Nerve growth factor inhibitor with novel-binding domain demonstrates nanomolar efficacy in both cell-based and cell-free assay systems

Article abstract of DOI:10.1002/prp2.339

Nerve growth factor (NGF), a member of the neurotrophin family, is known to regulate the development and survival of a select population of neurons through the binding and activation of the TrkA receptor. Elevated levels of NGF have been associated with painful pathologies such as diabetic neuropathy and fibromyalgia. However, completely inhibiting the NGF signal could hold significant side effects, such as those observed in a genetic condition called congenital insensitivity to pain and anhidrosis (CIPA). Previous methods of screening for NGF-inhibitors used labeling techniques which have the potential to alter molecular interactions. SPR spectroscopy and NGF-dependent cellular assays were utilized to identify a novel NGF-inhibitor, BVNP-0197 (IC₅₀?=?90?nmol/L), the first NGF-inhibitor described with a high nanomolar NGF inhibition efficiency. The present study utilizes molecular modeling flexible docking to identify a novel binding domain in the loop II/IV cleft of NGF.

Full text of DOI:10.1002/prp2.339

ORIGINAL ARTICLE
Nerve growth factor inhibitor with novel-binding domain
demonstrates nanomolar efficacy in both cell-based and
cell-free assay systems
Allison E. Kennedy1,2 , Corey A. Laamanen1,3, Mitchell S. Ross1, Rahul Vohra1,4
,
Douglas R. Boreham1, John A. Scott1,3 & Gregory M. Ross1
1Northern Ontario School of Medicine, Sudbury, Ontario, Canada
2Laurentian University, Biomolecular Sciences Program, Sudbury, Ontario, Canada
3Laurentian University, Bharti School of Engineering, Sudbury, Ontario, Canada
4Sussex Research Laboratories Inc., Ottawa, Ontario, Canada
Abstract
Keywords
NGF, pain, small molecule, SPR, TrkA
Nerve growth factor (NGF), a member of the neurotrophin family, is known to
regulate the development and survival of a select population of neurons
through the binding and activation of the TrkA receptor. Elevated levels of
NGF have been associated with painful pathologies such as diabetic neuropathy
and fibromyalgia. However, completely inhibiting the NGF signal could hold
significant side effects, such as those observed in a genetic condition called con-
genital insensitivity to pain and anhidrosis (CIPA). Previous methods of screen-
ing for NGF-inhibitors used labeling techniques which have the potential to
alter molecular interactions. SPR spectroscopy and NGF-dependent cellular
Correspondence
Gregory M. Ross: Northern Ontario School of
Medicine, Sudbury ON, Canada, P3E 2C6.
Tel: 705 662 7218; Fax: 705 675 4858;
E-mail: gross@nosm.ca
Received: 23 January 2017; Revised: 30
March 2017; Accepted: 20 April 2017
assays were utilized to identify
a
novel NGF-inhibitor, BVNP-0197
Pharma Res Per, 5(5), 2017, e00339
https://doi.org/10.1002/prp2.339
(IC50 = 90 nmol/L), the first NGF-inhibitor described with a high nanomolar
NGF inhibition efficiency. The present study utilizes molecular modeling flexi-
ble docking to identify a novel binding domain in the loop II/IV cleft of NGF.
doi: 10.1002/prp2.339
Abbreviations
ATCC, American type culture collection; FBS, fetal bovine serum; NGF, nerve
growth factor; NGF, Nerve growth factor; SPR, surface plasmon resonance; TLC,
thin layer chromatograph; TNF, anti-tumor necrosis factor.
et al. 1994; Zahn et al. 2004). Subcutaneous injection of
NGF into the forearm of healthy human adults induced
Introduction
Elevated levels of nerve growth factor (NGF) have been
implicated in several chronic pain syndromes such as
osteoarthritis (Kc et al. 2016) and diabetic neuropathy
(Malerba et al. 2015). Experimental evidence has shown
that NGF is released by several cell types including mast
cells (Bienenstock et al. 1987; Skaper et al. 2001), lym-
phocytes (Torcia et al. 1996), and monocytes/macro-
phages (Bracci-Laudiero et al. 2005) in response to tissue
inflammation. Interestingly, NGF has also been found to
produce hyperalgesia when administered in several animal
species (Brodie 1995; Hao et al. 2000; Cahill et al. 2003).
These pain-related behavioral responses to NGF in ani-
mals manifest within minutes, and can last anywhere
from several hours to days depending on the dose (Lewin
localized allodynia and hypersensitivity within minutes,
lasting for several hours (Dyck et al. 1997). In addition,
small intravenous NGF doses in healthy human adults are
responsible for widespread deep pain and tenderness
which persists for several days (Svensson et al. 2003). The
evidence of upregulated NGF in painful pathological con-
ditions, in addition to the evidence that NGF causes pain
in humans and in animals, have led to the rational for
developing therapeutics based on the inhibition of NGF
activity.
A growing body of evidence suggests that an anti-
hyperalgesia effect can be observed with pharmacological
interference of NGF–TrkA interactions in several neuro-
pathic pain models (Beglova et al. 2000; Hefti et al. 2006;
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
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This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited and
is not used for commercial purposes.
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NGF Inhibitor with Nanomolar Efficiency
A. E. Kennedy et al.
Wild et al. 2007). Monoclonal anti-NGF antibodies, such
as Tanezumab, have been used as NGF sequestering ther-
apy. Tanezumab binds to NGF with high selectivity thus
blocking NGF–TrkA interactions and inhibiting the sig-
naling of sensory neurons for the perception of pain
(Schnitzer et al. 2011). Despite the early clinical success
seen by Tanezumab, a clinical hold was placed on the
drug during Phase III trials when several individuals
developed joint damage, which progressed to a stage
where joint replacement was necessary. Even with the
apparent successes in the antibody therapeutics, there are
still potential drawbacks such as delivery challenges,
potential for autoimmune responses, capacity for produc-
tion and financial considerations (Samaranayake et al.
2009). Therefore, the generation of small molecule
antagonists which have the ability to selectively disrupt
NGF–TrkA interactions may have significant therapeutic
advantage.
novel small molecule analogs with specificity for NGF
that inhibit binding to TrkA. Our theoretical flexible
docking experiments revealed a novel-binding domain
in the loop II/IV cleft of NGF where a series of analogs
bind to inhibit NGF signaling. SPR spectroscopy was
also utilized to characterize novel small molecule inter-
actions with NGF and their inhibitory effect on TrkA
interactions. Analogs also demonstrated efficient inhibi-
tory activity of NGF in vitro, using an NGF-dependent
PC12 assay. Guided by receptor docking, we were able
to identify BVNP-0197 as a small molecule capable of
inhibiting NGF in vitro with greater potency than pre-
viously reported NGF inhibitors. The therapeutic poten-
tial of previously reported compounds in modulating
NGF signaling is still quite low as they bind to NGF
with micromolar affinity. The increase in inhibitory
potential and target binding affinity described by
BVNP-0197 to NGF could possibly translate into a
therapeutic option with reduced dosing and prolonged
biological effects compared to other small molecules
described.
A series of novel nonpeptidic small molecules have
been demonstrated to inhibit binding of NGF to TrkA.
Compounds such as ALE-0540 (Owolabi et al. 1999), PD
90780 (Colquhoun et al. 2004), Ro 08-2750 and (Nieder-
hauser et al. 2000) have been shown to inhibit NGF-TrkA
signal transduction pathways in vitro. However, the
mechanisms by which these described small molecules
exert their inhibitory effect remains speculative (Eibl et al.
2012). Historically, the identification of small molecule
NGF-inhibitors resulted from high-throughput receptor-
binding assays. However, recent advances in the
understanding of the structural biology of NGF–TrkA
interactions have allowed for rational development of
novel small molecules. PQC 083 is one example of a small
molecule inhibitor that was developed to target a specific
region on NGF to alter TrkA binding (Eibl et al. 2013a).
With newly identified crystal structures explaining the
interactions during NGF-TrkA binding (Wehrman et al.
2007), small molecules have been developed to alter the
molecular topology of NGF to inhibit TrkA binding.
Determining how potential therapeutic drugs modulate
analyte–ligand interactions and bind to target molecules will
help determine strategies for developing future therapeutics.
One such technique for investigating the strength and rate of
biomolecular interactions is surface plasmon resonance
(SPR) spectroscopy (Cooper 2002). SPR is advantageous
over other techniques because it monitors biomolecular
interactions in real time and is label-free, eliminating the
need for fluorescent reporter molecules or radioisotope tags
(Mir and Shinohara 2013). Not only is this advantageous in
saving time during labeling and reducing resources, but
more importantly it eliminates tags which can alter the
molecular interactions (Fraser et al. 2014).
Materials and Methods
Molecular modeling
Molecular modeling and in silico docking of bivalent
naphthalimide compounds to NGF (RCSB Protein Data
Bank ID 1BET) (McDonald et al. 1991) were completed
using Sybyl-X 2.1.1 software. The structure of NGF was
prepared for docking, using the Biopolymer suite of
Sybyl-X 2.1.1. Co-structures were deleted, hydrogens were
added and the appropriate formal charges on the C- and
N- termini were added. The structure was optimized
using the MMFF94 molecular mechanical force field.
Flexible docking of each compound was performed, using
the Surflex-Doc suite incorporated into Sybyl-X 2.1.1
(Jain 1996). The docking protomol was generated to
include the residues of the loop II/IV cleft (Glu41-Phe49
and Gln96-Trp99) with a bloat factor of 0 and a thresh-
old value of 0.5. For the Surflex-Doc function, the ang-
stroms to expand the search grid was set at 6 and the
maximum confirmations per fragment was set to 20.
Docking to proNGF (RCSB Protein Data Bank ID 3IJ2)
(Feng et al. 2010) was completed as described above with
a docking protomol generated with residues (Gly33-Cys39
and Gln90-Cys100) at the loop II/IV cleft.
SPR spectroscopy
Mouse NGF (Cedarlane, Burlington; Canada) was coupled
using the amine coupling method. Sensor Chip CM5 was
activated using 0.4 mol/L EDC/0.1 mol/L NHS at a flow
In the present study, we use a combination of
molecular modeling and SPR to identify a series of
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A. E. Kennedy et al.
NGF Inhibitor with Nanomolar Efficiency
rate of 10 lL/min for 7 min. NGF was dissolved in a
HBS-EP buffer, pH 4.5, to yield a 10 lg/mL solution. Fol-
lowing activation, the NGF solution was injected over the
activated sensor chip surface at a flow rate of 10 lL/min
for 9 min. After the injection, the surface was washed
with 1 mol/L NaCl to remove any uncoupled or noncova-
lently bound material from the surface. The excess
hydroxysuccinimidyl groups on the surface were deacti-
vated with 1M ethanolamine hydrochloride, pH 8.5, at a
flow rate of 10 lL/min for 7 min. The surface of a refer-
ence flow cell was activated with 0.4 mol/L EDC/0.1 mol/
L NHS and then deactivated with 1 mol/L ethanolamine,
with respective flow rates and times.
reaction was refluxed for 10 h and then cooled to room
temperature. The solid obtained was filtered and washed
with distilled water or dilute acid. It was reprecipitated
and/or recrystalized from the appropriate solvents. The
solid final product C/D was dried under a vacuum
(Fig. 1).
Compound sample preparation for SPR
analysis
Each compound of interest was weighed on an analytical
balance and dissolved in the appropriate amount of
DMSO to give a 10 mmol/L solution. The samples were
diluted with running buffer (0.01 mol/L HEPES,
0.15 mol/L NaCl, 3 mmol/L EDTA and 0.05% v/v Tween
20; pH 7.5) to yield solutions for the binding experiments
with concentrations varying from 200 lmol/L to
0.7 lmol/L and for IC50-binding experiments with con-
centrations varying from 3.16 nmol/L to 316 lmol/L.
Bivalent naphthalimide chemical synthesis
All chemicals and solvents were obtained from commer-
cial sources without further purification. Mass spectra
were recorded using Waters (Micromass ZQ) equipped
with a Thermo Betasil C18 (150 9 2.1 mm, 3l). 1H
NMR spectra were recorded on a Varian AS500 instru-
ment. Chromatography on silica gel columns were per-
formed, using Merck silica gel 60 (230-400 mesh), and
analytical thin layer chromatography (TLC) was con-
ducted on a glass plate coated with a 0.25 cm thin layer
Analog-binding affinity to immobilized NGF
Binding affinities of the bivalent naphthalimide analogs to
immobilized NGF were determined as previously
described, using a Biacore T200 SPR (Kennedy et al.
2016). Prior to the analyte injection, the series S CM5
chip was conditioned with three 30 sec cycles of running
buffer (0.01 mol/L HEPES, 0.15 mol/L NaCl, 3 mmol/L
EDTA and 0.05% v/v Tween 20; pH 7.5) followed by
three start up cycles, allowing the response to stabilize
before the analyte injection. Data were collected at a tem-
perature of 25 ᵒC. Individual compound samples were
tested from the lowest to the highest concentrations, sepa-
rated by a 15 sec stabilization period after each sample in
each compound series. During each sample cycle, the ana-
lyte was injected for 60 sec at a flow rate of 30 lL/min. A
dissociation period was monitored for 30 sec after analyte
injection before regeneration was induced with 1.0 mol/L
NaCl for 120 sec at a flow rate of 30 lL/min to wash any
remaining analyte from the sensor chip before running
the next sample.
of silica gel 60F254
.
General procedure for naphthalimide
derivatives
Under a nitrogen atmosphere, the primary amine (1
equiv) and sodium acetate (1 equiv) were added to a stir-
red solution of 1,4,5,8-naphthalene tetracarboxylic dian-
hydride (1 equiv) in glacial acetic acid. The reaction
mixture was refluxed and the progress of the reaction was
monitored by TLC. Upon completion of the reaction if
the resulting solution was clear, the solvent was evapo-
rated under reduced pressure and the residual solid was
reprecipitated and/or recrystalized from appropriate sol-
vent(s). If the reaction mixture contained precipitate then
the mixture was cooled to room temperature and the
solid was removed by filtration. The filtrate was concen-
trated and then diluted with water to precipitate the
crude intermediate product. The crude product was dis-
solved in ethanol and heated to reflux for 10 min, cooled
to room temperature, and filtered to remove any solid.
The filtrate was concentrated under reduced pressure to
get intermediate product A/B, which was dried in vacuum
for 24 h and used for the next steps (Fig. 1). Under a
nitrogen atmosphere, the appropriate amine (1 equiv)
and sodium acetate (1 equiv) or substituted diaminoben-
zene (1 equiv) was added to a stirred solution of interme-
diate compound A (1 equiv) in glacial acetic acid. The
PC12 cell culture
PC12 cells were obtained from the American Type Cul-
ture Collection (ATCC; USA). Cell passage 18–21 were
used for all experiments. PC12 cells were maintained in
Dulbecco’s Modified Eagles Medium supplemented with
10% fetal bovine serum (FBS) (Gibco; USA). Media was
supplemented with working stock concentration of peni-
cillin (0.1 U/mL) and streptomycin (0.1 U/mL). PC12
cells were seeded at 50% confluence and were passaged at
90% confluence in T25 cm2 tissue culture-treated flasks
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2017 | Vol. 5 | Iss. 5 | e00339
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NGF Inhibitor with Nanomolar Efficiency
A. E. Kennedy et al.
Figure 1. Synthesis of bivalent naphthalimide analog series. Final products (C/D) were screened for NGF inhibitory properties.
(Corning; USA). PC12 cells were incubated at 37°C in
5% CO2.
phase observation. Differentiation events were manually
scored when a cell developed a process with a constant
calibre from the origin to the terminal and its length was
equal or greater than 1 x cell body diameter. Wells with
fewer than 50 cells or greater than 350 cells were excluded
from the analysis. Three trials of each experiment were
performed, and each condition was tested in quadrupli-
cate.
PC12 toxicity assay
PC12 toxicity assay was performed by plating cells at a
density of 200 cells/well on Terasaki plates (Greiner Bio-
One; USA) at 37°C and 5% CO2. Cells were exposed to
100 lmol/L of compound of interest in the Terasaki well
for 72 h in the absence of NGF. Cells on the lower hori-
zontal surface of the well were imaged using an EVOS XL
Core cell imaging system at 20 9 phase observation. The
total cells per well were counted and compared to con-
trol. Three runs of each experiment were performed, and
each condition was tested in quadruplicate.
TrkA phosphorylation and western blotting
analysis
TrkA phosphorylation was conducted by the modification
of methods previously described (Ross et al. 1998). NGF
(50 pM) was incubated with the analog series compound
(10 to 1 lmol/L) for 1 h in HKR buffer. 1x106 cells/mL
PC12 cells suspended in HKR buffer were treated with
the NGF-analog complex for 15 min at 37°C with occa-
sional agitation. Following the incubation, an immuno-
precipitation was performed, using the Pierce Classic IP
Kit (Thermo, USA). The cells were lysed and exposed to
Protease Inhibitor Cocktail (Pierce, USA). Trk-antibody
(Cell Signaling, USA) was then incubated with the cell
lysate at 4°C overnight. Protein A/G coated agarose
matrix was used to precipitate the immune complex
which was then separated by electrophoresis, using an 8%
SDS-PAGE gel. A western blot was performed to transfer
the immune complex to PVDF membrane. The
PC12 NGF-dependent neurite outgrowth
assay
PC12 cells were plated 24 h prior to treatment at a den-
sity of 200 cells/well and were grown on Terasaki plates
(Greiner Bio-One; USA) at 37°C and 5% CO2. PC12 cells
were exposed to 500 pM of NGF (Life Technologies;
Canada) preincubated with varying concentrations of each
compound of interest (31.6 nmol/L – 100 lmol/L). Tera-
saki wells were imaged 72 h after exposure to NGF. Cells
on the lower horizontal surface of the well were imaged
using, an EVOS XL Core cell imaging system at 20 x
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A. E. Kennedy et al.
NGF Inhibitor with Nanomolar Efficiency
membrane was blocked in 5% skim milk TBS-T and
probed with phosphotyrosine primary antibody 4G10
(Millipore, USA) followed by the secondary HRP-linked
Goat Anti-Mouse (Santa Cruz Biotechnology, USA) anti-
body before being visualized by autoradiography.
targeting domain included NGF monomer residues
Glu41, Val42, Asn43, Ile44, Asn45, Asn46, Ser47, Val48,
Phe49, Gln96, Ala97, Ala98, and Trp99. Fig. 2 demon-
strates the protomol which was derived from the
described residues of NGF.
The results of the molecular modeling flexible dock-
ing score for each bivalent naphthalimide analog was
measured. The scoring function predicts the binding
affinity of analogs to NGF by counting the number of
interactions between entities (hydrophobic, hydrophilic,
hydrogen bonds and rotatable bonds in the complex).
The scoring output is represented in units of –log(KD).
The results of compound BVNP-0187 were chosen as a
representative bivalent naphthalimide scaffold analog
following flexible docking at the loop II/IV cleft of
NGF (Fig. 3). BVNP-0187, like previously reported
small molecule NGF inhibitors, has a ridged backbone
structure comprised of a bivalent naphthalimide motif
IC50 determination using SPR spectroscopy
TrkA was immobilized to a CM5 sensor surface as pre-
viously described (Forsell et al. 2013). Prior to analyte
injection, the Series S CM5 chip was conditioned with
three 30 sec cycles of running buffer (0.01 mol/L
HEPES, 0.15 mol/L NaCl, 3 mmol/L EDTA and 0.05%
v/v Tween 20; pH 7.5) followed by three start up
cycles, allowing the response to stabilize before analyte
injection. Data were collected at a temperature of 25°C.
Individual compound samples were tested from lowest
to highest concentrations, separated by a 15 sec stabi-
lization period after each sample in each compound
series. During each sample cycle, the analyte was
injected for 60 sec at a flow rate of 30 lL/min. A dis-
sociation period was monitored for 120 sec after the
(IUPAC
hexadeca-1,3,8,10,15-pentaene-5,7,12,14-tetrone moiety)
containing butanoic acid residue in –R1 and
name:
6,13-diazatetracyclo[6.6.2.04,16.011,15]
a
2-hydroxy-5-benzoic acid residue occupying –R2
(Table 1). Flexible docking determined that BVNP-0187
had the highest theoretical docking score of all analogs
screened. Flexible docking suggested that four hydrogen
bonds are formed between BVNP-0187 and NGF resi-
dues Asn43, Asn45, and Trp99 to stabilize within the
loop II/IV cleft and measured differences of electrostatic
potential were found at the BVNP-0187 docking
domain (Fig. 3).
analyte injection. Regeneration occurred with
a
10 mmol/L Glycine solution at pH 2.0 for 15 sec at a
flow rate of 30 lL/min to wash any remaining analyte
from the sensor chip before running the next sample.
Statistical analysis
Statistical analysis was performed, using Prism Graphpad
5.0 (San Diego; USA). Error bars are expressed as stan-
dard error of the mean with a sample size of n = 3. Inhi-
bition curves were fit using a nonlinear regression model.
Affinity of bivalent naphthalimide
compounds to immobilized NGF
The affinity of bivalent naphthalimide analogs with a
total score from flexible docking >3 (~mmol/L binding
affinity) was determined using a two-site steady-state
analysis to immobilized NGF. A concentration series
ranging from 0.70 lmol/L to 200 lmol/L was injected
over immobilized NGF for a 60 sec contact time fol-
lowed by a 30 sec dissociation phase. Concentration val-
ues were determined based on results from preliminary
binding experiments in which bivalent naphthalimide
compounds reached near-saturation levels of the immo-
bilized NGF on the sensor surface. The response
obtained from each bivalent naphthalimide sample was
plotted against concentration using the BIA evaluation
software (Version 2.0) and was evaluated using a two-
site binding model as previously described (Kennedy
et al. 2016). 17 bivalent naphthalimide analogs were
identified as specific binders of NGF (Fig. 4) with bind-
ing affinities ranging from 2.63 lmol/L to 118.10 lmol/
L. The 17 analogs showed no significant binding to
Results
Molecular modeling flexible docking
Previous studies have suggested that loop I, II, and IV of
each NGF monomer are responsible for binding to TrkA
(Barker 2007; Wehrman et al. 2007), whereas loop I and
IV are necessary for NGF binding to p75NTR (He and
Garcia 2004; Barker 2007) (Fig. 2). Previous drug discov-
ery efforts have been focused on a putative binding
domain for small molecule NGF inhibitors in the loop I/
IV cleft of the NGF monomer (Colquhoun et al. 2004;
Eibl et al. 2013a). Our flexible docking identified a novel-
binding domain located in the loop II/IV cleft of NGF for
a series of bivalent naphthalimide compounds which were
derived using similar molecular features to previously
reported small molecule NGF inhibitors. This novel-bind-
ing domain demonstrated higher binding capabilities than
previously suggested docking areas. The pharmacological
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2017 | Vol. 5 | Iss. 5 | e00339
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NGF Inhibitor with Nanomolar Efficiency
A. E. Kennedy et al.
Figure 2. (A)The dimeric structure of NGF (RCSB: 1BET) (McDonald et al. 1991) is presented in ribbon format. NGF monomers are colored blue
and yellow. Structural features of the yellow NGF monomer are labeled. Orange highlights represent structural features of the blue NGF
monomer which are responsible for TrkA binding. Green structural features highlighted represent areas responsible for p75NTR binding of the
yellow NGF monomer. (B) Theoretical docking experiments identified a docking site for bivalent naphthalimide scaffold at loop II/IV domain of
NGF. NGF is represented as a ribbon structure (RCSB: 1BET) (McDonald et al. 1991). Resides Glu41, Val42, Asn43, Ile44, Asn45, Asn46, Ser47,
Val48, Phe49, Gln96, Ala97, Ala98, and Trp99 were selected to create a protomol binding domain for bivalent naphthalimide analogs
represented in green.
Figure 3. Schematic representation of BVNP-0187 docked to NGF as determined by molecular modeling. Flexible theoretical docking experiments
predict that the bivalent naphthalimide scaffold is suited to bind at the loop II/IV cleft of NGF. (A) Hydrogen bonds are represented at residues
Asn43, Asn45, and Trp99 of NGF to stabilize BVNP-0187 within the loop II/IV cleft. (B) Differences in electrostatic potential at the docking
domain. Blue areas represent electron-poor regions where as a gradient to red represents electron-rich regions.
immobilized TrkA, nor to other neurotrophin family
Inhibitory effects of bivalent naphthalimide
members immobilized on the sensory surface, therefore
analogs on NGF-dependent PC12 cell
described specific for NGF (data not shown). Structures
differentiation
of these analogs are summarized in Table 1. Three previ-
ously reported compounds, ALE-0540, PD 90780 and Ro
08-2750 were also screened to determine the binding
affinities (KD) for NGF. The KD of ALE-0540, PD 90780
and Ro 08-2750 were determined to be 49.71 lmol/L,
35.83 lmol/L and 32.39 lmol/L, respectively, and none
showed binding to immobilized TrkA. The total binding
score measured during flexible docking was related to
the binding affinity measured with SPR binding (Fig. 5),
suggesting binding to immobilized NGF is occurring
similar to theoretical docking experiments.
In order to assess the toxicity of the bivalent naphthalim-
ide analogs in vitro, PC12 cells were exposed to
100 lmol/L of each compound for 72 h in the absence of
NGF. The cell count did not significantly alter from the
control conditions (Fig. 6) and the morphology of PC12
cells was also unaltered following the 72 h exposure.
The inhibitory effects of the 17 bivalent naphthalimide
analogs which were found to be specific NGF binders were
tested in the NGF-dependent PC12 cell differentiation
assay along with ALE-0540, PD 90780 and Ro 08-2750
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A. E. Kennedy et al.
NGF Inhibitor with Nanomolar Efficiency
Table 1. Residue description of bivalent naphthalimide analogs.
Compound
-R1
-R2
-R3
-R4
BVNP-0182
BVNP-0183
(CH2)3CO2H
(CH2)3CO2H
CO2H
CO2H
Cl
Cl
Cl
Cl
CO2H
Cl
CO2H
Cl
BVNP-0184
BVNP-0185
OH
OH
CO2H
CO2H
CO2H
CO2H
BVNP-0186
Br
Br
CO2H
BVNP-0187
BVNP-0188
BVNP-0189
(CH2)3CO2H
HO
Cl
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
BVNP-0190
BVNP-0191
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
H2N
H2N
BVNP-0192
BVNP-0193
O
C
O
CO2H
CO2H
Cl
BVNP-0194
(CH2)3CO2H
CO2H
(Continued)
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NGF Inhibitor with Nanomolar Efficiency
A. E. Kennedy et al.
Table 1. Continued.
Compound
BVNP-0195
-R1
-R2
-R3
-R4
CO2H
CO2H
CO2H
BVNP-0196
BVNP-0197
BVNP-0198
Cl
Cl
Cl
Cl
CO2H
CO2H
NO2
CO2Me
Analogs were identified as specific binders of NGF using SPR analysis, and were shown to exhibit inhibitory properties in NGF-TrkA-mediated sig-
naling. Analog series were synthesized around a bivalent naphthalimide scaffold with varied side chains substitutions at –R1, and –R2 or –R3 and
–R4 (Fig. 1).
(Table 2). The three previously reported compounds
demonstrated IC50 values of 2.44 lmol/L, 33.22 lmol/L,
and 11.96 lmol/L, respectively (Table 2). Of the 17 biva-
lent naphthalimide analogs, three compounds demon-
strated inhibitory effects more efficient than previously
NGF residues Asn43, Thr92 and Trp99 to stabilize BVNP-
0197 within the loop II/IV cleft, as well as measured elec-
trostatic potential differences at the docking domain
(Fig. 8). A total binding score of 5.5898 was measured for
BVNP-0197 docking to the loop II/IV cleft, estimating a
binding affinity of low micromolar range to NGF. Dock-
ing of BVNP-0197 to proNGF measured a binding score
of 3.1542, estimating a binding affinity of millimolar
range. This change in binding affinity is marked by a
decrease in hydrophobic contacts and a reduction in
hydrogen bonds between BVNP-0197 and proNGF as
compared to NGF.
reported NGF-inhibitors, which we defined as an IC50
<
2 lmol/L. BVNP-0188 and BVNP-0198 demonstrated IC50
values of 1.70 lmol/L and 1.08 lmol/L, respectively. The
most efficient analog in NGF-dependent PC12 cell differ-
entiation inhibition was BVNP-0197 which demonstrated
an IC50 value of 89.5 nmol/L (Fig. 7). BVNP-0197 has a
rigid backbone structure (IPUAC name: 3,10,17-Triaza-
hexacyclo[13.6.2.02,10.04,9.012,22.019,23]tricosa-1
To test the inhibitory effect of BVNP-0197 on NGF-
mediated signaling, the phosphorylation status of TrkA
was assessed when PC12 cells were incubated with control
media, NGF or NGF/BVNP-0197. Cells were treated with
their respective conditions for 15 min prior to cell lysis.
(21),2,4,6,12,14,19,22-nonaene-11,16,18-trione
moiety)
and contains a 2-chloro-5-benzoic acid residue in –R3 and
nitro group occupying –R4. Flexible docking molecular
modeling experiments predicted four hydrogen bonds at
Figure 4. SPR subtracted sensograms and specific binding curves for the 17 bivalent naphthalimide analog found to be specific binders of NGF,
as well as three previously reported compounds – ALE-0540, PD 90780 and Ro 08-2750 (1–3, respectively). 4–20 represent BVNP-0182-BVNP-
0198, respectively. Data points represent the mean of triplicate measure. Error bars represent the standard error of the mean with an n = 3. (A)
Blank subtracted sensograms which describe the association and dissociation of each compound. In each sensogram, the x-axis represents time.
0–60 sec represents the association of the compound to NGF; at 60 sec the analyte flow stops and a dissociation period is represented from 60
to 90 sec prior to chip regeneration at 90 sec. Y axis represents the response of binding in response units (RU). 1 RU = 1 pg/mm2. The lines
represent small molecule concentrations, from top to bottom: 200 lmol/L, 100 lmol/L, 50 lmol/L, 25 lmol/L, 12.5 lmol/L, 6.25 lmol/L,
3.13 lmol/L, 1.56 lmol/L and 0.78 lmol/L respectively. (B) Two-site steady-state binding models describing the specific binding of each
compound to NGF. X axis represents concentration (lmol/L) and Y axis represents subtracted response (response from active flow cell minus
response from reference flow cell) (RU). Each data point represents the subtracted response from the saturated association phase (last 10 sec) of
the subtracted sensograms from each compound concentration (refer to A). Binding affinity (KD) for each compound was calculated using the BIA
evaluation software using a 2:1 binding model.
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British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

A. E. Kennedy et al.
NGF Inhibitor with Nanomolar Efficiency
ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2017 | Vol. 5 | Iss. 5 | e00339
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NGF Inhibitor with Nanomolar Efficiency
A. E. Kennedy et al.
Table 2. Inhibitory effects of novel and previously reported com-
pounds.
Compound
IC50 PC12 neurite outgrowth
ALE-0540
2.44 lmol/L
33.22 lmol/L
11.96 lmol/L
>100 lmol/L
16.86 lmol/L
>100 lmol/L
11.08 lmol/L
15.18 lmol/L
5.45 lmol/L
1.70 lmol/L
>100 lmol/L
4.27 lmol/L
4.33 lmol/L
2.46 lmol/L
7.09 lmol/L
5.35 lmol/L
6.50 lmol/L
5.37 lmol/L
0.089 lmol/L
1.08 lmol/L
PD 90780
Ro 08-2750
BVNP-0182
BVNP-0183
BVNP-0184
BVNP-0185
BVNP-0186
BVNP-0187
BVNP-0188
BVNP-0189
BVNP-0190
BVNP-0191
BVNP-0192
BVNP-0193
BVNP-0194
BVNP-0195
BVNP-0196
BVNP-0197
BVNP-0198
Figure 5. The relationship between total score calculated during
molecular modeling flexible docking and the KD measured by SPR
spectroscopy for the 17 bivalent naphthalimide analogs and three
previously reported compounds (ALE-0540, PD 90780 and Ro 08-
2750). X-axis: KD (lmol/L) measured using SPR
– Data points
represent the mean of triplicate trials. A concentration range (0.70–
200 lmol/L) of each analog was injected over immobilized NGF. Y-
axis: total score (Àlog(KD)) measured during flexible docking. (F
(1,18) = 5.807; P = 0.0269; R2 = 0.2439).
A PC12 cell-based neurite outgrowth assay was used to score the rel-
ative inhibitory effects of each bivalent naphthalimide analog and
three previously reported compounds (ALE-0540, PD 90780 and Ro
08-2750) in the presence of 500 pM NGF for 72 h. PC12 cells were
treated with a concentration range from 100 lmol/L to 31.6 nmol/L.
Each trial condition was completed in quadruplicate wells, replicated
with an n = 3. Bold values represent analogues with a higher inhibi-
tory property than previously reported compounds.
in vitro and may be targeted for further therapeutic
development.
Inhibitory effects of bivalent naphthalimide
analogs
To determine the inhibitory properties of the 17 bivalent
naphthalimide analogs using SPR spectroscopy, a dilution
series of each analog (3.16 nmol/L to 316.23 lmol/L) was
incubated for 1 h with 10 nmol/L NGF prior to a 60 sec
injection over immobilized TrkA on the sensor surface.
Each injection over immobilized TrkA was followed by a
120 sec dissociation phase prior to regeneration before
the next injection. A response for each sample was
assessed using SPR spectroscopy and compared to a con-
trol sample of 10 nmol/L NGF with no analogpresent.
Dose response curves were generated to determine IC50
values using the BIA evaluation software (Version 2.0)
and were evaluated, using a one-site steady-state-binding
model. The 17 analogs demonstrated IC50 values ranging
from 18.5 nmol/L to 81.40 lmol/L, which were related to
the IC50 values calculated from neurite outgrowth assays
(Fig. 9).
Figure 6. In vitro toxicity of bivalent naphthalimide analog series.
PC12 cells were incubated in the absence (control) or presence of
100 lmol/L of each analog for 72 h. The average control cell count
per well was considered 100% viability. Each analog-treated sample is
represented by the average percentage of the control cell count.
Standard error of the mean count is shown with error bars (n = 3).
Treatment of PC12 cells with the analogs shown no significant
difference in cell count (P > 0.05) implicating no toxic effect of the
analogs in vitro.
The phosphorylated TrkA signal for NGF-treated cells
was distinctly increased over media control-treated cells
(Fig. 7). This increase in response was diminished by
both 10 lmol/L and 1 lmol/L BVNP-0197 incubated
with NGF for 1 h at 37°C (Fig. 7). These results confirm
that BVNP-0197 inhibits NGF-mediated TrkA signaling
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ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

A. E. Kennedy et al.
NGF Inhibitor with Nanomolar Efficiency
Figure 7. BVNP-0197 demonstrated inhibitory effects in the nanomolar range in the PC12 differentiation assay. (A) Detailed dose–response
experiments were conducted for all 17 bivalent naphthalimide compounds and three previously reported compounds. Previously reported
compounds ALE-0540, PD 90780 and Ro 08-2750 were determined to have IC50 values of 2.44 lmol/L, 33.22 lmol/L and 11.96 lmol/L,
respectively. Three bivalent naphthalimide compounds were shown to have inhibitory effects more efficient than the previously reported
compounds. BVNP-0188, and BVNP-0198 were determined to have IC50 values of 1.70 lmol/L and 1.08 lmol/L, respectively, whereas BVNP-0197
was found to have an IC50 value of 89.5 nmol/L. Previously reported compounds are depicted in red where novel compounds are in blue. Data
points represent a mean value of quadruplicate results with an n = 3. Error bars are represented as standard error of the mean with an n = 3. (B)
The chemical structure of BVNP-0197. IUPAC name 4-Chloro-3-{6-nitro-11,16,18-trioxo-3,10,17-triazahexacyclo[13.6.2.02,10.04,9.012 22.019 23
, , ]
tricosa-1(21),2,4,6,8,12,14,19,22-nonaen-17-yl}benzoic acid. (C) BVNP-0197 functionally inhibits NGF-mediated PC12 cell process formation. In
the presence of 500 pM NGF, PC12 cells were treated with varying concentrations of BVNP-0197 (100– 0 lmol/L). Images from 0, 1 and
100 lmol/L are shown. In the presence of NGF and the absence of inhibitor, PC12 cells underwent cell process formation (arrows). Process
formation significantly decreased with the addition of increasing concentrations of BVNP-0197. An unstimulated (0 pM NGF) sample was
prepared to demonstrate the NGF-dependence in cell process formation. (D) BVNP-0197 inhibits TrkA-mediated phosphorylation in PC12 cells
exposed to 50 pM NGF for 1 h at 37°C.
Figure 8. Schematic representation of BVNP-0197 docked to NGF as determined by molecular modeling. Flexible theoretical docking experiments
predict that the bivalent naphthalimide scaffold is suited to bind at the loop II/IV cleft of NGF. (A) Four hydrogen bonds are represented at
residues Asn43, Thr92 and Trp99 of NGF to stabilize BVNP-1097 within the loop II/IV cleft. (B) The differences in electrostatic potential at the
docking domain. Blue areas represent electron-poor regions where a gradient to red represents electron-rich regions.
neurite outgrowth and TrkA phosphorylation assays.
BVNP-0197 is also predicted to bind to NGF in a novel
binding domain found in the loop II/IV cleft.
Discussion
In the present study, we identified a novel bivalent naph-
thalimide compound, BVNP-0197, with a higher NGF-
inhibitory efficiency than previously described compounds
utilizing cell-free SPR technology and cell-based PC12
Historically, most drug discovery efforts in NGF-
mediated pain signaling therapeutics have been directed
toward identifying small molecules which inhibit signaling
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British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
2017 | Vol. 5 | Iss. 5 | e00339
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NGF Inhibitor with Nanomolar Efficiency
A. E. Kennedy et al.
through the receptor TrkA. Small molecules such as
K252a have been reported, however, pharmacological
specificity has limited the advancement of such com-
pounds into clinical trials (Watson et al. 2008). NGF-
mimetic peptides have also been evaluated, however no
peptide-based strategies has advanced into clinical trials
(Eibl et al. 2012). Mimetic strategies have seen success
in vivo neuropathic pain models, although their success
has been attributed to the beneficial effects of supple-
menting NGF in maintaining opiate receptors and may
see potential in the treatment of peripheral neuropathies
(Colangelo et al. 2008).
molecules such as ALE-0540 (Owolabi et al. 1999), Ro
08-2750 (Niederhauser et al. 2000), PD 90780 (Colqu-
houn et al. 2004), and PQC-083 (Eibl et al. 2013a) have
demonstrated efficiency in binding and inhibiting NGF
from interacting with its receptors. Another small mole-
cule Y1036 (Eibl et al. 2010), has been introduced as a
multipotent neurotrophin inhibitor by binding to not
only NGF, but also BDNF and inhibiting their action on
receptors. Compounds such as ALE-0540, Ro 08-2750,
PD 90780 and PQC-083 have been shown to bind to the
loop I/IV cleft and sufficiently alter the molecular topol-
ogy in a manner such that NGF can no longer bind effi-
ciently to its receptors (Eibl et al. 2010, 2013a). This loop
region of NGF has the highest degree of variance among
all the neurotrophins and is responsible for the selectivity
in receptor selection among the neurotrophin family
members. Alternatively Y1036 has been described to bind
at the neurotrophin hydrophobic interface, the region
conserved among all neurtrophin family members, and
therefore loses the specificity for NGF (Eibl et al. 2013b).
SPR spectroscopy has been used to study biomolecular
interactions and has proven effective in determining how
a potential drug therapeutic will interact with their target
receptor(s). Previously used screening strategies for NGF
inhibitors included the use of radioisotopes such as 125I
(Owolabi et al. 1999; Colquhoun et al. 2004; Eibl et al.
2013a). SPR is advantageous over labeling strategies
because it eliminates the need of fluorescent reporter
molecules or radioisotope tags which adds the concern of
altering the molecular interactions being examined.
Success of targeting a protein in a protein-receptor
system was first demonstrated with the approval of anti-
tumor necrosis factor (TNF) antibodies for the therapeu-
tic treatment of rheumatoid arthritis (Elliot et al. 2008)
ꢀ
and Crohn’s disease (Dezelak et al. 2016; Tolentino et al.
2016). Similar strategies have been explored with anti-
NGF antibodies, such as Tanezumab, for the treatment of
NGF-mediated pain. Although seemingly effective, anti-
NGF antibodies are still facing several therapeutic
drawbacks with potential serious side effects involving
autoimmune responses. Antibody-mediated pain manage-
ment strategies are also highly specific and are restricted
from crossing the blood brain barrier to the central ner-
vous system (Bannwarth and Kostine 2014).
Similar strategies of NGF inhibition have been intro-
duced using small molecule NGF-inhibitors. Small
Recently, there has been
a relationship established
between the inhibitory potential of NGF-inhibiting small
molecules described using 125I-NGF analysis and the high
affinity binding measured by SPR techniques (Kennedy
et al. 2016). This correlation between inhibitory potential
and binding affinity introduced SPR as a viable option
for efficient and rapid screening of small molecule entities
for identification of novel NGF-inhibitors.
The present study introduces SPR spectroscopy for
screening of a novel bivalent naphthalimide analog series
to determine a novel NGF-inhibitor, BVNP-0197, with a
higher potency than previously reported molecules.
Molecular modeling techniques were used to identify lead
analogs that targeted an area on NGF specific for TrkA
docking, in addition to reporting that NGF binding is of
higher affinity than binding to proNGF. These results are
of particular importance as it would be expected that the
bivalent naphthalimide analogs bind to both proNGF and
NGF in vivo. This model, supported by others character-
izing small molecule binding to NGF/proNGF (Sheffield
et al. 2016) suggests that the lead analogs would bind
both proteins, however have a higher affinity for NGF.
SPR screening of these lead analogs revealed 17 novel
Figure 9. The relationship of the IC50 measured by SPR spectroscopy
for the 17 bivalent naphthalimide analogs and the three previously
reported compounds (ALE-0540, PD 90780 and Ro 08-2750) and the
IC50 measured using the PC12 cell NGF-dependent differentiation
assay. X-axis: IC50 (lmol/L) measured using the SPR–Various
concentrations of analog were incubated for 1 h with 10 nmol/L NGF
prior to injection over immobilized TrkA. Y-axis: IC50 (lmol/L)
measured following PC12 cell treatment of increasing analog
concentration and 500 pM NGF for 72 h. Each data point represents
the mean of triplicate results. (F(1,18) = 22.69; P = 0.0002;
R2 = 0.5576).
2017 | Vol. 5 | Iss. 5 | e00339
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ª 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd,
British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

A. E. Kennedy et al.
NGF Inhibitor with Nanomolar Efficiency
compounds which specifically bind to NGF with binding
affinities between 3 lmol/L - 119 lmol/L. This study also
provides novel evidence that the bivalent naphthalimide
analogs described, as well as the three historic compounds
examined (ALE-0540, Ro 08-2750, and PD 90780) are
true NGF-inhibitors as they do not bind to TrkA to inhi-
bit NGF-TrkA-mediated signaling. The in vitro cell count
and morphology of PC12 cells treated with the bivalent
naphthalimide analog series, including BVNP-0197, did
not significantly differ from untreated cells after 72 h in
culture. The NGF-dependent cell assay determined that
three analogs had higher efficiency in NGF- inhibition
than previously reported molecules (IC50 < 2 lmol/L).
BVNP-0188, BVNP-0197 and BVNP-0198 demonstrated
IC50 values calculated from neurite outgrowth assays at
1.70 lmol/L, 89.5 nmol/L and 1.08 lmol/L respectively.
IC50 values determined using PC12 neurite outgrowth
assays were also related to the IC50 values determined for
each analog using SPR binding assays (Fig. 9) demon-
strating that SPR could be utilized in future therapeutic
screening strategies prior to entering in vitro studies.
It is expected that BVNP-0197 may have several poten-
tial applications in the field of neurotrophin research. For
example, BVNP-0197 may serve as a therapeutic with the
ability to inhibit NGF-TrkA-mediated pain signaling since
the predicted docking domain interferes with TrkA bind-
ing. BVNP-0197 and the other bivalent naphthalimide
analogs examined in this study may also serve as evidence
for the development of future compounds that inhibit
neurotrophin-mediated pain and other NGF-related dis-
orders. Due to similar homology, it is expected that
BVNP-0197 would also bind to proNGF and disrupt
binding to TrkA. Molecular modeling results suggest that
docking of BVNP-0197 to proNGF would occur at a
lower (millimolar) affinity than docking to NGF, how-
ever, further investigation with SPR, or using a cell-based
assay would be required to determine if BVNP-0197 holds
any inhibitory action for proNGF-TrkA binding. SPR
analysis of the bivalent naphthalimide analog–NGF inter-
action may allow for the development of compounds with
varied binding kinetics thus leading to more efficient
therapeutic options for NGF dysregulation. Further analy-
sis with animal models will be required to validate
BVNP-0197 as a potential therapeutic in clinical use for
pathologies associated to NGF dysregulation, such as
osteoarthritic pain and neurodegenerative diseases.
Performed data analysis: Kennedy, Laamanen.Wrote or
contributed to the writing of the manuscript: Kennedy, Laa-
manen, M. Ross Vohra, Boreham, Scott, G. Ross.
Disclosures
None declared.
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