Expert Opinion on Orphan Drugs

ISSN: (Print) 2167-8707 (Online) Journal homepage: http://www.tandfonline.com/loi/ieod20

Emerging drug targets in amyotrophic lateral

sclerosis

Michael P Bova PhD & Gene G Kinney PhD

To cite this article: Michael P Bova PhD & Gene G Kinney PhD (2013) Emerging drug targets in
amyotrophic lateral sclerosis, Expert Opinion on Orphan Drugs, 1:1, 5-20

To link to this article: http://dx.doi.org/10.1517/21678707.2013.744949

Published online: 17 Dec 2012.

Submit your article to this journal

Article views: 1209

View related articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=ieod20

Download by: [Jawaharlal Nehru University] Date: 09 February 2016, At: 22:22
 Review

Emerging drug targets in

amyotrophic lateral sclerosis
Michael P Bova† & Gene G Kinney
†
1. Introduction Department of Molecular Discovery, ELAN Pharmaceutical, South San Francisco, CA, USA

2. Genes associated with FALS

Introduction: Amyotrophic lateral sclerosis (ALS) is a progressive and devastat-
3. New targets for ALS ing neurodegenerative disease resulting from injury and death of upper and
4. Conclusions lower motor neurons. Symptoms initially include muscle weakness and twitch-
5. Expert opinion ing and subsequently progress to muscle atrophy, complete loss of limb use,
respiratory difficulties and ultimately death. Multiple biological mechanisms
have been implicated in ALS and a complex etiology has been described. As
a result, drug discovery researchers have few validated targets to pursue
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016

and patients have few therapeutic options.

Areas covered: Identification of new drug targets in ALS can be facilitated by
a detailed understanding of the processes and genes that contribute to path-
ogenesis. Accordingly, this review summarizes current hypotheses regarding
underlying mechanisms for motor neuron susceptibility in ALS. An overview
of emerging and tractable drug targets that could result in therapeutic
breakthroughs is provided.
Expert opinion: Despite the immense progress that has been made in under-
standing ALS over the last decade, riluzole remains the only approved drug
to treat ALS. Combining structure-guided drug design applied to validated
and pharmaceutically tractable targets with disease-relevant phenotypic
screens will allow for the identification of novel drug targets and potentially
breakthrough therapeutics for ALS.

Keywords: amyotrophic lateral sclerosis, motor neuron, phenotypic screening, structure-guided

drug design

Expert Opinion on Orphan Drugs (2013) 1(1):5-20

1. Introduction

1.1 Motor neuron susceptibility in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a pro-
gressive and devastating neurodegenerative disease resulting from injury and death
of upper and lower motor neurons. This degeneration of motor neurons causes
weakness and atrophy of limb musculature progressing to a complete loss of arm
and leg function, speech and swallowing difficulties and ultimately death, usually
from respiratory failure. The prognosis is bleak for ALS patients whose average
survival after first symptom onset is 3 years [1].
Multiple underlying factors responsible for the selective motor neuron degenera-
tion in ALS have been described. Motor neurons are large cells and have extensive
dendritic and axonal processes. While the cell body of an alpha motor neuron is
50 -- 70 µm in diameter, the axon length can oftentimes be greater than 1 m. Motor
neurons conduct action potentials to muscle fibers where larger axon diameters
increase the rate of neuronal conduction velocity. Motor neuron neuronal filaments
(NFs) function to increase axonal diameter in myelinated axons in a process known
as radial axonal growth [2]. A hallmark of both sporadic and familial ALS (FALS) is
the presence of cytoplasmic inclusions, many of which contain phosphorylated
NFs [3,4].

10.1517/21678707.2013.744949 © 2013 Informa UK, Ltd. e-ISSN 2167-8707 5

All rights reserved: reproduction in whole or in part not permitted
 M. P. Bova & G. G. Kinney
Article highlights.
. Overview of potential mechanisms underlying motor
neuron susceptibility in ALS is provided.
. Current genes linked to FALS including recently
identified Profilin 1 and C9Orf72 are covered.
.
In-depth discussion of the use of phenotypic screens to
identify new small molecules that could be of benefit in
ALS and new technology useful in deconvoluting hits
from phenotypic screens.
. Discussion of the tractability, from a drug discovery
perspective, of emerging targets for ALS such as EphA4,
ASK1, DJ-1 and HDAC6.
. Insight into the challenges facing those developing
therapeutics for ALS and how some of these challenges
may be mitigated from a clinical and research
perspective is provided.
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
This box summarizes key points contained in the article.
The metabolic demands in maintaining an axon of 1 m
length or more requires a high level of mitochondrial activity.
This high metabolic demand is consistent with the observa-
tion that motor neurons are particularly susceptible to
hypoxia. For example, deletion of the hypoxia responsive
element in the vascular endothelial growth factor (VEGF)
promoter caused a selective degeneration of motor neurons
and concomitant ALS-like phenotype in mice [5]. Motor
neurons are involved in neurotransmission to skeletal muscle
involving both glutamatergic and acetylcholine neurotrans-
mitter systems. Thus, motor neurons are susceptible to
oxidative stress. Elevated protein carbonyl levels, increased
3-nitrotyrosine levels and high 4-hydroxynonenal levels (an
indicator of lipid peroxidation) are observed in ALS
patients [6-8]. Despite high metabolic activity and exposure
to oxidative stress, motor neurons do not have high levels of
glutathione compared to hepatocytes or astrocytes [9] and
thus appear particularly vulnerable to oxidative stress [10].
High levels of oxidative stress can predispose proteins to
unfolding and aggregation, yet in motor neurons there is a
high threshold for induction of the stress response and expres-
sion of heat shock proteins (Hsps), which was associated with
a failure to activate HSF1 [11]. Similarly, challenge of spinal
motor neurons with ZnCl2, paraquat or toxic glutamate con-
centrations did not lead to induction of metallothioneins
(zinc-binding proteins with antioxidant properties) where, in
contrast, other neuronal populations did induce metallothio-
neins [12]. The attenuated Hsp response of motor neurons
could represent an opportunity for therapeutic intervention
as it has been shown that treatment with arimoclomol, a coin-
ducer of Hsps, delays disease progression in ALS mice [13].
Arimoclomol is now in the clinic for ALS patients [14]. It is
not clear why a neuronal type that is exposed to high levels
of stress has difficulty mounting a heat shock or other
protective responses to injury. Nonetheless, the high threshold
necessary to express Hsps may contribute, to some extent, to
6 Expert Opinion on Orphan Drugs (2013) 1(1)
the vulnerability of motor neurons in ALS and may represent
an opportunity for therapeutic intervention.
Lower motor neurons receive extensive glutamatergic input
from upper motor neurons and excitatory interneurons. Motor
neurons express alpha-amino-3-hydroxy-5-methylisoxazole-
4-propionic acid (AMPA), kainate and N-methyl-D-aspartate
(NMDA) glutamate receptors (GluRs) where glutamate is the
neurotransmitter used for fast synaptic transmission in the
CNS. AMPA receptors are highly expressed on motor neurons
and consist of four homo- or hetero-oligomeric receptor
subunits (GluR1, GluR2, GluR3, and GluR4). It has been
shown pharmacologically that there is a significant pool of
AMPA receptors expressed in motor neurons that are perme-
able to calcium [15-17]. Glutamate released into the synaptic
cleft stimulates AMPA receptors where signaling is terminated
through uptake of glutamate by the excitatory amino acid
transporter (EAAT2). It has been shown that there is a decrease
in EAAT2 levels in ALS patients [18,19], which may lead to over-
stimulation of AMPA receptors and excessive calcium influx,
initiating a number of detrimental processes and excitotoxicity
in motor neurons [20].
Natural cellular processes exist to counteract excessive
calcium influx from overstimulation of AMPA receptors.
Cytoplasmic calcium can be transported into the mitochon-
dria by the mitochondrial uniporter. Calcium is then slowly
released through the mitochondrial Na/Ca exchanger and
then ultimately leaves the cytoplasm through a number
of mechanisms including the plasma membrane Na/Ca
exchanger [21,22] and sarcoendoplasmic reticulum calcium
transport ATPase (SERCA) [23]. It has been shown that mito-
chondria from spinal cord have decreased calcium-buffering
capacity compared to other brain mitochondria [24] which
could be a result of a lower density of mitochondria in
motor neurons compared to other neurons.
The endoplasmic reticulum (ER) is also an important and
high capacity storage site for calcium. Calcium plays an
important role in protein folding and processing within the
ER [25,26]. Elevated intracellular calcium will cause calcium-
induced calcium release through the ER ryanodine and
triphosphoinositol (IP3) receptors and potentially deplete
ER calcium stores [21,27]. There is an important crosstalk
between ER and mitochondrial calcium stores as ER calcium
released into the cytosol can be uptaken by the mitochondria
and then ultimately reuptaken into the ER by SERCA
pumps [23,27]. This calcium signaling between compartments
could become disturbed in ALS where it is tempting to
speculate, that the depletion of ER calcium stores that could
happen with glutamate-receptor overstimulation will lead to
activation of the unfolded protein response (UPR) [27].
Although this seems to be a valid hypothesis, it has yet to be
unequivocally proven [27].
Intracellular calcium can also be buffered by calcium-
binding proteins (CaBPs). Of note, motor neuron popula-
tions, such as the oculomotor and abducens nuclei, which
express CaBPs such as parvalbumin, calbindin, calretinin,

and neurocalcin are spared in ALS patients, while other motor
neurons that do not express these proteins are not [28-31].
Expression of CaBPs in cultured motor neurons are also pro-
tective against mutant superoxide dismutase (SOD), excessive
glutamate and oxidative stress [32], which is consistent with the
clinical observation that neurons expressing CaBPs are spared
in ALS.
The ER orchestrates a complex symphony of protein distri-
bution for axonal transport, exocytosis, and for transport to
Golgi, mitochondria and nuclear compartments of the cell.
In the context of a motor neuron, with the demands of main-
taining large cells with long axons and extensive dendritic
morphology, it is not surprising that these cells could be sus-
ceptible to ER stress. It has been noted that ER stress, as indi-
cated by activation of the UPR, occurs in ALS [21,33]. The
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
UPR inhibits protein synthesis and results in the expression
of ER resident chaperones to assist in protein folding. Quan-
titative Western blotting of human spinal cord tissue from
sporadic ALS (SALS) patients showed that three UPR sensors,
inositol-requiring enzyme1 (IRE1), activating transcription
factor 6 (ATF6) and protein kinase RNA-like ER kinase
(PERK) were upregulated from fourfold to sixfold compared
to control patients [34]. Also in this study, the UPR chaperones
protein disulfide isomerase (PDI) and PDI-related protein
Erp57 were upregulated in ALS patients. These results were
consistent with an earlier study showing increased expression
of PDI and eukaryotic initiation factor 2-alpha (eIF2a) in
the spinal cord of ALS patients compared to control
patients [35]. Similarly, in 60-day-old mutant superoxide dis-
mutase 1 (SOD1) mice, before ALS symptoms emerged,
UPR sensor proteins IRE1, ATF6 and PERK were elevated
and a high level of expression of PDI was observed compared
to WT mice [21,34].
In summary, motor neuron susceptibility in ALS may have
many contributing factors. Motor neurons are large in size
and have long axons. The metabolic requirements to sustain
these cells are inherently large. Motor neurons express high
levels of calcium permeable AMPA receptors and by nature
of their function, are often exposed to elevated levels of cal-
cium, particularly during intense stimulation. Motor neurons
may have a limited ability to induce Hsps, reduced glutathi-
one levels, and many motor neurons do not express CaBPs
which reduce calcium-buffering capacity. In light of the
enhanced susceptibility of motor neurons to stress, it is per-
haps no surprise that there are more known mutated genes
causing FALS (Table 1) then there are for more common
neurodegenerative diseases such as Alzheimer’s disease (AD)
or Parkinson’s disease (PD).
1.2 Current and future therapy for ALS patients
Riluzole, a potent anticonvulsant drug [36], has been approved
to treat ALS since 1995, yet despite its approval for this use,
the efficacy of riluzole is modest. Riluzole will extend the life-
span of ALS patients by a few months on average [37]. Riluzole
may have several different mechanisms of action; however, the
Expert Opinion on Orphan Drugs (2013) 1(1)
Emerging drug targets in amyotrophic lateral sclerosis
biological relevance of these for its effects in ALS is not
completely understood. Riluzole noncompetitively blocks
excitatory amino acid receptors [38], inhibits glutamic acid
release and inactivates voltage-dependent sodium channels [39].
Some of the electrophysiological effects of riluzole can be
blocked by pertussis toxin suggesting that it may interact
with a G-protein driven mechanism and pathway [40].
There are a number of therapeutics that are currently in
clinical trials for ALS and these have been reviewed
elsewhere [37,41-43] and therefore will not be discussed in this
present report. Additional development candidates not
reviewed elsewhere include GSK1223249, an anti-Nogo-A
antibody [44]. Nogo-A is a high molecular weight membrane
protein of spinal cord myelin. Nogo-A functions to inhibit
neurite outgrowth and can trigger growth cone collapse [45,46]
which suggests that Nogo-A is an important factor involved in
limiting neurite regeneration and repair of injured axons. In
preclinical studies, Anti-Nogo-A antibody treatment pro-
motes recovery of manual dexterity after cervical lesion in pri-
mates [47]. Genetic ablation of Nogo-A in SOD mice resulted
in a moderate but significant increase in lifespan, increased
the number of motor neurons, and eliminated ubiquitin
inclusions (a marker of cell stress) [48]. Nogo-A expression
has also been shown to be a prognostic marker for ALS
early in the course of the disease [49]. A Phase I trial of
GSK1223249 in ALS patients has been completed where we
await safety and pharmacokinetic (PK) results of this
clinical study.
Tirasemtiv, formerly known as CK-2017357, is an
imidazo-pyrazine with a molecular mass of 230 Da. Tirasem-
tiv is a fast skeletal muscle troponin activator which increases
fast skeletal muscle sensitivity to calcium, resulting in
enhanced skeletal muscle force and slowing of time to muscle
fatigue [50]. Tirasemtiv demonstrated potentially clinically
relevant pharmacodynamic effects in a completed Phase IIa
evidence of effect clinical trial in ALS patients [51]. At 6 h after
dosing, patients demonstrated a positive change in overall sta-
tus and decreased muscle fatigability. Data also demonstrated
a statistically significant increase in the maximum volume of
air patients could inhale and exhale [51]. A Phase IIb clinical
trial for Tirasemtiv is expected in the near future as this looks
to be a promising, albeit palliative approach to treating ALS.
Histone deacetylase (HDAC) inhibitors have been dis-
cussed as potential therapeutics for neurodegenerative diseases
for some time where their limitation lies in their lack of selec-
tivity and concomitant toxicity [52]. Inhibition of histone
deacetylation can modulate gene expression and protein syn-
thesis [53] and thus presents a biological basis for potential
treatment of ALS. HDAC inhibitors have also been shown
to be efficacious in ALS mouse models in a number of
studies [54-58]. A Phase II clinical trial of sodium phenylbuty-
rate in patients with ALS demonstrated safety and tolerability
at high doses, as well as a trend toward an increase in histone
acetylation in patients’ blood samples [59]. As this clinical trial
was not powered to observe a potential clinical benefit, it
7
 M. P. Bova & G. G. Kinney

Table 1. Genes associated with FALS.

ALS subtype Gene Chromosome region % of FALS cases Alternative phenotype Function

ALS1 SOD1 21q22.1 20 None Oxidative stress

ALS2 Alsin 2q33 Rare Spastic paraplegia Vesicle trafficking
ALS3 Unknown 18q21 Rare Unknown Unknown
ALS4 SETX 9q34 Rare AOA2 RNA processing
ALS5 Unknown 15q15-21.1 Rare Unknown Unknown
ALS6 FUS/TLS 16q12 4 FTD RNA processing
ALS7 Unknown 20p13 Rare Unknown Unknown
ALS8 VAPB 20q13.33 Rare SMD Vesicle trafficking
ALS9 ANG 14q11 Rare FTD RNA processing
ALS10 TDP-43 1p36.22 5 FTD/FTLD-U RNA processing
ALS11 FIG4 6q21 Rare Unknown Vesicle trafficking
ALS12 OPTN 10p15-p14 Rare Unknown Vesicle trafficking
ALS13 ATXN2 12q24.12 Rare Unknown RNA processing
ALS14 VCP 9p13.3 Rare IBMPFD Vesicle trafficking/UPS
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016

ALS15 UBQLN2 Xp11.21 Rare Unknown UPS/Autophagy

Undesignated DAO 12q24 Rare Unknown Glutamate transmission
Undesignated DCTN1 2p13 Rare FTD Vesicle trafficking
Undesignated C9Orf72 9p21 23 -- 40 FTD RNA processing?
Undesignated PFN1 17p13.3 Rare Unknown Actin dynamics

ALS: Amyotrophic lateral sclerosis; ANG: Angiogenin; AOA2: Ataxia with oculomotor apraxia 2; ATXN2: Ataxin-2; DAO: D-amino oxidase; DCTN1: Dynactin 1;
FIG4: Factor induced gene 4; FTD: Frontotemporal dementia; FUS/TLS: Fused in sarcoma, translocated in liposarcoma; IBMPFD: Inclusion body myopathy,
Paget’s disease and frontotemporal dementia; OPTN: Optineurin; PFN1: Profillin 1; SETX: Sentaxin; SMD: Spinal muscular dystrophy; SOD1: Superoxide dismutase;
TDP-43: TAR DNA binding protein; UBQLN2: Ubiquilin-2; VAPB: Vesicle-associated membrane protein-associated protein B; VCP: Valosin containing protein.

remains to be seen whether phenylbutyrate will show efficacy
in ALS patients.
2. Genes associated with FALS
Approximately 5 -- 10% of ALS patients have a family history
and/or the presence of a known genetic mutation and thus has
familial ALS (FALS), while the remainder of patients are char-
acterized with SALS [60]. Symptoms first appear for patients
with FALS in the fourth to fifth decade of life, while features
of the condition for SALS usually appear later. FALS and
SALS are indistinguishable based on clinical symptoms.
Genes associated with FALS can be grouped into five catego-
ries: i) those that effect RNA processing, ii) those that effect
vesicle trafficking, iii) those that affect oxidative stress,
iv) those that effect the ubiquitin proteasome system (UPS)
and autophagy, and v) those that currently have an unknown
function (Table 1).
2.1 Genes associated with RNA processing
TAR DNA-binding protein of 43 kDa (TDP-43) is a major
component of ubiquitinated protein aggregates found in
the CNS of ALS patients and those suffering from frontotem-
poral lobar degeneration with ubiquitinated inclusions
(FTLD-U) [61,62]. To date, 44 different mutations of this
gene have been identified that account for close to 5% of all
FALS patients [53]. TDP-43 binds to an estimated one-third
of all mouse and human brain RNAs [63,64]. TDP-43 has a
profound effect on mRNA splicing and mature mRNA levels
as determined from TDP-43 knockdown studies [63,64].
TDP-43 participates in the processing of many mRNAs that
are involved in neuronal function, such as neurexins, the
NMDA receptor and NF-L [63]. Fused in sarcoma or translo-
cated in liposarcoma (FUS/TLS) is another gene involved in
RNA processing, which has structural and functional similar-
ities to TDP-43, where a total of 46 mutations have been
identified accounting for ~5% of all FALS patients [65]. In
ALS, wild type and mutant TDP-43, as well as FUS accumu-
late in the cytoplasm and lose their nuclear localization [65].
Interestingly, TDP-43 and FUS mislocalization and aggrega-
tion is observed in other diseases such as FTLD-U, AD and
Huntington’s disease (HD) [65]. One unanswered question is
why are TDP-43 and FUS so susceptible to aggregation?
They are certainly large, multi-domain proteins that have
poly-functional roles in mRNA processing. It is possible that
posttranslational modifications such as phosphorylation and
proteolysis may enhance the susceptibility of these proteins
to aggregation [65]. Interestingly, one ALS-associated mutation
in TDP-43 enhances its stability and promotes a complex
with FUS, suggesting that this mutation in TDP-43 may
perturb FUS function [66]. This result leaves the door open
for a potential toxic gain of function activity for some
TDP-43 mutations.
Other proteins that are genetically linked to ALS that are
involved in RNA processing include Senataxin (SETX) which
is a DNA/RNA helicase protein that affects gene transcrip-
tion [67]. It has been demonstrated that depletion of SETX
in HeLa cells causes an increase in readthrough RNA and
Pol II density downstream of the polyA site, suggesting an
involvement in transcriptional termination [68]. Mutations in

8 Expert Opinion on Orphan Drugs (2013) 1(1)


SETX account for <1% of FALS cases. Angiogenin (ANG) is
a secreted RNase that accounts for <1% of FALS cases. ANG
has been shown to induce RNA cleavage in astrocytes where
FALS associated mutations in ANG abrogate its RNase
activity [69].
2.2 SOD1
Cu/Zn SOD was the first gene to be linked to ALS. SOD is
responsible for the catabolism of superoxide radicals to hydro-
gen peroxide and oxygen. SOD1 is a 153 amino acid, func-
tional homodimer that binds to Cu and Zn. SOD is
ubiquitously expressed and comprises ~ 1% of all cytoplasmic
proteins [70]. Mutations that occur in the SOD gene account
for ~ 20% of all FALS cases [71]. The concept that mutant
SOD1 causes aberrant oxyradical reactions, explaining its
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
toxic gain of function, is controversial and has been reviewed
elsewhere [71,72]. However, of note, it has also been suggested
that the oxidative damage caused by mutant SOD1 may be
attributed to Cu binding outside of the active site [73].
2.3Genes involved in vesicle trafficking and other
genes of interest
The size of a motor neuron and length of its axon can cause
increased metabolic burden. Thus, it is not surprising that
mutations in genes involved in vesicle trafficking may be
linked to ALS. One such gene is Alsin (ALS2), which func-
tions as a Rab5 guanine nucleotide exchange factor [74].
Mice deficient in ALS2 exhibit age-dependent neurological
and motor neuron deficits that are associated with altered
endosome trafficking [75]. For example, in wild type cortical
neurons that were stimulated with brain-derived neurotrophic
factor (BDNF), we observed a typical accumulation of Trk-
B in the perinuclear area, which is indicative of retrograde
transport. In contrast, in neurons derived from Als2-/- mice,
this was not observed [75]. Vesicle-associated membrane
protein-associated protein B (VAPB), factor-induced gene
4 (FIG4), and Optineurin are other FALS genes that are
also involved in vesicle trafficking [76-78] and have been
similarly linked to FALS (Table 1).
Mutations in the C9Orf72 gene have been shown to cause
up to 40% of all FALS cases and are also associated with
FTLD-U [79]. Although the function of the C9Orf72 gene is
not known, the presence of RNA nuclear foci in C9Orf72
FALS patient brain suggests that alternative mRNA splicing
is dysregulated [80]. Most recently, mutations within the pro-
filin 1 (PFN1) gene have been shown to cause FALS [81].
PFN1 is an actin-binding protein that plays an important
role in regulating actin dynamics. PFN1 enhances the rate
of actin polymerization by binding to actin-ADP to catalyze
nucleotide exchange facilitating formation of actin-ATP.
PFN1 bound actin-ATP is a substrate of Formin which adds
actin-ATP to the barbed ends of nascent actin fibers, with a
subsequent release of PFN1 [82]. From a functional perspec-
tive, a mutation in PFN1 that blocked actin-binding activity
was shown to inhibit neurite outgrowth in a neuroblastoma
Expert Opinion on Orphan Drugs (2013) 1(1)
Emerging drug targets in amyotrophic lateral sclerosis
cell line [83]. In a follow-up study to determine the prevalence
of PFN1 gene mutations in FALS, this gene was sequenced in
a cohort of 94 FALS patients of European ancestry. No
PFN1 mutations were identified in this cohort suggesting
that PFN1 mutations are a rare cause of FALS [84]. Despite
that mutations in PFN1 may account for a low frequency of
FALS patients; PFN1 gene mutations and their linkage to
FALS have demonstrated a novel mechanism that can cause
the disease.
3. New targets for ALS
3.1 EphA4
Ephrin type-A receptor 4 (EphA4) is a receptor tyrosine
kinase that consists of an extracellular ligand-binding domain,
a transmembrane region, and an intracellular kinase domain
that can accommodate multiple docking sites for downstream
signaling [85]. Upon ligand binding, the EphA4 receptor
becomes activated and initiates signaling in the forward direc-
tion. However, ephrins, the ligands of the Eph receptor, are
attached to the cell membrane through a glycosylphosphatidy-
linositol link which enables signal transduction in the reverse
direction [86]. This bidirectional signaling is a key feature of
Ephrin biology.
EphA4 is enriched at excitatory synapses in motor neurons
and in the hippocampus [87,88]. Genetic knockdown of
EphA4 in the mouse suggests that ephrin-A:EphA forward
signaling is required for selection of lateral lower motor
column neurons toward the dorsal limb, thus contributing
to the topographic organization of motor neuron projec-
tions [87,89]. It has also been shown that the EphA4 receptor
tyrosine kinase regulates spine morphology where its activa-
tion reduces spine length and density in hippocampal sli-
ces [90]. Subsequently EphA4 forward signaling was shown
to decrease phosphorylation of focal adhesion kinase (FAK),
and proline-rich tyrposine kinase 2 (Pyk2), thus regulating
dendritic spine remodeling by affecting b1 integrin signaling
pathways [91]. In the hippocampus, EphA4 receptor reverse
signaling may overactivate its ligand glial ephrinA3 and
inhibit glutamate uptake by the glutamate transporter [88].
Recently, in a genetic screen using a SOD1 zebrafish model,
the most protective knockdown identified was of the gene
Rtk2, which has 67% identity to human EphA4 [92]. Further
validation of the importance of this target were the results in
SOD1G93A mice where a 50% knockdown of EphA4 expres-
sion significantly enhanced motor function (rotarod perfor-
mance) and increased survival time [92]. In patients with
ALS, EphA4 expression inversely correlated with disease onset
and survival where loss of function and EphA4 mutations are
associated with longer survival [92]. These findings suggest that
EphA4 represents a compelling biological target for ALS.
From a drug discovery perspective, EphA4 is a tractable
drug target. Compounds of the pyrrolyl benzoic acid scaffold
have been identified that block the interaction of ephrins with
the EphA4 receptor [93]. In addition, small molecules have
9
 M. P. Bova & G. G. Kinney
been identified that bind to the ATP pocket and block
EphA4 kinase activity with high affinity [94]. A crystal struc-
ture of the EphA4 tyrosine kinase domain in the apo- and
dasatinib-bound state has also been determined which will
facilitate structure-guided drug design [95]. Key insights from
the crystal structure suggest a path forward for kinase receptor
selectivity between EphA4 and other Ephrin receptors [95].
There are a number of commercially available EphA4
biochemical kinase assays that are amenable to high through-
put screening (HTS) which should facilitate the identification
of leads for drug discovery efforts. Further, a number of
antibody reagents are readily available suggesting that the
development of cell-based characterization assays will not
represent an obstacle for drug discovery. In addition to the
potential role for EphA4 as a drug target for ALS, it has
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
been implicated to play a role in gastric cancer [96] and
blocking EphA4 is effective in preclinical models of
spinal cord injury [97]. At present, however, it does not
appear that EphA4 has been intensely investigated in other
neurodegenerative indications.
3.2 Nuclear erythroid 2-related factor 2
Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription
factor, which under non-stressed resting conditions, is normally
located in the cytoplasm bound to Kelch-like ECH-associated
protein 1 (Keap1). Under conditions of oxidative stress, oxida-
tive modification of Keap1 leads to the dissociation of
Nrf2 from Keap1. Nrf2 can then translocate to the nucleus,
where Nrf2 binds the antioxidant response element (ARE)
and facilitate the expression of over 250 cytoprotective genes
including heme oxygenase-1 (HO-1), glutathione, thioredoxin
(TRX) and Hsps. The Nrf2/Keap1/ARE system is a major cel-
lular defense system for cells undergoing oxidative stress [98]. As
described above, oxidative stress has been implicated to play a
role in ALS [99] and the Nrf2/Keap1 system was shown to be
impaired in motor neurons in a SOD1 mouse model [100].
Interestingly, selective overexpression of Nrf2, in astrocytes,
using double transgenic SOD1G93A/GFAP-Nrf2 mice,
extended survival by over 20 days compared to SOD1G93A
mice [101]. This protective effect was mediated in part by an
increase of astrocyte-secreted glutathione [101]. Triterpenoid
small molecule activators of the Nrf-2/Keap1 system have
extended survival in SOD1G93A mice [102]. Bardoxolone
Methyl (formerly RTA402), a triterpenoid has completed a
Phase II clinical trial for stage 4 chronic kidney disease
(CKD) [103] and is currently in a Phase III trial for this indica-
tion. As there is good rationale that activation of Nrf2/Keap1
system would be helpful to ALS patients, clinical testing using
a CNS permeable Nrf2 activator could be considered.
3.3 DJ-1
DJ-1 is a 20 kDa protein that is ubiquitously expressed and
widely conserved throughout species [104]. DJ-1 was identified
as an oncogene and subsequently linked to familial PD [105].
DJ-1 is a multifunctional oxidative stress response protein that
10 Expert Opinion on Orphan Drugs (2013) 1(1)
confers protection to cells undergoing oxidative and or mito-
chondrial stress [106,107]. Multiple functions for DJ-1 have been
described. For example, DJ-1 has been shown to negatively reg-
ulate the apoptotic signaling kinase1 (ASK1) [108,109]. DJ-1 has
also shown to suppress the proapoptotic PTEN phosphatase
and thus is an enhancer of the Akt survival pathway [109,110].
DJ-1 is a redox sensitive protein where it has been shown that
oxidation of cysteine 106 on the DJ-1 protein is required for
its protective function [106,107]. Interestingly, excessive oxidation
of this cysteine to the sulfonic acid renders the protein inac-
tive [106]. DJ-1 is also linked to ALS where DJ-1 mutations in
one Italian family caused Parkinsonism-Dementia ALS
(PDALS) complex [111]. PDALS complex has been particularly
well characterized in patients in the Kii peninsula of Japan and
in Guam [112] where environmental as well as genetic factors
may play a role in the development of disease. Thus, ALS and
PD pathogenesis, may in some cases share a common etiology.
At the molecular level, DJ-1 forms complexes with mutant
SOD1 and ameliorates its toxicity [113]. As oxidative stress may
play a role in many neurodegenerative diseases, it is not surpris-
ing that a protein like DJ-1 could be implicated in both ALS and
PD. Although DJ-1 is protective in cellular and animal models
of oxidative stress, it has not proved simple to assay for small
molecule binders in a high-throughput biochemical format.
One virtual screen identified potential binders of DJ-1 whose
binding properties were confirmed using a quart crystal
microbalance [114]. These compounds exhibited specificity for
DJ-1 and prevented oxidative stress-induced cell death in
SH-SY5Y and in primary ventral mesencepahlon cells [114]. In
addition, these DJ-1-binding compounds were active in block-
ing dopaminergic cell death and blocked movement abnor-
malities in a 6-hydroxydopamine injected PD rat model [114].
It was reported that the protective effects of these compounds
were mediated through binding to an oxidized form of DJ-1.
It remains of interest to test these compounds in cellular and
animal models of ALS.
DJ-1 levels are reported to be lower in DJ-1 familial PD
patients and, as such, increasing levels of DJ-1 could be a
therapeutic strategy for familial and sporadic PD and
ALS [115,116]. Thus, compounds that bind and stabilize
DJ-1 may be useful therapeutics. Binding assays such as
those that use surface plasmon resonance (SPR) could also
be used to identify small molecules that interact with
DJ-1 in a medium throughput manner. A structure for
DJ-1 has been solved by X-ray crystallography which may
further facilitate a drug discovery program for this target [117].
Compounds of interest could be run through cellular oxida-
tive stress protection assays in cells expressing mutant or wild
type DJ-1. Although there are many inherent risks with this
approach, DJ-1 is a validated target for PD and may be of
relevance for ALS.
3.4Apoptotic signaling kinase1
Apoptotic signaling kinase1 (ASK1) is a 1374 amino acid poly-
peptide consisting of a central serine/threonine kinase domain

ROS
SH DJ-1
S
H Trx DJ-1*
ASK1
ASK1
S
S minary analysis suggests that selectivity between ASK1 and
Trx
DJ-1**
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
P Traf2
MKK4/7 MKK3/6
ASK1 Traf6
JNK p38
Apoptosis
Figure 1. Hypothetical scheme for activation of ASK1 by
oxidative stress. In resting conditions, ASK1 is bound to and
negatively regulated by TRX. Under conditions of oxidative
stress, TRX becomes oxidized and dissociates from the ASK1
signaling complex (signalosome). DJ-1 becomes oxidized
under conditions of oxidative stress (*) and subsequently
binds to ASK1, negatively regulating its activity. Continued
oxidation of DJ-1 can lead to a conformational change (**)
where it dissociates from the ASK1 signalosome, relieving the
negative inhibition. ASK1 becomes phosphorylated and is now
activated resulting in the activation of JNK and p38 pathways.
This schematic connects ASK1, DJ-1 and p38 targets in a
common pathway initiated by oxidative stress.
and coiled-coil domains at the N- and C-terminus [118,119].
ASK1 is a MAP3K that plays an essential role in cellular stress.
In its resting state, ASK1 is a homodimer stabilized by its C-
terminal coiled-coil domain. The N-terminal domain is bound
by the redox regulatory protein, thioredoxin, which prevents its
activation [120,121]. ASK1 is activated by ROS, ER stress, calcium
ion influx and other stressful stimuli [122]. ASK1 is a regulator of
the JNK and p38 MAP kinase cascades in stress signaling [118,122].
Oxidative stress leads to disulfide bride formation of thioredoxin
and dissociation from ASK1, triggering a conformational
change in the ASK1 dimer leading to phosphorylation and acti-
vation of its kinase activity [120,121]. DJ-1 can bind to ASK1 at its
N-terminal domain and negatively regulate ASK1 phosphoryla-
tion activity (Figure 1). Further levels of oxidative stress lead
to diminished DJ-1 levels, unregulated ASK1 activity, and
subsequent cell death (Figure 1). ASK1 knockout is protective
in ALS and PD mouse models [121,123]. Specifically, in
SOD1G93A/ASK1-/- mice, mean survival times were significantly
Expert Opinion on Orphan Drugs (2013) 1(1)
Emerging drug targets in amyotrophic lateral sclerosis
longer and neuronal cell death was ameliorated in the spinal cord
compared to SOD1G93A mice [123]. Furthermore, ASK1 has
been reported to negatively regulate the proteasome, possibly
by phosphorylating and inhibiting the ATPase activity of
Rpt5 [124]. Thus, there is good biological rationale suggesting
that well-tolerated ASK1 inhibitors may be useful for the
treatment of ALS [125].
From a drug discovery perspective, ASK1 appears to be a
tractable target. A crystal structure of ASK1 has been deter-
mined [119] which should facilitate structure-based drug
discovery. ASK has three isoforms (ASK1-3) and preli-
ASK2 is possible, yet selectivity between ASK1 and ASK3 is
unlikely [126]. It is not known whether selectivity between
the three ASK isoforms is necessary for an appropriate tolera-
bility/efficacy profile. Selectivity between ASK1 and the
broader kinome also appears possible from a preliminary anal-
ysis [126]. ASK1-/- mice are healthy and viable, yet studies have
shown that ASK1 does play a role in innate immunity [127].
Thus, the benefit to cost ratio of an ASK1 inhibitor may over-
ride any underlying safety concerns, particularly in ALS
patients, where there is a dire unmet medical need. Although
there have been reports of ASK1 inhibitors in development
for insulin resistance and cardiac indications, their use in
neurodegeneration seems relatively unexplored [128].
3.5 HDAC6 stabilizers/inhibitors
Currently the nonselective HDAC inhibitor, phenylbutyrate,
is undergoing clinical evaluation for the treatment of
ALS [59]. HDAC6, a class II HDAC, represents a particularly
intriguing target for ALS as there appears to be rationale for
both a HDAC6 stabilizer/activator and a HDAC6 inhibitor
for therapeutic application to ALS. HDAC6 has been shown
to facilitate the formation of aggresomes, activate autophagy,
and may be involved in the expression of Hsps [129,130].
Some of these neuroprotective functions may be independent
of the deacetylating activity of HDAC6 [129-131]. TDP-43 and
FUS may function in a complex to regulate HDAC6 mRNA
expression where a loss of TDP-43 function may decrease
levels of HDAC6 expression in motor neurons [132]. In this
manner, a HDAC6 stabilizer, which could increase HDAC6
levels, would be helpful in ALS. Small molecule chaperones or
stabilizers have been successfully pursued for some targets [133].
However, this approach is not universally applicable to
all proteins.
There is also rationale for potential therapeutic role for a
HDAC6 inhibitor in ALS. HDAC6 is the major a-tubulin
deacetylating enzyme [134-136]. HDAC6 inhibition protects
against oxidative stress-induced neurodegeneration and pro-
motes neurite outgrowth in cortical neurons co-cultured with
Chinese hamster ovary (CHO) cells expressing myelin-
associated glycoprotein (MAG) [137]. In addition, HDAC6
inhibition promotes neurite outgrowth in dorsal root ganglion
neurons exposed to MAG [137]. Furthermore, in a mouse model
of mutant Hsp27-induced Charcot-Marie-Tooth (CMT)
11
 M. P. Bova & G. G. Kinney
disease (a peripheral neuropathy), HDAC6 inhibitors restored
axonal transport, and partially restored the CMT phenotype
both at the behavioral and electrophysiological levels [138].
From a pharmacological perspective, HDAC6 represents a
tractable target. The HDAC6 knockout mice are fully func-
tional and viable, suggesting that the reduction of HDAC6
activity may be less likely to induce mechanism-based toxic-
ity [139]. In addition, selective HDAC6 inhibitors have been
synthesized [140,141], thereby obviating the potential for nonspe-
cific toxicity that may be observed following treatment with
CNS penetrant pan-HDAC inhibitors.
3.6 Nuclear factor-kappaB
Nuclear factor-kappaB (NF-kB) consists of a dimer of the
p65 and p50 proteins. In the inactivated state, NF-kB is
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
kept in the cytoplasm complexed with the inhibitory protein
inhibitor of kappaB (IkBa). Extracellular stimuli such as
TNF, reactive oxygen species and cytokines can activate
many transmembrane receptors which can then activate IkB
kinase which then phosphorylates the inhibitory protein
IkBa. After phosphorylation, IkBa becomes ubiquitinated
and then degraded. The NF-kB transcription factor then
translocates to the nucleus to turn on protein expression [142].
Aberrant regulation of NF-kB has been linked to cancer,
inflammatory disease, septic shock and autoimmune dis-
ease [142,143]. Recently, TDP-43 was shown to interact
with the p65 subunit of NF-kB in mouse microglia cells
after LPS simulation [144]. From a functional perspective, a
gene reporter assay was used to show that TDP-43 acts as a
coactivator of p65. Furthermore, this interaction was shown
to occur in the context of human ALS patients where
TDP-43 and p65 were coimmunoprecipitated from spinal
cord extracts of ALS patients but not from spinal cord extracts
of control patients. Consistent with an inflammatory
component of ALS, it was also demonstrated that TDP-43
overexpression in glia or macrophages causes hyperactive
inflammatory responses following a LPS challenge. Treatment
of TDP-43 transgenic mice with Withaferin-A (WA), an
NF-kB inhibitor, significantly improved motor function
as demonstrated by enhanced rotorod performance and
WA-treated mice had a 40% reduction in the number of
partially denervated neuromuscular junctions [144]. Corrobo-
rating the results from this study, high levels of circulating
TNFa have also been observed in other cohorts of ALS
patients and in other animal models of ALS [145-148]. Using
spinal cord organotypic cultures, it was shown that TNFa
potentiates glutamate-induced motor neuron cell death which
was blocked by inhibition of the NF-kB pathway [149]. Thus,
chronic NF-kB activation may be a contributing factor to
ALS disease and or disease progression and as such is a rele-
vant therapeutic target. There are many approaches to success-
fully inhibit the NF-kB pathway with small molecules and
these have been thoroughly reviewed elsewhere [150,151]. The
advancement of these compounds toward clinical testing for
ALS may offer hope to patients in the future.
12 Expert Opinion on Orphan Drugs (2013) 1(1)
3.7 P38a
The P38a is a member of the mitogen-activated protein
kinase (MAPK) family of serine/threonine kinases. The
p38a is widely expressed in endothelial, inflammatory cells,
microglia and in the spinal cord [152]. The p38a is activated
in response to stressful stimuli such as osmotic stress, oxida-
tive stress, LPS, growth factors and inflammatory cytokines
(TNFa, IL-1b, IL6) [152,153]. The p38 is activated in motor
neurons and microglia of SOD1G93A mice [154-157] and in
ALS patients [157,158]. The p38a directly phosphorylates NFs
and aberrant accumulation of phosphorylated NFs are hall-
mark pathological features of ALS [157]. Deficits in axonal
transport are an early event observed in the disease course of
SOD1 mutant transgenic mice [159]. Additional substrates
for p38a include the heavy chain of the molecular motor
kinesin [160]. In a series of studies, it was shown that one
functional consequence of p38-mediated phosphorylation of
kinesin was to inhibit fast axonal transport, an effect that
was blocked by pharmacological p38 inhibition [160,161]. Of
interest, p38 inhibition had a marked effect on motor neuron
survival in the SOD1G93A mouse model, yet only elicited a
modest effect on survival [162]. It has been shown that riluzole
protects against glutamate-induced deficits in NF axonal
transport [163,] and it is therefore tempting to speculate that
a combination of riluzole with a p38 inhibitor may work syn-
ergistically to reverse axonal transport deficits. There are a
number of p38 inhibitors that have now entered into clinical
testing for inflammatory indications and these have been
reviewed extensively elsewhere [164,165]. It would be of interest
to understand if a p38 inhibitor in combination with riluzole
could provide significant benefit to ALS patients.
3.8Activation of autophagy and the UPS for protein
clearance
The UPS and autophagy/lysosome pathways are the two major
cellular systems that degrade and clear proteins. In ALS, cyto-
plasmic inclusions consisting of hyperphosphorylated neurofi-
laments, TDP-43 and other proteins are a hallmark of the
disease [61,62]. Clearance of these protein inclusions could be
helpful in ameliorating symptoms of the disease as we do not
know for certain whether inclusions are directly toxic them-
selves or represent an attempt to package aberrantly folded pro-
teins that may not be cleared otherwise, into an innocuous
compartmentalized form. Proteasomes predominantly degrade
shorter half life nuclear and cytoplasmic proteins that need to
be unfolded. In contrast, lysosomes degrade larger membrane
proteins, oligomers and aggregates, and organelles in a process
called autophagy. When proteins oligomerize, aggregate and
form large inclusions, they become inaccessible to the protea-
some. HD is a neurodegenerative disorder caused by a long
CAG repeat in the Huntington (Htt) protein. Thus this
mutant protein contains an abnormally long polyglutamine
tract where after cleavage of the mutant protein, fragments
containing the polyglutamine have a tendency to aggregate,
forming inclusions. These inclusions, which are a hallmark of

HD, can interfere with neuronal function, ultimately leading to
cell death [166]. It was shown that inhibition of mTOR by rapa-
mycin induces autophagy and reduces toxicity of mutant Htt in
fly and mouse models of HD [167]. In addition, a screen for
autophagy enhancers was performed which identified a number
of small molecule modulators of autophagy [168]. It remains to
be determined whether a similar approach will yield benefits in
ALS. However, evidence of the relevance of enhancing autoph-
agy as a potential therapeutic approach for ALS comes from a
report by Hetz et al. [33]. In this study, they investigated the
contribution of X-box binding protein-1 (XBP-1), a transcrip-
tion factor involved in the UPR, for its contribution to FALS.
They observed that knockdown of XBP1 in NSC34 cells tran-
siently transfected with mutant SOD enhanced clearance of
mutant SOD aggregates by macroautophagy [33]. This finding
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
in a cellular system was validated in vivo where genetic knock-
down of XBP1 in female mutant SOD mice prolonged survival
by 10 days and increased autophagy and SOD1 degrada-
tion [33]. Thus, we look forward to the results of testing small
molecule enhancers of autophagy in ALS animal models.
It has similarly been postulated that enhancing proteasomal
clearance of proteins would have broad benefit across neurode-
generative diseases. Proteasomal degradation is a complex and
highly regulated process. Proteins are brought to the
proteasome after ubiquitination for degradation. Once at the
proteasome, proteins must be deubiquitinated and unfolded
before entry into the proteasome. The deubiquitinating
function is performed by a family of proteins termed deubiqui-
tinating enzymes (DUBs). There are three resident DUBs that
are associated with the proteasome: Usp14, Uch37 and Rpn11.
Usp14 has an ubiquitin chain trimming activity which in bio-
chemical experiments slowed down proteasomal degradation of
ubiquitinated protein substrates [169]. In addition, it was shown
that small molecule inhibitors of Usp14 enhanced the clearance
of TDP-43, Ataxin-3 and other proteins of relevance for neuro-
degenerative diseases in cellular assays [169]. It remains to be
seen whether small molecule inhibitors of Usp14 will be
efficacious in in vivo models of neurodegeneration.
Phenotypic screens aimed at identifying novel
3.9
ALS targets
Despite some potentially promising clinical candidates cur-
rently under evaluation and some of the new promising
drug targets listed above, a lack of a detailed understanding
of the underlying mechanisms that cause ALS has hampered
drug discovery. Cellular phenotypic screens can help facilitate
the identification of new drug targets for ALS. Phenotypic
screening is a type of screening used in drug discovery to iden-
tify small molecules that alter a cellular phenotype such as
neurite outgrowth, expression of a particular protein or cell
death. In a blinded phenotypic screen of 1040 FDA-approved
drugs, it was discovered that many b-lactam antibiotics are
potent stimulators of the glutamate transporter, EAAT2
(also called GLT1) expression [170]. Glutamate uptake by the
transporter is one of the major modes of inactivation of
Expert Opinion on Orphan Drugs (2013) 1(1)
Emerging drug targets in amyotrophic lateral sclerosis
glutamate signaling and thus increased expression of this
transporter may be of benefit for ALS. Ceftriaxone, one of
the b-lactam antibiotics identified in that study, elevated
GLT1 expression threefold in cellular models and also raised
GLT1 expression in rodents. In SOD1G93A mice, administra-
tion of Ceftriaxone significantly spared motor neurons
and had a modest effect on prolonging survival [170]. In a
Phase II study for ALS, Ceftriaxone demonstrated good
tolerability and is now in an ongoing Phase III study [171].
Similarly, cellular phenotypic screens can be used to iden-
tify compounds that enhance vesicle trafficking, RNA proc-
essing and clearance of aberrantly folded proteins. Not only
can previously approved drugs be used in these screens, but
screening could also be performed on larger and more diverse
collections of compounds. One limitation of phenotypic
screens is the lack of identification of the molecular drug tar-
get. This lack of understanding may make a traditional medic-
inal chemistry-based structure activity relationship (SAR)
effort more difficult. However, novel chemical proteomics
technologies have emerged that could help in target deconvo-
lution following a phenotypic screen [172]. Stable isotope
labeling for cell culture (SILAC) is one such technique based
on mass spectrometry that allows for identification and quan-
tification of proteins. Of relevance for identifying targets from
phenotypic screens, using SILAC, cells can be metabolically
labeled with different isotopes of arginine and lysine. The
compound of interest can then be chemically tethered to
sepharose beads and added to the cellular lysate. Specifi-
cally bound proteins can be identified, and their Kd values
determined using relative quantitative mass spectrometry.
Although careful controls must be considered with this tech-
nique, it has been successful in identifying novel binding
targets of small molecule compounds.
One potential limitation in the development of new ther-
apeutics targeting ALS is the observed discordance between
interventions that demonstrate activity in ALS animal mod-
els and efficacy in the clinical setting. The reasons for this
discrepancy are manyfold and have been highlighted else-
where and are therefore not in the scope for this review [173].
As a potential mechanism for mitigating this issue, a
drug repositioning approach has been suggested [173]. This
approach would test as many FDA approved drugs as possi-
ble, in a small number of patients to observe if a marked
clinical benefit was noted in individual patients. Endpoints
in such a trial would include improvement of strength or
cessation of disease progression. Using cellular phenotypic
screens or directly testing a number of previously approved
drugs in a smaller number of ALS patients may decrease
the overall time to realize success in the search for new
therapeutics to treat this disease.
4. Conclusions
Numerous mechanisms have been described that may con-
tribute to the pathogenesis of ALS. The primary target of
13
 M. P. Bova & G. G. Kinney
degeneration in ALS, the motor neuron, is large and has a
high metabolic requirement. Motor neurons are exposed to
a high level of oxidative stress as a result of primary signaling
using excitatory neurotransmitters; yet these neurons appear
particularly vulnerable in that additional protective mecha-
nisms to counterbalance this stress appear to be lacking.
Many motor neurons do not express high levels of calcium-
buffering proteins which likely also contribute to their suscep-
tibility in ALS.
Identification of mutations in TDP-43, FUS and most
recently C9Orf72 that cause FALS have changed the way
the field thinks about this disease from a mechanistic and clin-
ical perspectives. There are currently 15 described ALS gene
subtypes (Table 1) and many more unnamed genes that con-
tribute to FALS. As this field of study progresses, it appears
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
probable that many more genes will be categorized in the
near future. Many of the ALS-associated genes can be broadly
categorized into those that effect RNA processing, vesicular
trafficking and oxidative stress. The recently discovered
FALS gene PFN1 has illustrated a new mechanism of motor
neuron degeneration. As additional genes are identified, these
will further help to advance our understanding of ALS in
the future.
As described above, there are a number of promising
novel ALS drug targets on the horizon that may lead to
breakthrough therapeutics. Genetic knockdown studies eval-
uating EphA4 and ASK1 kinases have significant biological
effects in prolonging survival in the SOD animal models.
Inhibition of HDAC6 has also shown efficacy in CMT
animal models. New technology platforms such as relative
quantitative mass spectrometry using SILAC will further
help to deconvolute hits from phenotypic screens which
should lead to additional ALS drug targets in the future, as
well as provide further mechanistic understanding around
this complex, multifactorial disease.
5. Expert opinion
There are many challenges facing researchers attempting to
identify and develop new therapeutics for ALS. First, as dem-
onstrated from ALS genetic studies, it is clearly a multifacto-
rial disease with multiple potential underlying pathological
mechanisms that can converge at the common endpoint of
motor neuron degeneration. Despite the potential multifacto-
rial nature of the pathogenesis of the disease, clinical studies
typically utilize a heterogeneous group of ALS patients for
clinical trials. Thus, it remains formally possible that com-
pounds that have previously failed in clinical trials may
exhibit efficacy in a small subset of ALS patients that are
appropriately stratified. Further complicating the ability to
translate nonclinical findings to effective treatments is the
lack of universal concordance between results using current
animal models and clinical outcomes. Given the large number
of molecules that are reported to provide efficacy in nonclin-
ical models, it may be argued that these models and/or the
14 Expert Opinion on Orphan Drugs (2013) 1(1)
endpoints used for assessment of efficacy are set at a lower
hurdle relative to clinical studies in patients. Another possibil-
ity that must be considered is whether the current clinical
study paradigms are sufficiently sensitive and/or whether it
is too late to therapeutically intervene with current treatment
approaches once patients are symptomatic. Similar arguments
have been forwarded in the treatment of other neurodegener-
ative disorders such as AD, where it has been suggested that
earlier intervention may be needed to effectively impact the
course of the disease. Treatment studies that allow for earlier
intervention in the course of ALS, before unequivocal diagno-
sis, could result in an enhanced efficacy profile for the current
standard of care, riluzole, as well as potentially allow
compounds that have previously failed in clinical trials to
demonstrate efficacy. In order to enable this approach, new
biomarkers that can accurately predict disease progression
need to be identified.
Despite the fact that genetics play a prominent role in
ALS research, particularly with the discovery of mutations
in TDP-43 and FUS that cause FALS and more recently
with C9Orf72 and PFN1, these discoveries have not yet
translated into new therapeutic approaches. New animal
models such as the TDP-43 transgenic mouse should allow
us to test new hypotheses and gain additional insight into
mechanisms underlying ALS pathology. Such models may
also prove to be more predictive for clinical efficacy. The
zebrafish model of ALS allows for both small molecule and
genetic screens directly in a medium throughput mode
in vivo. Such a model system provides the opportunity to
screen low-to-medium-sized compound or siRNA libraries
in a reasonable time frame. Drug screening using ALS
patient-specific induced pluripotent stem cells represents
an alternate, albeit exciting approach that has tremendous
potential from both a research and clinical perspective. Use
of stem cells in this manner could allow for the high-
throughput screening of large chemical libraries to identify
compounds of relevance. Once a target is identified, the
combination of traditional drug discovery approaches such
as structure-guided drug design on compelling new targets
like EphA4, ASK1, Nrf2 and HDAC6 with phenotypic
screening approaches holds the promise to enhance the rate
at which new drug targets for ALS are discovered and trans-
lated into clinical testing paradigms. Phenotypic screening
historically has been less well utilized due to the fact that it
is often difficult to deconvolute the molecular target for
compounds of interest that emerge after a screening cam-
paign. However, with new technologies such as relative
quantitative mass spectrometry using SILAC, the ability to
identify a compound’s cellular-binding partners in a reason-
able time frame is significantly enhanced. Phenotypic screens
with endpoints focused on protein clearance, RNA process-
ing, vesicle trafficking and motor neuron survival have the
potential to result in multiple new targets for ALS.
Finally, the growing emphasis on drug repositioning
approaches for the treatment of ALS represents a significant
 Emerging drug targets in amyotrophic lateral sclerosis

opportunity to expedite possible treatments to patients. Acknowledgments

Testing previously FDA-approved compounds in smaller
numbers of ALS patients has the potential to lead to exciting
new findings. Also, using combination therapy, taking
advantage of pharmacological synergy, as is increasingly
accepted as standard of care for cancer and hypertension, is
an additional approach that should be considered. By com-
bining the recent advances in genetics, chemical proteomics
and informatics, we expect a number of new and exciting
drug targets for this indication, and hope for ALS patients
in the future.
The authors thank E Johnson and D Walker for helpful
discussions and Z Ren and R Artis for the critical reading of
this manuscript. This manuscript is dedicated to the memory
of L Gehrig and H Katz, may we all follow our dreams.
Declaration of interest
M Bova and G Kinney are both employees of ELAN
Pharmaceutical.

Bibliography
Papers of special note have been highlighted as cerebrospinal fluid of patients with 17. Van Damme P, Van Den BL,
either of interest () or of considerable interest sporadic amyotrophic lateral sclerosis. Van Houtte E, et al. GluR2-dependent
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016

() to readers. Ann Neurol 1998;44:696-9 properties of AMPA receptors determines

1. del Aguila MA, Longstretch WT Jr,
McGuire V, et al. Prognosis in
amyotrophic lateral sclerosis:
a population-based study. Neurology
2003;60(5):813-19
2. Lee MK, Cleveland DW. Neurofilament
function and dysfunction:involvement in
axonal growth and neuronal disease.
Curr Opin Cell Biol 1994;6(1):34-40
3. Manetto V, Sternberger NH, Perry G,
et al. Phosphorylation of neurofilaments
is altered in amyotrophic lateral sclerosis.
J Neuropathol Exp Neurol
1988;47:642-53
4. Itoh T, Sobue G, Ken E, et al.
Phosphorylated high molecular weight
neurofilament protein in the peripheral
motor, sensory and sympathetic neuronal
perikarya: system-dependent normal
variations and changes in amyotrophic
lateral sclerosis and multiple system
atrophy. Acta Neuropathol
1992;83(3):240-5
5. Oosthuyse B, Moons L, Storkebaum
et al. Deletion of the hypoxia-response
element in the vascular endothelial
growth factor promoter causes motor
neuron degeneration. Nat Genet
2001;28:131-8
6. Shaw PJ, Ince PG, Falkous G, et al.
Oxidative damage to protein in sporadic
motor neuron disease spinal cord.
Ann Neurol 1995;38:691-5
7. Abe K, Pan LH, Watanabe M, et al.
Induction of nitrotyrosine-like
immunoreactivity in the lower motor
neuron of amyotrophic lateral sclerosis.
Neurosci Lett 1995;199:152-4
8. Smith RG, Henry YK, Mattson MP,
et al. Presence of 4-hydroxynonenal in
9. Durham HD. In: Shaw PJ, Strong MJ,
editors. Motor neuron disorders:
bluebooks of practical aneurology.
Butterworth-Heinemann; Philadelphia:
2003. p. 379-400
10. Wang X, Michaelis EK. Selective
vulnerability to oxidative stress in the
brain. Front Aging Neurosci
2010;2(12):1-13
11. Batulan Z, Shinder GA, Minotti S, et al.
High threshold for induction of the stress
response in motor neurons is associated
with failure to activate HSF1. J Neurosci
2003;23(13):5789-98
12. Taylor DM, Minotti S, Agar JN, et al.
Overexpression of metallothionein
protects cultured motor neurons against
oxidative stress, but not mutant Cu/Zn-
superoxide dismutase toxicity.
Neurotoxicology 2004;25:779-92
13. Kiernan D, Kalmar B, Dick JR, et al.
Treatment with arimoclomol, a
coinducer of heat shock proteins, delays
disease progression in ALS mice.
Nat Med 2004;10(4):402-5
14. Arimoclomol clinical trials information.
Available from: www.Clinical.Trials.gov
[Last accessed 24 October 2012]
15. Williams TS, Day NC, Ince PG, et al.
Calcium permeable alpha-amino-3-
hydroxy-5-methyl-4-isoxazole propionic
acid receptors: a molecular determinant
of selective vulnerability in amyotrophic
lateral sclerosis. Ann Neurol
1997;42:200-7
16. Greig A, Donevan SD, Mujtaba TJ,
et al. Characterization of the
AMPA-activated receptors present on
motoneurons. J Neurochem
2000;74:179-91
the selective vulnerability of motor
neurons to excitotoxicity. J Neurophysiol
2002;88:1279-87
18. Rothstein JD, Van Kammen M,
Levey AI, et al. Selective loss of glial
glutamate transporter GLT-1 in
amyotrophic lateral sclerosis. Ann Neurol
1995;38:73-84
19. Ferraiuolo L, Kirby J, Grierson AJ, et al.
Molecular pathways of motor neuron
injury in amyotrophic lateral sclerosis.
Nat Rev Neurol 2011;7:616-30
20. Carriedo SG, Sensi SL, Yin JH, et al.
AMPA exposures induce mitochondrial
Ca2+ overload and ROS generation in
spinal motor neurons in-vitro. J Neurosci
2000;20:240-50
21. Lautenschlaeger J, Prell T, Grosskreutz J.
Endoplasmic reticulum stress and the ER
mitochondrdia calcium cycle in
amyotrophic lateral sclerosis.
Amyotroph Lateral Scler 2012;13:166-77
. Excellent review on the role that
calcium homeostasis plays in ER stress
as indicated by activation of the UPR.
22. Arnaudeau S, Kelley WL, Walsh JV,
et al. Mitochondria recycle Ca2+ to the
endoplasmic reticulum and prevent the
depletion of neighboring endoplasmic
reticulum regions. J Biol Chem
2001;276:29430-9
23. Brini M, Carafoli E. Calcium pumps in
Health and Disease. Physiol Rev
2009;89:1341-78
24. Panov AV, Kubalik N, Zinchenko N,
et al. Metabolic and functional
differences between brain and spinal cord
mitochondria underlie different
predisposition to pathology. Am J
Physiol Regul Integr Comp Physiol
2011;300:R844-54

Expert Opinion on Orphan Drugs (2013) 1(1) 15

 M. P. Bova & G. G. Kinney
25. Lodish HF, Kong N, Wikstrom L.
Calcium is required for folding of newly
made subunits of the asialoglycoprotein
receptor within the endoplasmic
reticulum. J Biol Chem
1992;267:12753-60
26. Kuznetsov G, Brostrom MA,
Brostrom CO. Demonstration of a
calcium requirement for secretory protein
processing and export. Differential effects
of calcium and dithiothreitol.
J Biol Chem 1992;267:3932-9
27. Grosskreutz J, Van Den Bosch L,
Keller BU. Calcium dysregulation in
amyotrophic lateral sclerosis.
Cell Calcium 2010;47:165-74
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
28. Ince P, Stout N, Shaw P, et al.
Parvalbumin and calbindin D-28K in the
human motor system and in motor
neuron disease.
Neuropathol Appl Neurobiol
1993;19:291-9
29. Alexianu ME, Ho B-K, Mohamed AH,
et al. The role of calcium binding
proteins in selective motor neuron
vulnerability in amyotrophic lateral
sclerosis. Ann Neurol 1994;36:846-58
30. Elliott JL, Snider WD. Parvalbumin is a
marker of ALS-resistant motor neurons.
Neuroreport 1995;6:449-52
31. Shaw PJ, Eggett CJ. Molecular factors
underlying selective vulnerability of
motor neurons to neurodegeneration in
amyotrophic lateral sclerosis. J Neurol
2000;247(Suppl):I17-27
32. Roy J, Minotti S, Dong L, et al.
Glutamate potentiates the toxicity of
mutant Cu/Zn-superoxide dismutase in
motor neurons by post-synaptic calcium-
dependent mechanisms. J Neurosci
1998;18:9673-84
33. Hetz C, Thielen P, Matus S, et al.
XBP-1 deficiency in the nervous system
protects against amyotrophic lateral
sclerosis by increasing autophagy.
Genes Dev 2009;23:2294-306
34. Atkin JD, Farg MA, Walker AK, et al.
Endoplasmic reticulum stress and
induction of the unfolded protein
response in human sporadic amyotrophic
lateral sclerosis. Neurobiol Dis
2008;30:400-7
35. Ilieva EV, Ayala V, Jove M, et al.
Oxidative and endoplasmic reticulum
stress interplay in sporadic amyotrophic
lateral sclerosis. Brain 2007;30:3111-23
16
36. Mizoule J, Meldrum B, Mazadier M,
et al. 2-amino-6-
trifluoromethoxybenzothiazole, a possible
antagonist of excitatory amino acid
neurotransmission. I. Anticovulsant
properties. Neuropharmacology
1985;24:767-83
37. Habib AA, Mitsumoto H. Emerging
drugs for amyotrophic lateral sclerosis.
Expert Opin Emerg Drugs
2011;16(3):537-58
.. Excellent review on current drugs in
clinical trials for ALS.
38. Debono MW, Le Guern J, Canton T,
et al. Inhibition by riluzole of
electrophysiological responses mediated
by rat kainite and NMDA receptors
expressed in Xenopus oocytes.
J Pharmacol 1993;235:283-9
39. Doble A. The pharmacology and
mechanism of action of riluzole.
Neurology 1996;47(Suppl4):S233-41
. Summarizes the putative mechanisms
of action of riluzole highlighting both
pre-synaptic and post-synaptic effects
on glutamate neurotransmission.
40. Doble A, Hubert JP, Blanchard JC.
Pertussis toxin pretreatment abolishes the
inhibitory effect of riluzole and carbachol
on D-[3H]-aspartate release from
cerebellar granule cells. Neurosci Lett
1992;140:251-4
41. Siciliano G, Carlesi C, Pasquali L, et al.
Clinical trials for neuroprotection in
ALS. CNS Neurol Disord Drug Targets
2010;9(3):305-13
42. Traynor BJ, Bruijn L, Conwit R, et al.
Neuroprotective agents for clinical trials
in ALS: a systematic assessment.
Neurology 2006;67:20-7
43. Bruijn LI, Cudkowicz M. Therapeutic
targets for amyotrophic lateral sclerosis:
current treatments and prospects for
more effective therapies.
Expert Rev Neurother 2006;6:417-28
44. GSK1223249 clinical trials information.
Available from: www.Clinical.Trials.gov
[Last accessed 17 September 2012]
45. Prinja R, Moore SE, Vinson M, et al.
Inhibitor of neurite outgrowth in
humans. Nature 2000;403:383-4
46. Oertle T, van der Haar ME,
Bandtlow CE, et al. Nogo-A inhibits
neurite outgrowth and cell spreading
with three discrete regions. J Neurosci
2003;23(13):5393-406
Expert Opinion on Orphan Drugs (2013) 1(1)
47. Freund P, Schmidlin E, Wannier T,
et al. Anti-Nogo-A antibody treatment
promotes recovery of manual dexterity
after unilateral cervical lesion in adult
primates-re-examination and extension of
behavioral data. Eur J Neurosci
2009;29:983-96
48. Jokic N, de Aguilar J-LG, Dimou L,
et al. The neurite outgrowth inhibitor
Nogo-A promotes denervation in an
amyotrophic lateral sclerosis model.
EMBO Rep 2006;7:1162-7
49. Pradat P-F, Bruneteau G,
de Aguilar J-LG, et al. Muscle
Nogo-A expression is a prognostic
marker in lower motor neuron
syndromes. Ann Neurol 2007;62:15-20
50. Russell AJ, Hartman JJ, Hinken AC,
et al. Activation of fast skeletal muscle
troponin as a potential therapeutic
approach for treating neuromuscular
diseases. Nat Med 2012;3:452-6
51. Shefner J, Cedarbaum JM,
Cudkowicz ME, et al. Safety, tolerability
and pharmacodynamics of a skeletal
muscle activator in amyotrophic lateral
sclerosis. Amyotroph Lateral Scler
2012;13(5):430-8
52. Schmalbach S, Petri S. Histone
deacetylation and motor neuron
degeneration. CNS Neurol Disord
Drug Targets 2010;9(3):279-84
53. Echaniz-Laguna A, Bousiges O,
Loeffler JP, et al. Histone deacetylase
inhibitors: therapeutic agents and
research tools for deciphering motor
neuron diseases. Curr Med Chem
2008;15:1263-73
54. Yoo Y-E, Ko CP. Treatment with
trichostatin A initiated after disease onset
delays disease progression and increases
survival in a mouse model of
amyotrophic lateral sclerosis. Exp Neurol
2011;231:147-59
55. Ryu H, Smith K, Camelo SI, et al.
Sodium phenylbutyrate prolongs survival
and regulates expression of anti-apoptotic
genes in transgenic amyotrophic lateral
sclerosis mice. J Neurochem
2005;93:1087-98
56. Petri S, Kiaei M, Kipiani K, et al.
Additive neuroprotective effects of a
histone deacetylase inhibitor and a
catalytic antioxidant in a transgenic
mouse model of amyotrophic lateral
sclerosis. Neurobiol Dis 2006;22:40-9

57. Del Signore SJ, Amante DJ, Kim J, et al.
Combined riluzole and sodium
phenylbutyrate therapy in transgenic
amyotrophic lateral sclerosis mice.
Amyotroph Lateral Scler 2009;10:85-94
58. Chuang D-M, Leng Y, Marinova Z,
et al. Multiple roles of HDAC inhibition
in neurodegenerative conditions.
Trends Neurosci 2009;32:591-601
59. Cudkowicz ME, Andres PL,
Macdonald SA, et al. Phase 2 study of
sodium phenylbutyrate in ALS.
Amyotroph Lateral Scler 2009;10:99-106
60. Siddique T, Ajroud-Driss S. Familial
ALS, a historical perspective. Acta Myol
2011;30:117-20
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
61. Lagier-Tourenne C, Polymedidou M,
Cleveland DW. TDP-43 and FUS/TLS:
emerging roles in RNA processing and
neurodegeneration. Hum Mol Genet
2010;19:R46-64
62. Lagier-Tourenne C, Cleveland DW.
Rethinking the fuss about TDP-43. Cell
2009;136:1001-4
63. Tollervey JR, Curk T, Rogelj B, et al.
Characterizing the RNA targets and
position-dependent splicing regulation by
TDP-43. Nat Neurosci 2011;14(4):452-8
. Seminal work identifying
mRNA-binding partners of
TDP-43 that may have relevance to
neurodegenerative disease.
64. Polymenidou M, Lagier-Tourenne C,
Hutt KR, et al. Long
pre-mRNA depletion and
RNA missplicing contribute to the
neuronal vulnerability from loss of
TDP-43. Nat Neurosci 2011;14:459-68
. Seminal work identifying
mRNA-binding partners of
TDP-43 that may have relevance to
neurodegenerative disease.
65. Da Cruz S, Cleveland DW.
Understanding the role of TDP-43 and
FUS/TLS in ALS and beyond.
Curr Opin Neurobiol 2011;21:904-19
. Discusses the broadening of
TDP-43 and FUS proteinopathies
beyond ALS to other diseases such as
HD, PD and AD.
66. Ling S-C, Albuququerque CP, Han JS,
et al. ALS-associated mutations in
TDP-43 increase its stability and
promote TDP-43 complexes with FUS/
TLS. PNAS 2010;107:13318-23
67. Kawauchi J, Mischo H, Braglia P, et al.
Budding yeast RNA polymerases I and II
Emerging drug targets in amyotrophic lateral sclerosis
employ parallel mechanisms of
transcriptional termination. Genes Dev
2008;22:1082-92
68. Skourti-Stathaki K, Proudfoot NJ,
Gromak N. Human Senataxin resolves
RNA/DNA hybrids formed at
transcriptional pause sites to promote
Xrn2-dependent termination. Mol Cell
2011;42:794-805
69. Skorupa A, King MA, Aparicio IM.
Motoneurons secrete angiogenin to
induce RNA cleavage in astroglia.
J Neurosci 2012;32:5024-38
70. Ticozzi N, Tiloca C, Morelli C, et al.
Genetics of familial amyotrophic lateral
sclerosis. Arch Ital Biol 2011;149:65-82
71. Boillee S, Velde CV, Cleveland DW.
ASL: a disease of motor neurons and
their nonneuronal neighbors. Neuron
2006;52:39-59
72. Pasinelli P, Brown RH. Molecular
biology of amyotrophic lateral sclerosis:
insights from genetics. Nat Rev Neurosci
2006;7:710-23
73. Bush AI. Is ALS caused by an altered
oxidative activity of mutant superoxide
dismutase? Nat Neurosci 2002;5:919
. Discusses potential mechanism to
explain SOD mutant gain of function.
74. Hadano S, Benn SC, Kakuta S, et al.
Mice deficient in the Rab5 guanine
nucleotide exchange factor ALS2/alsin
exhibit age-dependent neurological
deficits and altered endosome trafficking.
Hum Mol Genet 2006;15:233-50
75. Devon RS, Orban PC, Gerrow K, et al.
Als2-deficient mice exhibit disturbances
in endosome trafficking associated with
motor behavioral abnormalities. PNAS
2000;103:9595-600
76. Morotz GN, De Vos KJ, Vagnoni A,
et al. Amyotrophic lateral
sclerosis-associated mutant VABP56S
perturbs calcium homeostasis to disrupt
axonal transport of mitochondria.
Hum Mol Genet 2012;21(9):1979-88
77. Ferguson CJ, Lenk GM, Meisler MH.
Defective autophagy in neurons and
astrocytes from mice deficient in PI(3,5)
P2. Hum Mol Genet
2009;18(24):4868-78
78. Nagabhushana A, Chalasani ML, Jain N,
et al. Regulation of endocytic trafficking
of transferring receptor by optineurin
and its impairment by a
glaucoma-associated mutant.
BMC Cell Biol 2010;11(4):1-19
Expert Opinion on Orphan Drugs (2013) 1(1)
79. Renton AE, Majounie E, Waite A, et al.
A hexanucleotide repeat expansion in
C9ORF72 is the cause of chromosome
9p21-linked ALS-FTD. Neuron
2011;72(2):257-68
80. Polymenidou M, Lagier-Tourenne C,
Hutt KR, et al. Misregulated
RNA processing in amyotrophic lateral
sclerosis. Brain Res 2012;1462:3-15
81. Wu C-H, Fallini C, Ticozzi N, et al.
Mutations in the profilin 1 gene cause
familial amyotrophic lateral sclerosis.
Nature 2012;488:499-505
82. Horrevoets AJG. Profilin-1:
an unexpected molecule linking vascular
inflammation to the actin cytoskeleton.
Circ Res 2007;101:328-30
83. Suetsugu S, Miki H, Takenawa T. The
essential role of profilin in the assembly
of actin for microspike formation.
EMBO J 1998;17:6516-26
84. Daoud H, Dobrzeniecka S, Camu W,
et al. Mutation analysis of PFN1 in
familial amyotrophic lateral sclerosis
patients. Neurobiol Aging
2012;12:In press
85. Hirai H, Maru Y, Hagiwara K, et al.
A novel putative tyrosine kinase receptor
encoded by the eph gene. Science
1987;238:1717-20
86. Qin H, Noberini R, Huan X, et al.
Structural characterization of the
EphA4-Ephrin-B2 complex reveals new
features enabling Eph-ephrin binding
promiscuity. J Biol Chem
2010;285:644-54
87. Kao T-J, Law C, Kania A. Eph and
ephrin signaling: lessons learned from
spinal motor neurons. Semin Cell
Dev Biol 2012;23:83-91
88. Chen Y, Fu AKY, Ip NY. Eph receptors
at synapses: implications in
neurodegenerative diseases. Cell Signal
2012;24:606-11
89. Helmbacher F, Schneider-Maunoury S,
Topilko P, et al. Targeting of
EphA4 tyrosine kinase receptor affects
dorsal/ventral pathfinding of limb motor
axons. Development 2000;127:3313-24
90. Murai KK, Nguyen LN, Irie F, et al.
Control of hippocampal dendritic spine
morphology through ephrin-A3/
EphA4 signaling. Nat. Neurosci
2003;6:153-60
91. Bourgin C, Murai KK, Richter M, et al.
The EphA4 receptor regulates dendritic
spine remodeling by affecting
17
 M. P. Bova & G. G. Kinney
beta1-integrin signaling pathways.
J Cell Biol 2007;178:1295-307
92. Van Hoecke A, Schoonaert L,
Lemmens R, et al. EPHA4 is a disease
modifier of amyotrophic lateral sclerosis
in animal models and in humans.
Nat Med 2012
.. Demonstrated that EphaA4 was a
disease modifier in a zebrafish model
of ALS and that EphA4 expression
inversely correlated with disease onset
and survival in ALS patients.
93. Noberini R, Koolpe M, Peddibhotla S,
et al. Small molecules can selectively
inhibit ephrin binding to the EphA4 and
EphA2 receptors.
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
2008;283(43):29461-72
94. Noberini R, Lamberto I, Pasquale EB.
Targeting Eph receptors with peptides
and small molecules: progress and
challenges. Semin Cell Dev Biol
2012;23:51-7
95. Farenc C, Celie PNH, Tensen CP, et al.
Crystal structure of the EphA4 protein
tyrosine kinase domain in the apo- and
dasatinib-bound state. FEBS Lett
2011;585:3593-9
96. Oki M, Yamamoto H, Taniguchi H,
et al. Overexpression of the
receptor-tyrosine kinase EphA4 in human
gastric cancers. World J Gastroenterol
2008;14(37):5650-6
97. Goldshmit Y, Spanevello MD, Tajouri S,
et al. EphA4 blockers promote axonal
regeneration and functional recovery
following spinal cord injury in mice.
PLoS ONE 2011;6(9):e24636
98. Kobayashi M, Yamamoto M.
Nrf2-Keap1 regulation of cellular defense
mechanisms against electrophiles and
reactive oxygen species.
Adv Enzyme Regul 2006;46:113-20
99. Barber SC, Shaw PJ. Oxidative stress in
ALS: Key role in motor neuron injury
and therapeutic target. Free Radic
Biol Med 2010;48:629-41
100. Mimoto T, Miyazaki K, Morimoto N,
et al. Impaired antioxydative Keap1/
Nrf2 system and the downstream stress
protein responses in the motor neuron of
ALS model mice. Brain Res
2012;1446:109-18
101. Vargas MR, Johnson DA, Sirkis DW,
et al. Nrf2 activation in astrocytes
protects against neurodegeneration in
mouse models of familial amyotrophic
18
lateral sclerosis. J Neurosci
2008;28(50):13574-81
102. Neymotin A, Calingasan NY, Willie E,
et al. Neuroprotective effect of Nrf2/ARE
activators, CDDO-ethylamide and
CDDO-trifluoroethylamide in a mouse
model of amyotrophic lateral sclerosis.
Free Radic Biol Med 2011;51(1):88-96
103. Pergola PE, Raskin P, Toto RD, et al.
Bardoxolone methyl and kidney function
in CKD with Type 2 diabetes. NEJM
2011;365(4):327-36
. Nrf2 activator Bardoxolone methyl was
associated with an improvement in
kidney function in patients with
advanced CKD where this result paves
the way for use of Nrf2 activators in
other diseases linked to
oxidative stress.
104. Bandyopadhyay S, Cookson MR.
Evolutionary and functional relationships
within the DJ1 superfamily.
BMC Evol Biol 2004;4:6
105. Bonifati V, Rizzu P, van Baren MJ, et al.
Mutations in the DJ-1 gene associated
with autosomal recessive early-onset
parkinsonism. Science 2003;299:256-9
106. Wilson MA. The role of cysteine
oxidation in DJ-1 function and
dysfunction. Antioxid Redox Signal
2011;15:111-22
107. Canet-Aviles RM, Wilson MA,
Miller DW, et al. The Parkinson’s
disease protein DJ-1 is neuroprotective
due to cysteine-sulfinic acid-driven
mitochondrial localization. PNAS
2004;101:9103-8
108. Im JY, Lee KW, Junn E, et al.
DJ-1 protects against oxidative damage
by regulating the thioredoxin/
ASK1 complex. Neurosci Res
2010;67:203-8
109. Gorner K, Holtorf E, Waak J, et al.
Structural determinants of the C-terminal
helix-kink-helix motif essential for
protein stability and survival promoting
activity of DJ-1. J Biol Chem
2007;282:13680-91
110. Kim YC, Kitaura H, Taira T, et al.
Oxidation of DJ-1 dependent cell
transformation through direct binding of
DJ-1 to PTEN. Int J Oncol
2009;35(6):1331-41
111. Annesi G, Savettieri G, Pugliese P, et al.
DJ-1 mutations and
Parkinsonism-Dementia-Amyotrophic
Expert Opinion on Orphan Drugs (2013) 1(1)
Lateral Sclerosis complex. Ann Neurol
2005;58:803-7
112. Kaji R, Izumi Y, Adachi Y, Kuzuhara S.
ALS-Parkinsonism-Dementia complex of
Kii and other related diseases in Japan.
Parkinsonism Relat Disord
2012;18S1:S190-1
113. Yamashita S, Mori A, Mita S, et al.
DJ-1 forms complexes with mutant
SOD1 and ameliorates its toxicity.
J Neurochem 2010;113:860-70
114. Miyazaki S, Yanagida T, Nunome K,
et al. DJ-1 binding compounds prevent
stress-induced cell death and movement
defect in Parkinson’s disease model rats.
J Neurochem 2008;105:2418-34
. Brain penetrant DJ-1 binding
compounds were shown to protect in
cellular and animal models of PD.
115. Macedo MG, Anar B, Bronner IF, et al.
The DJ-1L166P mutant protein
associated with early onset Parkinson’s
disease is unstable and forms
higher-order protein complexes.
Hum Mol Genet 2003;12(21):2807-16
116. Alvarez-Castelao B, Munoz C, Sanchez I,
et al. Reduced protein stability of human
DJ-1/Park7 L166P, linked to autosomal
recessive Parkinson disease, is due to
direct endoproteolytic cleavage by the
proteasome. Biochim Biophys Acta
2012;1823(2):524-33
117. Wilson MA, Collins JL, Hod Y, et al.
The 1.1 A resolution crystal structure of
DJ-1, the protein mutated in autosomal
recessive early onset Parkinson’s disease.
PNAS 2003;100(16):9256-61
118. Ichijo H, Nishida E, Irie K, et al.
Induction of apoptosis by ASK1, a
mammalian MAPKKK that activates
SAPK/JNK and p38 signaling pathways.
Science 1997;275:90-4
119. Bunkoczi G, Salah E, Filippakopoulos P,
et al. Structural and functional
characterization of the human protein
kinase ASK1. Structure 2007;15:1215-26
120. Saitoh M, Nishitoh H, Fujii M, et al.
Mammalian thioredoxin is a direct
inhibitor of apoptosis signal-regulating
kinase (ASK) 1. EMBO J
1998;17(9):2596-606
121. Hu X, Weng Z, Chu CT, et al.
Peroxiredoxin-2 protects against
6-Hydroxydopamine-induced
dopaminergic neurodegeneration via
attenuation of the apoptosis

signal-regulating kinase (ASK1) signaling
cascade. J Neurosci 2011;31(1):247-61
122. Tobiume K, Matsuzawa A, Takahashi T,
et al. ASK1 is required for sustained
activations of JNK/p38 MAP kinases and
apoptosis. EMBO Rep 2001;2(3):222-8
123. Nishitoh H, Kadowaki H, Nagai A,
et al. ALS-linked mutant SOD1 induces
ER stress- and ASK1-dependent motor
neuron death by targeting Derlin-1.
Genes Dev 2008;22:1451-64
. ASK1 knockout in SOD1G93A mice
significantly prolongs survival and
preserves motor neurons.
124. Um JW, Im E, Park J, et al.
ASK1 negatively regulates the 26S
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
proteasome. J Biol Chem
2010;285(47):36434-46
125. Cuny GD. Kinase inhibitors as potential
therapeutics for acute and chronic
neurodegenerative conditions.
Curr Pharm Des 2009;15:3919-39
126. Personal communication from Marc
Adler, based on homology modles of
ASK2 and ASK3 built on the
ASK1 structure 2CLQ
127. Matsuzawa A, Saegusa K, Noguchi T,
et al. ROS-dependent activation of the
TRAF6-ASK1-p38 pathway is selectively
required for TLR4-mediated innate
immunity. Nat Immunol
2005;6(6):587-92
128. Norman P. Evaluation of
WO2012003387, Gilead’s
ASK1 inhibitors. Expert Opin
Ther Patents 2012;22(4):455-9
129. Boyault C, Zhang Y, Fritah S, et al.
HDAC6 controls major cell response
pathways to cytotoxic accumulation of
protein aggregates. Genes Dev
2007;21:2172-81
130. Matthias P, Yoshida M, Khochbin S.
HDAC6 a new cellular stress surveillance
factor. Cell Cycle 2008;7(1):7-10
131. Li G, Jiang H, Chang M, et al.
HDAC6 alpha-tubulin deacetylase:
A potential therapeutic target in
neurodegenerative diseases. J Neurol Sci
2011;304:1-8
132. Kim SH, Shanware NP, Bowler MJ,
et al. Amyotrophic lateral
sclerosis-associated proteins TDP-43 and
FUS/TLS function in a common
biochemical complex to co-regulate
HDAC-6 mRNA.
2010;285(44):34097-105
Emerging drug targets in amyotrophic lateral sclerosis
133. Yu Z, Sawkar AR, Whalen LJ, et al.
Isofogamine- and 2,5-Anhydro-2,5-
Imino-D-Glucitol-based
Glucocerebrosidase pharmacological
chaperones for Gaucher disease
intervention. J Med Chem
2007;50(1):94-100
. This paper highlights one of the
success stories of a pharmacological
chaperone approach in drug discovery.
134. Hubbert C, Guardiola A, Shao R, et al.
HDAC6 is a microtubule-associated
deacetylase. Nature 2002;417:455-8
135. Boyault C, Sadoul K, Pabion M, et al.
HDAC6, at the crossroads between
cytoskeleton and cell signaling by
acetylation and ubiquitination. Oncogene
2007;26:5468-76
136. Zilberman Y, Ballestrem C,
Carramusa L, et al. Regulation of
microtubule dynamics by inhibition of
the tubulin deacetylase HDAC6.
J Cell Sci 2009;122:3531-41
137. Rivieccio MA, Brochier C, Willis DE,
et al. HDAC6 is a target for protection
and regeneration following injury in the
nervous system. PNAS
2009;106(46):19599-604
138. d’Ydewalle C, Krishnan J, Chiheb DM,
et al. HDAC6 inhibitors reverse axonal
loss in a mouse model of mutant
HSPB1-induced Charcot-Marie-Tooth
disease. Nat Med 2011;17(8):968-75
.. Exciting finding of significant efficacy
of a HDAC6 inhibitor in a rodent
model of CMT disease.
139. Zhang Y, Kwon S, Yamaguchi T, et al.
Mice lacking histone deacetylase 6 have
hyperphosphorylated tubulin but are
viable and develop normally.
Mol Cell Biol 2008;28(5):1688-701
140. Butler KV, Kalin J, Brochier C, et al.
Rational design and simple chemistry
yield a superior, neuroprotective
HDAC6 inhibitor, Tubastatin A. J Am
Chem Soc 2010;132(31):10842-6
. Paper demonstrated that selective and
efficacious HDAC6 inhibitors can
be synthesized.
141. Inks ES, Josey BJ, Jesinkey SR, et al.
A novel class of small molecule inhibitors
of HDAC6. ACS Chem Biol
2012;7:331-9
142. Gilmore TD. Introduction to NF-
kappaB: players, pathways, perspectives.
Oncogene 2006;25:6680-4
Expert Opinion on Orphan Drugs (2013) 1(1)
143. DiDonato JA, Mercurio F, Karin M.
NF-kappaB and the link between
inflammation and cancer. Immunol Rev
2012;246:379-400
144. Swarup V, Phaneuf D, Dupre N, et al.
Deregulation of TDP-43 in amyotrophic
lateral sclerosis triggers nuclear factor
kappaB-mediated pathogenic pathways.
J Exp Med 2011;208(12):2429-47
. TDP-43 was shown to be a coactivator
of p65 and NF-kB inhibitors
demonstrated efficacy in a
TDP-43 mouse transgenic model
of ALS.
145. Babu GN, Kumar A, Chandra R, et al.
Elevated inflammatory markers in a
group of amyotrophic lateral sclerosis
patients from northern India.
Neurochem Res 2008;33:1145-9
146. Cereda C, Baiocchi C, Bongioanni P,
et al. TNF and sTNFR1/2 plasma levels
in ALS patients. J Neuroimmunol
2008;194:123-31
147. Poloni M, Facchetti D, Mai R, et al.
Circulating levels of tumour necrosis
factor-alpha and its soluble receptors are
increased in the blood of patients with
amyotrophic lateral sclerosis.
Neurosci Lett 2000;287:211-14
148. Ghezzi P, Bernardini R, Giuffrida R,
et al. Tumor necrosis factor is increased
in the spinal cord of an animal model of
motor neuron degeneration.
Eur Cytokine Netw 1998;9:139-44
149. Tolosa L, Caraballo-Miralles V,
Olmos G, Llado J. TNF-alpha
potentiates glutamate-induced spinal cord
motoneuron death via NF-kappaB.
Mol Cell Neurosci 2011;46:176-86
150. Gupta SC, Sundaram C, Reuter S,
Aggarwal BB. Inhibiting NF-kappaB
activation by small molecules as a
therapeutic strategy.
Biochim Biophys Acta 2010;1799:775-87
151. Kwak J-H, Jung J-K, Lee H. Nuclear
factor-kappa B inhibitors: a patent review
(2006-2010). Expert Opin Ther Patents
2011;21(12):1897-910
152. Schieven GL. The p38alpha kinase plays
a central role in inflammation. Curr Top
Med Chem 2009;9(11):1038-48
153. Coulthard LR, White DE, Jones DL,
et al. p38MAPK: stress responses from
molecular mechanisms to therapeutics.
Trends Mol Med 2009;15(8):369-79
154. Hu JH, Chernoff K, Pelech S, et al.
Protein kinase and protein phosphatase
19
 M. P. Bova & G. G. Kinney
expression in the central nervous system
of G93A mSOD over-expressing mice.
J Neurochem 2003;85:422-31
155. Tortarolo M, Veglianese P, Calvaresi A,
et al. Persistent activation of
p38 mitogen-activated protein kinase in a
mouse model of familial amyotrophic
lateral sclerosis correlates with disease
progression. Mol Cell Neurosci
2003;23:180-92
156. Xu L, Guo Y-S, Liu Y-L, et al. Oxidative
stress in immune-mediated motoneuron
destruction. Brain Res 2009;1302:225-32
157. Ackerley S, Grierson AJ, Banner S, et al.
p38alpha stress-activated protein kinase
phosphorylates neurofilaments and is
Downloaded by [Jawaharlal Nehru University] at 22:22 09 February 2016
associated with neurofilament pathology
in amyotrophic lateral sclerosis.
Mol Cell Neurosci 2004;26:354-64
158. Hu JH, Zhang H, Wagey R, et al.
Protein kinase and protein phosphatase
expression in amyotrophic lateral sclerosis
spinal cord. J Neurochem
2003;85:432-42
159. Williamson TL, Cleveland DW. Slowing
of axonal transport is a very early event
in the toxicity of ALS-linked
SOD1 mutants to motor neurons.
Nat Neurosci 1999;2:50-6
160. Morfini GA, Burns M, Binder LI, et al.
Axonal transport defects in
neurodegenerative diseases. J Neurosci
2009;29(41):12776-86
161. Bosco DA, Morfini G, Karabacak NM,
et al. Wild type and mutant SOD1 share
an aberrant conformation and a common
pathogenic pathway in ALS.
Nat Neurosci 2010;13(11):1396-403
162. Dewil M, dela Cruz VF,
Van Den Bosch L, Robberecht W.
20
Inhibition of p38 mitogen activated
protein kinase activation and mutant
SOD1G93A-induced motor neuron
death. Neurobiol Dis 2007;26:332-41
163. Stevenson A, Yates DM, Manser C, et al.
Riluzole protects against
glutamate-induced slowing of
neurofilament axonal transport.
Neurosci Lett 2009;454:161-4
164. Yasuda S, Tanaka H, Takigami S,
Yamagata K. p38 MAP kinase inhibitors
as potential therapeutic drugs for neural
disease. Cent Nerv Syst Agents
Med Chem 2011;11(1):45-59
165. Goldstein DM, Kuglstatter A, Lou Y,
Soth MJ. Selective p38alpha inhibitors
clinically evaluated for the treatment of
chronic inflammatory disorders.
J Med Chem 2010;53:2345-53
166. Bano D, Zanetti F, Mende Y,
Nicotera P. Neurodegenerative processes
in Huntington’s disease.
Cell Death Disease 2011;2:e228
167. Ravikumar B, Vacher C, Berger Z, et al.
Inhibition of mTOR induces autophagy
and reduces toxicity of polyglutamine
expansions in fly and mouse models of
Huntington’s disease. Nat Genet
2004;36(6):585-95
168. Sarkar S, Peristein EO, Imarisio S, et al.
Small molecules enhance autophagy and
reduce toxicity in Huntington’s disease
models. Nat Chem Biol 2007;3(6):331-8
.
Small molecule phenotypic screen
identified novel modulators of
autophagy which could have broad
relevance for
neurodegenerative diseases.
169. Lee B-H, Lee MJ, Park S, et al.
Enhancement of proteasome activity by a
Expert Opinion on Orphan Drugs (2013) 1(1)
small-molecule inhibitor of Usp14.
Nature 2010;467:179-87
170. Rothstein JD, Patel S, Regan MR, et al.
beta-lactam antibiotics offer
neuroprotection by increasing glutamate
transporter expression. Nature
2005;433:73-7
171. Ceftriaxone clinical trial information.
Available from: www.ClinicalTrials.gov
[Last accessed 25 October 2012]
172. Sharma K, Weber C, Bairlein M, et al.
Proteomics strategy for quantitative
protein interaction profiling in cell
extracts. Nat Meth 2009;6(10):741-4
173. Glass JD. New drugs for ALS: how do
we get there? Exp Neurol
2012;233(1):112-17
. Excellent review on drug repositioning
approaches for ALS.
Affiliation
Michael P Bova†1 PhD & Gene G Kinney2 PhD
†
Author for correspondence
1
Senior Director of Target Advancement,
Department of Molecular Discovery,
ELAN Pharmaceutical,
180 Oyster Point Boulevard,
South San Francisco,
CA 94080, USA
Tel: +1 650 794 4284;
E-mail: Michael.bova@elan.com
2
Senior Vice President,
Department of Pharmacology,
ELAN Pharmaceutical,
180 Oyster Point Boulevard,
South San Francisco,
CA 94080, USA