Neurotrophins 1

REVIEWS
Small-molecule modulation of
neurotrophin receptors: a strategy for
the treatment of neurological disease
Frank M.Longo1 and Stephen M.Massa2
Abstract | Neurotrophins and their receptors modulate multiple signalling pathways to

regulate neuronal survival and to maintain axonal and dendritic networks and synaptic
plasticity. Neurotrophins have potential for the treatment of neurological diseases.
However, their therapeutic application has been limited owing to their poor plasma
stability, restricted nervous system penetration and, importantly, the pleiotropic actions
that derive from their concomitant binding to multiple receptors. One strategy to
overcome these limitations is to target individual neurotrophin receptors such as
tropomyosin receptor kinase A (TRKA), TRKB, TRKC, the p75 neurotrophin receptor or
sortilin with small-molecule ligands. Such small molecules might also modulate various
aspects of these signalling pathways in ways that are distinct from the programmes
triggered by native neurotrophins. By departing from conventional neurotrophin
signalling, these ligands might provide novel therapeutic options for a broad range
of neurological indications.
Department of Neurology
and Neurological Sciences,
Stanford University,
300 Pasteur Drive, Stanford,
California 94305, USA.
2
Department of Neurology,
San Francisco Veterans
Affairs Medical Center,
University of California San
Francisco, 4150 Clement
Street, San Francisco,
California 94121, USA.
e-mails: longo@stanford.edu;
stephen.massa@ucsf.edu
doi:10.1038/nrd4024
1
The mammalian neurotrophin family comprises nerve

growth factor (NGF), brain-derived neurotrophic factor
(BDNF), neurotrophin3 (NT3) and neurotrophin4
(NT4; also known as NT5)1,2. Since the discovery of NGF
more than 50years ago, the ability of neurotrophins to
prevent or reverse neuronal degeneration, to promote
neurite regeneration and to enhance synaptic plasticity
in various invitro and invivo models has offered the
promise that they might provide primary or adjunctive
therapy for a number of neurological disorders3,4.
However, as is the case with proteins in general,
neurotrophins have suboptimal pharmacological properties, such as low stability in serum (with half-lives of
a few minutes or less) and negligible oral bioavailability.
In addition, they have minimal bloodbrain barrier
penetration and restricted diffusion within central
nervous system (CNS) parenchyma57. The pleiotropic
actions of neurotrophins that are triggered by the
activation of their multi-receptor signalling networks
(FIG.1) could lead to adverse ontarget effects such
as the promotion of neurodegeneration8,9 or pain10,11
thereby further limiting their clinical application.
Indeed, these limiting factors probably contributed
to the lack of efficacy seen in early clinical trials that
investigated the therapeutic efficacy of exogenously
administered neurotrophins in various neurodegenerative

diseases, including Alzheimers disease and amyotrophic
lateral sclerosis (BOX1).
Various strategies for addressing the pharmacokinetic limitations of native neurotrophins have therefore
been pursued. These include the development of small
molecules that target specific neurotrophin receptors
such as the tropomyosin receptor kinase (TRK) receptors and the p75 neurotrophin receptor (p75NTR; also
known as NGFR) (discussed in more detail below).
Other approaches include the development of mutant
neurotrophins12,13, neurotrophin receptor-specific antibodies (that act through individual neurotrophin receptors to modulate signalling processes that are relevant to
pathological states)1417 and localized expression of neurotrophins via viral and cell-based delivery systems1821.
In addition to having more favourable pharmacokinetic
profiles, several of these alternatives may differentially
stimulate neurotrophin receptors to achieve distinct signalling profiles, thus resulting in more selective effects
than those elicited by native neurotrophins.
In this Review, after providing a brief overview of
the latest understanding of neurotrophins, their receptors and their effects, we discuss the rationale underlying the selective targeting of neurotrophin receptors
NATURE REVIEWS | DRUG DISCOVERY
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Transactivating activators
Ligands that indirectly activate
a given receptor through the
activation of another receptor.
Secretagogues
Exogenous agents that increase
the production or secretion
of an endogenous agent.
by small molecules and the strategies being pursued to

achieve this. Although there are pharmacological efforts
underway towards the development of indirect receptor
activators (so-called transactivating activators) and agents
that promote the increased expression of neurotrophins
(so-called secretagogues), this Review focuses on the
development of small molecules that interact directly
with neurotrophin receptors. Neurotrophin receptors
Mature neurotrophin
also have key roles outside the nervous system but

here we focus on neurological and neuropsychiatric
disorders.
Neurotrophins, their receptors and signalling

The current understanding of the molecular biology of
neurotrophins, their receptors and downstream signalling pathways, as well as their normal and pathological
180
Pro-neurotrophin
1
Saddle
TRK
N
p75NTR
Pro
Pro
Sortilin
p75NTR
SHC
Tyr490
Tyr670
NADE
NRAGE
IRAK
NRIF
TRAF6
FAP1
RHOA
Tyr674
PI3K
Tyr675
Tyr695
AKT
Tyr751
RIP2
MAPK
JNK
NF-B
PKC
GSK3
PLC1
Cell death or
degeneration
Survival
Tyrosine
kinase
activation
Tyr785
Increase in
endocytosis
and
degradation
Neurite growth
Figure 1 | Neurotrophins, their receptors and signalling pathways. The figure shows a highly simplified schematic
of the signalling pathways that are regulated by neurotrophins and their receptors. Secreted neurotrophins bind to
two principal receptor types in an antiparallel fashion: tropomyosin receptor kinase (TRK)
receptors
theDiscovery
p75
Nature
Reviews and
| Drug
neurotrophin receptor (p75NTR). In TRK complexes, the neurotrophin apical region containing turn loops 1, 2 and 4 is
near the membrane, whereas in the p75NTR complex it faces away from the membrane (loops 14 and the amino terminus
are labelled on the ribbon structure). Pro-neurotrophins are proteolytically processed intra- or extracellularly to remove
the Pro-region, which is the principal site of interaction with the coreceptor sortilin (the Pro-crystal structure is not
available but shown schematically in its approximate location relative to the mature (ribbon structure) domain).
Neurotrophin signalling proceeds through preformed or induced receptor dimers, and the binding of p75NTR stimulates
extracellular domain shedding and regulated intramembrane proteolysis involving - and secretases (not shown),
which releases intracellular domains that are important for signalling and for interacting with intracellular adaptor proteins.
TRK ligand binding by mature neurotrophins results in the phosphorylation of an array of intracellular domain tyrosine
residues (illustrated with the numbering scheme of TRKA), which activate kinase activity (Tyr670, Tyr674 and Tyr675 are
shown in the activation domain), resulting in further receptor autophosphorylation. Phosphorylation at Tyr490, Tyr785 and
possibly Tyr751 (or their equivalent residues in other TRK receptors), forms adaptor binding sites that couple the receptor
to mitogen-activated protein kinases (MAPKs), phosphoinositide 3kinase (PI3K) and phospholipase C1 (PLC1)
pathways, which may act locally and/or via signalling endosomes that are transported to the nucleus, to ultimately
promote neurite outgrowth, differentiation and cell survival. Mature neurotrophins binding to p75NTR, depending on
the context, may augment neurotrophin binding to TRK receptors, reinforce TRK signalling through AKT and MAPKs,
and further promote survival through the nuclear factorB (NFB) pathway, or antagonize the actions of TRK through
the activation of JUN N-terminal kinase (JNK) and RHOA pathways. Pro-neurotrophin binding in complex with sortilin
selectively activates cell-death-related pathways. FAP1, FAS-associated phosphatase1; GSK3, glycogen synthase
kinase3; IRAK, interleukin1 receptor-associated kinase; NADE, p75NTR-associated cell death executor; NRAGE,
neurotrophin receptor-interacting MAGE homologue; NRIF, neurotrophin receptor-interacting factor; RIP2, receptorinteracting protein2; SHC, SRC homology domain-containing protein; TRAF6, TNF receptor-associated factor 6.
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effects in the brain, is evolving rapidly. Here, we provide
a brief overview of neurotrophin receptors and their signalling pathways, particularly in the context of creating
novel ligands; these advances in our understanding have
recently been highlighted in the literature (reviewed in
REFS2225).
Signalling adaptors
Proteins binding to activated
receptors that mediate
the activation of further
intracellular signalling events.
Dominant negative
A term used to describe
a situation whereby one
protein isoform interferes
with the effects of another.
Signalling endosomes
Endosomes containing
ligandreceptor complexes
that remain active, transducing
cytoplasmic signals as they
are transported within the cell.
Direct mechanisms
Interactions between
transmembrane receptors
that are mediated by direct
contact with each other.
Bridged mechanisms
that are mediated through
an intermediary structure.
Proteolytic peptidemediated mechanisms

that are mediated through the
cleavage, translocation and
binding of a portion of one
protein to the other.
Neurotrophin synthesis and secretion. NGF, BDNF,

NT3 and NT4 are initially synthesized in the endoplasmic reticulum as pre-proproteins, and cleavage of the
signal peptide of pre-proproteins converts these into
pro-neurotrophins. In the trans-Golgi network and
in secretory vesicles, pro-neurotrophins dimerize and
are proteolytically processed by proprotein convertase
subtilisin kexin (PCSK) enzymes to their mature forms
before their release from the cell22.
The amount of mature neurotrophin secreted from
the cell depends on the range of convertases expressed
in different cell types; furin (also known as PCSK3) is
expressed in most cells, whereas PCSK1 (also known as
NEC1) and PCSK2 (also known as NEC2) are principally
expressed in neurons. The expression of convertases
is physiologically regulated and in some scenarios a
considerable proportion of pro-neurotrophins may
be secreted into the extracellular space (reviewed in
REFS22,26). In the extracellular space, pro-neurotrophin
cleavage may be catalysed by plasmin27. The secreted
~120amino-acid mature forms of neurotrophins exist
in solution as dimers22,26 (FIG.1).
Neurotrophin receptors. Pro- and mature neurotrophins
both bind to and signal through two principal receptor
types: p75NTR and the TRK receptors. p75NTR is a tumour
necrosis factor (TNF) receptor family member that
unselectively binds all of the neurotrophins and lacks
known intrinsic enzymatic activity but recruits signalling
adaptors and modulates TRK signalling.
The pro-domain of pro-neurotrophins can concurrently bind to the vacuolar protein sorting 10 (VPS10)
family member sortilin (also known as NTR3), which
allows the formation of a ternary complex with p75NTR
(REF.28). Among the numerous pathways and proteins
that are regulated by p75NTR are the phosphoinositide
3kinase (PI3K)AKT pathway 29, nuclear factor-B
(NF-B)30, mitogen-activated protein kinase (MAPK)31,
JUN Nterminal kinase (JNK)32, RHOA33, the cyclic
AMPprotein kinase A (PKA) pathway 34, hypoxiainducible factor (HIF)35 and the ceramide signalling
pathway 36. The p75NTR-induced effects are diverse and
include cell survival, cell death, regulation of proliferation and inhibition of neurite outgrowth, depending on
the expression of the TRK receptors, sortilin and various
intracellular signalling adaptors (FIG.1).
There are three TRK receptors: TRKA (also known
as NTRK1), TRKB (also known as NTRK2) and TRKC
(also known as NTRK3). The full-length versions of these
transmembrane receptors contain an intracellular tyro
sine kinase domain and an extracellular neurotrophinbinding region that comprises tandem immunoglobulin
domains and a leucine-rich domain. Each TRK receptor selectively binds to different neurotrophin family
members: NGF binds to TRKA; BDNF and NT4 bind

to TRKB; and NT3 binds to TRKC37,38. In addition, there
is heterologous binding, with NT3 and NT4 both eliciting
some activation of TRKA, and NT3 eliciting some activation of TRKB37,39. TRK signalling occurs through
three principal tyrosine kinase-mediated pathways: the
MAPKERK (extracellular signal-regulated kinase)
pathway, the PI3KAKT pathway and the phospholipase
C1 (PLC1)PKC pathway. The effects elicited through
these signalling pathways predominantly promote cell
survival and differentiation2,38. In addition, several truncated isoforms of TRKB and TRKC exist, which lack
the tyrosine kinase domain. These isoforms may have
dominant negative effects, sequester neurotrophins and/
or signal through alternative mechanisms compared to
full-length neurotrophins25,40.
Both TRK receptors and p75NTR are subject to endo
cytosis, participate in the formation of signalling endosomes
and may elicit effects at the ends of neuronal processes,
along axons and in the nucleus4143. Although several
functional interactions between TRK receptors and
p75NTR are well established for example, the formation
of a neurotrophin binding site with increased affinity,
as well as reciprocal modulation of signalling 4447 the
nature of their physical associations and the formation of
complexes with neurotrophins remain areas of ongoing
debate and study. Evidence has been presented for and
against various possible models, including the formation of stable p75NTRneurotrophinTRK extracellular
domain (ECD) complexes48,49, as well as intracellular
associations mediated by direct mechanisms50, bridged
mechanisms50 or proteolytic peptide-mediated mechanisms51,
and the transient transfer of a neurotrophin from p75NTR
to a TRK49. The complexity of neurotrophin receptor
ligand interfaces and the resulting extensive effects that
are elicited through their signalling pathways create a
broad range of possibilities for small-molecule binding
and modulation of function.
Potential pharmacological targets

Although there is an expanding number of neurotrophin
receptor-mediated physiological effects and mechanisms
of action, the currently available structural and mechanistic information related to the actions of pro- and mature
neurotrophins provides substantial opportunities for
pharmacological exploration, as discussed below.
Pro-neurotrophin signalling. The principal mode by
which pro- and mature neurotrophins bind to p75NTR
appears to involve the formation of a complex containing
a neurotrophin dimer and a p75NTR dimer with its elongated extracellular domains flanking the neurotrophin28,52
(FIG.1). p75NTR dimers may be preformed and they are
connected together through a cysteine bond in the
transmembrane domain. This cysteine bond is important for neurotrophin-mediated signal transduction (via
JNK activation) and the transmission of steric changes
to the intracellular domains53. The extracellular region
of p75NTR contains four cysteine-rich domains that are
arranged linearly; the second and third of these cysteinerich domains bind to neurotrophins at two major sites:
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Box 1 | Clinical trials of neurotrophins
Systemic delivery of exogenous neurotrophin protein has been used in clinical trials
of several neurological conditions. The results of these trials have been largely
disappointing with nominal therapeutic effects and/or dose-limiting side effects
(reviewed in REF.203). Below, we discuss three neurological conditions in which
therapeutic administration of neurotrophins has been tested.
Alzheimers disease
Intraventricular administration of high doses of nerve growth factor (NGF) in three
patients with Alzheimers disease was associated with improved cognition in two out
of the three treated patients. However, all three patients experienced an induction of
back pain, and two experienced sustained weight loss, which was attributed to
NGF infusion; consequently, the trial was discontinued204. Subsequent studies in
rats demonstrated that intrathecal administration of NGF causes the sprouting of
sympathetic fibres, which is known to contribute to various pain syndromes that
may involve tropomyosin receptor kinase A signalling205,206.
Neuropathies
Clinical trials involving the subcutaneous administration of NGF207 or brain-derived
neurotrophic factor (BDNF)208 to patients with diabetic neuropathy showed no overall
efficacy. However, the adequacy of dosing was not determined, and the maximum dose
was limited by pain in the NGF study207. In studies of subcutaneously administered NGF
for the treatment of HIV-associated neuropathy (at similar doses), the treatment was
well tolerated with improvements in pain symptoms but no objective improvement in
the neuropathy209,210.
Amyotrophic lateral sclerosis
In studies of intrathecal or subcutaneous administration of BDNF to patients with
amyotrophic lateral sclerosis there was no effect on clinical outcomes203,211. However,
the extent to which BDNF penetrated the brain and spinal cord parenchyma to reach
motor neurons was unknown.
Although these outcomes have contributed to a shift in the focus of neurotrophin
therapies, from systemic delivery of exogenous neurotrophins to local or invasive
delivery methods with sustainable sources (cell or viral vector-based), it should be
noted that it remains unknown whether adequate target engagement was achieved
in any of these clinical trials.
one involving the hairpin loops (in particular loop 1)

and the other at the amino terminus of mature neurotrophins52. The neurotrophin pro-domain, which is the
principal binding domain for the coreceptor sortilin,
has not been visualized crystallographically ostensibly
owing to its high flexibility and lack of ordered structure28. However, the pro-domain does appear to have a
role in altering the structure of the mature neurotrophin
domain in the p75NTRpro-NGF complex such that loop 2
of NGF and subjacent residues are exposed28. The physio
logical role of this NGF structure and the possibility of
utilizing it pharmacologically remain to be determined.
Proteolytic processing of p75NTR involves - and
secretases (reviewed in REF.24), which also cleave amyloid precursor protein. secretase mediates the generation
of amyloid- peptides, whereas secretases cleave within
the amyloid- domain, thus disallowing amyloid- peptide production. Therapeutics for Alzheimers disease that
target these enzymes (to increase -secretase or decrease
secretase expression) might therefore also modulate
neurotrophin signalling.
Status epilepticus
Prolonged epileptic
seizures that may result
in excitotoxic injury.
Do pro-neurotrophins invariably cause cell death? The

concomitant binding of sortilin and p75 NTR to proneurotrophin selectively promotes cell death54, which
requires the regulated shedding of the extracellular

domain of p75NTR by the metalloproteinase ADAM17 (a
disintegrin and metalloproteinase domain-containing
protein 17; also known as TACE) and regulated intra
membrane proteolysis of the receptor by secretase
(reviewed in REF.24). Regulated intramembrane proteo
lysis results in the release of a p75NTR intracellular domain
that mediates apoptotic cell death via JNK and caspase
activation (reviewed in REF.24). Pro-neurotrophins have
also been reported to stimulate TRK receptors, although
they are less effective than their mature counterparts5557.
In cells with high TRK/p75NTR ratios (for example, in
superior cervical ganglia, in PC12 cells and, following
seizures, in hippocampal neurons)5558, pro-neurotrophins
may promote, or at least not impair, cell survival. Together,
these observations suggest that pro-neurotrophin signalling through TRK receptors may counter their signalling
through p75NTR, allowing cells with high TRK/p75NTR
ratios to survive while those with low TRK/p75NTR ratios
undergo apoptotic death5560.
Cells with high p75NTR expression are most readily
detectable in basal forebrain cholinergic, dorsal root
ganglion and sympathetic neuron populations; however,
p75NTR is constitutively expressed by various other diseaserelated neurons, including those in the cortex, hippo
campus, basal ganglia and several brainstem nuclei6163.
In addition, although p75NTR expression may be increased
in disease and injury states6469, it may not always be associated with increased cell death64. Moreover, the balance
between mature and pro-neurotrophin signalling may
regulate local processes such as neurite outgrowth and
retraction as well as dendritic spine formation without
affecting cell survival7074. Overall, accumulating evidence
suggests that there is a functional balance of neurotrophin signalling in the central nervous system regulated
by the TRK/p75NTR ratio and the ratio of pro- to mature
neurotrophins7274 which may become disequilibrated
in disease states75.
Excess pro-neurotrophin levels may be associated
with conditions including Alzheimers disease, status
epilepticus, spinal cord injury and ageing, leading to
pathological effects such as a loss of neurites as well as
apoptotic neuronal or oligodendrocyte death8,9,58,67,7678.
Pro-neurotrophin levels may increase as a result of
increased neurotrophin synthesis and/or, importantly, as
a result of deficits in the conversion of pro-neurotrophins
to mature neurotrophins, which has been proposed as
a contributing factor in Alzheimers disease progression27,79,80 (FIG. 2). Following from these observations,
targeting pro-neurotrophinsortilinp75NTR interactions
could constitute a therapeutic strategy for conditions that
are characterized by excess levels of pro-neurotrophin.
The 13-amino-acid peptide sortilin ligand, neuro
tensin, inhibits pro-neurotrophinsortilin binding and
pro-neurotrophin-mediated neuronal cell death54. In
addition, a short sortilin peptide has been discovered
that is crucial for pro-neurotrophinsortilin binding
and apoptotic signalling, and that does not substantially
affect other functions of sortilin. These observations hold
the possibility of finding small molecules that selectively
inhibit pro-neurotrophin-induced cell death81.
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Mature neurotrophins signalling through TRK receptors.
Structurally, mature neurotrophins contain a central
saddle region49,82,83 that forms a principal TRK binding
site, as well as three hairpin turn loops (loops 1, 2 and
4), which display a relatively low degree of amino acid
similarity across the neurotrophin family and represent
three of the five neurotrophin variable regions84. These
variable regions contribute to multidomain receptor
interfaces and receptor specificity, and have therefore
had a key role in the structure-based development of
receptor-specific small-molecule ligands. Notably, current crystallographic data suggest that there are only
minor contacts between neurotrophin loop 1 and TRK
receptors; interactions with loops 2 and 4 have not been
visualized. These interactions may occur in the extracellular juxtamembrane portion of the receptor 49, but a
detailed structure of this region of the complex is not
yet available.
Neurotrophin binding induces TRK dimerization85,86,
although preformed TRK multimers may occur 8789,
and autophosphorylation of TRK receptors at multiple
tyrosine residues leads to the recruitment of different
intracellular signalling components and the activation
of downstream pathways2,38 (FIG.1). The existence of preformed TRK dimers is a crucial issue in the question of
whether a monovalent small-molecule ligand would be
capable of activating a TRK receptor, and is discussed further below. The multiplicity of ligand-induced TRK phosphorylation sites creates the opportunity for differential
patterns of phosphorylation induced by different ligands,
resulting in differential downstream signalling patterns
and potential outcomes. For example, within TRKA the
phosphorylation of Tyr490 is associated with the activation of the MAPKERK and PI3KAKT pathways,
whereas the phosphorylation of Tyr785 is associated with
the activation of the PLC PKC signalling pathway 2,38.
Signalling through TRK receptors largely promotes cell
survival and differentiation, although when unliganded it
may promote cell death2,38,90. TRKB and TRKC are found
in a large number of neuronal populations throughout the
central and peripheral nervous system in humans, whereas
the distribution of TRKA is more restricted to basal forebrain cholinergic, dorsal root ganglion and sympathetic
neurons, among others9193,217. Although neurotrophin and
neurotrophin receptors have overlapping expression patterns and functions in many areas of the brain, some neural
mechanisms are modulated by specific systems, including: NGFTRKA-mediated upregulation of cholinergic
function in the basal forebrain94; BDNFTRKB-mediated
promotion of synaptic plasticity 95,96; and NT3TRKCmediated survival of peripheral proprioceptive neurons97,98.
Consequently, the identification of agents that modulate
specific TRK receptors would potentially provide opportunities for more selective treatment of neurological disorders: for example, the activation of TRKA and TRKB
for Alzheimers disease, TRKB for stroke and trauma, and
TRKC for some forms of peripheral neuropathy.
Mature neurotrophins signalling through p75NTR. Unlike
the pro-neurotrophinp75NTRsortilin interaction, which
generally elicits strong death signalling, the interactions
of mature neurotrophin with p75NTR produce highly variable results depending on the cellular context24,99. In the
absence of a cognate TRK, mature neurotrophins may
promote cell death. For example, this has been observed
in oligodendrocyte cultures expressing p75NTR but not
TRKA32 that have been treated with NGF, and in sympathetic neurons expressing TRKA and p75NTR but not
TRKB that have been treated with BDNF100. Conversely,
in other cell types or under different experimental conditions, the binding of mature neurotrophins to p75NTR
may increase cell survival via the inhibition of constitutive p75NTR apoptotic signalling101,102 and/or the activation
of pro-survival factors such as NFB and AKT103,104.
Further complications and opportunities for pharmacological targeting arise in consideration of the associations and functional interactions of TRK receptors and
p75NTR. As noted above, numerous studies have identified
potential points of interaction, from direct interactions
at the cell surface to distal signalling overlaps. TRK activation modulates the uptake, processing and signalling
of p75NTR, and p75NTR has important effects on TRK
neurotrophin binding and signalling 46,47,49,51,105110. This
crosstalk, to some extent, constrains the responses of
systems to a single neurotrophin, and provides the possibility that a pharmacological intervention that decouples
the receptors (either TRK receptors or p75NTR) activities
could produce effects that are not achievable with native
ligands. In addition, p75NTR functions as a coreceptor
for the myelin-associated neurite outgrowth inhibitors
Nogo receptor and LINGO1 (leucine-rich repeat and
immunoglobulin domain-containing 1)111113. The presence of such coreceptors would be expected to locally
modify the availability and functions of p75NTR and,
consequently, the effects of p75NTR-targeted compounds.
Thus, the complex landscape of p75NTR signalling mechanisms allows a broad range of possibilities by which specific ligands might influence p75NTR in a manner distinct
from native neurotrophins.
Further considerations for drug discovery. In addition
to the diseases mentioned above, there are several dis
orders in which TRK or p75NTR signalling mechanisms
are directly linked to processes underlying disease onset
or progression, and representative disorders with nervous
system relevance are listed in Supplementary information
S1 (table). In terms of drug discovery, disease processes
that may be amenable to treatment via neurotrophin
receptor modulation fall into three categories: those in
which impaired neurotrophic signalling is a significant
pathogenic factor; those in which such a deficiency is
not causative or necessarily present but in which neurotrophin signalling counteracts or favourably modulates
pathological signalling; and conditions involving excess
neurotrophin activity (FIG.2).
For many complex disorders, several of these possibilities for modulating neurotrophin receptors may be
applicable during the course of disease pathogenesis,
and they may function only in specific anatomic loci
or cell types. An example of this is Alzheimers disease,
in which amyloid- can have both direct and indirect
effects on the pathogenesis of the disease (BOX2). Impaired
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neurotrophin synthesis or transport, as occurs in Rett syndrome and Huntingtons disease, for example, may lead to
shrinkage and dysfunction of neuronal cell populations
through a primary loss of trophic signalling 114117. In addition, diminished anterograde transport of neurotrophins
may deprive remote target cell populations of support,
producing a degeneration of neuronal networks114117.
Similar processes may occur in pericontusional areas
following traumatic brain injury 118. Excessive neurotrophic
signalling may lead to aberrant dendritic sprouting,
decreased pruning and altered electrophysiological properties119. The formation of abnormal neuronal networks
engendered by these effects has been implicated in the
production of pain and in epileptogenesis120122.
As a result, the underlying cause of the disease or
condition needs to be sufficiently understood to enable the appropriate corrective modulatory effect to be
elicited when targeting neurotrophin receptors. In the
majority of cases involving TRK receptors, increases
in TRK activity are sought for therapeutic effects.
Conversely, in other cases such as in the control of
pain, in which NGFTRKA signalling is a contributing
factor inhibition of signalling is desired123. For example, small-molecule TRKA antagonists might provide a
means for modulating TRKA activity as an alternative
or adjunctive therapy to NGF-specific antibody treatment 124,125. Similarly, as excess levels of neurotrophins
such as BDNF might promote certain forms of epilepsy,
the application of TRKB antagonists could be considered in this setting 126. In the case of p75NTR, therapeutic
efforts may involve the inhibition of its degenerative signalling and/or the activation of associated pro-survival
pathways51.
Below, we discuss the structural aspects of modulating
neurotrophin receptors and outline the strategies that
are currently being pursued.
Small ligands for neurotrophin receptors?

In consideration of the interaction of small molecules
with large, multidomain, multifunctional receptor systems, many traditional pharmacological concepts and
the associated terminology (that is, describing a ligand as
an agonist, partial agonist or antagonist) must be revisited in the context of neurotrophins and their receptors.
Given that the crosswise looptoloop dimensions of
neurotrophin dimers (>25) are beyond those that are
typically bridged by monovalent small molecules (molecular mass <500 Da), if induced dimerization at neuro
trophin binding sites was required for activation then
the development of monovalent small-molecule ligands
activating TRK receptors would seem infeasible. However,
alternative models of dimeric receptor activation, elucidated by studying other receptor systems, have emerged.
In one model, spontaneous receptor dimerization occurs
(resulting in preformed dimers) and dimeric or even
monomeric ligands serve to stabilize these complexes
and thereby favour activation states127130. In another
model, dimeric or monovalent ligand binding to monomeric receptors induces a conformational change in the
receptor, which in turn leads to dimerization and receptor
activation131,132. Finally, small-molecule ligands might bind
to a site outside the ligand-binding domain and thereby

induce dimerization (via allosteric mechanisms)133,134.
Both the TRK and p75 NTR populations may be
partially dimerized in the unliganded state, with the
potential for further complex formation induced by
neurotrophin binding 53. In the case of TRKA, single
amino acid changes in the extracellular domain lead to
spontaneous dimerization and activation, presumably
through a conformational change135, which suggests that
conformational changes induced by monovalent small
molecules might achieve receptor activation. Each of
these alternative models is compatible with the possibility of developing small-molecule ligands for inducing
the dimerization and/or activation of TRK receptors and
p75NTR. However, further considerations regarding the
interactions of small molecules with macromolecular
complexes need to be taken into account when planning
a search for or designing such ligands.
Neurotrophins, like many protein ligands, interact
with their receptors through multiple domains, some of
which may principally mediate binding, whereas others
recognize and/or promote receptor conformations associated with activation. Moreover, within the same ligand
molecule, some domains may mediate interactions that
are specific to a single ligandreceptor pairing, whereas
others may mediate molecular family or broader interactions. Given the limited number and spacing of the
chemical motifs present in a single monomeric small
molecule, it is unlikely that it can emulate the full set of
multidomain receptor interactions exhibited by native
neurotrophins. Therefore, it is highly unlikely in most
cases that small molecules targeting neurotrophin receptors would function as full neurotrophin mimics. Indeed,
experiences with neurotrophin-domain peptidomimetics
and early-generation non-peptide small molecules (discussed below) support this view. Hence, rather than using
terms such as neurotrophin mimetic we prefer to use
descriptive terms such as neurotrophin receptor ligands
or neurotrophin receptor-modulating ligands.
Small molecules may bind to receptors at sites that
would otherwise be occupied by only one of the several
domains within a bound protein ligand. Depending on
the contribution of that site to the total binding energy
of the ligandreceptor complex, and the flexibility of the
ligand, such binding might or might not result in substantial displacement of the native ligand from the receptor.
Moreover, small molecules that bind with lower affinity
at receptor-activating sites within the canonical binding site or other locations may promote conformational
changes that activate the receptor. Because of these factors,
traditional approaches that involved screening for smallmolecule ligands of neurotrophin receptors, and that relied
primarily on competitive binding assays, largely failed to
identify novel ligands. Interestingly, one such screen to
identify molecules that target the NGFp75NTR interaction
yielded a binding inhibitor that bound to NGF rather than
to the receptor 136. Functional and/or biological screens
have been more successful, including high-throughput cell
survival screens for TRKB ligands137,138 and coupled insilico
low-throughput cell survival screens for p75NTR ligands139,
which are described in more detailbelow.
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Decreased
synthesis and/or
transport
Amyloid-
Excitotoxicity
Cytokines
Chemotherapy
Ischaemia
Trauma, and so on
Pro-neurotrophin
Increased
synthesis,
decreased
degradation
Decreased
proteolytic
conversion
Pro-neurotrophin
Neurotrophin
Neurotrophin
Status epilepticus
Ageing
Rett syndrome
Huntingtons
disease
Alzheimers
disease
Pro-neurotrophin
Neurotrophin
Neurotrophin
Activated
microglia
Deleterious signalling
RHOA
JNK
Calpain
Caspases
GSK3, and so on
Pro-survival signalling
AKT
NF-B
ERK, and so on
Astrocytes
Sprouting
Pruning
Pain
Epilepsy
Altered synaptic function

Decreased neurite integrity
Demyelination
Decreased survival
Figure 2 | Neurotrophin signalling and pathological effects. Changes in absolute neurotrophin levels or in the
ratio of pro-neurotrophin to mature neurotrophin can cause and/or contribute to numerous
pathogenic
Nature Reviews
| Drugstates.
Discovery
Decreases in neurotrophin synthesis or transport (as in Rett syndrome or Huntingtons disease) may activate or
coactivate, along with other toxic stimuli (for example, amyloid- or excess glutamate), signalling that results in
cellular injury. Increased pro-neurotrophin/mature neurotrophin ratios (either owing to decreased conversion as
observed to occur in ageing, or owing to overt increases in pro-neurotrophin secretion, as hypothesized to occur
in status epilepticus) may also contribute to these pathological effects. Similar injury mechanisms are activated
following trauma and ischaemia as well as in multiple sclerosis. The activation of signalling pathways that counter
these mechanisms via pharmacological modulation of neurotrophinreceptor systems therefore has the potential
to have broad therapeutic effects. In addition, excess or misplaced neurotrophic or neuritogenic signalling, owing to
increased synthesis or decreased degradation of mature neurotrophins, might occur in an attempt to compensate
for deficits; this can lead to pathological states such as chronic pain and epilepsy, and may contribute to abnormalities
in cortical migration. See the main text and Supplementary information S1 (table) for further details of aberrant
neurotrophin signalling and its pathological effects.
The multi-receptor neurotrophin system and its context-dependent activity profiles complicate the application
of traditional pharmacological terms such as full agonist,
partial agonist and antagonist. For instance, a small
molecule might bind to TRKB to promote the survival of
TRKB- and p75NTR-coexpressing neurons with a lower
efficacy than BDNF, and it may appear to function as a
partial agonist. By contrast, the application of the same
small molecule and BDNF to neurons in which p75NTR
expression has been eliminated might result in similar
efficacies, leading to the conclusion that the compound
is a full agonist. In another case, a small-molecule p75NTR
ligand might promote neuronal survival with an efficacy
similar to NGF in a particular assay, resembling the profile
of a full agonist; however, in another assay, in which NGF
promotes cell death, the same small-molecule ligand may
continue to promote survival and be considered a reverse
agonist.
Comparative signalling studies involving the various

neurotrophins and their receptors support the possibility
that small-molecule ligands targeting these receptors may
trigger signalling patterns that are distinct from those promoted by the native ligands. One study 140 demonstrated
that NGF and NT3 both triggered prolonged TRKA activity; however, in the same assay system NGF caused an
acute TRKA activation that was several times higher than
that stimulated by NT3. Moreover, under conditions that
equalized the acute activation, NGF was markedly more
effective in supporting neuronal survival. These findings
were consistent with a mechanism in which NGF and
NT3 differentially interacted with or promoted the formation of distinct TRKA conformations, dimerization
states and/or interactions with p75NTR, resulting in different signalling patterns140.
The potential for differential signalling by TRKA
was also highlighted by the observation that NGF and
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Box 2 | Multiple pathways that can be targeted in Alzheimers disease
In Alzheimers disease, the pathogenic molecule amyloid- can have both direct
and indirect effects on the pathogenesis of the disease, which can have
implications in terms of determining which pathways to target for therapeutic
purposes.
First, amyloid- can interact directly with p75 neurotrophin receptor (p75NTR),
leading to aberrant signalling and cell death212214. Second, amyloid- can interact
with various cellular receptors unrelated to neurotrophins to initiate pathogenic
signalling, including the activation of JUN N-terminal kinase (JNK), calpains,
caspases and glycogen synthase kinase 3 (GSK3), leading to excessive tau
phosphorylation215. These alterations in signalling can result in structural and
functional changes in the brain, including decreased branching, retraction of
dendritic trees, loss of synaptic boutons and dendritic spines as well as deficits
in long-term responses to stimuli196,216.
Downstream signalling pathways that may be activated by neurotrophin receptors,
including those involving AKT, nuclear factor-B (NFB) and extracellular
signal-regulated kinase (ERK), may interact with injurious pathways activated by
amyloid- at multiple points, counteract the deleterious signalling and thereby
improve neuronal function and integrity.
Finally, Alzheimers disease is associated with impaired proteolytic processing of
neurotrophins, leading to an unmasking of degenerative signalling that is mediated
by excess levels of the precursor (pro-neurotrophin) and a deficit in mature forms of
the protein (FIG.2).
These mechanisms suggest that modulating neurotrophic receptor signalling
could have potential benefits in Alzheimers disease. Indeed, the effects of such
modulation on multiple mechanisms that contribute to the development of
Alzheimers disease may be synergistic and therefore lead to particularly effective
therapeutic outcomes.
an NGF antibody complex composed of NGF and an

NGF-specific antibody directed to the carboxyl terminus (NGF-mAb) bound to TRKA with high affinity,
but NGF stimulated longer-lasting MAPK activation
resulting in both neuronal survival and neurite outgrowth, whereas the NGF-mAb promoted survival but
not neurite outgrowth141. In another example, mutation
of the SHC binding site in TRKB led to decreased neuro
trophic activity of NT4 with a relatively spared BDNF
response142, which suggests that NT4 and BDNF may
differentially activate TRKB signalling.
Thus, the pharmacological profiling of small-molecule
neurotrophin receptor ligands should include a range of
assays that allow a broader degree of discrimination that
goes beyond simple classifications limited to agonist
versus antagonist activity. Moreover, the potential to
create novel neurotrophin receptor signalling patterns
with small-molecule ligands provides a rationale for their
development that exceeds the goal of simply producing
neurotrophin mimetics and also involves improving
the pharmacokinetic features of neurotrophins. Indeed,
many ligands with so-called partial agonist and other
profiles may present pharmacological advantages that
make them more effective in a disease setting.
Kindling-induced mossy
fibre sprouting
Repetitive small seizures or
other abnormal electrical
activity leading to the growth
of hippocampal dentate output
axons, which enhance the
likelihood of seizure activity.
Peptide ligands
The identification of synthetic peptides corresponding
to specific domains of neurotrophin ligands (FIG.1) with
antagonist or agonist activity established the vital proof
of concept that small molecules, including those that
bind monomerically, might be capable of modulating
neurotrophin receptor function and, in some cases, provide a useful basis for the development of non-peptide
small-molecule ligands. The development of most of
these first-generation synthetic ligands targeting neurotrophin receptors focused on the modelling of domains
within NGF, BDNF orNT3.
NGF peptides. An early approach for the development of
ligands targeted to neurotrophin receptors consisted
of creating small synthetic peptides with amino acid
residues corresponding to various domains of NGF and
assessing them for their ability to inhibit or mimic the
neurotrophic function of NGF (reviewed in REFS143145).
Before the crystal structure of NGF had been derived, a
peptide scanning strategy of mouse NGF demonstrated
that small linear synthetic peptides containing the LysGly-Lys-Glu (KGKE) sequence which was later
found to comprise the core of the NGF loop 1 domain
inhibited NGF activity invitro146.
The first small peptide corresponding to a specific
NGF domain to demonstrate neurotrophic activity consisted of a cyclized dimeric form of a KGKE-containing
peptide (peptide P7)194. The addition of a p75NTR-specific
antibody that was capable of blocking NGF binding
and activity, or the use of neurons lacking p75NTR expression (Ngfr/ neurons), eliminated P7 activity, whereas
the pan-TRK inhibitor K252a had no effect, which suggested that this peptide acted through p75NTR. These
data are consistent with site-directed mutagenesis
studies indicating that the Lys32 and Lys34 residues
of NGF are crucial for the binding of NGF to p75NTR
(REF.147). Peptides containing KGKE or a homologous
sequence have since been shown to: block the binding
of amyloid- to p75NTR and its ability to induce neuronal
death148; inhibit apoptosis-driven hair follicle involution, a process that is thought to involve p75NTR signalling 149; modulate kindling-induced mossy fibre sprouting in
a rat model of epilepsy 150; and decrease post-axotomy
retinal ganglion cell death in rats, which is another
p75NTRdependent process151. This spectrum of actions
highlights the biological and therapeutic potential of
developing non-peptide small molecules that incorporate features of the loop 1 domain and are capable of
modulating p75NTR function.
Site-directed mutagenesis studies indicated that the
loop 4 domain of NGF was one of several domains that
was likely to interact with TRKA, and peptide mimetics
of this domain had an important role in developing
early perspectives of howsmall-molecule ligands might
engage TRK receptors. Cyclized, monomeric loop 4
peptides (C(9296) and C(9297)) blocked NGFinduced PC12 cell neurite outgrowth, and when they
were assayed in the absence of NGF they had no neurotrophic activity, which suggests that they had antagonist
activity but no agonist activity 152. However, under conditions in which p75NTR was bound by a monoclonal antibody, monomeric C(9296) supported the survival of
PC12 cells and other cells (at ~3050% of maximal NGF
efficacy), demonstrating a partial agonist profile and
illustrating the principle that agonism and antagonism
are context-dependent 153.
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A cyclized dimeric peptide, C(9297)dimer, induced
TRKA dimerization, phosphorylation and internalization in neuroblastoma cells overexpressing TRKA153.
C(9297)dimer promoted the survival of neuroblastoma
cells at ~20% of NGF efficacy and of PC12 pheochromo
cytoma-derived cells at 5% of NGF efficacy 153. This profile further indicated that synthetic peptides could, at
least partially, activate TRKA survival signalling. In a
concomitant study 154, an NGF loop 4 cyclized dimeric
peptide (P9297) supported neuronal survival and
neurite outgrowth of dorsal root ganglion neurons (with
an efficacy of ~30% of that of NGF)154 and stimulated
TRKA autophosphorylation (F.M.L. and Y. Xie, unpublished observations), thereby offering a parallel line
of evidence that NGF loop 4 peptides are capable of
inducing TRKA-mediated survival signalling.
The screening of turn peptidomimetics that contained a macrocyclic ring incorporating amino acid
side chains corresponding to NGF turn loops led to
the identification of a peptidic monovalent compound
termed compound D3 that bound to TRKA,
promoted the formation of TRKA homodimers and
achieved ~40% of the maximum efficacy of NGF in promoting the survival of dorsal root ganglion neurons155.
This finding is particularly noteworthy because it demonstrated that a monovalent compound (compound D3;
TABLE1) was capable of inducing TRKA dimerization
and thereby suggested that non-peptide monovalent
small molecules might also be found that induce TRK
receptor dimerization and activation. In subsequent
studies, compound D3 was shown to: rescue basal
forebrain cholinergic neuron degeneration and spatial
memory in aged rats when administered by intracerebroventricular minipump156; prevent the death of retinal
ganglion cells in a rat model of glaucoma when administered by intraocular injection157; and prevent retinal
ganglion cell degeneration in a post-axotomy rat model
following intravitreal injection151.
In the J20 APPSwe/Ind mouse model of Alzheimers
disease, which develops amyloid plaques and memory
impairment, the administration of compound D3 by
intracerebroventricular minipump led to improved learning andshort-term memory but caused persistent deficits
in long-term memory 12. In addition, a related peptidomimetic called MIMD3 increased glycoconjugate secretion
and improved a measure of corneal injury in a rat model
of dry eye syndrome218. MIMD3 completed PhaseII
clinical trials for the treatment of dry eye syndrome
(ClinicalTrials.gov identifier: NCT01257607).
In another peptide approach that is relevant to TRKA,
a bicyclic peptide containing components of both loop
1 and loop 4 of NGF had neurotrophic activity in the
micromolar range and induced phosphorylation of TRKA
but notof TRKB158. These peptide studies demonstrated
that TRKA ligands could elicit a range of biological outcomes and encouraged the search for active non-peptide
monomeric small molecules targeting TRKA.
BDNF and NT3 peptides. Synthetic peptides corresponding to loop 2 and loop 4 of BDNF have also been characterized for their effects on cell survival alone and in the
presence of BDNF. Loop 2 monomeric cyclized peptides

antagonized the binding of BDNF to receptors and had
no intrinsic activity 159, whereas cyclized dimeric loop 2
peptides promoted survival but these peptides had less
efficacy than BDNF160. Whether these peptides promoted
TRKB activation was not directly established. Synthetic,
linear, monomeric tetrapeptides derived from the loop 4
and carboxyterminal regions of BDNF stimulated
phosphorylation of TRKB but not TRKC, and exhibited
neurotrophic activity in hippocampal neuron cultures161.
Importantly, in addition to directly activating TRKB,
these peptides promoted BDNF expression, thus highlighting the importance of considering a secretagogue
effect in neurotrophin small-molecule studies.
In another study, a cyclic pentapeptide derived from
a putative p75NTR-binding region of BDNF loop 4 had
neurotrophic effects without activating TRKB162. In addition, this peptide produced p75NTR-dependent increases
in the myelination of dorsal root ganglion neurons in
culture162,163. A cyclized peptide termed cyclotraxinB,
which encompasses nine residues from the highly variable region III of BDNF (adjacent to the loop 3 domain),
inhibited both BDNF-mediated and basal TRKB phosphorylation invitro164. A Tat-fused form of cyclotraxin,
which was designed to allow CNS delivery after intra
venous administration, was given to mice and resulted in
decreased TRKB activation in the brain as well as reduced
anxiety behaviour. In NT3related studies, turn peptido
mimetics that are designed to mimic ligand-interacting
regions of NT3 have been developed that selectively bind
to and activate TRKC165,166.
Together, these studies show that peptidomimetics are
capable of modulating neurotrophin receptor function.
However, given the limitations of peptide compounds for
drug development, including their stability, bioavailability
and bloodbrain barrier penetration, a higher priority
has been placed on identifying and creating non-peptide
small molecules that are capable of modulating neurotrophin receptors.
Small-molecule ligands for neurological diseases

TRKA ligands. The screening of combinatorial compound libraries identified several TRKA activators.
These include an asterriquinone (1H5) and a monoindolyl-quinone (5E5) that activated TRKA, possibly by
binding to an intracellular site, and promoted PC12 cell
survival at low micromolar concentrations167. Another
screen identified gambogic amide (MW 628), which
prevented the death of a TRKA-expressing cell line168
(TABLE1). Interestingly, gambogic amide bound to the
intracellular juxtamembrane domain of TRKA rather
than the extracellular ligand-binding region. This finding
suggests that the compound causes allosteric activation
of the receptor, and is consistent with studies of other
receptor kinases (including the insulin receptor) that
have identified small molecules that activate receptors
through interactions with regions that are not involved
in the binding of the physiological ligand169. Additional
studies will be necessary to establish the degree of
specificity of gambogic amide for TRKA. Nevertheless,
gambogic amide activated TRKA and its downstream
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Table 1 | Ligands targeting neurotrophin receptors and invitro activity
Compound
Structure
In vitro activity
Refs
TRKA
Compound D3
NH2
HO
N
H
NH
Inhibits death of dorsal root

ganglion neurons at 10M
155
Inhibits death of hippocampal

neurons at 100250nM
168

neurons at 530M
124

neurons at 10250nM
170

neurons at 10250nM
137

neurons at 100250nM
Inhibits death of cortical
neurons at 500nM
138
O
HN
HO
NH+
HO
HN
HO
Gambogic amide
O
H2N
O
H
OH
MT2
N O
TRKA and TRKB

Amitriptyline
TRKB
7,8dihydroxyflavone
HO
OH
Deoxygedunin
O
O
H
O
H
O
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Table 1 (cont.) | Ligands targeting neurotrophin receptors and invitro activity
Compound
Structure
In vitro activity
Refs
TRKB (cont.)
LM22A4
O
HO
N
H

neurons at 0.5500nM
183
Inhibits BDNF-induced
neurite outgrowth of
TRKBPC12 cells at
0.01100M
188

neurons at 1001,000pM
139

neurons at 1001,000pM
Inhibits binding of NGF to
p75NTR in ELISA at 50nM
139,202
OH
N
H
NH
OH
ANA12
S
O
NH
O
HN
O
N
H
p75NTR
LM11A31
H2N
HN
N
O
LM11A24
N
HN
O
N
N
N
THXB
HN
N
O
N
N
O
Inhibits pro-neurotrophininduced death of B104 and

PC12 NNR5 cells at 20M
Inhibits binding of NGF to
p75NTR in ELISA at 50nM
202
BDNF, brain-derived neurotrophic factor; ELISA, enzyme-linked immunosorbent assay; NGF, nerve growth factor; p75NTR, p75
neurotrophin receptor; TRK, tropomyosin receptor kinase.
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signalling partners AKT and ERK, and promoted the
survival of cultured hippocampal cells, which under
the conditions utilized were reported to express
TRKA168. In mouse studies, subcutaneous administration
of gambogic amide prevented neuronal death induced by
kainic acid or ischaemic stroke.
From the same screening programme that identified
gambogic amide, but published separately 170, the antidepressant drug amitriptyline (TABLE1) was shown to
bind the extracellular domains of TRKA and TRKB and
induce their activation. Interestingly, amitriptyline promoted TRKATRKB heterodimerization (which does not
occur with NGF or BDNF), which further suggests that
this compound induces alternative signalling outcomes170.
Amitriptyline protected cultured hippocampal cells from
apoptosis, stimulated neurite outgrowth in PC12 cells and,
in invivo studies, reduced kainic acid-triggered neuronal
cell death170. Studies in inducible TRKA-null mice have
supported a key role for TRKA in mediating the effects
of amitriptyline170; however, given the broad spectrum of
mechanisms affected by this compound, the issue of target
specificity needs to be carefully considered.
Another approach for creating TRKA ligands incorporates the principle that most proteinprotein interactions,
including the binding of NGF to TRKA, involve two or
more binding sites known as hotspots171,172. Therefore,
a small molecule is more likely to be capable of activating
TRKA if it includes two pharmacophores corresponding to such regions173. With this in mind, bivalent turn
mimics were created as minimalist mimics of the peptidic turn structures that are present in NGF, and four
compounds were found to selectively bind to the TRKA
receptor 173. Additional studies will be required to determine the extent to which these compounds might promote or inhibit receptor function.
Several small bicyclic peptidomimetic compounds
were identified from a chemical library to interact with the
immunoglobulin-like domain D5 (an area that interacts
with the saddle region of NGF) of TRKA124. Interestingly,
although the lead compound MT2 (TABLE1) produced a
robust effect on the survival of PC12 cells, similar to that
of NGF, it was substantially less capable of inducing TRKA
phosphorylation, the expression of the NGF-inducible
gene VGF and differentiation of PC12 cells124. Further
analysis showed that compound MT2 and NGF stimulated TRKATyr490 phosphorylation to a similar degree,
whereas compound MT2 induced significantly less phosphorylation at Tyr674, Tyr675 and Tyr785, which suggests
that there is differential activation of signalling between
the compound and NGF124. Whether this distinctive signalling pattern provides any therapeutic advantages or
disadvantages relative to NGF remains to be determined.
Taken together, these studies demonstrate that small
molecules can be created, or otherwise identified, that
are capable of activating TRKA, in some cases through
non-ligand receptor sites. A remaining challenge in most
cases is demonstrating the degree of specificity forTRKA.
TRKB ligands. A cell line-based assay to screen compounds for their ability to inhibit apoptosis in TRKBtransfected cells relative to the TRKB-negative parental
line has yielded various interesting TRKB ligands137. One

compound, 7,8dihydroxyflavone (7,8DHF; TABLE1),
induced the phosphorylation of TRKB as well as its
downstream targets AKT and ERK. 7,8DHF inhibited
neuronal death invitro, with an efficacy approximately
equal to that of BDNF, and its activity was blocked by
K252a. Whether the phosphorylation of TRKBTyr490
or other tyrosine residues was stimulated in the cells
screened was not determined. Nevertheless, 7,8DHF
binds to the cysteine cluster 2 (CC2) and leucine-rich
region (LRR) in the extracellular domain of TRKB.
As the LRRs have been implicated in BDNF binding, this
finding suggests that 7,8DHF might produce its effects
at least partially through mechanisms that are similar to
those of BDNF137. Of note, this TRK binding localization
is similar to that of amitryiptyline170, but differs from
that of gambogic amide (which binds to the intracell
ular domain)168 and compound MT2 (which binds to the
immunoglobulinlike domain D5)124, and illustrates the
diversity of activation modes that are identifiable using
functionalassays.
Interestingly, diosmetin, a compound related to
7,8DHF, had similar anti-apoptotic effects but induced
only weak phosphorylation of TRKB, yet it upregulated
phosphorylated AKT and phosphorylated ERK to a
similar degree137. Furthermore, other related compounds
including pinocembrin and quercetin had minimal effects on TRKB, and upregulated phosphorylated
ERK but not phosphorylated AKT137, again illustrating the different patterns of TRK signalling that can be
induced by small-molecule ligands. Studies in TrkbF616Amutant knockin mice in which TRKB activity can be
selectively eliminated by the kinase inhibitor analogue
1NMPP1 (4amino1tert-butyl3-(1-naphthylmethyl)
pyrazolo(3,4d)pyrimidine) supported the notion
that the effects of 7,8DHF were dependent on TRKB
tyrosine kinase activity, although direct detection of
TRKB activation was not demonstrated invivo137. A subsequent study using a cell line lacking TRKB found that
7,8DHF protected against glutamate-, hydrogen peroxide- and menadione-induced toxicity while increasing
glutathione levels and reducing levels of reactive oxygen
species a profile that is consistent with an antioxidant
effect 174. Thus, the mechanisms of action of 7,8DHF
in a given setting may be complex, and may include
mechanisms that are independent of TRKB activation.
The efficacy of 7,8DHF (administered either orally
or parenterally) has been examined in several disease
models in which BDNFTRKB mechanisms are thought
to have a role. These include the neuroprotective effects
of BDNFTRKB signalling in mouse models of kainic
acid-, ischaemia- and MPTP (1methyl-4phenyl
1,2,3,6tetrahydropyridine)-induced injury 137 and in
the 5XFAD transgenic mouse model of Alzheimers disease175. Improved learning and behavioural outcomes
were also elicited with 7,8DHF in rodent models with
prior exposure to traumatic stress176,177. Morphologically,
7,8DHF partially reversed age-related dendritic spine
loss in an invitro hippocampal slice model178, and prevented increased dendritic length in the amygdala and
the occurrence of a depressive profile in a rat model of
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depression179. Along with a more potent derivative
4dimethylamino7,8dihydroxyflavone 7,8DHF
also promoted TRKBTyr816 phosphorylation in the
dentate gyrus along with progenitor cell proliferation
and antidepressant effects in the forced swim test 180.
A follow-on programme of pharmacological optimization yielded a derivative of 7,8DHF, 2methyl8(4-(pyrrolidin-1yl)phenyl)chromeno[7,8d]imidazol
6(1H)-one, which showed similar behavioural effects but
had improved pharmacokinetic and metabolism profiles181. 7,8DHF may also ameliorate certain phenotypical features of genetic diseases. In a mouse model of Rett
syndrome, in which lossoffunction mutations in the
gene encoding methyl CpG-binding protein 2 (MECP2)
lead to reduced BDNF levels and abnormal motor, respiratory and cognitive function, 7,8DHF increased survival
and reduced abnormal breathing patterns182.
Another compound that was identified from smallmolecule screens using TRKB-expressing cell lines is
deoxygedunin (TABLE1), which is a derivative of gedunin
and a tetranortriterpenoid with antimalarial, insecticidal and anticancer activity 138. Deoxygedunin binds
to the ECD of TRKB to stimulate its dimerization and
autophosphorylation without the activation of TRKA or
TRKC. It inhibited the death of cultured hippocampal
neurons (induced by oxygen and glucose deprivation)
and the glutamate-induced death of cultured cortical
neurons in a TRKB-dependent manner 138. Deoxygedunin
also rescued vestibular ganglion neurons in Bdnf/ mice;
in wild-type mice the compound exhibited antidepressant activity and enhanced the acquisition of conditioned
fear a BDNF-dependent learning process138.
Distinct from high-throughput small-molecule
screening, the insilico screening of small-molecule libraries with a pharmacophore modelled on the loop 2 domain
of BDNF was used to identify TRKB agonists183. These
TRKB agonists were then screened for their ability to
prevent the death of primary hippocampal neurons. One
compound, LM22A4 (TABLE1), bound to TRKB but not
to TRKA, TRKC or p75NTR, and inhibited the binding of
BDNF but not of NGF or NT3 to their cognate receptors.
Further evidence of the specificity of LM22A4 was demonstrated in the following ways: through the inhibition
of its neurotrophic activity by K252a; using an antibody
directed to the ECD of TRKB; and through the lack of
binding in a receptor selectivity screen183.
Upon closer examination of its activity, LM22A4 was
shown to promote the survival of hippocampal neurons
(at ~85% of the efficacy of BDNF) and induce the phosphorylation of TRKBTyr490 (at ~30% of the efficacy of
BDNF)183. The phosphorylation of other tyrosine moieties was not examined. Interestingly, similar to the effects
noted with compound MT2 on TRKA and the effects
noted with diosmetin on TRKB, LM22A4 promoted
robust upregulation of ERK and AKT phosphorylation
at lower levels of activation of TRKB phosphorylation compared to the levels induced by native neurotrophins183. Other compounds that were identified in
the screening, and that were chemically distinct from
LM22A4, produced even lower and more delayed
TRKB phosphorylation, again with strong upregulation
of ERK and AKT phosphorylation183. Notably, stimulation

of AKT and ERK was completely reversed by K252a or
an antibody targeting the ECD of TRKB, which suggests
that interaction with the ECD of TRKB and the phosphorylation of TRKB were essential components of the
response183.
In invitro models of Alzheimers, Huntingtons and
Parkinsons disease, LM22A4 prevented neuronal death
at efficacies equal to that of BDNF. In wild-type mice,
LM22A4 stimulated the activation of TRKB along with
AKT and ERK in hippocampal and cortical tissue; furthermore, in rats inflicted with traumatic brain injury,
treatment with the compound led to enhanced recovery
of motor function in rotarod testing 183. In other pathological contexts that are thought to involve BDNFTRKB
signalling mechanisms, LM22A4 (administered parenterally) led to a normalization of brainstem TRKB activation and respiratory patterns in the Mecp2mutant mouse
model of Rett syndrome184, and increased neurogenesis
in the subventricular zone and enhanced the recovery of
motor function in a mouse model of post-stroke recovery 185. Cortical inhibitory interneurons express TRKB
and are supported by BDNF that is derived from target
neurons and transported in a retrograde manner 186; in
a rat model of post-traumatic epilepsy, in which these
neurons undergo axotomy and subsequent degeneration,
intranasal application of LM22A4 prevented post-injury
loss of expression of the 3 subtype (Na++K+) ATPase in
interneuron terminals187.
Another virtual screening approach188 modelled the
interaction of the Nterminal region of BDNF with the
immunoglobulin-like domainD5 of TRKB to screen
a database of commercially available drug-like compounds. Hits were then screened for their ability to
inhibit BDNF-induced TRKB activation. Compound
ANA12 (TABLE1) inhibited TRKB phosphorylation with
submicromolar potency and bound to TRKB noncompetitively with BDNF. In PC12nnr5 cells (which lack
TRKA) that were transfected to express specific TRK
receptors, ANA12 inhibited BDNF-induced neurite
outgrowth at concentrations as low as 10nM, with full
inhibition in the micromolar range. No inhibitory activities for TRKA or TRKC were detected, which suggests
that ANA12 is specific to TRKB; however, the results of
a broad screen for receptor binding will be of interest.
In invivo studies, intraperitoneal administration of
ANA12 inhibited TRKB phosphorylation in the striatum, cortex and hippocampus. Importantly, ANA12
did not appear to significantly promote neuronal
death, which is one potential consequence of inhibiting BDNFTRKB signalling; however, a small increase
in the number of apoptotic cells was observed in the
dentate gyrus of mice that received the highest dose of
ANA12 (REF.188). Excessive BDNFTRKB signalling in
reward circuitry promotes the development of anxiety
and depression in rodent models, and the administration
of ANA12 was able to substantially reduce anxiety and
depression-related behaviour 188. Subsequent studies have
demonstrated that ANA12 may also be a useful tool for
examining the involvement of BDNFTRKB signalling
in physiological processes; for example, the compound
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inhibits the reduction in food intake caused by BDNF
delivery to the medial nucleus tractus solitarius189 and
reverses the 7,8DHF-mediated improvements in methamphetamine-induced deficits in prepulse inhibition of
acoustic startle190.
Nacetylserotonin (NAS), a compound that is normally
expressed in the pineal gland and retina, has antidepressant effects, and many antidepressant compounds are
associated with elevations in BDNF levels; these findings
have led to the testing of NAS for its TRK-activating properties. NAS activated TRKB, but not TRKA, and achieved
antidepressant effects in a TRKB-dependent manner 191.
A NAS derivative, N-[2-(5hydroxy-1Hindol3yl)
ethyl]-2oxopiperidine-3carboxamide (HIOC), was subsequently identified, which activates the TRKB receptor
with greater potency than NAS and inhibits light-induced
retinal degeneration192. The extent to which NAS and
HIOC bind specifically to TRKB to function as direct
agonists will need to be established through additional
studies.
Together, these TRKB ligand studies show the feasibility of developing non-peptide small molecules that are
capable of binding to and activating TRKB at nanomolar
concentrations, without the requirement of a divalent
structure. However, in some cases, receptor specificity
needs to be examined more closely, as studies vary in
terms of the extent to which specificity is established.
As K252a is not specific to TRK receptors, other
approaches that are recommended for future studies
include the use of TRKB-specific ECD-blocking antibodies, the removal of TRKB, broad-binding screens and
approaches to rule out a BDNF secretagogueeffect.
Prepulse inhibition
A sequence of responses to
stimuli in which a weaker
stimulus inhibits the response
to a subsequent stronger
stimulus.
Acoustic startle
A reflexive motor response
to a sudden, unexpected
auditory stimulus.
TRKC ligands. Two sets of compounds, each containing

an array of four peptide side-chain mimics with differential TRKC activation, were identified in a programme
to find agents that have fewer peptidic features than
the first-generation compound D3 turn peptidomimetic193. In assays with a concentration of 20M, one
group of compounds promoted survival but not neurite
outgrowth of TRKC-expressing PC12 cells, whereas the
other group of compounds promoted neurite outgrowth
but not survival, thereby demonstrating that small molecules could be synthesized to elicit different TRKC
activation profiles. The latter group (which elicited neurite outgrowth but not survival) was found to stimulate
TRKC phosphorylation, whereas the receptor activation
status of the former group (which promoted survival
but not neurite outgrowth) remains to be established.
The neurite-promoting structures were distinguished
by the inclusion of a lysine-like moiety, but further exploration of structureactivity relationships in this system will
be required to delineate the detailed structural requirements that are necessary for the observed segregation of
TRKC activities. Moreover, these compounds provide an
important platform that will enable the mechanisms of
differential signalling to be studied in moredetail.
p75NTR ligands. The first non-peptide small-molecule
ligands that were found to bind to and modulate p75NTR
signalling were identified through insilico screening 139.
The pharmacophores used in insilico screening were

designed to capture key structural and physical chemical features of NGF loop 1. These features were based on
prior mutational analyses of neurotrophin, experiences
with synthetic peptides modelled on the loop 1 domain
of NGF146,194, and molecular dynamics simulations and
comparative features of the loop 1 domain across the
neurotrophin family 139.
The screening of small-molecule libraries with such
pharmacophores identified a series of non-peptide small
molecules that were capable of inhibiting the death of
cultured neurons at picomolar concentrations139. Two
compounds, LM11A31 and LM11A24 (TABLE1), were
further characterized as prototypes owing to calculations
predicting their greater drug-like characteristics and
preliminary measurements of bloodbrain barrier penetration. The specificity of these compounds was confirmed when their neurotrophic activity and signalling
was abolished in Ngfr/ neurons and using an antibody
specific for the p75NTR extracellular domain. Moreover,
each compound, like NGF, induced the recruitment of
the interleukin1 receptor-associated kinase (IRAK)
survival adaptor to p75NTR and activated downstream
AKT and NFB signalling. Although p75NTR ligands
are known to induce the death of oligodendrocytes,
LM11A31 and LM11A24 did not induce the death of
these cells. Indeed, these compounds inhibited the binding of pro-NGF to the extracellular domain of p75NTR
and diminished pro-NGF-induced apoptosis of oligodendrocytes in culture. Increased levels of p75NTR and
pro-NGF contribute to the death of oligodendrocytes
and loss of myelin following spinal cord injury 65; consistent with these invitro studies, oral administration of
LM11A31 to mice, starting 3hours after a spinal cord
contusion injury, led to decreased binding of pro-NGF
to p75NTR (REF.67). LM11A31 also decreased JNK activation, decreased oligodendrocyte death and demyelination and improved functional outcomes in these mice67.
Importantly, this functional improvement was observed at
a dose of 100mg per kg per day, but not at a dose of 25mg
per kg per day. The ability to decrease JNK activation was
lost in Ngfr/ mice, further confirming the specificity of
LM11A31 forp75NTR.
In a model of motor neuron degeneration involving
cultured embryonic motor neurons, LM11A24 prevented
p75NTR-dependent motor neuron death that was induced
by three different insults, including the addition of: NGF;
spinal cord extracts from superoxide dismutase mutant
mice (Sod1G93A-mutant mice); and NGF-producing reactive astrocytes195. In contrast to these invitro results,
preliminary studies suggest that the administration of
LM11A31 (at doses of up to 20mg per kg per day) to
Sod1G93A-mutant mice had no effect on functional motor
end points (F.M.L., unpublished observations). However,
the results of Tep etal.67 in the models of spinal cord injury
described above suggest that higher doses of LM11A31
should be investigated.
Signalling systems that are linked to p75NTR are
substantially integrated with signalling pathways that
are implicated in Alzheimers disease196. In a model of
Alzheimers disease in which amyloid- oligomers are
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Box 3 | Potential limitations of small-molecule neurotrophin receptor ligands
Although small-molecule ligands of neurotrophin receptors have numerous advantages over native neurotrophins,
there are potential limitations that must be considered during their development. Some of these limitations are
outlined below.
Insufficient receptor specificity

Protein interfaces generally contain several interaction hotspots comprising groups of amino acid residues.
The structures and chemical constituencies of these hotspots are not necessarily unique, but their combination in a
three-dimensional structure produces larger interaction regions with the potential for high degrees of specificity.
Small molecules can capture only a limited number of the motifs that are present in protein interaction regions;
this may therefore lead to a substantial likelihood of an identical epitope occurring in another protein interface,
which could produce potentially deleterious off-target effects.
Continuous dosing required
Unlike nucleic acids and proteins, which may be permanently transduced with viral vectors, small molecules cannot
be readily produced endogenously.
Consequently, continued exogenous administration may be needed to maintain therapeutic efficacy.
Effects not readily anatomically restricted
The production of macromolecules may be genetically engineered to occur in specific cell types and anatomic loci.
At best, small molecules may be somewhat site-restricted through the use of indwelling catheter and reservoir
systems.
Neurotrophin receptor-mediated side effects
Even highly specific small molecules may produce aberrant patterns of signalling through neurotrophin receptors by
bypassing the homeostatic mechanisms (for example, proteolysis and endocytosis) that would ordinarily limit the
extent and duration of receptor activation.
These considerations, along with the potential for broad tissue exposure, suggest that some small molecules may
have a propensity to produce ontarget side effects in neural and non-neuronal tissues: for example, pain, epilepsy,
the promotion of neoplasia or hypertension.
Long-term potentiation
Prolonged strengthening of
synaptic signalling between
neurons; induced by repetitive
stimulation.
Mller glia
Radial support cells located
in the retina.
added to cultured hippocampal neurons or slices,

LM11A31 and LM11A24 inhibited amyloid-induced
deleterious signalling, including the activation of calpain
CDK5 (cyclin-dependent kinase 5), glycogen synthase
kinase 3 (GSK3) and JNK signalling. They also prevented amyloid-induced excessive tau phosphorylation and the inactivation of AKT and cAMP-responsive
element-binding protein (CREB)196. Moreover, both
compounds blocked amyloid-induced neuritic dystrophy, death of cultured neurons and amyloid-induced
impairment of hippocampal long-term potentiation.
Highlighting the distinction between these p75NTRtargeting small-molecule ligands and an NGF mimetic,
native mature NGF failed to protect neurons from amyloidinduced degeneration196. Finally, once-daily administration of LM11A31 over a 3month period corrected
behavioural deficits and inhibited neurodegenerative
pathology in the hAPPLond/Swe mouse model of Alzheimers
disease, which is characterized by amyloid plaques, neuritic degeneration and cognitive impairments197.
Neurotrophins are also thought to have a role in HIVinduced neurodegeneration198. In a mixed cortical feline
immunodeficiency virus (FIV) culture model, the addition of LM11A31 substantially reduced or eliminated
neuronal pathology, the effects of FIV on microglia and
astrocytes, and prevented FIV-induced pathological
aberrant calcium regulation199.
The neuropathy induced by chemotherapy agents
has been linked to excess activation of RHOA GTPase200.
p75NTR has been shown to modulate RHOA activation33,
and the application of LM11A31 in dorsal root ganglion

cultures in the presence of cisplatin and methotrexate
inhibited RHOA activation and neuronal degeneration201. p75NTR is expressed by retinal Mller glia; furthermore, in rat models of glaucoma and optic nerve axotomy
it is upregulated and mediates the release of TNF and
2macroglobulin, which injure retinal ganglion cells202.
LM11A24 and a closely related derivative, THXB
(TABLE1), demonstrated substantial inhibition of retinal
ganglion cell degeneration and decreased the expression
of TNF and 2macroglobulin in these models202.
Together, these studies support the proposal104 that
non-peptide small-molecule p75NTR ligands might offer
therapeutic approaches for a range of disorders.
Conclusions and future directions

The development of small-molecule neurotrophin
receptor ligands has only recently begun and so only a
limited number of ligands have been created and characterized. Nevertheless, observations invitro and invivo
using prototype compounds have indicated important
mechanistic principles that can be used to inform the
future development of such compounds. These include
the finding that small molecules might achieve patterns
of signalling and biological end points that are distinct
from those induced by the native neurotrophins, and the
finding that monovalent small molecules are capable of
activating TRK receptors or modulating p75NTR processes that are typically associated with dimeric native
ligands and receptor dimerization.
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These capabilities, along with the fundamental roles of
neurotrophin receptors in several neurological disorders,
will encourage the development and broad application
of many more ligands. Moreover, several of the recently
described compounds noted above or their derivatives (including compound D3, LM11A-24, LM11A-31,
LM22A-4 and 7,8-DHF) have favourable pharmacological characteristics indicating that they could be advanced
to clinical studies. However, the potential limitations of
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Acknowledgements
Work by the authors has been supported by grants from the

following bodies: NIA UO1 AG032225 (to F.M.L.);
the Alzheimers Drug Discovery Foundation (to F.M.L.); the
Alzheimers Association (to F.M.L.); the Eastern Chapter of
the North Carolina Alzheimers Association (to F.M.L.); the
Koret Foundation (to F.M.L.); the Taube Philanthropies (to
F.M.L.); the Jean Perkins Foundation (to F.M.L.); the Horngren
Family Alzheimers Research Fund (to F.M.L.); the Richard M.
Lucas Foundation (to F.M.L.); and the US Department
of Veterans Affairs, Veterans Health Administration, Office of
Research and Development (to S.M.M).
Competing interests statement
The authors declare competing financial interests: see Web

version for details.
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Neurotrophins 1

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REVIEWS

Abstract | Neurotrophins and their receptors modulate multiple signalling pathways to

The mammalian neurotrophin family comprises nerve

administered neurotrophins in various neurodegenerative

NATURE REVIEWS | DRUG DISCOVERY

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by small molecules and the strategies being pursued to

also have key roles outside the nervous system but

Neurotrophins, their receptors and signalling

508 | JULY 2013 | VOLUME 12

Proteolytic peptidemediated mechanisms

Neurotrophin synthesis and secretion. NGF, BDNF,

members: NGF binds to TRKA; BDNF and NT4 bind

Potential pharmacological targets

NATURE REVIEWS | DRUG DISCOVERY

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one involving the hairpin loops (in particular loop 1)

Do pro-neurotrophins invariably cause cell death? The

requires the regulated shedding of the extracellular

510 | JULY 2013 | VOLUME 12

NATURE REVIEWS | DRUG DISCOVERY

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Small ligands for neurotrophin receptors?

to a site outside the ligand-binding domain and thereby

512 | JULY 2013 | VOLUME 12

Altered synaptic function

Comparative signalling studies involving the various

NATURE REVIEWS | DRUG DISCOVERY

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an NGF antibody complex composed of NGF and an

514 | JULY 2013 | VOLUME 12

presence of BDNF. Loop 2 monomeric cyclized peptides

Small-molecule ligands for neurological diseases

NATURE REVIEWS | DRUG DISCOVERY

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Inhibits death of dorsal root

Inhibits death of hippocampal

Inhibits death of hippocampal

Inhibits death of hippocampal

Inhibits death of hippocampal

Inhibits death of hippocampal

TRKA and TRKB

516 | JULY 2013 | VOLUME 12

Inhibits death of hippocampal

Inhibits death of hippocampal

Inhibits death of hippocampal

Inhibits pro-neurotrophininduced death of B104 and

NATURE REVIEWS | DRUG DISCOVERY

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line has yielded various interesting TRKB ligands137. One

518 | JULY 2013 | VOLUME 12

of ERK and AKT phosphorylation183. Notably, stimulation

NATURE REVIEWS | DRUG DISCOVERY

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TRKC ligands. Two sets of compounds, each containing

The pharmacophores used in insilico screening were

520 | JULY 2013 | VOLUME 12

Insufficient receptor specificity

added to cultured hippocampal neurons or slices,

and the application of LM11A31 in dorsal root ganglion

Conclusions and future directions

NATURE REVIEWS | DRUG DISCOVERY

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Chao,M.V. Neurotrophins and their receptors: a