neurotrophins 1

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The mammalian neurotrophin family comprises nervegrowth factor (NGF), brain-derived neurotrophic factor(BDNF), neurotrophin 3 (NT3) and neurotrophin 4(NT4; also known as NT5)1,2. Since the discovery of NGFmore than 50 years ago, the ability of neurotrophins toprevent or reverse neuronal degeneration, to promoteneurite regeneration and to enhance synaptic plasticityin various in vitro and in vivo models has offered thepromise that they might provide primary or adjunctivetherapy for a number of neurological disorders3,4.

However, as is the case with proteins in general,neurotrophins have suboptimal pharmacological prop-

erties, such as low stability in serum (with half-lives ofa few minutes or less) and negligible oral bioavailability.In addition, they have minimal blood–brain barrierpenetration and restricted diffusion within centralnervous system (CNS) parenchyma5–7. The pleiotropicactions of neurotrophins that are triggered by theactivation of their multi-receptor signalling networks(FIG. 1) could lead to adverse on-target effects — suchas the promotion of neurodegeneration8,9 or pain10,11 — thereby further limiting their clinical application.Indeed, these limiting factors probably contributedto the lack of efficacy seen in early clinical trials thatinvestigated the therapeutic efficacy of exogenously

administered neurotrophins in various neurodegenerativediseases, including Alzheimer’s disease and amyotrophiclateral sclerosis (BOX 1).

Various strategies for addressing the pharmacoki-netic limitations of native neurotrophins have thereforebeen pursued. These include the development of smallmolecules that target specific neurotrophin receptorssuch as the tropomyosin receptor kinase (TRK) recep-tors and the p75 neurotrophin receptor (p75NTR ; alsoknown as NGFR) (discussed in more detail below).Other approaches include the development of mutantneurotrophins12,13, neurotrophin receptor-specific anti-

bodies (that act through individual neurotrophin recep-tors to modulate signalling processes that are relevant topathological states)14–17 and localized expression of neu-rotrophins via viral and cell-based delivery systems18–21.In addition to having more favourable pharmacokineticprofiles, several of these alternatives may differentiallystimulate neurotrophin receptors to achieve distinct sig-nalling profiles, thus resulting in more selective effectsthan those elicited by native neurotrophins.

In this Review, after providing a brief overview ofthe latest understanding of neurotrophins, their recep-tors and their effects, we discuss the rationale under-lying the selective targeting of neurotrophin receptors

1Department of Neurology

and Neurological Sciences,

Stanford University,

300 Pasteur Drive, Stanford,

California 94305, USA.2Department of Neurology,

San Francisco Veterans

Affairs Medical Center,

University of California San

Francisco, 4150 Clement

Street, San Francisco,

California 94121, USA.

e-mails: [email protected];

[email protected]

doi:10.1038/nrd4024

Small-molecule modulation ofneurotrophin receptors: a strategy forthe treatment of neurological diseaseFrank 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.

REVIEWS

NATURE REVIEWS | DRUG DISCOVERY VOLUME 12 | JULY 2013 | 507

© 2013 Macmillan Publishers Limited. All rights reserved


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Pro

Pro

NADE

Cell death ordegeneration Survival Neurite growth

RHOANRAGE

NRIF

JNK

FAP1

IRAK

TRAF6

NF-κB

PI3K

AKT

GSK3β

SHC

Pro-neurotrophin

p75NTRp75NTR

Sortilin

TRK

Mature neurotrophin 180°

1

24

N

3

Increase inendocytosisanddegradation

MAPK PKC

PLCγ1

Tyr490

Tyr670

Tyr674

Tyr675

Tyr695

Tyr751

Tyr785

Tyrosinekinaseactivation

Saddle

RIP2

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 toachieve this. Although there are pharmacological effortsunderway towards the development of indirect receptoractivators (so-called transactivating activators) and agentsthat promote the increased expression of neurotrophins(so-called secretagogues), this Review focuses on thedevelopment of small molecules that interact directlywith neurotrophin receptors. Neurotrophin receptors

also have key roles outside the nervous system buthere we focus on neurological and neuropsychiatricdisorders.

Neurotrophins, their receptors and signalling

The current understanding of the molecular biology ofneurotrophins, their receptors and downstream signal-ling pathways, as well as their normal and pathological

Figure 1 | Neurotrophins, their receptors and signalling pathways. The figure shows a highly simplified schematicof 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 and the p75

neurotrophin receptor (p75NTR). In TRK complexes, the neurotrophin apical region containing β-turn loops 1, 2 and 4 isnear the membrane, whereas in the p75NTR complex it faces away from the membrane (loops 1–4 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 co-receptor 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 α- and γ-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 3-kinase (PI3K) and phospholipase Cγ1 (PLCγ1)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 factor-κB (NF-κB) pathway, or antagonize the actions of TRK throughthe 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 phosphatase 1; GSK3β, glycogen synthasekinase 3β; IRAK, interleukin-1 receptor-associated kinase; NADE, p75NTR-associated cell death executor; NRAGE,neurotrophin receptor-interacting MAGE homologue; NRIF, neurotrophin receptor-interacting factor; RIP2, receptor-interacting protein 2; SHC, SRC homology domain-containing protein; TRAF6, TNF receptor-associated factor 6.

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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

ligand–receptor 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 peptide-

mediated mechanisms



that are mediated through the

cleavage, translocation and

binding of a portion of one

protein to the other.

effects in the brain, is evolving rapidly. Here, we providea brief overview of neurotrophin receptors and their sig-nalling pathways, particularly in the context of creatingnovel ligands; these advances in our understanding haverecently been highlighted in the literature (reviewed inREFS 22–25).

Neurotrophin synthesis and secretion. NGF, BDNF,NT3 and NT4 are initially synthesized in the endoplas-mic reticulum as pre-proproteins, and cleavage of thesignal peptide of pre-proproteins converts these intopro-neurotrophins. In the trans-Golgi network andin secretory vesicles, pro-neurotrophins dimerize andare proteolytically processed by proprotein convertasesubtilisin kexin (PCSK) enzymes to their mature formsbefore their release from the cell22.

The amount of mature neurotrophin secreted fromthe cell depends on the range of convertases expressedin different cell types; furin (also known as PCSK3) isexpressed in most cells, whereas PCSK1 (also known asNEC1) and PCSK2 (also known as NEC2) are principally

expressed in neurons. The expression of convertasesis physiologically regulated and in some scenarios aconsiderable proportion of pro-neurotrophins maybe secreted into the extracellular space (reviewed inREFS 22,26). In the extracellular space, pro-neurotrophincleavage may be catalysed by plasmin27. The secreted~120-amino-acid mature forms of neurotrophins existin solution as dimers22,26 (FIG. 1).

Neurotrophin receptors. Pro- and mature neurotrophinsboth bind to and signal through two principal receptortypes: p75NTR and the TRK receptors. p75NTR is a tumournecrosis factor (TNF) receptor family member thatunselectively binds all of the neurotrophins and lacksknown intrinsic enzymatic activity but recruits signallingadaptors and modulates TRK signalling.

The pro-domain of pro-neurotrophins can concur-rently bind to the vacuolar protein sorting 10 (VPS10)family member sortilin (also known as NTR3), whichallows the formation of a ternary complex with p75NTR (REF. 28). Among the numerous pathways and proteinsthat are regulated by p75NTR are the phosphoinositide3-kinase (PI3K)–AKT pathway 29, nuclear factor-κB(NF-κB)30, mitogen-activated protein kinase (MAPK)31,JUN N-terminal kinase (JNK)32, RHOA33, the cyclicAMP–protein kinase A (PKA) pathway 34, hypoxia-inducible factor (HIF)35 and the ceramide signalling

pathway 36. The p75NTR -induced effects are diverse andinclude cell survival, cell death, regulation of prolifera-tion and inhibition of neurite outgrowth, depending onthe expression of the TRK receptors, sortilin and variousintracellular signalling adaptors (FIG. 1).

There are three TRK receptors: TRKA (also knownas NTRK1), TRKB (also known as NTRK2) and TRKC(also known as NTRK3). The full-length versions of thesetransmembrane receptors contain an intracellular tyro-sine kinase domain and an extracellular neurotrophin-binding region that comprises tandem immunoglobulindomains and a leucine-rich domain. Each TRK recep-tor selectively binds to different neurotrophin family

members: NGF binds to TRKA; BDNF and NT4 bindto TRKB; and NT3 binds to TRKC37,38. In addition, thereis heterologous binding, with NT3 and NT4 both elicitingsome activation of TRKA, and NT3 eliciting some acti-

vation of TRKB37,39. TRK signalling occurs throughthree principal tyrosine kinase-mediated pathways: theMAPK–ERK (extracellular signal-regulated kinase)pathway, the PI3K–AKT pathway and the phospholipaseCγ1 (PLCγ1)–PKC pathway. The effects elicited throughthese signalling pathways predominantly promote cellsurvival and differentiation2,38. In addition, several trun-cated isoforms of TRKB and TRKC exist, which lackthe tyrosine kinase domain. These isoforms may havedominant negative effects, sequester neurotrophins and/or signal through alternative mechanisms compared tofull-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 nucleus41–43. Although severalfunctional interactions between TRK receptors and

p75NTR are well established — for example, the formationof a neurotrophin binding site with increased affinity,as well as reciprocal modulation of signalling44–47 — thenature of their physical associations and the formation ofcomplexes with neurotrophins remain areas of ongoingdebate and study. Evidence has been presented for andagainst various possible models, including the forma-tion of stable p75NTR –neurotrophin–TRK extracellulardomain (ECD) complexes48,49, as well as intracellularassociations mediated by direct mechanisms50, bridgedmechanisms50 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 thatare elicited through their signalling pathways create abroad range of possibilities for small-molecule bindingand modulation of function.

Potential pharmacological targets

Although there is an expanding number of neurotrophinreceptor-mediated physiological effects and mechanismsof action, the currently available structural and mechanis-tic information related to the actions of pro- and matureneurotrophins provides substantial opportunities forpharmacological 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 containinga neurotrophin dimer and a p75NTR dimer with its elon-gated extracellular domains flanking the neurotrophin28,52 (FIG. 1). p75NTR dimers may be preformed and they areconnected together through a cysteine bond in thetransmembrane domain. This cysteine bond is impor-tant for neurotrophin-mediated signal transduction (viaJNK activation) and the transmission of steric changesto the intracellular domains53. The extracellular regionof p75NTR contains four cysteine-rich domains that arearranged linearly; the second and third of these cysteine-rich domains bind to neurotrophins at two major sites:

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Status epilepticus

Prolonged epileptic

seizures that may result

in excitotoxic injury.

one involving the β-hairpin loops (in particular loop 1)and the other at the amino terminus of mature neuro-trophins52. The neurotrophin pro-domain, which is theprincipal binding domain for the co-receptor sortilin,has not been visualized crystallographically ostensiblyowing to its high flexibility and lack of ordered struc-ture28. However, the pro-domain does appear to have arole in altering the structure of the mature neurotrophindomain in the p75NTR –pro-NGF complex such that loop 2of NGF and subjacent residues are exposed28. The physio-logical role of this NGF structure and the possibility ofutilizing it pharmacologically remain to be determined.

Proteolytic processing of p75NTR involves α- andγ-secretases (reviewed in REF. 24), which also cleave amy-loid precursor protein. γ-secretase mediates the generationof amyloid-β peptides, whereas α-secretases cleave withinthe amyloid-β domain, thus disallowing amyloid-β pep-tide production. Therapeutics for Alzheimer’s disease thattarget these enzymes (to increase α-secretase or decreaseγ-secretase expression) might therefore also modulateneurotrophin signalling.

Do pro-neurotrophins invariably cause cell death? Theconcomitant binding of sortilin and p75NTR to pro-neurotrophin selectively promotes cell death54, which

requires the regulated shedding of the extracellulardomain of p75NTR by the metalloproteinase ADAM17 (adisintegrin and metalloproteinase domain-containingprotein 17; also known as TACE) and regulated intra-membrane proteolysis of the receptor by γ-secretase(reviewed in REF. 24). Regulated intramembrane proteo-lysis results in the release of a p75NTR intracellular domainthat mediates apoptotic cell death via JNK and caspaseactivation (reviewed in REF. 24). Pro-neurotrophins havealso been reported to stimulate TRK receptors, althoughthey are less effective than their mature counterparts55–57.In cells with high TRK/p75NTR ratios (for example, insuperior cervical ganglia, in PC12 cells and, followingseizures, in hippocampal neurons)55–58, pro-neurotrophinsmay promote, or at least not impair, cell survival. Together,these observations suggest that pro-neurotrophin signal-ling through TRK receptors may counter their signallingthrough p75NTR , allowing cells with high TRK/p75NTR ratios to survive while those with low TRK/p75NTR ratiosundergo apoptotic death55–60.

Cells with high p75NTR expression are most readily

detectable in basal forebrain cholinergic, dorsal rootganglion and sympathetic neuron populations; however,p75NTR is constitutively expressed by various other disease-related neurons, including those in the cortex, hippo-campus, basal ganglia and several brainstem nuclei61–63.In addition, although p75NTR expression may be increasedin disease and injury states64–69, it may not always be asso-ciated with increased cell death64. Moreover, the balancebetween mature and pro-neurotrophin signalling mayregulate local processes such as neurite outgrowth andretraction as well as dendritic spine formation withoutaffecting cell survival70–74. Overall, accumulating evidencesuggests that there is a functional balance of neurotro-phin signalling in the central nervous system — regulatedby the TRK/p75NTR ratio and the ratio of pro- to matureneurotrophins72–74 — which may become disequilibratedin disease states75.

Excess pro-neurotrophin levels may be associatedwith conditions including Alzheimer’s disease, statusepilepticus, spinal cord injury and ageing, leading topathological effects such as a loss of neurites as well asapoptotic neuronal or oligodendrocyte death8,9,58,67,76–78.Pro-neurotrophin levels may increase as a result ofincreased neurotrophin synthesis and/or, importantly, asa result of deficits in the conversion of pro-neurotrophinsto mature neurotrophins, which has been proposed asa contributing factor in Alzheimer’s disease progres-

sion27,79,80 (FIG. 2). Following from these observations,targeting pro-neurotrophin–sortilin–p75NTR interactionscould constitute a therapeutic strategy for conditions thatare characterized by excess levels of pro-neurotrophin.

The 13-amino-acid peptide sortilin ligand, neuro-tensin, inhibits pro-neurotrophin–sortilin binding andpro-neurotrophin-mediated neuronal cell death54. Inaddition, a short sortilin peptide has been discoveredthat is crucial for pro-neurotrophin–sortilin bindingand apoptotic signalling, and that does not substantiallyaffect other functions of sortilin. These observations holdthe possibility of finding small molecules that selectivelyinhibit pro-neurotrophin-induced cell death81.

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.

Alzheimer’s disease

Intraventricular administration of high doses of nerve growth factor (NGF) in threepatients with Alzheimer’s 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 inthe 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.

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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 bindingsite, as well as three hairpin β-turn loops (loops 1, 2 and4), which display a relatively low degree of amino acidsimilarity across the neurotrophin family and representthree of the five neurotrophin variable regions84. These

variable regions contribute to multidomain receptorinterfaces and receptor specificity, and have thereforehad a key role in the structure-based development ofreceptor-specific small-molecule ligands. Notably, cur-rent crystallographic data suggest that there are onlyminor contacts between neurotrophin loop 1 and TRKreceptors; interactions with loops 2 and 4 have not been

visualized. These interactions may occur in the extra-cellular juxtamembrane portion of the receptor49, but adetailed structure of this region of the complex is notyet available.

Neurotrophin binding induces TRK dimerization85,86,although preformed TRK multimers may occur 87–89,and autophosphorylation of TRK receptors at multiple

tyrosine residues leads to the recruitment of differentintracellular signalling components and the activationof downstream pathways2,38 (FIG. 1). The existence of pre-formed TRK dimers is a crucial issue in the question ofwhether a monovalent small-molecule ligand would becapable of activating a TRK receptor, and is discussed fur-ther below. The multiplicity of ligand-induced TRK phos-phorylation sites creates the opportunity for differentialpatterns of phosphorylation induced by different ligands,resulting in differential downstream signalling patternsand potential outcomes. For example, within TRKA thephosphorylation of Tyr490 is associated with the acti-

vation of the MAPK–ERK and PI3K–AKT pathways,whereas the phosphorylation of Tyr785 is associated withthe activation of the PLCγ– PKC signalling pathway 2,38.

Signalling through TRK receptors largely promotes cellsurvival and differentiation, although when unliganded itmay promote cell death2,38,90. TRKB and TRKC are foundin a large number of neuronal populations throughout thecentral and peripheral nervous system in humans, whereasthe distribution of TRKA is more restricted to basal fore-brain cholinergic, dorsal root ganglion and sympatheticneurons, among others91–93,217. Although neurotrophin andneurotrophin receptors have overlapping expression pat-terns and functions in many areas of the brain, some neuralmechanisms are modulated by specific systems, includ-ing: NGF–TRKA-mediated upregulation of cholinergic

function in the basal forebrain94; BDNF–TRKB-mediatedpromotion of synaptic plasticity 95,96; and NT3–TRKC-mediated survival of peripheral proprioceptive neurons97,98.Consequently, the identification of agents that modulatespecific TRK receptors would potentially provide oppor-tunities for more selective treatment of neurological dis-orders: for example, the activation of TRKA and TRKBfor Alzheimer’s disease, TRKB for stroke and trauma, andTRKC for some forms of peripheral neuropathy.

Mature neurotrophins signalling through p75 NTR. Unlikethe pro-neurotrophin–p75NTR –sortilin interaction, whichgenerally elicits strong death signalling, the interactions

of mature neurotrophin with p75NTR produce highly vari-able results depending on the cellular context24,99. In theabsence of a cognate TRK, mature neurotrophins maypromote cell death. For example, this has been observedin oligodendrocyte cultures expressing p75NTR but notTRKA32 that have been treated with NGF, and in sym-pathetic neurons expressing TRKA and p75NTR but notTRKB that have been treated with BDNF100. Conversely,in other cell types or under different experimental con-ditions, the binding of mature neurotrophins to p75NTR may increase cell survival via the inhibition of constitu-tive p75NTR apoptotic signalling101,102 and/or the activationof pro-survival factors such as NF-κB and AKT103,104.

Further complications and opportunities for pharma-cological targeting arise in consideration of the associa-tions and functional interactions of TRK receptors andp75NTR . As noted above, numerous studies have identifiedpotential points of interaction, from direct interactionsat the cell surface to distal signalling overlaps. TRK acti-

vation modulates the uptake, processing and signallingof p75NTR , and p75NTR has important effects on TRK–

neurotrophin binding and signalling46,47,49,51,105–110. Thiscrosstalk, to some extent, constrains the responses ofsystems to a single neurotrophin, and provides the possi-bility that a pharmacological intervention that decouplesthe receptor’s (either TRK receptors or p75NTR ) activitiescould produce effects that are not achievable with nativeligands. In addition, p75NTR functions as a co-receptorfor the myelin-associated neurite outgrowth inhibitorsNogo receptor and LINGO-1 (leucine-rich repeat andimmunoglobulin domain-containing 1)111–113. The pres-ence of such co-receptors would be expected to locallymodify the availability and functions of p75NTR and,consequently, the effects of p75NTR -targeted compounds.Thus, the complex landscape of p75NTR signalling mecha-nisms allows a broad range of possibilities by which spe-cific ligands might influence p75NTR in a manner distinctfrom native neurotrophins.

Further considerations for drug discovery. In additionto the diseases mentioned above, there are several dis-orders in which TRK or p75NTR signalling mechanismsare directly linked to processes underlying disease onsetor progression, and representative disorders with nervoussystem relevance are listed in Supplementary information S1 (table). In terms of drug discovery, disease processesthat may be amenable to treatment via neurotrophinreceptor modulation fall into three categories: those in

which impaired neurotrophic signalling is a significantpathogenic factor; those in which such a deficiency isnot causative or necessarily present but in which neuro-trophin signalling counteracts or favourably modulatespathological signalling; and conditions involving excessneurotrophin activity (FIG. 2).

For many complex disorders, several of these possi-bilities for modulating neurotrophin receptors may beapplicable during the course of disease pathogenesis,and they may function only in specific anatomic locior cell types. An example of this is Alzheimer’s disease,in which amyloid-β can have both direct and indirecteffects on the pathogenesis of the disease (BOX 2). Impaired

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neurotrophin synthesis or transport, as occurs in Rett syn-drome and Huntington’s disease, for example, may lead toshrinkage and dysfunction of neuronal cell populationsthrough a primary loss of trophic signalling114–117. In addi-tion, diminished anterograde transport of neurotrophinsmay deprive remote target cell populations of support,producing a degeneration of neuronal networks114–117.Similar processes may occur in pericontusional areasfollowing traumatic brain injury 118. Excessive neurotrophicsignalling may lead to aberrant dendritic sprouting,decreased pruning and altered electrophysiological prop-erties119. The formation of abnormal neuronal networksengendered by these effects has been implicated in theproduction of pain and in epileptogenesis120–122.

As a result, the underlying cause of the disease orcondition needs to be sufficiently understood to ena-ble the appropriate corrective modulatory effect to beelicited when targeting neurotrophin receptors. In themajority of cases involving TRK receptors, increasesin TRK activity are sought for therapeutic effects.Conversely, in other cases — such as in the control of

pain, in which NGF–TRKA signalling is a contributingfactor — inhibition of signalling is desired123. For exam-ple, small-molecule TRKA antagonists might provide ameans for modulating TRKA activity as an alternativeor adjunctive therapy to NGF-specific antibody treat-ment124,125. Similarly, as excess levels of neurotrophinssuch as BDNF might promote certain forms of epilepsy,the application of TRKB antagonists could be consid-ered in this setting126. In the case of p75NTR , therapeuticefforts may involve the inhibition of its degenerative sig-nalling and/or the activation of associated pro-survivalpathways51.

Below, we discuss the structural aspects of modulatingneurotrophin receptors and outline the strategies thatare currently being pursued.

Small ligands for neurotrophin receptors?

In consideration of the interaction of small moleculeswith large, multidomain, multifunctional receptor sys-tems, many traditional pharmacological concepts andthe associated terminology (that is, describing a ligand asan agonist, partial agonist or antagonist) must be revis-ited in the context of neurotrophins and their receptors.

Given that the crosswise loop-to-loop dimensions ofneurotrophin dimers (>25 Å) are beyond those that aretypically bridged by monovalent small molecules (molec-ular mass


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• Amyloid-β• Excitotoxicity• Cytokines• Chemotherapy• Ischaemia• Trauma, and so on

Rett syndrome

Huntington’sdisease

Alzheimer’sdisease

AgeingStatus epilepticus

Pain Epilepsy

Pro-survival signalling• AKT• NF-κB• ERK, and so on

Deleterious signalling• RHOA• JNK• Calpain• Caspases• GSK3β, and so on

• Altered synaptic function• Decreased neurite integrity• Demyelination• Decreased survival

↓Pro-neurotrophin

↑Pro-neurotrophin

↑Pro-neurotrophin

↓Neurotrophin

↑Neurotrophin

↓Neurotrophin

↑Neurotrophin

↑Sprouting↓Pruning

Increasedsynthesis,decreaseddegradation

Decreasedproteolyticconversion

Activatedmicroglia

Astrocytes

Decreasedsynthesis and/ortransport

The multi-receptor neurotrophin system and its con-text-dependent activity profiles complicate the applicationof traditional pharmacological terms such as ‘full agonist’,‘partial agonist’ and ‘antagonist’. For instance, a smallmolecule might bind to TRKB to promote the survival of

TRKB- and p75NTR -co-expressing neurons with a lowerefficacy than BDNF, and it may appear to function as a‘partial agonist’. By contrast, the application of the samesmall molecule and BDNF to neurons in which p75NTR expression has been eliminated might result in similarefficacies, leading to the conclusion that the compoundis a ‘full agonist’. In another case, a small-molecule p75NTR ligand might promote neuronal survival with an efficacysimilar to NGF in a particular assay, resembling the profileof a ‘full agonist’; however, in another assay, in which NGFpromotes cell death, the same small-molecule ligand maycontinue to promote survival and be considered a ‘reverseagonist’.

Comparative signalling studies involving the variousneurotrophins and their receptors support the possibilitythat small-molecule ligands targeting these receptors maytrigger signalling patterns that are distinct from those pro-moted by the native ligands. One study 140 demonstrated

that NGF and NT3 both triggered prolonged TRKA activ-ity; however, in the same assay system NGF caused anacute TRKA activation that was several times higher thanthat stimulated by NT3. Moreover, under conditions thatequalized the acute activation, NGF was markedly moreeffective in supporting neuronal survival. These findingswere consistent with a mechanism in which NGF andNT3 differentially interacted with or promoted the for-mation of distinct TRKA conformations, dimerizationstates and/or interactions with p75NTR , resulting in differ-ent signalling patterns140.

The potential for differential signalling by TRKAwas also highlighted by the observation that NGF and

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 states.

Decreases in neurotrophin synthesis or transport (as in Rett syndrome or Huntington’s disease) may activate or

co-activate, along with other toxic stimuli (for example, amyloid-β or excess glutamate), signalling that results incellular 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 neurotrophin–receptor systems therefore has the potentialto 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.

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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.

an NGF antibody complex composed of NGF and anNGF-specific antibody directed to the carboxyl termi-nus (NGF-mAb) bound to TRKA with high affinity,but NGF stimulated longer-lasting MAPK activationresulting in both neuronal survival and neurite out-growth, whereas the NGF-mAb promoted survival butnot neurite outgrowth141. In another example, mutationof the SHC binding site in TRKB led to decreased neuro-trophic activity of NT4 with a relatively spared BDNFresponse142, which suggests that NT4 and BDNF maydifferentially activate TRKB signalling.

Thus, the pharmacological profiling of small-moleculeneurotrophin receptor ligands should include a range ofassays that allow a broader degree of discrimination thatgoes beyond simple classifications limited to ‘agonist’

versus ‘antagonist’ activity. Moreover, the potential tocreate novel neurotrophin receptor signalling patternswith small-molecule ligands provides a rationale for their

development that exceeds the goal of simply producing‘neurotrophin mimetics’ and also involves improvingthe pharmacokinetic features of neurotrophins. Indeed,many ligands with so-called ‘partial agonist’ and otherprofiles may present pharmacological advantages thatmake them more effective in a disease setting.

Peptide ligands

The identification of synthetic peptides correspondingto specific domains of neurotrophin ligands (FIG. 1) with‘antagonist’ or ‘agonist’ activity established the vital proofof concept that small molecules, including those thatbind monomerically, might be capable of modulating

neurotrophin receptor function and, in some cases, provide a useful basis for the development of non-peptidesmall-molecule ligands. The development of most ofthese first-generation synthetic ligands targeting neuro-trophin receptors focused on the modelling of domainswithin NGF, BDNF or NT3.

NGF peptides. An early approach for the development ofligands targeted to neurotrophin receptors consistedof creating small synthetic peptides with amino acidresidues corresponding to various domains of NGF andassessing them for their ability to inhibit or mimic theneurotrophic function of NGF (reviewed in REFS 143–145).Before the crystal structure of NGF had been derived, apeptide scanning strategy of mouse NGF demonstratedthat small linear synthetic peptides containing the Lys-Gly-Lys-Glu (KGKE) sequence — which was laterfound to comprise the core of the NGF loop 1 domain— inhibited NGF activity in vitro146.

The first small peptide corresponding to a specificNGF domain to demonstrate neurotrophic activity con-

sisted of a cyclized dimeric form of a KGKE-containingpeptide (peptide P7)194. The addition of a p75NTR -specificantibody that was capable of blocking NGF bindingand activity, or the use of neurons lacking p75NTR expres-sion (Ngfr –/– neurons), eliminated P7 activity, whereasthe pan-TRK inhibitor K252a had no effect, which sug-gested that this peptide acted through p75 NTR . Thesedata are consistent with site-directed mutagenesisstudies indicating that the Lys32 and Lys34 residuesof NGF are crucial for the binding of NGF to p75NTR (REF. 147). Peptides containing KGKE or a homologoussequence have since been shown to: block the bindingof amyloid-β to p75NTR and its ability to induce neuronaldeath148; inhibit apoptosis-driven hair follicle involu-tion, a process that is thought to involve p75NTR signal-ling149; modulate kindling-induced mossy fibre sprouting ina rat model of epilepsy 150; and decrease post-axotomyretinal ganglion cell death in rats, which is anotherp75NTR -dependent process151. This spectrum of actionshighlights the biological and therapeutic potential ofdeveloping non-peptide small molecules that incorpo-rate features of the loop 1 domain and are capable ofmodulating p75NTR function.

Site-directed mutagenesis studies indicated that theloop 4 domain of NGF was one of several domains thatwas likely to interact with TRKA, and peptide mimeticsof this domain had an important role in developing

early perspectives of how small-molecule ligands mightengage TRK receptors. Cyclized, monomeric loop 4peptides (C(92–96) and C(92–97)) blocked NGF-induced PC12 cell neurite outgrowth, and when theywere assayed in the absence of NGF they had no neuro-trophic activity, which suggests that they had ‘antagonist’activity but no ‘agonist’ activity 152. However, under con-ditions in which p75NTR was bound by a monoclonal anti-body, monomeric C(92–96) supported the survival ofPC12 cells and other cells (at ~30–50% of maximal NGFefficacy), demonstrating a ‘partial agonist’ profile andillustrating the principle that agonism and antagonismare context-dependent153.

Box 2 | Multiple pathways that can be targeted in Alzheimer’s disease

In Alzheimer’s disease, the pathogenic molecule amyloid-β can have both directand 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-β can interact directly with p75 neurotrophin receptor (p75NTR),leading to aberrant signalling and cell death212–214. Second, amyloid-β can interact

with various cellular receptors unrelated to neurotrophins to initiate pathogenicsignalling, including the activation of JUN N-terminal kinase (JNK), calpains,

caspases and glycogen synthase kinase 3β (GSK3β), leading to excessive tauphosphorylation215. 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-κB (NF-κB) and extracellularsignal-regulated kinase (ERK), may interact with injurious pathways activated by

amyloid-β at multiple points, counteract the deleterious signalling and therebyimprove neuronal function and integrity.

Finally, Alzheimer’s 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 Alzheimer’s disease. Indeed, the effects of such

modulation on multiple mechanisms that contribute to the development of

Alzheimer’s disease may be synergistic and therefore lead to particularly effective

therapeutic outcomes.

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A cyclized dimeric peptide, C(92–97)dimer

, inducedTRKA dimerization, phosphorylation and internaliza-tion in neuroblastoma cells overexpressing TRKA153.C(92–97)

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 pro-file further indicated that synthetic peptides could, atleast partially, activate TRKA survival signalling. In aconcomitant study 154, an NGF loop 4 cyclized dimericpeptide (P92–97) supported neuronal survival andneurite outgrowth of dorsal root ganglion neurons (withan efficacy of ~30% of that of NGF)154 and stimulatedTRKA autophosphorylation (F.M.L. and Y. Xie, unpub-lished observations), thereby offering a parallel lineof evidence that NGF loop 4 peptides are capable ofinducing TRKA-mediated survival signalling.

The screening of β-turn peptidomimetics that con-tained a macrocyclic ring incorporating amino acidside chains corresponding to NGF β-turn loops led tothe identification of a peptidic monovalent compound— termed compound D3 — that bound to TRKA,

promoted the formation of TRKA homodimers andachieved ~40% of the maximum efficacy of NGF in pro-moting the survival of dorsal root ganglion neurons155.This finding is particularly noteworthy because it dem-onstrated that a monovalent compound (compound D3;TABLE 1) was capable of inducing TRKA dimerizationand thereby suggested that non-peptide monovalentsmall molecules might also be found that induce TRKreceptor dimerization and activation. In subsequentstudies, compound D3 was shown to: rescue basalforebrain cholinergic neuron degeneration and spatialmemory in aged rats when administered by intracere-broventricular minipump156; prevent the death of retinalganglion cells in a rat model of glaucoma when admin-istered by intraocular injection157; and prevent retinalganglion cell degeneration in a post-axotomy rat modelfollowing intravitreal injection151.

In the J20 APPSwe/Ind mouse model of Alzheimer’sdisease, which develops amyloid plaques and memoryimpairment, the administration of compound D3 byintracerebroventricular minipump led to improved learn-ing and short-term memory but caused persistent deficitsin long-term memory 12. In addition, a related peptidomi-metic called MIM-D3 increased glycoconjugate secretionand improved a measure of corneal injury in a rat modelof dry eye syndrome218. MIM-D3 completed Phase IIclinical 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 loop1 and loop 4 of NGF had neurotrophic activity in themicromolar range and induced phosphorylation of TRKAbut not of TRKB158. These peptide studies demonstratedthat TRKA ligands could elicit a range of biological out-comes and encouraged the search for active non-peptidemonomeric small molecules targeting TRKA.

BDNF and NT3 peptides. Synthetic peptides correspond-ing to loop 2 and loop 4 of BDNF have also been charac-terized for their effects on cell survival alone and in the

presence of BDNF. Loop 2 monomeric cyclized peptidesantagonized the binding of BDNF to receptors and hadno intrinsic activity 159, whereas cyclized dimeric loop 2peptides promoted survival but these peptides had lessefficacy than BDNF160. Whether these peptides promotedTRKB activation was not directly established. Synthetic,linear, monomeric tetrapeptides derived from the loop 4and carboxy-terminal regions of BDNF stimulatedphosphorylation of TRKB but not TRKC, and exhibitedneurotrophic activity in hippocampal neuron cultures161.Importantly, in addition to directly activating TRKB,these peptides promoted BDNF expression, thus high-lighting the importance of considering a secretagogueeffect in neurotrophin small-molecule studies.

In another study, a cyclic pentapeptide derived froma putative p75NTR -binding region of BDNF loop 4 hadneurotrophic effects without activating TRKB162. In addi-tion, this peptide produced p75NTR -dependent increasesin the myelination of dorsal root ganglion neurons inculture162,163. A cyclized peptide termed cyclotraxin-B,which encompasses nine residues from the highly vari-

able region III of BDNF (adjacent to the loop 3 domain),inhibited both BDNF-mediated and basal TRKB phos-phorylation in vitro164. A Tat-fused form of cyclotraxin,which was designed to allow CNS delivery after intra-

venous administration, was given to mice and resulted indecreased TRKB activation in the brain as well as reducedanxiety behaviour. In NT3-related studies, β-turn peptido-mimetics that are designed to mimic ligand-interactingregions of NT3 have been developed that selectively bindto and activate TRKC165,166.

Together, these studies show that peptidomimetics arecapable of modulating neurotrophin receptor function.However, given the limitations of peptide compounds fordrug development, including their stability, bioavailabilityand blood–brain barrier penetration, a higher priorityhas been placed on identifying and creating non-peptidesmall molecules that are capable of modulating neuro-trophin receptors.

Small-molecule ligands for neurological diseases

TRKA ligands. The screening of combinatorial com-pound libraries identified several TRKA activators.These include an asterriquinone (1H5) and a mono-indolyl-quinone (5E5) that activated TRKA, possibly bybinding to an intracellular site, and promoted PC12 cellsurvival at low micromolar concentrations167. Anotherscreen identified gambogic amide (MW 628), which

prevented the death of a TRKA-expressing cell line168 (TABLE 1). Interestingly, gambogic amide bound to theintracellular juxtamembrane domain of TRKA ratherthan the extracellular ligand-binding region. This findingsuggests that the compound causes allosteric activationof the receptor, and is consistent with studies of otherreceptor kinases (including the insulin receptor) thathave identified small molecules that activate receptorsthrough interactions with regions that are not involvedin the binding of the physiological ligand169. Additionalstudies will be necessary to establish the degree ofspecificity of gambogic amide for TRKA. Nevertheless,gambogic amide activated TRKA and its downstream

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Table 1 | Ligands targeting neurotrophin receptors and in vitro activity

Compound Structure In vitro activity Refs

TRKA

Compound D3

NH

O

HO

O

O

NH

NH2

O

HN

O

HN

OHO

ONH

+

HO

HO

• Inhibits death of dorsal rootganglion neurons at 10 μM

155

Gambogic amide

O O

OOH

O

O

H

O

H2N

• Inhibits death of hippocampalneurons at 100–250 nM

168

MT2

ON

O

O

O

O

• Inhibits death of hippocampalneurons at 5–30μM

124

TRKA and TRKB

Amitriptyline

N


170

TRKB

7,8-dihydroxyflavone

O

HO

O

OH


137

Deoxygedunin

O

H

O

O

O

H

O

O


• Inhibits death of corticalneurons at 500 nM

138

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Table 1 (cont.) | Ligands targeting neurotrophin receptors and in vitro activity

Compound Structure In vitro activity Refs

TRKB (cont.)

LM22A-4

OO

N

H

HO

O NH

OH

N

H

OH

• Inhibits death of hippocampalneurons at 0.5–500 nM

183

ANA-12

NH

HN

O

NH

O

S

O

• Inhibits BDNF-inducedneurite outgrowth ofTRKB-PC12 cells at0.01–100μM

188

p75NTR

LM11A-31

H2N

OHN

N

O

• Inhibits death of hippocampalneurons at 100–1,000 pM

139

LM11A-24

O

NO

N

N

N

HN

N

O

• Inhibits death of hippocampalneurons at 100–1,000 pM

• Inhibits binding of NGF top75NTR in ELISA at 50 nM

139,202

THX-B

O

NO

N

N

N

N

HN

O

O

• Inhibits pro-neurotrophin-induced death of B104 andPC12 NNR5 cells at 20 μM

• Inhibits binding of NGF top75NTR in ELISA at 50 nM

202

BDNF, brain-derived neurotrophic factor; ELISA, enzyme-linked immunosorbent assay; NGF, nerve growth factor; p75 NTR, p75neurotrophin receptor; TRK, tropomyosin receptor kinase.

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signalling partners AKT and ERK, and promoted thesurvival of cultured hippocampal cells, which — underthe conditions utilized — were reported to expressTRKA168. In mouse studies, subcutaneous administrationof gambogic amide prevented neuronal death induced bykainic acid or ischaemic stroke.

From the same screening programme that identifiedgambogic amide, but published separately 170, the anti-depressant drug amitriptyline (TABLE 1) was shown tobind the extracellular domains of TRKA and TRKB andinduce their activation. Interestingly, amitriptyline pro-moted TRKA–TRKB heterodimerization (which does notoccur with NGF or BDNF), which further suggests thatthis compound induces alternative signalling outcomes170.Amitriptyline protected cultured hippocampal cells fromapoptosis, stimulated neurite outgrowth in PC12 cells and,in in vivo studies, reduced kainic acid-triggered neuronalcell death170. Studies in inducible TRKA-null mice havesupported a key role for TRKA in mediating the effectsof amitriptyline170; however, given the broad spectrum ofmechanisms affected by this compound, the issue of target

specificity needs to be carefully considered.Another approach for creating TRKA ligands incorpo-

rates the principle that most protein–protein interactions,including the binding of NGF to TRKA, involve two ormore binding sites — known as hotspots171,172. Therefore,a small molecule is more likely to be capable of activatingTRKA if it includes two pharmacophores correspond-ing to such regions173. With this in mind, bivalent β-turnmimics were created as ‘minimalist’ mimics of the pep-tidic β-turn structures that are present in NGF, and fourcompounds were found to selectively bind to the TRKAreceptor173. Additional studies will be required to deter-mine the extent to which these compounds might pro-mote or inhibit receptor function.

Several small bicyclic peptidomimetic compoundswere identified from a chemical library to interact with theimmunoglobulin-like domain D5 (an area that interactswith the saddle region of NGF) of TRKA124. Interestingly,although the lead compound MT2 (TABLE 1) produced arobust effect on the survival of PC12 cells, similar to thatof NGF, it was substantially less capable of inducing TRKAphosphorylation, the expression of the NGF-induciblegene VGF and differentiation of PC12 cells124. Furtheranalysis showed that compound MT2 and NGF stimu-lated TRKA-Tyr490 phosphorylation to a similar degree,whereas compound MT2 induced significantly less phos-phorylation at Tyr674, Tyr675 and Tyr785, which suggests

that there is differential activation of signalling betweenthe compound and NGF124. Whether this distinctive sig-nalling pattern provides any therapeutic advantages ordisadvantages relative to NGF remains to be determined.Taken together, these studies demonstrate that smallmolecules can be created, or otherwise identified, thatare capable of activating TRKA, in some cases throughnon-ligand receptor sites. A remaining challenge in mostcases is demonstrating the degree of specificity for TRKA.

TRKB ligands. A cell line-based assay to screen com-pounds for their ability to inhibit apoptosis in TRKB-transfected cells relative to the TRKB-negative parental

line has yielded various interesting TRKB ligands137. Onecompound, 7,8-dihydroxyflavone (7,8-DHF; TABLE 1),induced the phosphorylation of TRKB as well as itsdownstream targets AKT and ERK. 7,8-DHF inhibitedneuronal death in vitro, with an efficacy approximatelyequal to that of BDNF, and its activity was blocked byK252a. Whether the phosphorylation of TRKB-Tyr490or other tyrosine residues was stimulated in the cellsscreened was not determined. Nevertheless, 7,8-DHFbinds to the cysteine cluster 2 (CC2) and leucine-richregion (LRR) in the extracellular domain of TRKB.As the LRRs have been implicated in BDNF binding, thisfinding suggests that 7,8-DHF might produce its effectsat least partially through mechanisms that are similar tothose of BDNF137. Of note, this TRK binding localizationis similar to that of amitryiptyline170, but differs fromthat of gambogic amide (which binds to the intracell-ular domain)168 and compound MT2 (which binds to theimmunoglobulin-like domain D5)124, and illustrates thediversity of activation modes that are identifiable usingfunctional assays.

Interestingly, diosmetin, a compound related to7,8-DHF, had similar anti-apoptotic effects but inducedonly weak phosphorylation of TRKB, yet it upregulatedphosphorylated AKT and phosphorylated ERK to asimilar degree137. Furthermore, other related compounds— including pinocembrin and quercetin — had mini-mal effects on TRKB, and upregulated phosphorylatedERK but not phosphorylated AKT137, again illustrat-ing the different patterns of TRK signalling that can beinduced by small-molecule ligands. Studies in TrkbF616A-mutant knock-in mice — in which TRKB activity can beselectively eliminated by the kinase inhibitor analogue1NMPP1 (4-amino-1-tert-butyl-3-(1ʹ-naphthylmethyl)pyrazolo(3,4-)pyrimidine) — supported the notionthat the effects of 7,8-DHF were dependent on TRKBtyrosine kinase activity, although direct detection ofTRKB activation was not demonstrated in vivo137. A sub-sequent study using a cell line lacking TRKB found that7,8-DHF protected against glutamate-, hydrogen per-oxide- and menadione-induced toxicity while increasingglutathione levels and reducing levels of reactive oxygenspecies — a profile that is consistent with an antioxidanteffect174. Thus, the mechanisms of action of 7,8-DHFin a given setting may be complex, and may includemechanisms that are independent of TRKB activation.

The efficacy of 7,8-DHF (administered either orallyor parenterally) has been examined in several disease

models in which BDNF–TRKB mechanisms are thoughtto have a role. These include the neuroprotective effectsof BDNF–TRKB signalling in mouse models of kainicacid-, ischaemia- and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced injury 137 and inthe 5XFAD transgenic mouse model of Alzheimer’s dis-ease175. Improved learning and behavioural outcomeswere also elicited with 7,8-DHF in rodent models withprior exposure to traumatic stress176,177. Morphologically,7,8-DHF partially reversed age-related dendritic spineloss in an in vitro hippocampal slice model178, and pre-

vented increased dendritic length in the amygdala andthe occurrence of a depressive profile in a rat model of

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depression179. Along with a more potent derivative —4ʹ-dimethylamino-7,8-dihydroxyflavone — 7,8-DHFalso promoted TRKB-Tyr816 phosphorylation in thedentate gyrus along with progenitor cell proliferationand ‘antidepressant’ effects in the forced swim test180.

A follow-on programme of pharmacological opti-mization yielded a derivative of 7,8-DHF, 2-methyl-8-(4-(pyrrolidin-1-yl)phenyl)chromeno[7,8-]imidazol-6(1H )-one, which showed similar behavioural effects buthad improved pharmacokinetic and metabolism pro-files181. 7,8-DHF may also ameliorate certain phenotypi-cal features of genetic diseases. In a mouse model of Rettsyndrome, in which loss-of-function mutations in thegene encoding methyl CpG-binding protein 2 ( MECP2)lead to reduced BDNF levels and abnormal motor, respir-atory and cognitive function, 7,8-DHF increased survivaland reduced abnormal breathing patterns182.

Another compound that was identified from small-molecule screens using TRKB-expressing cell lines isdeoxygedunin (TABLE 1), which is a derivative of geduninand a tetranortriterpenoid with antimalarial, insecti-

cidal and anticancer activity 138. Deoxygedunin bindsto the ECD of TRKB to stimulate its dimerization andautophosphorylation without the activation of TRKA orTRKC. It inhibited the death of cultured hippocampalneurons (induced by oxygen and glucose deprivation)and the glutamate-induced death of cultured corticalneurons in a TRKB-dependent manner138. Deoxygeduninalso rescued vestibular ganglion neurons in Bdnf –/– mice;in wild-type mice the compound exhibited antidepres-sant activity and enhanced the acquisition of conditionedfear — a BDNF-dependent learning process138.

Distinct from high-throughput small-moleculescreening, the in silico screening of small-molecule librar-ies with a pharmacophore modelled on the loop 2 domainof BDNF was used to identify TRKB agonists183. TheseTRKB agonists were then screened for their ability toprevent the death of primary hippocampal neurons. Onecompound, LM22A-4 (TABLE 1), bound to TRKB but notto TRKA, TRKC or p75NTR , and inhibited the binding ofBDNF but not of NGF or NT3 to their cognate receptors.Further evidence of the specificity of LM22A-4 was dem-onstrated in the following ways: through the inhibitionof its neurotrophic activity by K252a; using an antibodydirected to the ECD of TRKB; and through the lack ofbinding in a receptor selectivity screen183.

Upon closer examination of its activity, LM22A-4 wasshown to promote the survival of hippocampal neurons

(at ~85% of the efficacy of BDNF) and induce the phos-phorylation of TRKB-Tyr490 (at ~30% of the efficacy ofBDNF)183. The phosphorylation of other tyrosine moie-ties was not examined. Interestingly, similar to the effectsnoted with compound MT2 on TRKA and the effectsnoted with diosmetin on TRKB, LM22A-4 promotedrobust upregulation of ERK and AKT phosphorylationat lower levels of activation of TRKB phosphoryla-tion compared to the levels induced by native neuro-trophins183. Other compounds that were identified inthe screening, and that were chemically distinct fromLM22A-4, produced even lower and more delayedTRKB phosphorylation, again with strong upregulation

of ERK and AKT phosphorylation183. Notably, stimulationof AKT and ERK was completely reversed by K252a oran antibody targeting the ECD of TRKB, which suggeststhat interaction with the ECD of TRKB and the phos-phorylation of TRKB were essential components of theresponse183.

In in vitro models of Alzheimer’s, Huntington’s andParkinson’s disease, LM22A-4 prevented neuronal deathat efficacies equal to that of BDNF. In wild-type mice,LM22A-4 stimulated the activation of TRKB along withAKT and ERK in hippocampal and cortical tissue; fur-thermore, in rats inflicted with traumatic brain injury,treatment with the compound led to enhanced recoveryof motor function in rotarod testing 183. In other patho-logical contexts that are thought to involve BDNF–TRKBsignalling mechanisms, LM22A-4 (administered paren-terally) led to a normalization of brainstem TRKB activa-tion and respiratory patterns in the Mecp2-mutant mousemodel of Rett syndrome184, and increased neurogenesisin the subventricular zone and enhanced the recovery ofmotor function in a mouse model of post-stroke recov-

ery 185. Cortical inhibitory interneurons express TRKBand are supported by BDNF that is derived from targetneurons and transported in a retrograde manner186; ina rat model of post-traumatic epilepsy, in which theseneurons undergo axotomy and subsequent degeneration,intranasal application of LM22A-4 prevented post-injuryloss of expression of the α3 subtype (Na++K+) ATPase ininterneuron terminals187.

Another virtual screening approach188 modelled theinteraction of the N-terminal region of BDNF with theimmunoglobulin-like domain D5 of TRKB to screena database of commercially available drug-like com-pounds. Hits were then screened for their ability toinhibit BDNF-induced TRKB activation. CompoundANA-12 (TABLE 1) inhibited TRKB phosphorylation withsubmicromolar potency and bound to TRKB noncom-petitively with BDNF. In PC12-nnr5 cells (which lackTRKA) that were transfected to express specific TRKreceptors, ANA-12 inhibited BDNF-induced neuriteoutgrowth at concentrations as low as 10 nM, with fullinhibition in the micromolar range. No inhibitory activi-ties for TRKA or TRKC were detected, which suggeststhat ANA-12 is specific to TRKB; however, the results ofa broad screen for receptor binding will be of interest.

In in vivo studies, intraperitoneal administration ofANA-12 inhibited TRKB phosphorylation in the stria-tum, cortex and hippocampus. Importantly, ANA-12

did not appear to significantly promote neuronaldeath, which is one potential consequence of inhibit-ing BDNF–TRKB signalling; however, a small increasein the number of apoptotic cells was observed in thedentate gyrus of mice that received the highest dose ofANA-12 (REF. 188). Excessive BDNF–TRKB signalling inreward circuitry promotes the development of anxietyand depression in rodent models, and the administrationof ANA-12 was able to substantially reduce anxiety anddepression-related behaviour188. Subsequent studies havedemonstrated that ANA-12 may also be a useful tool forexamining the involvement of BDNF–TRKB signallingin physiological processes; for example, the compound

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inhibits the reduction in food intake caused by BDNFdelivery to the medial nucleus tractus solitarius189 andreverses the 7,8-DHF-mediated improvements in meth-amphetamine-induced deficits in prepulse inhibition ofacoustic startle190.

N -acetylserotonin (NAS), a compound that is normallyexpressed in the pineal gland and retina, has antidepres-sant effects, and many antidepressant compounds areassociated with elevations in BDNF levels; these findingshave led to the testing of NAS for its TRK-activating prop-erties. NAS activated TRKB, but not TRKA, and achievedantidepressant effects in a TRKB-dependent manner 191.A NAS derivative, N -[2-(5-hydroxy-1H -indol-3-yl)ethyl]-2-oxopiperidine-3-carboxamide (HIOC), was sub-sequently identified, which activates the TRKB receptorwith greater potency than NAS and inhibits light-inducedretinal degeneration192. The extent to which NAS andHIOC bind specifically to TRKB to function as directagonists will need to be established through additionalstudies.

Together, these TRKB ligand studies show the feasi-

bility of developing non-peptide small molecules that arecapable of binding to and activating TRKB at nanomolarconcentrations, without the requirement of a divalentstructure. However, in some cases, receptor specificityneeds to be examined more closely, as studies vary interms of the extent to which specificity is established.As K252a is not specific to TRK receptors, otherapproaches that are recommended for future studiesinclude the use of TRKB-specific ECD-blocking anti-bodies, the removal of TRKB, broad-binding screens andapproaches to rule out a BDNF secretagogue effect.

TRKC ligands. Two sets of compounds, each containingan array of four peptide side-chain mimics with differ-ential TRKC activation, were identified in a programmeto find agents that have fewer peptidic features thanthe first-generation compound D3 β-turn peptidomi-metic193. In assays with a concentration of 20 μM, onegroup of compounds promoted survival but not neuriteoutgrowth of TRKC-expressing PC12 cells, whereas theother group of compounds promoted neurite outgrowthbut not survival, thereby demonstrating that small mol-ecules could be synthesized to elicit different TRKCactivation profiles. The latter group (which elicited neu-rite outgrowth but not survival) was found to stimulateTRKC phosphorylation, whereas the receptor activationstatus 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 explo-ration of structure–activity relationships in this system willbe required to delineate the detailed structural require-ments that are necessary for the observed segregation ofTRKC activities. Moreover, these compounds provide animportant platform that will enable the mechanisms ofdifferential signalling to be studied in more detail.

p75 NTR ligands. The first non-peptide small-moleculeligands that were found to bind to and modulate p75NTR signalling were identified through in silico screening139.

The pharmacophores used in in silico screening weredesigned to capture key structural and physical chemi-cal features of NGF loop 1. These features were based onprior mutational analyses of neurotrophin, experienceswith synthetic peptides modelled on the loop 1 domainof NGF146,194, and molecular dynamics simulations andcomparative features of the loop 1 domain across theneurotrophin family 139.

The screening of small-molecule libraries with suchpharmacophores identified a series of non-peptide smallmolecules that were capable of inhibiting the death ofcultured neurons at picomolar concentrations139. Twocompounds, LM11A-31 and LM11A-24 (TABLE 1), werefurther characterized as prototypes owing to calculationspredicting their greater ‘drug-like’ characteristics andpreliminary measurements of blood–brain barrier pen-etration. The specificity of these compounds was con-firmed when their neurotrophic activity and signallingwas abolished in Ngfr –/– neurons and using an antibodyspecific for the p75NTR extracellular domain. Moreover,each compound, like NGF, induced the recruitment of

the interleukin-1 receptor-associated kinase (IRAK)survival adaptor to p75NTR and activated downstreamAKT and NF-κB signalling. Although p75NTR ligandsare known to induce the death of oligodendrocytes,LM11A-31 and LM11A-24 did not induce the death ofthese cells. Indeed, these compounds inhibited the bind-ing of pro-NGF to the extracellular domain of p75NTR and diminished pro-NGF-induced apoptosis of oligo-dendrocytes in culture. Increased levels of p75NTR andpro-NGF contribute to the death of oligodendrocytesand loss of myelin following spinal cord injury 65; con-sistent with these in vitro studies, oral administration ofLM11A-31 to mice, starting 3 hours after a spinal cordcontusion injury, led to decreased binding of pro-NGFto p75NTR (REF. 67). LM11A-31 also decreased JNK acti-

vation, decreased oligodendrocyte death and demyelina-tion and improved functional outcomes in these mice67.Importantly, this functional improvement was observed ata dose of 100 mg per kg per day, but not at a dose of 25 mgper kg per day. The ability to decrease JNK activation waslost in Ngfr –/– mice, further confirming the specificity ofLM11A-31 for p75NTR .

In a model of motor neuron degeneration involvingcultured embryonic motor neurons, LM11A-24 preventedp75NTR -dependent motor neuron death that was inducedby three different insults, including the addition of: NGF;spinal cord extracts from superoxide dismutase mutant

mice (Sod1G93A-mutant mice); and NGF-producing reac-tive astrocytes195. In contrast to these in vitro results,preliminary studies suggest that the administration ofLM11A-31 (at doses of up to 20 mg per kg per day) toSod1G93A-mutant mice had no effect on functional motorend points (F.M.L., unpublished observations). However,the results of Tepet al.67 in the models of spinal cord injurydescribed above suggest that higher doses of LM11A-31should be investigated.

Signalling systems that are linked to p75NTR aresubstantially integrated with signalling pathways thatare implicated in Alzheimer’s disease196. In a model ofAlzheimer’s disease in which amyloid-β oligomers are

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.

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added to cultured hippocampal neurons or slices,LM11A-31 and LM11A-24 inhibited amyloid-β-induceddeleterious signalling, including the activation of calpain–CDK5 (cyclin-dependent kinase 5), glycogen synthasekinase 3β (GSK3β) and JNK signalling. They also pre-

vented amyloid-β-induced excessive tau phosphoryla-tion and the inactivation of AKT and cAMP-responsiveelement-binding protein (CREB)196. Moreover, bothcompounds blocked amyloid-β-induced neuritic dystro-phy, death of cultured neurons and amyloid-β-inducedimpairment of hippocampal long-term potentiation.Highlighting the distinction between these p75NTR -targeting small-molecule ligands and an ‘NGF mimetic’,native mature NGF failed to protect neurons from amyloid-β-induced degeneration196. Finally, once-daily adminis-tration of LM11A-31 over a 3-month period correctedbehavioural deficits and inhibited neurodegenerative

pathology in the hAPPLond/Swe mouse model of Alzheimer’sdisease, which is characterized by amyloid plaques, neu-ritic degeneration and cognitive impairments197.

Neurotrophins are also thought to have a role in HIV-induced neurodegeneration198. In a mixed cortical felineimmunodeficiency virus (FIV) culture model, the addi-tion of LM11A-31 substantially reduced or eliminatedneuronal pathology, the effects of FIV on microglia andastrocytes, and prevented FIV-induced pathologicalaberrant calcium regulation199.

The neuropathy induced by chemotherapy agentshas been linked to excess activation of RHOA GTPase200.p75NTR has been shown to modulate RHOA activation33,

and the application of LM11A-31 in dorsal root ganglioncultures in the presence of cisplatin and methotrexateinhibited RHOA activation and neuronal degenera-tion201. p75NTR is expressed by retinal Müller glia; further-more, in rat models of glaucoma and optic nerve axotomyit is upregulated and mediates the release of TNF andα2-macroglobulin, which injure retinal ganglion cells202.LM11A-24 and a closely related derivative, THX-B(TABLE 1), demonstrated substantial inhibition of retinalganglion cell degeneration and decreased the expressionof TNF and α2-macroglobulin in these models202.

Together, these studies support the proposal104 thatnon-peptide small-molecule p75NTR ligands might offertherapeutic 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 alimited number of ligands have been created and char-acterized. Nevertheless, observations in vitro and in vivo using prototype compounds have indicated importantmechanistic principles that can be used to inform thefuture development of such compounds. These includethe finding that small molecules might achieve patternsof signalling and biological end points that are distinctfrom those induced by the native neurotrophins, and thefinding that ‘monovalent’ small molecules are capable ofactivating TRK receptors or modulating p75NTR — pro-cesses that are typically associated with dimeric nativeligands and receptor dimerization.

Long-term potentiation

Prolonged strengthening of

synaptic signalling between

neurons; induced by repetitive

stimulation.

Müller glia

Radial support cells located

in the retina.

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 on-target side effects in neural and non-neuronal tissues: for example, pain, epilepsy,

the promotion of neoplasia or hypertension.

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These capabilities, along with the fundamental roles ofneurotrophin receptors in several neurological disorders,will encourage the development and broad applicationof many more ligands. Moreover, several of the recentlydescribed compounds noted above or their deriva-tives (including compound D3, LM11A-24, LM11A-31,LM22A-4 and 7,8-DHF) have favourable pharmacologi-cal characteristics indicating that they could be advancedto clinical studies. However, the potential limitations of

small-molecule modulation of neurotrophin receptorsneed to be taken into consideration (BOX 3), and it will becrucial to better characterize in vivo target binding andestablish the pharmacodynamic properties of these com-pounds. Nevertheless, as neurotrophin receptor signallingmechanisms and pathways are better understood, it maybe possible to design small molecules to achieve tailoredsignalling profiles, which could lead to the development of‘designer ligands’ for specific disease applications.

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