Neuroscience & Medicine, 2013, 4, 290-298
Published Online December 2013 (
Open Access NM
Affinity Crosslinking of Y1036 to Nerve Growth Factor
Identifies Pharmacological Targeting Domain for Small
Molecule Neurotrophin Antagonists
Joseph K. Eibl1, Zouleika Abdallah1, Allison E. Kennedy2, John A. Scott1, Gregory M. Ross1,2
1Northern Ontario School of Medicine, Sudbury, Canada; 2Biology Department, Laurentian University, Sudbury, Canada.
Received October 24th, 2013; revised November 20th, 2013; accepted December 10th, 2013
Copyright © 2013 Joseph K. Eibl et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Classically, small molecule antagonists have targeted membrane bound receptors and intracellular enzyme targets.
While this drug discovery strategy is extremely successful, the number of new chemical entities in the pharmaceutical
pipeline is diminishing and complementary strategies are in need. A particularly attractive therapeutic strategy is to
neutralize soluble signalling proteins using small molecules. Small molecule-based technologies have the potential to
sufficiently alter the molecular topology of a given ligand and inhibit ligand/receptor interactions—effectively neutral-
izing the ligand’s signalling capacity. Recent technical advances in the field of structural biology have enabled the elu-
cidation of ligand/receptor complexes at atomic resolution enabling a detailed appreciation of the molecular interactions
governing ligand-mediated receptor activation. Exploiting molecular modeling techniques to study these signalling
complexes allows for a paradigm shift from “receptorcentric” to “ligandcentric” screening strategies. Nerve growth fac-
tor (NGF) is a prototypical protein signalling ligand, which binds two receptors, TrkA and p75NTR. We first explore the
molecular landscape governing the ligand/receptor interactions of NGF/TrkA and NGF/p75 structures. Next, we use the
recently reported NGF neutralizing small-molecule, Y1036, as an affinity probe to determine residues in proximity to
the pharmacological targeting domain of NGF and perform theoretical docking experiments to predict the residues
which comprise distinct pharmacological targeting domains on the surface of NGF. Exploiting such strategies may fa-
cilitate “ligandcentric” drug discovery and could further the development of a trophic-factor-selective compound such
as a BDNF-selective antagonist.
Keywords: NGF; BDNF; Inhibitor; Antagonist; Pain
1. Introduction
Historically, small molecule-based therapeutics have
generally focused on compounds directed towards mem-
brane bound receptors, channels and intracellular enzyme
targets. However, the physiological role of several of
these targets is governed by soluble protein ligands such
as cytokines or growth factors. Traditional “receptorcen-
tric” screening strategies yielded clinically effective
therapeutics for most of the past century [1]. Obvious
examples in the field of pain research include targets
such as cyclooxygenase (Cox1/2), canabinoid and opioid
receptors, and a variety of ion-channel inhibitors [2].
However, in the last decade, the success of these strate-
gies has not kept pace with the demand for new chemical
entities and, complementary approaches may help to re-
vitalize lead identification in many drug discovery pro-
In an attempt to counteract declining productivity, re-
search and development in the pharmaceutical industry
are moving towards unprecedented targets and novel
technologies [3]. By definition, unprecedented targets are
proteins or enzymes for which a pharmacological (or
biological) therapy has yet to be approved in a clinical
setting [3]. In the field of neuropharmacology, a prime
example of an unprecedented target is nerve growth fac-
tor (NGF).
NGF is a soluble ~26 kDa homodimeric protein which
is known to be critical for the development and mainte-
nance of the central and peripheral nervous system [4].
NGF is a member of the neurotrophin family of proteins
and mediates its physiological role by binding the com-
Affinity Crosslinking of Y1036 to Nerve Growth Factor Identifies Pharmacological
Targeting Domain for Small Molecule Neurotrophin Antagonists
mon low-affinity receptor p75NTR [5] and the selective
high-affinity receptor TrkA [6]. Interestingly, the dys-
regulation of NGF has also been implicated in several
disease states of the nervous system [4,7]. Thus, identi-
fying mechanisms to inhibit pathological signalling may
have significant therapeutic potential in the field of neu-
The therapeutic efficacy of targeting the ligand in a
ligand/receptor system has been demonstrated by the
neutralizing effect of monoclonal antibodies in the clinic
[8]. However, antibody-mediated therapies have several
technical, practical, and economical limitations that re-
strict their widespread application in many clinical set-
tings. A similar strategy using small-molecules to neu-
tralize soluble peptide ligands in a pathological setting is
just starting to be explored.
NGF neutralizing small-molecules (which bind NGF
rather than the TrkA or p75NTR receptors) has been de-
scribed by our group and others. For example, ALE-
0540 [9], Ro 08-2750 [10], PD90780 [11] and Y1036 [12]
are known to effectively neutralize NGF activity. Inter-
estingly, Y1036 is reported to also bind the related neu-
rotrophin brain-derived neurotrophic factors. Better un-
derstanding the high resolution of structural biology gov-
erning the mode-of-action of these molecules may facili-
tate the identification and optimization of such com-
pounds in drug discovery programs directed at unprece-
dented targets.
In this study, we describe the identification of a puta-
tive pharmacological targeting domain on the structure of
NGF. Beginning with high-resolution structural biology
data, we dissect the molecular interactions governing the
NGF/p75NTR and NGF/TrkA interface. Next, we use the
NGF-binding antagonist Y1036 as an affinity probe to
identify the putative pharmacological targeting domain.
From these data, we perform theoretical docking experi-
ments which demonstrate that the binding of Y1036 can
substantially alter the molecular topology of NGF. The
results of this study provide valuable information to-
wards the identification of a pharmacological targeting
domain on the surface of a soluble signalling ligand and
may prove usefulness in rationally modifying small
molecules directed towards the neutralization of NGF.
2. Methods
2.1. Molecular Modeling
All molecular modeling was performed in the Sybyl 8.0
environment (Tripos; St. Louis, MO). The 3D coordi-
nates of the protein backbone were kept fixed at their X-
ray geometry, and a water environment was not included
in the model. Ribbon structures of NGF [RCSB ID-1BET
[13]], NGF/TrkA [RCSB ID-2IFG [6]] and NGF/p75NTR
[RCSB ID-1SG1[5]] were generated using the Molcad
rendering suite of Sybyl 8.0. External surfaces were cal-
culated using the Fast Connoly approximation of the
MolCad suite. Surface to charge ratios were approxi-
mated using the MMFF94 molecular mechanical force
field [14].
2.2. NGF Crosslinking and Digest
NGF was purchased from Cedarlane Laboratories (Bur-
lington, ON, Canada). NGF (1 mg/mL) and Y1036 (50
μM) were incubated for 1 hour in 25 mM phosphate
buffer (pH 7.4) at room temperature in a volume of 100
μl. Covalent crosslinking of NGF to Y1036 was per-
formed via 1-ethyl-3-(3-dimethyl amino-propyl) carbo-
diimide (EDC) and N-hydroxysuccinimide (NHS)-me-
diated reaction (Pierce; Rockford, IL). 5 mM EDC and 2
mM NHS were added to the solution and incubated at
25˚C temperature for 30 minutes. Crosslinked NGF was
reduced in 200 mM DTT in 100 mM ammonium bicar-
bonate buffer (pH = 8.0) for 1hr at room temperature.
The reaction mixture was then acetylated with iodoace-
tamide (1 M) via 1hr incubation at room temperature in
the dark. Excess iodoacetamide was neutralized using
dithiotheritol (DTT) for 1hr at room temperature. A pro-
tein digest was then performed by incubating 1 μg of se-
quencing grade trypsin (Madison, WI, USA) with 50 μg
of NGF or crosslinked NGF-Y1036 at 37˚C for 18 hrs.
2.3. Mass Spectroscopy
Peptide analysis was performed at the Biological Mass
Spectrometry Laboratory at the University of Western
Ontario (London, ON, Canada). Peptides were separated
on a CapLC high-performance liquid chromatography
system using a Nano-Acquity C18 column and analyzed
using Q-TOF Micro mass spectrometer (Waters, Missis-
sauga ON, Canada). NGF-peptides were identified via
the MASCOT analysis (Matrix Science; Boston MA).
Analysis was then used to identify peptides which ex-
actly matched the m/z of a given NGF peptide + Y1036 –
2.4. Theoretical Docking
Molecular modeling and in silico docking of Y1036 to
NGF [RCSB ID-1BET (Wehrman et al., 2007)] was
carried out using the software program Sybyl 8.0 (Tripos,
St. Louis, MO). The structure of NGF was prepared for
docking using the Biopolymer suite of Sybyl 8.0. Co-
structures were deleted and hydrogens were added, the
appropriate formal charges were applied to the N- and
C-termini and the structure was optimized using the
MMFF94 molecular mechanical force field [14]. Flexible
docking of Y1036 was performed using the Surflex-Doc
suite [15] incorporated into Sybyl 8.0. The docking
Open Access NM
Affinity Crosslinking of Y1036 to Nerve Growth Factor Identifies Pharmacological
Targeting Domain for Small Molecule Neurotrophin Antagonists
Open Access NM
rate was 1 mL/min. Samples (100 μL) were injected and
separated using the following gradient system: 5% B to
55% B (over 40 min) then to 100% B in 10 min. The
eluent was monitored by photodiode array detection at
280 nm and 450 nm.
protmol (molecular space) was generated to include the
residues using a 5Å radius from the lysines of interest
(K57) with a bloat factor = 0 and a threshold value of 0.5.
For the Surflex-Doc function, the angstroms to expand
search grid was set at 6 and the maximum confirmations
per fragment was set to 20. 3. Results
2.5. HPLC Analysis 3.1. Molecular Modeling Identifies Putative NGF
Targeting Domains
High-performance liquid chromatography (HPLC) was
carried out using a System Gold Microbore HPLC with
32 Karat Software from Beckman Coulter including a
delivery module pump and a diode array detector. An
Inertsil ODS-3 column (150 × 4.6 mm, 5 μm) with an In-
ertsil ODS-3 guard cartridge (10 × 4 mm, 5 μm) was
used for separation. A mixture of two solvents consti-
tuted the mobile phase: solvent A [0.1% concentrated
trifluoroacetic acid (TFA) in deionized water] and sol-
vent B [acetonitrile (MeCN) with 0.1 % TFA]. The flow
Recently, atomic resolution structures have been reported
which describe the interactions between NGF and its
receptors, TrkA [6] and p75NTR [5]. Using structural
modeling techniques, we examined the molecular topol-
ogy of the NGF/p75NTR-dependent and NGF/Trk-de-
pendent binding. With respect to ligand/receptor binding
mode, it is important to note that the orientation in which
p75NTR binds NGF is opposite to that of TrkA (Figure 1).
(a) (b) (c)
(d) (e) (f)
Figure 1. Footprinting of the molecular interactions governing NGF/receptor binding. (a) The asymmetrical NGF/p75NTR
complex is presented as ribbon structure (RCSB ID-1S1G). Two NGF dimers (NGF monomer presented in white; NGF’
monomer presented in yellow) are bound by p75NTR (blue). Panel (b) illustrates the NGF/p75NTR orientation rotated by ap-
proximately 90˚. (c) Residues of NGF w hich participate directly in NGF/p75NTR complex are highlighted in blue. Region i) is
composed of residues 9 - 23 and region ii) is composed of residues 48 - 55 of the NGF monomer. Region iii) is comprised of
residues 30 - 32; region iv) is comprised of residue 88 and region v) is comprised of 95 - 100 of the NGF’ monomer. (d) The
symmetrical NGF/TrkA complex is illustrated as ribbon structure (RCSB ID – 2IFG). Two NGF dimers (NGF monomer
presented in white; NGF’ monomer presented in yellow) are bo und by TrkA (red). Panel (e) illustr ates the NGF/TrkA orien-
tation rotated by approximately 90˚. (f) Residues of NGF which participate directly in NGF/TrkA complex are highlighted in
Red. Region i) is comprised of residues 1 - 23 of the NGF monomer. Region ii) is composed of residues 30 - 32 and region iv)
is composed of residues 83 - 86 of the NGF’ monomer.
Affinity Crosslinking of Y1036 to Nerve Growth Factor Identifies Pharmacological
Targeting Domain for Small Molecule Neurotrophin Antagonists
Interestingly, select regions participating in p75NTR
binding of NGF are shared with TrkA binding while
other regions are primarily distinct from those involved
in TrkA interactions. For instance, the N-terminal resi-
dues of NGF participate in binding with both receptors,
but the loop I/IV region is of critical importance to
p75NTR interactions. The NGF/TrkA complex reported by
Wehrmann et al. [6] involves the residues of loop IV, but
it is unclear if loop I participates in binding. NGF/TrkA
interactions also occur at the C-terminal domain of NGF
which are outside the contact region of p75NTR. Figure 1
illustrates the NGF ligand/receptor footprint with respect
to p75NTR and TrkA. The molecular topology of NGF’s
ligand/receptor complex suggests that an inhibiting small
molecule would likely bind in proximity to these regions
of interaction.
3.2. Proximity Cross-Linking Identifies
Pharmacological Targeting Domains
Y1036 is a small molecule which neutralizes the signal-
ling activity of NGF and inhibits NGF interaction with
the p75NTR and TrkA receptors [12]. The minimized en-
ergy structure of Y1036 is presented in Figure 2. The
free carboxyl group of Y1036 has the potential to form
H-bonds with the ε-amine of a given lysine residue on
the surface of NGF. Importantly, this pharmacological
property may allow Y1036 to be used as chemical probe
to identify a functional pharmacological targeting do-
main(s) on the surface of NGF. Thus, we performed a
zero-distance EDC/NHS crosslinking proximity assay.
Recombinant NGF was incubated in the presence of ex-
cess Y1036 at room temperature for 1 hr. Pre-activation
of Y1036 or NGF with EDC/NHS did not affect the effi-
ciency of the crosslinking reaction. Crosslinking was
then performed via an EDC/NHS reaction. Native NGF
and crosslinked NGF-Y1036 then underwent tryptic di-
Using time-of-flight mass spectrometry, we were able
to resolve the full sequence coverage of NGF via Mascot
analysis of tryptic peptides as summarized in Figure 3(a).
Similar analysis of NGF crosslinked to Y1036 identified
a principal peak corresponding in mass to the Y1036-
modified peptide QYFFETK(-Y1036) (Figures 3(b) and
(c)). As EDC/NHS crosslinking occurs between the car-
boxyl group of Y1036 and primary amines of lysine resi-
dues these data suggests that Y1036 crosslinked with
residues K57 (Figure 3(c)). Figure 4 illustrates the spa-
tial orientation of the QYFFETK(-Y1036) on the struc-
ture of NGF.
Theoretical docking experiments support the involve-
ment of K57 in pharmacological targeting domains.
To evaluate the potential contribution of K57 to the
binding mode of Y1036, we created an ab initio docking
Figure 2. Structure of Y1036. (a) The structure of Y1036, 3-
thioxo-thiazolidin-3-yl]propanoic acid. (b) The minimized
energy conformation of Y1036.
site biased to the residues within a 5Å radius of the K57
crosslinking site. We then performed flexible docking
experiments to obtain a theoretical binding mode for
Y1036 in the proximity to the dimer interface (K57 site).
A favorable docking mode for Y1036 was obtained with
a consensus score of 4.
In the case of the pharmacological targeting domain
near the hydrophobic interface, the docking results pre-
dicted the formation of three stabilizing H-bonds: the
first H-bond is formed between the ε-amine of Lys57 and
the carboxyl group of Y1036; another H-bond is shared
by the aminosulfonyl group of Y1036 and the α-carbonyl
group of Trp21; and a third H-bond with the double
bonded oxygen of the sulfonamyl group of Y1036 and
the α-amide of Gly23 (Figure 5(a)). Accordingly, the
docking mode also demonstrates a favorable pose with
little conformational strain consistent with the minimized
energy conformation of Y1036.
Importantly, the results of the docking experiments
suggest the formation of an H-bond between the carboxyl
group of Y1036 and the ε-amine of the lysine (K57).
Thus, these theoretical docking experiments are consis-
tent with the mass spectra data obtained the Y1036
proximity crosslinking assay. These results narrow the
possible pharmacological targeting domains of NGF to
the hydrophobic interface with involvement of K57.
In order to rule out non-specific crosslinking/binding
activity, we attempted to crosslink a non-active analogue
of Y1036 (Figure 6(a)). Y410 (Figure 6(b) shares struc-
tural homology to Y1036, but is not an active NGF an-
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Affinity Crosslinking of Y1036 to Nerve Growth Factor Identifies Pharmacological
Targeting Domain for Small Molecule Neurotrophin Antagonists
Figure 3. Proximity cross-linking of Y1036 identifies putative docking site(s) on NGF. LCMS identified peptides of (a) trypsin
digested NGF and (b) trypsin digested NGF-Y1036 are aligned to the consensus sequence of NGF. Mass spectra of the peptide
corresponding to (c) QYFFETK(-Y1036). The peptide sequence represented in violet corresponds to the peptide which cross-
linked at K57 (colored cyan).
Figure 4. Landmarking of pharmacological targeting do-
mains on the structure of NGF. The symmetrical ribbon
structure of the NGF dimer is composed of two monomers
(NGF, white; NGF’, yellow). The results of Y1036 proximity
assay suggest crosslinking occurs at K57. The regions cor-
responding to the QYFFETK(-Y1036) crosslinked peptide
is highlighted in violet or green, respectively. The location
of K57 is highlighted in cyan. The structure and amino acid
sequence of NGF were obtained from (RCSB ID-2IFG).
tagonist (IC50 > 50 μM; unpublished data). Following
EDC/NHS crosslinking and tryptic digestion, we ob-
tained evidence for crosslinked peptides of Y1036 and
NGF by HPLC-UV analysis (Figure 6(c)). Using a simi-
lar experimental approach, no crosslinking was observed
between Y410 and NGF (Figure 6(d)). Similarly, Y410
also did not yield a favorable docking mode at the K57
site with consensus score < 2 (data not shown). The re-
sults of this control experiment provide further support
for the proposed crosslinking site and docking model.
4. Discussion
NGF-dependent signalling has been identified as a po-
tential pharmacological target for therapeutic intervene-
tion in several neurological disorders, including chronic
pain, Parkinson’s disease, and Alzheimer’s disease [4].
In the past, small molecule kinase inhibitors, such as
K252a [16,17] and the isothiazole family of compounds
[18], have functionally limited TrkA signalling in ex-
perimental models, but their partial specificity has pre-
vented clinical translation.
An alternative strategy which shows much promise is
the use of therapeutic biologicals. For example, mono-
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Affinity Crosslinking of Y1036 to Nerve Growth Factor Identifies Pharmacological
Targeting Domain for Small Molecule Neurotrophin Antagonists
Figure 5. Theoretical docking experiments are consistent
with the involvement of K57 in the binding mode of Y1036
to NGF. Schematic representations of Y1036 docked to
NGF as determined by molecular modeling. (a) Flexible
theoretical docking experiments predict that Y1036 is able
to bind at the K57 pharmacological targeting domain in a
favorable mode by forming three H-bonds with Trp21,
Gly23 and K57. Y1036 is rendered as a ball and stick for-
mation. Residues of the NGF pharmacological targeting do-
mains are rendered as capped stick formations. The relative
location of K57 (purple asterisk) is illustrated on the crystal
structure of (b) NGF/p75NTR (1SG1) and (c) NGF/TrkA
(2I FG). Ato ms are colored according to their types: C, gray;
N, blue; O, red; H, cyan. Hydroge n bonds are illustrated as
dashed yellow lines. The location of residues participating
forming H-bond are highlighted on the ribbon structure in
clonal antibodies directed towards NGF effectively neu-
tralize its signalling activity [8,19]. Despite the efficacy
of such antibody mediated therapies, technical, practical
and economical issues may limit their application in
many clinical settings. To this end, several groups have
been exploiting peptidomimetic approaches directed to-
wards modulating neurotrophin receptors [21-23].
A complementary approach to receptor-centric strate-
gies is to develop ligand-centric small molecule antago-
nists which are bound to and sufficiently alter the mo-
lecular topology of the ligand (NGF) effectively neutral-
izing its signalling activity. Our group has previously
shown that Y1036, a small molecule which are bound to
NGF can effectively neutralize NGF’s signalling activity
[12]. In this study, we use available high-resolution
structural data to identify the pharmacological targeting
domain of NGF’s signal neutralizing small molecules.
Such findings may facilitate the rational development of
small molecules directed towards NGF and other soluble
signalling proteins.
The elucidations of the NGF/TrkA and the NGF/p75NTR
complexes now allow for the evaluation and prediction
of molecular interactions which govern efficient neuro-
trophin signalling. However, it is also important to un-
derstand the caveats of these structures. For example,
Figure 1 nicely illustrates the location, proximity, and
footprint of ligand/receptor interaction of both TrkA and
p75NTR binding to NGF. However, some regions of the
NGF/TrkA crystal proved difficult to resolve, and data
are not available for NGF residues 61-68 [5,6]. Impor-
tantly, in order to facilitate crystallization of the NGF/
TrkA complex, Wehrman truncated the structure of TrkA
by a 32 residue sequence which joins the Ig-C2 domain
TrkA to the cell surface [6]. This sequence likely plays a
very important role for TrkA’s ability to resolve one
neurotrophin ligand from another. Thus, the contribution
of loop domains I and II of NGF cannot be analyzed with
respect to TrkA binding from crystal structure analysis. It
is also important to note that the binding epitope of αD11
NGF-neutralizing antibody has been demonstrated to
bind at the loop II and loop IV regions of NGF [24].
In the case of the NGF/p75NTR, the ligand/receptor
binding mode more completely encompasses the whole
of NGF, but some very important questions still surround
the structure of the NGF/p75NTR complex. For instance,
the structure put forth by He and Garcia [5] proposes an
asymmetrical binding mode with one p75NTR receptor
bound to an NGF dimer. Follow-up work demonstrated
the importance of a disulfide bond in arranging symmet-
rical p75NTR receptor [25], and the related neurotrophin
NT-3 indeed binds NGF in a symmetrical 1:1 manner
[26]. Similarly, p75NTR has been found to also symmetri-
cally bind the pro-NGF [27]. Understanding these subtle-
ties may aid in the rationalization and design of novel
ligandcentric inhibitors.
It may also be worth considering the orientation of the
NGF ligand with respect to the membrane in binding to
p75NTR or TrkA. NGF is believed to bind p75NTR with the
N- and C-terminal domains oriented towards the mem-
brane. When bound to TrkA, the loop regions of NGF are
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Affinity Crosslinking of Y1036 to Nerve Growth Factor Identifies Pharmacological
Targeting Domain for Small Molecule Neurotrophin Antagonists
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Y1036 Y410
(a) (b)
Figure 6. Y410 does not crosslink to NGF. (a) The structure of Y1036 and (b) Y410. (c) HPLC-UV analysis confirms NGF-
Y1036 crosslinked peptides following tryptic digest. Crosslinked peptides were monitored at 280 nm (red trace) and 450 nm
(black trace). Chromatograms are zoomed such that larger peaks may exceed the 50 m Au displayed on the Y-axis. The large
peak at 34.5 minutes corresponds to the elution of free Y1036. (d) No peptides eluted with a signal at 280 nm and 450 nM
following incubation and crosslinking of NGF and Y410. The large peak eluting at 46.5 min corresponds to free Y410.
oriented towards the plane of the membrane with the N-
and C-terminal domains interacting with the ectodomain.
Currently, two hypotheses exist to reconcile this spatial
phenomenon. First, it has long been hypothesized that the
p75NTR and TrkA form “high-affinity” binding complex
whereby both receptors interact with NGF simultane-
ously [28]. Analysis by Wehrman et al. [6], demonstrated
that the footprints of p75NTR and TrkA in NGF may al-
Affinity Crosslinking of Y1036 to Nerve Growth Factor Identifies Pharmacological
Targeting Domain for Small Molecule Neurotrophin Antagonists
low for simultaneous binding. Cross-linking experiments
have demonstrated that a NGF/p75NTR/TrkA complex
forms [29], but it’s not clear if this is a stable or transient
complex. Alternatively, a “hand-over” mechanism has
also been proposed whereby p75NTR first binds NGF and
a hand-to-hand exchange from p75NTR to TrkA occurs
[28]. It is thought that this mechanism may account for
the binding of NGF in opposite orientation. For the pur-
poses of rational drug design, it is important to consider
the dynamics of such receptor interplay. With respect to
the pharmacological targeting domains put forward in
this study, the potential mechanism at the hydrophobic
interface (K57 site) is not obvious from crystallography
data. The molecular topology altered by Y1036 binding
at the K57 site is in proximity, but outside of the static
interaction surfaces of NGF/p75NTR or NGF/TrkA. The
static nature of crystallography provides very useful in-
sights, but further real-time high-resolution techniques
will undoubtedly refine our current understanding of
these events.
5. Conclusion
The results of our molecular modeling suggest that the
binding of Y1036 to NGF is sufficient to alter the mo-
lecular topology such that the resultant change in sur-
face charge density may alter the receptor: neurotrophin
interface and prevent neurotrophin mediated signalling.
However, there are limitations to the interpretation of our
data. In order to have definitive proof of binding me-
chanism of Y1036, co-crystallization experiments will be
required. The docking model presented in this study is
consistent with our experimental observations. Under-
standing the structural and molecular features of the
residues in proximity to the NGF crosslinking site now
gives researchers the ability to modify chemical scaffolds
in a rational manner. Taking advantage of similar high
resolution structural biology, it may be possible to apply
this strategy to soluble protein ligands more generally.
Adopting such emerging high resolution strategies may
ultimately enhance drug discovery activities and aid in
revitalizing the collective pharmaceutical pipeline.
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