg i.p.) (e), and MK-801 (0.1 mg/kg) (f). Scale bar is equal to
100 mm.
Lee et al. 9
of time spent responding to cold, cold responsiveness
differed across test days, and the interaction between
treatment and test day was significant (F1,22¼ 43.491,
p< 0.0001 (treatment); F4,88¼ 34.02, p< 0.0001 (day);
F4,88¼ 13.31, p< 0.0001 (interaction); Figure 7(b)).
Mechanical and cold hypersensitivity was present on
day 7, was maintained throughout the observation
interval, and remained ongoing on day 15, prior
to initiation of pharmacological manipulations
(p< 0.0001, Bonferroni’s post hoc test for both mechan-
ical and cold assessment), relative to day 0 prepaclitaxel
baseline response (Figure 7(a) and (b)).
Paclitaxel lowered mechanical paw withdrawal
thresholds (t11¼ 9, p< 0.0001; Figure 7(c)) and
increased duration of time spent responding to cold
(t11¼ 8.28, p< 0.0001; Figure 7(d)) relative to prepacli-
taxel baseline responses. In paclitaxel-treated mice,
ZLc002 (10 mg/kg, i.p.) increased postinjection mechan-
ical paw withdrawal thresholds, mechanical paw with-
drawal thresholds differed across postinjection times,
and the interaction between drug treatment and injection
time was significant (F1,10¼ 14.81, p¼ 0.0032 (drug);
F2,20¼ 9.05, p¼ 0.0016 (time); F2,20¼ 4.20, p¼ 0.030
(interaction); two-way repeated measures ANOVA;
(a) (b)
(c) (d)
(e) (f)
Figure 7. The putative nNOS–NOS1AP disruptor ZLc002 suppresses mechanical and cold allodynia induced by paclitaxel treatment.
Paclitaxel (a) lowers mechanical paw withdrawal thresholds consistent with the development of mechanical allodynia and (b) increases cold
response time, consistent with the development of cold allodynia. ZLc002 (10 mg/kg i.p.) increases (c) mechanical paw withdrawal
thresholds and (d) lowers duration of response to cold in paclitaxel-treated mice. ZLc002 (10 mg/kg i.p.) does not alter responsiveness to
(e) mechanical or (f) cold stimulation in control mice receiving the cremophor vehicle in lieu of paclitaxel. Data are mean S.E.M.
*p< 0.05; **p< 0.01; ***p< 0.001 (two-way repeated measures ANOVA followed by Bonferroni’s post hoc test); #paired sample t-test
baseline vs. postpaclitaxel predrug response.
BL: baseline; PTX: paclitaxel.
10 Molecular Pain
Figure 7(c)). In paclitaxel-treated mice, ZLc002 elevated
mechanical paw withdrawal thresholds relative to vehi-
cle treatment from 30 min (p< 0.01, Bonferroni’s post
hoc test; Figure 7(c)) to 90 min postinjection (p< 0.05,
Bonferroni’s post hoc test; Figure 7(c)). In paclitaxel-
treated mice, ZLc002 (10 mg/kg, i.p.) decreased postin-
jection cold responsiveness, cold responsiveness did not
differ reliably across postinjection times, and the inter-
action between drug treatment and injection time was
not significant (F1,10¼ 5.46, p¼ 0.0415 (drug);
F2,20¼ 1.99, p¼ 0.1632 (time); F2,20¼ 0.16, p¼ 0.8556
(interaction); two-way repeated measures ANOVA;
Figure 7(d)). Thus, ZLc002 attenuated paclitaxel-
induced cold responsiveness throughout the observation
interval (Figure 7(d)).
In mice that received the cremophor-based vehicle in
lieu of paclitaxel, ZLc002 treatment did not reliably alter
postinjection mechanical or cold responsiveness, behav-
ioral responsiveness was stable across postinjection
times, and the interaction between drug treatment and
time was not significant (Mechanical: F1,10¼ 3.30,
p¼ 0.0992 (drug); F2,20¼ 0.84, p¼ 0.4469 (time);
F2,20¼ 0.16, p¼ 0.8565 (interaction); Cold: F1,10¼ 2.40,
p¼ 0.1522 (drug); F2,20¼ 1.51, p¼ 0.2441 (time);
F2,20¼ 2.15, p¼ 0.1430 (interaction); two-way repeated
measures ANOVA; Figure 7(e) to (f)).
Effects of repeated dosing with nNOS–NOS1AP
disruptor in a mouse model of paclitaxel-induced
neuropathic pain
In paclitaxel-treated mice, once daily dosing with
ZLc002 (10 mg/kg i.p. x 8 days) increased mechanical
paw withdrawal thresholds relative to the vehicle-treated
group across the observation interval (F1,10¼ 26.59,
p¼ 0.0004 (drug)) (Figure 8(a)). Mechanical paw with-
drawal thresholds also differed across injection days,
and the interaction between treatment and injection
day was significant (F2,20¼ 4.13, p¼ 0.0316 (day);
F2,20¼ 8.25, p¼ 0.0024 (interaction)) (Figure 8(a)).
ZLc002 increased mechanical paw withdrawal thresh-
olds relative to vehicle in paclitaxel-treated mice on
day 1 (p< 0.01; Bonferroni’s post hoc test) and day 4
(p< 0.001; Bonferroni’s post hoc test) but not on day
8 of chronic dosing (p> 0.05 for each comparison;
Bonferroni’s post hoc test).
Once daily dosing with ZLc002 (10 mg/kg i.p. 8
days) also lowered paclitaxel-induced cold hypersensitiv-
ity relative to the vehicle-treated group across the obser-
vation interval (F1,10¼ 12.5, p¼ 0.0054 (drug)), and
these effects were also time dependent (F2,20¼ 8.20,
p¼ 0.0025 (interaction)) (Figure 8( b)). Cold responsive-
ness did not differ across injection days (F2,20¼ 0.15,
p¼ 0.8579 (day)) (Figure 8(a)). ZLc002 lowered cold
response times relative to vehicle in paclitaxel-treated
mice on day 1 (p< 0.05; Bonferroni’s post hoc test)
and day 4 (p< 0.001; Bonferroni’s post hoc test) but
not on day 8 (p> 0.05 for each comparison;
Bonferroni’s post hoc test) of chronic dosing (Figure 8
(a) and (b)).
Impact of ZLc002 in the presence and absence of
paclitaxel on breast and ovarian tumor cell
line viability
The impact of ZLc002 and paclitaxel over a wide range
of molar ratios (i.e. dose–response matrix between eight
concentrations of ZLc002 and eight concentrations of
paclitaxel) on 4T1 and HeyA8 tumor cell line cytotoxic-
ity is shown in Figures 9 and 10, respectively. ZLc002
BL PTX 1 4 8
0
2
4
6
8
Paclitaxel-ZLc002Paclitaxel-Vehicle
#
** ***
                         Treatment  day
Th
re
sh
ol
d
(g
)
BL PTX 1 4 8
0
2
4
6
8
Paclitaxel-ZLc002Paclitaxel-Vehicle
* ***
#
                         Treatment day
Ti
m
e
(s
ec
)
(b)(a) Mechanical Cold
Figure 8. Effects of repeated sytemic dosing with nNOS–NOS1AP disruptor ZLc002 on paclitaxel-induced mechanical and cold allodynia.
In paclitaxel-treated mice, ZLc002 (10 mg/kg i.p. 8 days) increased mechanical paw withdrawal threshold (a) and reduced cold response
durations (b) relative to vehicle. ZLc002 reduced paclitaxel-induced allodynia on days 1 and 4 but not on day 8 of repeated dosing. Data are
mean S.E.M. *p< 0.05; **p< 0.01; ***p< 0.05 vs. corresponding vehicle condition (two-way repeated measures ANOVA, followed by
Bonferroni’s post hoc test). #paired sample t-test baseline vs. postpaclitaxel predrug response.
PTX: paclitaxel; BL: baseline.
Lee et al. 11
alone had no effect on the viability of either 4T1
(Figure 9(a)) or HeyA8 (Figure 10(a)) tumor cells,
which was markedly inhibited by paclitaxel in each
case (Figures 9(b) and 10(b)). Nonetheless, quantifica-
tion of the drug combination responses indicates that
the combination between ZLc002 and paclitaxel is syn-
ergistic using the Bliss model (4T1: Figure 9(e); HeyA8:
Figure 10(e)), Loewe additivity model (4T1: Figure 9(f);
HeyA8: Figure 10(f)), and HSA model (4T1: Figure 9(g);
HeyA8: Figure 10(g)). The synergy maps showed that
ZLc002 and paclitaxel have synergistic effects (blue
areas in the model graph) on inhibiting tumor cell pro-
liferation at a wide range of drug combination ratios in
both 4T1 (Figure 9(e) to (g)) and HeyA8 (Figure 10(e) to
(g)) cells.
Discussion
Our studies support a role for disruption of nNOS–
NOS1AP protein–protein interactions downstream of
NMDARs as a therapeutic strategy for suppressing
inflammatory and neuropathic pain. We verified that
the putative small-molecule nNOS–NOS1AP inhibitor
ZLc002 disrupts the NMDA-induced interaction
between full-length nNOS and NOS1AP proteins in pri-
mary cortical neurons. We also showed that ZLc002
Figure 9. ZLc002 synergizes with paclitaxel in 4T1 cells to reduce breast cancer tumor cell line viability. Dose–response matrix for the
effect of (a) ZLc002 and (b) paclitaxel in 4T1 cells. Abs EC50, absolute EC50> 50 mM, reflects little or no inhibition of 4T1 tumor cell
viability by ZLc002; Abs EC50, absolute EC50¼ 33.5 nM, reflects efficacy of paclitaxel in reducing 4T1 tumor cell viability by 50% of
maximum. (c–d) Single-agent and combination responses determined by an MTT viability assay in 4T1 cells. The landscape of the com-
bination responses for ZLc002 and paclitaxel based on the (e) Bliss model, (f) Loewe model, and (g) highest single agent (HSA) model. Each
model supports synergism of the combination of ZLc002 with paclitaxel in reducing tumor cell line viability. (n¼ 7 experiments).
HSA: highest single agent.
12 Molecular Pain
selectively disrupted the preestablished interaction in
HEK293T cells transfected with full-length nNOS and
NOS1AP proteins without altering the interactions
between full-length nNOS and PSD95-PDZ2. These
observations are consistent with a direct action on the
nNOS–NOS1AP protein–protein interaction itself rather
than on the upstream, NMDA-evoked mechanism of
interaction. Our studies suggest that the nNOS–
NOS1AP interface is a previously unrecognized target
for inflammatory pain. We revealed, for the first time,
that a small-molecule nNOS–NOS1AP disruptor exhib-
its anti-allodynic efficacy in models of inflammatory and
neuropathic pain. Importantly, ZLc002, administered
systemically, suppressed both formalin-evoked pain
behavior as well as inflammation-evoked neuronal acti-
vation in lumbar spinal dorsal horn of the same subjects,
similar to the NMDAR antagonist MK-801. Moreover,
ZLc002 suppressed both mechanical and cold allodynia
in a mouse model of neuropathic pain induced by pac-
litaxel treatment and enhanced the ability of paclitaxel to
reduce tumor cell viability in vitro.
ZLc002 was previously shown to disrupt nNOS–
NOS1AP interactions, as judged by the observation of
reduced co-immunoprecipitation of NOS1AP with
Figure 10. ZLc002 synergizes with paclitaxel in HeyA8 cells to reduce ovarian tumor cell line viability. Dose–response matrix delineating
the effect of (a) ZLc002 and (b) paclitaxel in HeyA8 cells. Rel EC95, Relative EC95¼ 0 mM, reflects absence of inhibition of HeyA8 tumor
cell viability by ZLc002; Abs EC50, Absolute EC50¼ 10.2 nM, reflects efficacy of paclitaxel in reducing HeyA8 tumor cell viability by 50% of
maximum. (c–d) Single-agent and combination responses determined by an MTT viability assay in 4T1 cells. The landscape of the com-
bination responses for ZLc002 and paclitaxel based on the (E) Bliss model, (F) Loewe model, and (G) highest single agent (HSA) model.
Each model supports synergism of the combination of ZLc002 with paclitaxel in reducing tumor cell line viability. (n¼ 3 experiments).
HSA: highest single agent.
Lee et al. 13
nNOS measured in hippocampal cells.14 However, such
actions in a complex cellular system could occur through
direct or indirect mechanisms. Our studies verify and
extend these published observations by showing that
ZLc002 reduces the co-immunoprecipitation of
NOS1AP but not PSD95-PDZ2 with nNOS in
HEK293T cells. Although both of these interactions
involve the extended PDZ domain at the first 130
N-terminal amino acids of nNOS, the stable NOS1AP
interaction requires the docking of a PDZ ligand motif
into the nNOS-PDZ pocket,15 whereas PSD95-PDZ2
interacts with a distinct b-finger extension of the core
nNOS-PDZ domain.36 The two sites of interaction are,
therefore, spatially very close to one another but are
distinct,15,16,18 making this comparison a good evalua-
tion of selectivity. Importantly, because endogenous
nNOS, PSD95, or NMDAR subunits can be expected
to be absent in HEK293T cells and ZLc002 disrupts the
constitutive nNOS–NOS1AP interaction in this system,
ZLc002 is unlikely to reduce co-immunoprecipitation of
nNOS and NOS1AP in cultured neurons by acting
through NMDA-evoked mechanisms involving extrane-
ous targets that are present in neurons but not mea-
sured herein.
To determine whether disruption of nNOS–NOS1AP
interactions induced by ZLc002 resulted from a direct
mechanism, we used a cell-free AlphaScreen biochemical
binding assay employing purified nNOS and NOS1AP
fragments containing the interacting interface.15,16,18
Intriguingly, we failed to detect the disruption by
ZLc002 of His-nNOS1-299-GST-NOS1AP400-506 binding
with the fragments that are critical for nNOS–NOS1AP
interactions15 in this reductionist assay. By contrast, the
peptide nNOS–NOS1AP disruptor TAT-GESV dis-
rupted these interactions in the same AlphaScreen
assay, whereas a putative inactive peptide TAT-
GESVD1, lacking the terminal valine residue, failed to
do so, consistent with the known critical role of the pep-
tide ligand terminal valine in target recognition.37
Therefore, the potency of ZLc002 for disrupting binding
of nNOS–NOS1AP could not be determined using
AlphaScreen, which uses a cell-free system consisting
of only a single pair of purified protein fragments. It is
plausible that ZLc002 disrupts the interactions through
a mechanism distinct from the direct disruption at this
interacting interface (e.g. allosteric mechanisms and/or
via an active metabolite of ZLc002 that is produced
in cells).
ZLc002 has recently been hypothesized to act as
a pro-drug.14 This observation could account for
our observation that ZLc002 disrupted the
co-immunoprecipitation of NOS1AP with nNOS from
primary cortical neurons expressing enzymes such as
esterases that would, presumably, be available to trans-
form ZLc002 into other bioactive mediators, but it failed
to disrupt the interaction between nNOS and NOS1AP
in our cell-free AlphaScreen assay even though the pep-
tide nNOS–NOS1AP disruptor TAT-GESV was able to
potently disrupt this interaction. Support for this
hypothesis is derived from the fact that ZLc002 also
disrupted co-immunoprecipitation between full-length
nNOS and NOS1AP in transfected HEK293T cells
in the immunoprecipitation assay. In our study,
ZLc002 disrupted nNOS–NOS1AP interactions in
HEK293T cells transfected with full-length nNOS and
full-length NOS1AP but not between full-length nNOS
and PDZ2 of PSD95. This finding is consistent with
previous work showing that ZLc002 inhibits the
co-immunoprecipitation of NOS1AP with nNOS but
not of PSD95 with nNOS.14 More work is needed to
determine the precise location at which ZLc002 binds
within the complex, if indeed it binds at all, and whether
the currently identified two nNOS interacting sites on
NOS1AP may affect ZLc002’s binding differentially
from TAT-GESV, therefore, giving us different results
in different protein–protein disruption assays. Our
observations, nonetheless, suggest that ZLc002 produces
a functional disruption of nNOS–NOS1AP interactions
in intact cells.
ZLc002 exhibits anxiolytic efficacy in mice and dis-
rupts co-immunoprecipitation of nNOS and NOS1AP in
ZLc002-treated hippocampal cells without changing
PSD95 expression or its association with NMDARs.14
Increases in association of nNOS and NOS1AP also
accompany anxiogenic-like behaviors.14 However, prior
to the present report, whether this small molecule dis-
rupts nNOS–NOS1AP binding through a direct mecha-
nism or suppresses pathological pain was unknown.14
Our studies demonstrate, for the first time, that disrup-
tion of nNOS–NOS1AP protein–protein interactions
suppresses inflammatory nociception. ZLc002, adminis-
tered systemically, produced antinociceptive efficacy in
the formalin test and reduced the number of formalin-
evoked Fos-like immunoreactive cells in the lumbar
spinal dorsal horn. ZLc002 selectively suppressed
phase 2, but not phase 1, of formalin-induced pain
behavior, similar to the NMDAR antagonist MK-801.
These observations are consistent with the role of
NMDARs in contributing to central nervous system sen-
sitization and, specifically, phase 2 of formalin-induced
pain behavior.2,6,38–40 Moreover, ZLc002 selectively sup-
pressed the number of formalin-evoked Fos protein-like
immunoreactive cells, a marker of neuronal activation,
in dorsal horn regions implicated in nociceptive process-
ing but did not reliably alter Fos protein expression in
ventral horn regions typically associated with motor
function. Notably, ZLc002 suppressed formalin-evoked
Fos protein expression in the superficial dorsal horn
(lamina I, II) and neck region (lamina V, VI) of the
dorsal horn but not in the nucleus proprius (lamina
14 Molecular Pain
III, IV) or ventral horn. The ZLc002-induced suppres-
sion of Fos protein expression was observed in the same
subjects that exhibited ZLc002-induced antinociception
in the formalin test. These observations are consistent
with the hypothesis that the suppression of
inflammation-evoked Fos protein expression induced
by antinociceptive doses of ZLc002 reflects a suppression
of nociceptive processing. Moreover, the pattern of
changes in both pain behavior and Fos protein expres-
sion was similar to those observed previously by
our group with PSD95-nNOS inhibitors IC87201
and ZL006.6
We previously demonstrated that the nNOS–
NOS1AP inhibitor TAT-GESV, but not the inactive
peptide TAT-GESVD1, reversed established neuropathic
pain due to paclitaxel treatment.11 Anti-allodynic effica-
cy of TAT-GESV (i.t.) was preserved following repeated
intrathecal injection of the peptide inhibitor.11 Here, we
show that ZLc002, administered systemically, reduces
the maintenance of paclitaxel-evoked mechanical and
cold allodynia. The anti-allodynic effects of ZLc002
were preserved for at least four days of repeated
dosing, although loss of anti-allodynic efficacy was
observed by day 8 of repeated dosing. More work is
necessary to determine whether differences in the route
of administration contributed to loss of efficacy (i.e. tol-
erance) observed with repeated systemic, but not intra-
thecal, administration. A compensatory mechanism in
NMDAR-PSD95-nNOS–NOS1AP-mediated nocicep-
tive signaling could be engaged following repeated sys-
temic dosing of the small molecule but not following
repeated intrathecal dosing with the peptide nNOS–
NOS1AP disruptor. Tolerance was not observed with
repeated intrathecal dosing of either TAT-GESV or
MK-801 in the same paclitaxel model of neuropathic
pain in our previous work.9,11 More work is necessary
to establish the site of action of systemically adminis-
tered small-molecule nNOS–NOS1AP disruptors and
determine whether mechanisms of anti-allodynic efficacy
and tolerance could differ at spinal and supraspi-
nal levels.
The anti-allodynic effects of ZLc002 observed herein
were selective for the pathological pain state; ZLc002 did
not alter responsiveness to either mechanical or cold
stimulation in control animals that received the
cremophor-based vehicle in lieu of paclitaxel. Our find-
ings reveal, for the first time, that a functional
small-molecule inhibitor of nNOS–NOS1AP interac-
tions produces anti-allodynic efficacy in rodent models
of both neuropathic and inflammatory pain.
ZLc002 failed to impede, and in fact, was synergistic
with paclitaxel in reducing tumor cell line viability, with-
out itself producing tumor cell cytotoxicity. Similar syn-
ergistic effects of ZLc002 with paclitaxel were observed
in both breast cancer (4T1) and ovarian (HeyA8) tumor
cell lines. The same conclusions were obtained using
Combenefit analysis applied to different classical syner-
gy models (i.e. the Bliss model, Loewe model, and HSA
model). The fact that identical conclusions were derived
from separate experiments employing distinct tumor cell
lines and three different synergy models further validate
our findings, although synergism appeared more robust
in the breast cancer 4T1 cell line. These observations
raise the possibility that nNOS–NOS1AP disruption
may be highly efficacious clinically for suppressing
chemotherapy-induced neuropathic pain in breast
cancer patients without impeding the anti-tumor efficacy
of paclitaxel. More work is necessary to evaluate wheth-
er ZLc002 could enhance the anti-cancer effects of pac-
litaxel in vivo and whether such effects translate to
different chemotherapeutic agents.
We recently reported that nNOS–NOS1AP interac-
tions are involved in pro-nociceptive signaling using
a peptide nNOS–NOS1AP disruptor.11 TAT-GESV,
administered i.t., attenuated mechanical and cold allody-
nia in two mechanistically distinct neuropathic pain
models (i.e. chemotherapy-induced peripheral neuropathy
produced by paclitaxel and traumatic nerve injury
induced by partial sciatic nerve ligation).11 TAT-GESV,
administered i.t., also reduced paclitaxel-evoked phos-
phorylation of p53 in the lumbar spinal cord, consistent
with a spinal site of anti-allodynic efficacy.11 Because p53
is a downstream substrate of proinflammatory p38
MAPK that is known to be activated upon nNOS–
NOS1AP association, it was used as a surrogate marker
of p38 activation.18 More work is necessary to determine
whether p38MAPK could also be activated by patholog-
ical pain and disrupted by ZLc002 at its site of action.
Elevated NMDAR activity contributes to central sen-
sitization.2 However, targeting NMDAR produces
unwanted side effects, such as motor and memory
impairment rendering NMDAR antagonism undesir-
able.3 Thus, it is noteworthy that ZLc002 does not pro-
duce motor impairment,14 consistent with similar
observations made by our group using the peptide
nNOS–NOS1AP disruptor TAT-GESV11 and
nNOS-PSD95 inhibitors IC87201 and ZL006.6–9,11
Novel strategies disrupting protein–protein interactions
downstream of NMDARs—including NR2B-PSD95,
PSD95-nNOS, and nNOS–NOS1AP interactions—thus
remain promising alternative approaches capable of sup-
pressing elevated NMDAR activity-mediated pro-
nociceptive signaling and allodynia without unwanted
on-target side effects of NMDAR antagonists.6–9,11
Further studies are required to determine whether ther-
apeutic strategies disrupting nNOS–NOS1AP interac-
tions are superior to those targeting NR2B-PSD95 or
PSD95-nNOS interactions in the quest to develop safe
and effective anti-hyperalgesic and anti-allodynic agents
for the treatment of pain.
Lee et al. 15
In conclusion, our findings collectively suggest that
the putative nNOS–NOS1AP small-molecule inhibitor,
ZLc002, disrupts nNOS–NOS1AP interactions in prima-
ry cortical neurons and in HEK293T cells transfected
with the full-length proteins but not in a cell-free
Alphascreen biochemical binding assay. ZLc002 sup-
presses neuropathic and inflammatory pain as well as a
neurochemical marker of inflammation-evoked neuronal
activation in pain processing regions of the spinal dorsal
horn. ZLc002 reduces paclitaxel-induced neuropathic
pain in vivo and synergized with paclitaxel to reduce
both breast and ovarian tumor cell line viability in
vitro. ZLc002 did not alter mechanical or cold sensitivity
in the absence of paclitaxel, suggesting that the nNOS–
NOS1AP disruptor reversed the sensitized responses to
cutaneous (mechanical and cold) stimulation in a
manner that was selective for the pathological pain
state. Future medicinal chemistry efforts are required
to identify small-molecule nNOS–NOS1AP inhibitors
that themselves disrupt nNOS–NOS1AP in a cell-free
system and exhibit better drug-like properties (i.e.
enhanced duration of action, lack of tolerance, and
show improved drug-like properties).
Author contributions
WL conducted the paclitaxel studies and drafted the ini-
tial manuscript. LLL conducted the immunoprecipitation
experiments and prepared the expression constructs.
LMC conducted the formalin and immunohistochemical
experiments. ZX conducted the AlphaScreen and tumor
cell viability assays. WL, LMC, LLL, ZX, and AGH
analyzed data. MJC and AGH designed the study.
AGH and MJC oversaw the project and wrote the man-
uscript with WL, LMC, ZX, LLL, and YYL.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of
interest with respect to the research, authorship, and/or publi-
cation of this article: YYL is partially employed at Anagin, Inc.
Funding
The author(s) disclosed receipt of the following financial sup-
port for the research, authorship, and/or publication of this
article: Supported by CA200417 and DA041229 and
DA042584 (to AGH) (to AGH and MJC). LMC is supported
by NIDA T32 training grant DA024628 and the Harlan
Scholars Research Program.
ORCID iD
Michael J Courtney http://orcid.org/0000-0001-8693-3933
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