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Developing therapeutic approaches

for metachromatic leukodystrophy

This article was published in the following Dove Press journal:

Drug Design, Development and Therapy
7 August 2013
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Shilpa A Patil 1 Abstract: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal dis-
Gustavo HB Maegawa 1,2 order caused by the deficiency of arylsulfatase A (ASA), resulting in impaired degradation of
sulfatide, an essential sphingolipid of myelin. The clinical manifestations of MLD are character-
1
McKusick-Nathans Institute
of Genetic Medicine, 2Department ized by progressive demyelination and subsequent neurological symptoms resulting in severe
of Pediatrics, The Johns Hopkins debilitation. The availability of therapeutic options for treating MLD is limited but expanding
School of Medicine, Baltimore,
MD, USA with a number of early stage clinical trials already in progress. In the development of therapeutic
approaches for MLD, scientists have been facing a number of challenges including blood–brain
barrier (BBB) penetration, safety issues concerning therapies targeting the central nervous
system, uncertainty regarding the ideal timing for intervention in the disease course, and the
lack of more in-depth understanding of the molecular pathogenesis of MLD. Here, we discuss
the current status of the different approaches to developing therapies for MLD. Hematopoietic
stem cell transplantation has been used to treat MLD patients, utilizing both umbilical cord
blood and bone marrow sources. Intrathecal enzyme replacement therapy and gene therapies,
administered locally into the brain or by generating genetically modified hematopoietic stem
cells, are emerging as novel strategies. In pre-clinical studies, different cell delivery systems
including microencapsulated cells or selectively neural cells have shown encouraging results.
Small molecules that are more likely to cross the BBB can be used as enzyme enhancers of
diverse ASA mutants, either as pharmacological chaperones, or proteostasis regulators. Specific
small molecules may also be used to reduce the biosynthesis of sulfatides, or target different
affected downstream pathways secondary to the primary ASA deficiency. Given the progressive
neurodegenerative aspects of MLD, also seen in other lysosomal diseases, current and future
therapeutic strategies will be complementary, whether used in combination or separately at
specific stages of the disease course, to produce better outcomes for patients afflicted with this
devastating inherited disorder.
Keywords: metachromatic leukodystrophy, arylsulfatase A, enzyme replacement therapy, gene
therapy, enzyme enhancement therapy, small molecules

Introduction
Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal stor-
age disorder caused by the deficiency of arylsulfatase A (ASA; EC [Link]),
Correspondence: Gustavo HB Maegawa a lysosomal sulfatase that hydrolyzes the 3-O ester bond from sulfatides including
McKusick-Nathans Institute of Genetic 3-O-sulfogalactosylceramide and 3-O-sulfolactosylceramide.1,2 These sulfatides are
Medicine, Department of Pediatrics,
The Johns Hopkins School of
expressed in specific tissues and are mostly abundant in the central (CNS) and periph-
Medicine, Baltimore, MD 21205, USA eral nervous systems (PNS). Sulfatides are important components of myelin, which
Tel +1 443 287 3505
Fax +1 410 502 5677
is a crucial insulator sheath that surrounds axons as a bilayer membrane.3 An intact
Email gmaegaw1@[Link] myelin sheath is important for the proper conduction of nerve impulse in both CNS

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and PNS.3 The inappropriate recycling of sulfatides results in of these ARSA pseudodeficiency alleles is up to 5% in the
alterations of the myelin structure, affecting the physiological European population.13–17 The carrier frequency of the ARSA
transmission of electrical impulse between nerve cells.4 This pseudodeficiency alleles in Australia is estimated to be 20%.16
process of degeneration of the myelin sheath surrounding Its correlation with occurrence of other neurodegenerative
axons, mostly known as demyelination, is the hallmark of disorders that occur later in life remains debatable.14 The
MLD. Patients afflicted with this condition present with a existence of the ARSA pseudodeficiency alleles demonstrates
broad spectrum of neurological manifestations centered on that the sulfatide degradation can occur normally in the
the impairment of CNS and PNS, including progressive presence of ASA variants functioning at 10–15% of the wild
coordination and speech problems, seizures, and behavioral type ASA enzymatic activity. This biochemical observation
disturbances.5 In the design and development of disease- has crucial implications in the development of therapeutic
specific therapies for MLD, the biochemical pathogenesis strategies for MLD.
and secondary molecular events to the ASA deficiency are
important to be considered. Biochemistry and molecular
genetics of MLD
Epidemiology and pseudodeficiency As with other lysosomal hydrolases, ASA is synthesized in
ARSA alleles the rough endoplasmic reticulum (ER) and co-translationally
Inherited disorders affecting the lysosomes have a collective transported to the lumen of ER.18–20 Once properly folded, ASA
incidence of 1  in 2,300–7,000 live births, making them a is targeted to the trans-Golgi network where it becomes a sub-
prevalent class of inborn organelle disorders.6,7 Among dif- strate of uridine diphosphate (UDP)-N-Acetylglucosamine-1
ferent lysosomal disorders, the general prevalence of MLD phosphotransferase (EC [Link]) and N-acetylglucosamine-
varies from 1:40,000 to 1:100,000.8 In the Polish population, 1-phosphodiester alpha-N-acetylglucosaminidase (EC
its incidence was reported as 4:100,000.9 MLD is a pan-ethnic [Link]). These two enzymes catalyze the binding and
lysosomal storage disease with affected patients described formation of mannose-6-phosphate residue, the recognition
in several populations including European, Japanese, Jew- marker of lysosomal hydrolases. This modification is impor-
ish, Lebanese, Muslim Arab, South African, Iranian, Indian, tant to allow ASA to interact with the mannose-6-phosphate
Polynesian, Algerian, Habbanite Jew, Navajo Indian, Alaskan receptor, and be targeted to the lysosomal compartment.21
Eskimo, and Christian Arab, with ranging from mild to severe In terms of its molecular structure, 2.1-Å resolution crystal
forms of MLD.8 The deficiency of ASA is caused by muta- X-ray structure showed that ASA is a homo-octamer com-
tions in the ARSA gene in homo- or heterozygosity. Over posed of a tetramer of dimers (α2)4.22 In fact, the formation
150 mutations have been reported in MLD patients.10 Some of ASA homodimers (α2) occurs in the ER. Subsequently,
specific alterations in the ARSA gene sequence result in an ASA only assumes the homo-octomeric conformation at the
enzymatic activity of 10%–15% of the normal (wild-type) acidic pH of the lysosomal compartment.22 The identification
ASA, which is sufficient to physiologically hydrolyze sulfati- of the c.1277T.G/p.P426L mutation showed it preventing
des, maintaining their recycling process and avoiding their the formation of homo-octomers, as the P426 residue locates
accumulation in the lysosomal compartment. These non-del- proximal to the E424 residue, whose protonation is necessary
eterious ARSA gene alterations resulting in reduction of ASA for the octomerization process.23 The mutant ASA-P426L
enzymatic activity are known as pseudodeficiency alleles. is degraded by cathepsin-L, a lysosomal protease, which
These ARSA alleles are considered polymorphisms, with no recognizes a cleavage site in the ASA homo-dimer interface
disease-associated symptoms either in hetero or homozygous that is normally protected by the octomerization conforma-
states. Two most common ARSA pseudodeficiency alleles are tion seen in wild-type ASA.23,24 Functionally, ASA catalyzes
c.1049A.G/p.N350S and c.*96A.G. As they occur in cis- the desulfation of 3-O-sulfogalactosyl-bound sphingolipids.
trans, they are described as c.[1049A.G; c.*96A.G].11,12 Sulfatides, or 3-O-galactosylceramides, are the main sphin-
The c.1049A.G/p.N350S results in modification of one of golipids containing sulfate residues. Sulfatides are abundant
the N-glycosylation sites of ASA, affecting the structural in both the CNS and PNS. These glycosphingolipids are the
stability of ASA and its targeting to the lysosomes.12 The main components of the myelin sheath. In the CNS, oligo-
c.*96A.G is found in the 3′ non-translated region that alters dendrocytes synthesize sulfatides, which are located in the
the signaling of the polyadenylation of mRNA, substantially myelin surrounding neural axons. In the PNS, the Schwann
reducing the amount of ASA produced.13 The frequency cells are the myelin-producing cells. Interestingly, in the gall

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bladder of MLD-affected patients, sulfatides accumulate ASA-p.I179S presents the higher residual ASA enzymatic
in the epithelium cells, resulting in sludge, gallstones, activity, and is correlated with later onset and more protracted
hemobilia, cholecystitis, polyposis, papillomatosis, and small MLD course.41 The ARSA c.536T.G/p.I179S allele is pres-
or enlarged gallbladder.25–29 Sulfatides are also present, but ent in 12%–13% of the total European population of mutant
at low levels, in the respiratory tract,30 gastric mucosa,31 and alleles.41,42 Though several missense mutations have been
uterine endometrium.32 The 3-O-sulfatelactosyl ceramide, identified, few of them have been biochemically character-
which is another sulfatide substrate of ASA, is found in ized.10,43 Most missense mutations result in misfolded mutant
the renal tubules and liver.33 Another deacylated sulfatide, ASA that is early selected for the ER-associated degradation
known as lysosulfatide or psychosine sulfatide, is present in (ERAD) pathway and targeted to the ubiquitin-proteasome
the CNS and renal tubules.34,35 The presence of sulfatides in system.44 Therefore, small amounts of partially folded and/
the kidney is the cornerstone of the main diagnostic tool for or misfolded mutant ASA pass the ER post-translational
MLD, which is based on measuring urinary sulfatides.36 ASA quality control and ultimately reach the lysosomal compart-
is also responsible for catalyzing the desulfation of glycerol- ment, resulting in the residual ASA enzymatic activity.45 Such
containing sulfated sphingolipids, which have a sulfate bond molecular characterizations, and their correlation with the
to the galactose C-3 hydroxyl residue. These 3-O-sulfaga- MLD pathogenesis, may assist in delineating the molecular
lactosyl sphingolipids are present in the mammalian testes mechanism to correct the lysosomal enzymatic deficiencies
and sperm, which are mostly known as seminolipids.37 These at genetic and biochemical levels.
sphingolipids are produced by spermatocytes, and their
roles in different stages of the spermatogenesis are still to Clinical presentations
be determined.37 MLD is a neurological lysosomal storage disorder with a
In MLD, the deficiency of ASA results in sulfatide accu- broad and heterogeneous clinical spectrum characterized by
mulation in the myelin-producing cells: oligodendrocytes CNS and PNS demyelination resulting in a progressive demy-
in CNS, and Schwann cells in PNS. With the progressive elination and encephalopathy. The clinical presentation and
accumulation of sulfatides, the lysosomal-endosomal system disease course of MLD can be classified into three clinical
becomes dysfunctional, and other secondary pathogenic cas- forms. The three clinical forms are determined on the basis
cades occur, ultimately resulting in apoptosis.38 This results of the age of onset of first symptoms, the rate of progression
in progressive demyelination in both CNS and PNS, which of the neurological symptoms, and their severity. The late
correlates with the major clinical manifestations of MLD.39 infantile form is the most severe form, in which patients
However, cellular inclusions called membranous cytoplasmic develop first symptoms within a few months to 2 years of
bodies (MCBs), which can correspond to accumulation of age.46 The juvenile MLD clinical form includes patients who
sulfatides, are observed in pyramidal neurons and Purkinje have the onset of symptoms occur between 3 and 16 years
cells in the brain of MLD patients.40 of age. Patients with the adult MLD clinical form have the
The deficiency of ASA is caused by mutations in the onset of disease when older than 16 years. The two late-onset
ARSA gene. Amongst the over 150  mutations reported in forms of MLD, juvenile and adult, sometimes overlap, and
the ARSA gene, more than 70% are missense mutations.10 they usually present with a more insidious manifestation
The most common mutation is the splice donor site muta- of a wide range of neurological symptoms, offering ample
tion of the exon2/intron2 border, IVS459+1G.A, which opportunity for possible therapeutic interventions.
occurs in 15%–43% of MLD alleles, and is associated with Patients with the late infantile form of MLD present
the late infantile clinical form of MLD.41 The second most with delays in psychomotor development characterized by
common ARSA mutation is c.1277C.T/p.P426L, which, impairment of speech, gross, and fine motor development.
when in homozygosity or heterozygosity with an infantile Additionally, patients can present with muscular hypotonia,
mutation (eg, IVS459+1G.A), is associated with juvenile hyporreflexia, lately followed by pyramidal signs character-
onset of MLD.41 Each of these two common mutant alleles ized by spasticity, and hypereflexia. Ataxia, spastic paresis,
represents approximately 25% of all ARSA mutant alleles and optic dystrophy were also seen. The juvenile form of
in MLD patients with European ancestry.42 The third most MLD is characterized by later onset of the above symptoms,
prevalent ARSA mutation is c.536T.G/p.I179S, which, when which are usually accompanied by poor school performance,
in heterozygosity with the c.1277C.T/p.P426L mutation, is and some behavioral disturbance and peripheral neuropathies.
associated with adult onset of the disease.41,42 Interestingly, Later on, MLD patients become debilitated, losing gait

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independence and speech. As disease progresses, seizures hydrophilic environment around its structure, represented
and recurrent pulmonary infections occur frequently.8,43 The by cytosol and extracellular fluids. Second, the same
adult form of MLD is characterized by gradual impairment hydrophobic structures have strong attractive forces among
of intellectual function, emotional instability, and behavioral/ them, making myelin a compact structure.3 The sulfatide and
psychiatric disturbances. Polyneuropathy occurs later and galactosylceramide structures are major contributors to these
does not usually present symptoms in this clinical form of attractive forces through formation of hydrogen bonds.48 In
MLD. In general, the course of disease is slower than the MLD, the excess of sulfatides and deficiency of cerebrosides
juvenile and late infantile clinical forms.8 can lead to abnormal myelin composition and possibly affect
the formation of a stable lipid bilayer.49 Further, the sulfatide
Pathogenesis metabolite galactosylsphingosine can also cause cytotoxicity
Sulfatide formation and its role in myelin and lead to death and dysfunction of neuronal cells.50
Sulfatides are membrane sphingolipids that show a cell Sulfatides are formed by the action of a series of enzymes,
type-specific expression.3 They are expressed in the kidney as shown involved in the biosynthesis and degradation of
and bile duct, in addition to brain tissue. Sulfatides are the sulfatides in Figure 1. The importance of different myelin
most abundant sphingolipid in myelin, accounting for 4% components has been investigated in specific mutant murine
of its composition. The stability of the myelin structure is models with deficiencies in genes encoding the enzymes in
one of its distinct features, as demonstrated in the intact the sulfatide biosynthetic pathway. UDP-galactose:ceramide
myelin found in the 5,000-year old Tyrolean Iceman.47 Two galactosyltransferase (CGT) knockout mice (cgt−/−) were
main factors confer stability of the myelin structure. First, unable to synthesize the galactosylceramide or sulfatide
the hydrophobic components of myelin work to repel the structures.51 Though the myelin structure seems normal,

L-Serine + palmitoyl-CoA

Serine
palmitoyltransferase 3-Dehydrosphinganine
reductase
3-Dehydro-sphinganine Dihydro-sphingosine

Ceramide
SRT Ceramidase
synthase 1

Dihydro-ceramide eliglustat
tartrate
Sphingolipid delta-4-
desaturase NB-DNJ

Ceramide
Galactosylceramidase glucosyltransferase
Gal-ceramide Ceramide Glc-ceramide

N-acylsphingosine Beta-1,4-galactosyl Glucosyl

galactosyltransferase transferase 6 ceramidase
Galactosyl
sulfotransferase
Lactosylceramide Ceramide Glc

Galactosyl
sulfotransferase
HSCT
SGal-Cer
SGal-Glc-cer
(Sulfatide)

ASA ASA

Gal-ceramide Lactosylceramide

SO42− SO42−
EET ERT

Figure 1 Sulfatide metabolic pathway.

Notes: The figure shows the biosynthetic pathways of sulfatide, the sphingolipid that accumulates during metachromatic leukodystrophy MLD. The specific enzymes described in
each step of the pathway are in italics. The rounded boxes depict the products or substrates of the reactions. The green shaded square shapes show different therapeutic approaches
for MLD treatment. Two current substrate reduction therapies for another lysosomal disease (Gaucher disease) are also shown: N-Butyldeoxynojirimycin (NB-DNJ) miglustat
(Zavesca®, Actelion Pharmaceuticals Ltd, Allschwil, Switzerland), and eliglustat tartrate. Both of these small molecules competitively inhibit ceramide glucosyltransferase.
Abbreviations: ASA, arylsulfatase A; EET, enzyme enhancement therapy; ERT, enzyme replacement therapy; Gal, galactose; HSCT, hematopoietic stem cell transplantation;
Glc, glucose; SGal-Glc-cer, sulfo-galactose-glucosylceramide; MLD, metachromatic leukodystrophy; SRT, substrate reduction therapy.

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the cgt−/− mice develop progressive hind limb paralysis and the function of the renal tubular, respiratory epithelium, or
extensive vacuolation of the ventral region of the spinal cord endometrium as it does in the CNS and PNS.58 Of note is that
as they age.2,51,52 Thus, these results indicate that the sulfati- some degree of accumulation of sulfatides in the mucosa of
des are important in myelin stability and its physiological the biliary tract is observed in MLD, resulting in mild to mod-
function. In the absence of CGT, the cgt−/− mice synthesize erate clinical manifestations.26–29 This observation is explained
2-OH glucocerebroside as the main component of myelin, because, physiologically, the levels of sulfatides present in the
a glycosphingolipid that is absent in the normal myelin nervous system are much higher, reflecting its crucial func-
composition. The 2-OH glucocerebroside gives a seemingly tion in the composition of myelin. In the arsa−/− MLD mouse
normal structure to myelin, and was hypothesized to prevent model generated by Hess et al, mild neurological symptoms
the severe effects of the loss of hydroxylated fatty acids are only observed by the end of a normal life span.59 As the
such as galactosylceramide.52,53 To validate this hypothesis, arsa−/− mice fail to show sulfatide accumulation, no signs
a CGT/FA2H double knockout mouse (cgt−/−/fa2h−/−) was of demyelination are observed.59 However, the arsa−/− mice
generated with an additional deletion of the FA2H gene showed neuromotor incoordination, suggesting that such
encoding the fatty acid 2-hydroxylase.54 These mice showed neurological signs might occur before demyelination.59 The
myelin comprised of non-hydroxylated sphingomyelin.54 No lack of a complete recapitulation of the clinical and pathologi-
phenotypic differences were observed between cgt−/−/fa2h−/− cal MLD phenotype led to the generation of the arsa−/− mice
and cgt−/− knockout mice.54 Thus, these results suggest that with neural cells overexpressing the sulfatide synthesizing
the presence of glucosylceramide and hydroxylated sphin- enzymes, including UDP-galactose:ceramide galactosyl-
gomyelin in cgt−/− mice does not functionally compensate transferase (CGT) and cerebroside sulfotransferase (CST).60
for the loss of hydroxylated fatty acid galactosylceramide.54 These CGT/arsa−/− and CST/arsa−/− mice showed increased
On the other hand, the fa2h−/− mice with intact CGT showed sulfatide storage in myelin-forming cells, resulting in axonal
absence of 2-OH galactosylceramide and sulfatide in normal degeneration and development of neurological symptoms
myelin.48 However, myelin sheath degeneration was observed similar to MLD.60 The hallmark of the disease is progressive
with aging.48,55 These studies of different murine knockout demyelination with resulting neurological manifestations.
models deprived of specific myelin sphingolipids demonstrate The precise pathogenic mechanism of the demyelination is
that myelin biogenesis in the nervous system remains intact, still not clearly understood.43
likely due to the redundancies of these myelin components.
The murine model with ceramide synthase 2 (CerS2) also Pathogenic cascades
showed interesting observations with regard to the impor- In lysosome storage disorders (LSDs), several pathogenic
tance of galactosylceramide and sulfatides in myelin.56 The cascades have been described secondary to the substrate
CerS2 knockout mice showed decreased levels of galactosyl- accumulation in the lysosomal-endosomal system.38,61,62
ceramide, sulfatide, and very long-chain fatty acids.56 From These cascades include calcium signaling, altered lipid
early adulthood, the CerS2 null mice showed progressive trafficking, oxidative stress, ER stress/unfolded protein
myelin instability, with 50% loss of the compacted myelin response, autophagy, and inflammation.38 The role of these
pattern and ∼80% loss of myelin-basic protein.56 In a differ- different pathogenic cascades in MLD is largely unchar-
ent study of the hepatic biochemical aspects of CerS2-null acterized, but the cascades in some lysosomal disorders
mice, the biophysical properties of lipid extracts isolated presenting with neurodegenerative processes are well
from liver microsomes were shown to be altered, indicating studied. Calcium signaling is essential for cell motility and
higher membrane fluidity and morphological changes. 57 myelination of neurons. Therefore, perturbations of cal-
Taken as a whole, these observations on Cer2S knockout cium signaling are likely to contribute to the demyelination
mice reiterate the role of the sulfatides in the maintenance observed in MLD.63,64 Studies in brain tissue from Gaucher
of myelin stability, which seems to be of crucial importance disease patients indicate that defective calcium homeostasis
during the aging process. may be a mechanism that leads to neuropathophysiology
in the acute and chronic neuronopathic forms of Gaucher
Mechanism of leukodystrophy disease.65 Studies in mice models of mucopolysacchari-
In MLD, ASA deficiency causes impairment of sulfatide dosis type III showed that neurodegeneration proceeds
degradation, and its accumulation mostly in nervous sys- independently of the signaling cascade associated with
tem (Figure  1). Sulfatide accumulation does not impair heparin sulfate.66 Signaling cascades may direct cells towards

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autophagy, as observed in a lysosomal disease characterized showed inflammatory response characterized by the presence
by neuromuscular presentation known as Pompe disease. of vacuolated macrophages and activation of microglia in
This inborn organelle disease is caused by deficiency of different regions of the brain.75 In sumf1−/− mice between 4
lysosomal acid α-glucosidase (GAA). In the fibroblasts from and 6 months of age, elevated expression of inflammatory
Pompe disease patients carrying c.546G.T mutation in the cytokines and apoptotic markers were observed in the CNS
GAA gene, the misfolded mutant GAA was shown to induce and liver, indicating that inflammation and apoptosis occur
ER stress, and subsequently elicit autophagy through activa- later in the disease and play an important role in the neuro-
tion of the p38 mitogen-activated protein kinase pathway.67 logical phenotype.75 Thus, arresting the disease after these
Autophagy is the pathway through which a cell can recycle processes occur becomes very difficult as, by this point in
defective proteins in the system. Thus, autophagy is a key time, permanent and irreversible neural damage is already
cleansing and regenerating pathway, in which products of established in the CNS.77
protein breakdown can make amino acids available to the
cell again. Also, autophagy and apoptosis have been shown Challenges in development
previously to be inversely related in hematopoietic stem of treatment options
cells.68 In another study in cultured Pompe patient fibro- Blood-brain barrier
blasts, the inactivation of Akt can lead to autophagy, in a ER The blood-brain barrier (BBB) controls the migration of
stress-independent manner, via the suppression of mTOR components from circulating blood to brain tissue. The BBB
signaling.69 In the absence of ER stress in Pompe disease is formed by several types of cells, including endothelial
fibroblasts, the inactivated Akt signaling cascade drives the cells with tight junctions, pericytes, astrocytes with foot
cells to autophagy, which can be reversed with insulin, which processes, and perivascular neurons. Thus, the arterial
activates Akt signaling.69 In Niemann-Pick disease type C capillaries in the CNS form a permeability barrier distinct
(NPC), sphingosine storage inhibits calcium uptake into from any other tissue, which harbors endothelial fenestra-
the lysosomal acidic compartment, leading to insufficient tions between cells. The tight junctions found in the BBB
calcium release for function of the endocytic pathway.70 generate high transendothelial electrical resistances of
In this lysosomal disease, the altered lipid trafficking was 1,500–2,000 Ω cm2. As a comparison, in tissues other than
demonstrated using fluorescent analogues of different lipids. brain, the transendothelial electrical resistance ranges from
Fluorescent dye-labeled sulfatides were shown to accumulate 3–33 Ω cm2.78 Thus, the BBB contributes with the essential
in lysosomes from fibroblasts obtained from MLD patients.71 protection of neural structures preventing cellular damage
Studies have shown evidence that altered chain length of the from extra-cellular insults.
lipids can affect its trafficking along the endocytic pathway.72 However, from the standpoint of the therapeutic devel-
Further, the storage of such metabolites in lysosomes is opment for inherited neurological disorders such as MLD,
linked with defective autophagosome-lysosome fusion,73 the BBB represents “a problem standing before the major
ultimately resulting in impaired autophagy. The processes problem”. In other words, BBB constitutes the major obstacle
of autophagy and apoptosis have been shown to be comple- to novel therapeutic agents that require reaching the affected
mentary in some cell types.68 Thus, the impaired autophagy neural tissue to treat the neurological manifestations of the
pathway in lysosomal disorders can lead to apoptosis. Inflam- disease. Amongst the drugs in the current medicinal chemis-
mation is associated with the development of neurodegen- try database, only a few – approximately 5%–6% – cross the
erative diseases like multiple sulfatase deficiency (MSD).74 BBB and are able to produce CNS effects.79 These are small
MSD is pathogenically similar to MLD, since in both disor- molecules with high Log P coefficient of lipophilicity, and
ders sulfatide accumulation is noted. In MSD, inflammation molecular weight ranging from 400 to 450 D.79 These drugs
was observed in the mouse model with null mutation in the include antipsychotics, antidepressants, and hypnotics.79 The
SUMF1 gene,75 which encodes the formylglycine-generating BBB imposes the main obstacle to delivery of several of the
enzyme that is responsible for the oxidation of cysteine and novel therapies developed for MLD, including the recombi-
formation of formylglycine (alanine-semialdehyde), the nant enzyme, which is a large molecular weight therapeutic
critical catalytic residue present in the active site of eukary- agent (∼52 kD). Additionally, certain cells of hematopoietic
otic sulfatases.76 The sumf1−/− mice demonstrated complete origin, including macrophages, activated T-cells, and micro-
loss of sulfatase activities and, consequently, early death, glial cells, are capable of crossing the BBB and migration
growth retardation, and neurological defects.75 These mice to the CNS.80 Thus, both specific strategies, utilizing small

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molecules and specific cells, constitute potential novel pathogenic process may occur that can result in neurological
avenues to deliver CNS disease-modifying agents for MLD symptoms, beyond demyelination. 83 Therefore, a better
patients. understanding of the molecular pathogenesis of MLD is a
substantial challenge in developing therapies for MLD.
Incomplete understanding
of MLD pathogenesis Time of intervention in the course
Sulfatides account for 4%–6% of the lipids forming of disease
the myelin and, as above, are essential sphingolipids In case of MLD, as in others lysosomal disorders, the classifi-
in the myelin sheath surrounding the axons. They modulate cation of patients in clinical forms such as late infantile, juve-
the differentiation of myelinating cells, and are required nile, and adult is based on the age of onset of first symptoms.58
for axon-glial interactions. Sulfatides are extracted from In general, the earlier the onset of the disease, the more rapid
the myelin by saposin B (SapB), a heat-stable sphingolipid is the disease progression. The different treatment options,
activator protein that is necessary to solubilize sulfatides and though currently limited, can produce a better outcome when
make them accessible to ASA (Figure 1). Consequently, the started in the earlier stages of the disease.84 Generally, the ear-
molecular and clinical manifestations of MLD can also be lier the intervention, the better the responses to that specific
resultant from defects in SapB, which was the first saposin therapy observed. The importance of earlier intervention can
described.81 The arsa−/− mouse model showed a similar be noted in MLD patients who underwent hematopoietic stem
pattern of accumulation of sulfatides in the CNS and other cell transplantation (HSCT).85 MLD patients who received
organs.59 However, the levels of sulfatide accumulation were HSCT earlier usually had a better outcome than those trans-
lower, which resulted in mild phenotype with no myelin loss planted at later stages of the disease. This can considerably
until the second year of mouse life, when progressive astro- increase an already high risk of mortality strictly related to
gliosis and microgliosis in the CNS and peripheral nerves the procedure.85–89 Some studies have shown that lysosomal
were noticeable.59 In the arsa−/− mouse, reduction of the storage in neuronal cells can be reduced to some extent in
levels of membrane raft-associated sulfatide-binding myelin different LSDs. However, the cellular changes occurring
and myelin and lymphocyte (MAL) protein were observed downstream of storage are usually not reversible.90 Thus, it
in the oligodendrocyte-generated myelin. This phenomenon is necessary to arrest such a primary cellular event at earlier
indicates the presence of a compensatory mechanism to stages of the natural history of the disease. Hence, the time of
limit MAL-mediated delivery of sulfatide to the myelin intervention becomes an important parameter in determining
membrane.82 The sulfatide in the mice CNS has a long half- the prognosis and outcome.
life (.6 months). Therefore, a short lifespan of the MLD
mice model can limit the extent of sulfatide accumulation. Current treatments for MLD
How the accumulation of sulfatides causes the disruption of Since MLD is caused by defective ASA, most therapeutic
myelin sheath resulting in apoptosis of the myelin-forming approaches have tried to correct the biochemical defect by
cells, as observed in older arsa−/− mice, is still unclear.59 In providing wild-type ASA (Figure 1). The different methods
ultrastructural transmission electron microscopy studies of and sources of wild-type ASA constitute distinct therapeutic
neurons from the arsa−/− mice, several MCBs were notice- approaches. Enzyme replacement therapy (ERT) and HSCT
able, indicating that sulfatides are likely accumulated in rely on providing normal ASA to the deficient cells, while
these cells.83 The accumulation of sulfatides is possible gene therapy approaches are based on the overexpression of
in oligodendrocytes and Schwann cells of affected MLD wild-type ASA in different cell types (Figure 1).
patients, resulting in failure to produce and maintain myelin
in axons located in the CNS and PNS, respectively. Thus, Enzyme replacement therapy
the complete inhibition of sulfatide biosynthesis is not an ERT has been a therapeutic modality used with remarkable
option in treatment, considering its importance as a myelin success in treating some LSDs. Its applicability to MLD is
component. In addition, the biological effects of sulfatide challenged by the BBB. Lysosomal ASA is a high molecu-
accumulation in neural cells other than oligodendrocytes lar weight therapeutic agent, and consequently is unable to
and Schwann cells are still unknown.83 The arsa−/− mouse penetrate the BBB to reach the affected brain tissue. In mice
model of MLD showed incoordination signs in the absence models, some studies have shown positive results,91 leading
of pathological signs of demyelination59, alluding that other to early phase clinical trials in MLD-affected patients. The

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efficacy of this treatment modality is inversely related to the One of the limitations of HSCT is the rate of repopulation
age of mice.92 The ERT approach also faces the difficulty of microglia into the recipient CNS, which should be a
of producing the recombinant enzyme at a large scale and sufficient cell number to provide an adequate amount of
in high purity, which has been overcome already by several wild-type ASA for the affected neural cells of the recipient.
pharmaceuticals.93 In this setting, the BBB limitation has HSCT was shown to be efficacious in the treatment of MLD
been addressed with different routes of administration, as early as 1990.84 However, HSCT failed to show efficacy
including intracerebroventricular or intrathecal ERT agent in the late infantile clinical form of MLD.96 Though HSCT
delivery. These approaches have been carefully evaluated is a well-established therapeutic approach for a few LSDs,
as modes of delivery for ERT. The current ongoing clinical including mucopolysaccharidosis type I and globoid-cell
trials using Metazym, (Shire Human Genetics Therapeutics, leukodystrophy (Krabbe disease), this treatment modality is
Inc., Lexington, MA, USA) administered intrathecally as an limited only to MLD patients who are diagnosed at earlier
ERT agent, are listed in Table 1. The final results of these stages of the disease course (Table  1).97–99 HSCT brings
clinical studies will be important to determine the efficacy other potential factors to be considered, such as identifi-
of this mode of ERT delivery for MLD and other lysosomal cation of matched related donors, the rate of repopulation
diseases. of microglial cells, along with specific transplant-related
complications including acute and chronic graft-versus host
Hematopoietic stem cell therapy diseases, and the level of engraftment.100,101 An alternative
As described earlier, the BBB prevents the delivery of ASA approach to HSCT is using umbilical cord blood as a source
enzyme to the affected neural cells. This sets the scene of hematopoietic and mesenchymal stem cells, as reviewed
for the development of various strategies that focus on elsewhere.102 Recently, the outcomes of 27 patients with
overcoming the BBB and making the enzyme available to different clinical forms of MLD were reported.85 In this
CNS cells. HSCT is one such approach, where hematopoi- study, an inverse relationship between time of intervention
etic stem cells (HSCs) cross the BBB and differentiate to and outcome was observed. In addition, children with the
microglial cells.94 In principle, the donor microglial cells juvenile form of MLD showed better outcomes than those
are able to secrete the wild-type ASA enzyme, which is with late infantile onset.85
uptaken by the recipient neural cells that are ASA-deficient.
Uptake of the secreted wild-type ASA occurs through Gene therapy
the mannose-6-phosphate receptor system, which brings Types of gene therapies have been studied for the treatment
the enzyme directly to the lysosomes, where sulfatide is of MLD. Autologous HSCs obtained from the affected
accumulated in the recipient ASA-deficient neural cells.95 MLD patient can be genetically modified to overexpress the
ARSA gene using retrovirus gene transfer.103 This approach
Table 1 Current therapeutic modalities in metachromatic
has been shown to be highly successful in the arsa−/− mice
leukodystrophy (MLD) model, as it prevents the development of motor conduc-
Treatment Outcome Limitations
tion impairment, learning and coordination deficits, and
Hematopoietic Stabilization or reduction Donor availability,
neuropathological abnormalities typical of the disease.104
stem cell therapy of disease progression risk and complications Thus, genetically modified HSCs can help prevent neurode-
(HSCT) post-HSCT97–99 related to the HSCT generation or slow its rate of progression. The synthesized
Ongoing clinical procedure
wild-type ASA secreted by these genetically modified
trials147,148
Enzyme Completed clinical Blood-brain barrier, HSCs is uptaken by the adjacent ASA-deficient cells via the
replacement trials149–153 non-homogeneous mannose-6-phosphate receptor system.95 The overexpression
therapy (ERT) distribution of ERT of ARSA gene by the autologous HSCs before transplant is
agent to CNS
performed using retro- or lentivirus vectors.105,106 Different
Gene therapy Completed and ongoing Safety and
clinical trials154,155 oncogenecity studies using this strategy have been discussed elsewhere.103
Specific cell Studies in vitro110–112 Production in culture The advantage of this therapeutic approach is the reduction
therapy (SCT) and in mice113 systems of specific in risk of graft-versus host disease. The major limitation of
cell types
Small molecule No effect on patients Toxicity and off-target
this approach is safety consideration, because of unknown
therapy with late infantile MLD156 effects risk of mutagenesis of cancer-related genes or oncogenes.107
Abbreviation: CNS, central nervous system. Currently, a phase I/II clinical study is ongoing with MLD

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patients receiving autologous HSCT with genetically tested in the murine model for Tay-Sachs disease (hexa−/−),
modified HSCs (Table 1). and was showed to reduce the accumulation of GM2 ganglio-
side.119 However, the hexa−/− mouse lacks an overt Tay-Sachs
Specific cell therapy disease phenotype, as mice can partially bypass the blocked
In addition to HSCT, various other cell-based approaches catabolic pathway and escape disease.119 In an early clinical
can be used to deliver ASA to affected cells.102 Briefly, trial with patients with the juvenile clinical form of GM2 gan-
microencapsulated recombinant cells that overexpress the gliosidosis, miglustat showed adequate pharmacokinetics
ARSA gene can be directly delivered. These cells offer and safety profiles,120 but failed to arrest some aspects of
substantial flexibility in terms of the dosage and control of disease progression.121 However, patients affected by NPC,
delivery, since they can be easily retracted.108,109 Similarly, a lysosomal disorder resulting in intra-cellular lipid traffick-
oligodendrocyte progenitor cells110 and neural progenitor ing defect and secondary glycosphingolipid storage, showed
cells111,112 are other cell types that can be used for cell- improvement and stabilization of a number of clinical end-
based therapy. Embryonic stem cells constitute another cell points, including swallowing capacity and ambulatory index,
source for ARSA overexpression and delivery. Studies in the when receiving miglustat in a clinical trial.122
arsa−/− mice, using embryonic stem cells, have demonstrated Small molecule-based therapy can potentially overcome
significant decreases in sulfatide accumulation.113 These limitations of the current therapies for MLD and may also
specific cell therapies are attractive options for the treatment tackle, at multiple levels, different pathogenic and molecular
of neurological manifestations of MLD. All these studies mechanisms involved in MLD. Small molecules can eas-
have demonstrated considerable clearance of the storage of ily cross the BBB, which is a major hindrance in the ERT
sulfatides in animal models.111–113 However, their efficacy strategy. Warfarin (coumadin) is a classical anti-coagulant
and (of utmost importance) safety and tolerability are still that functions by competitively inhibiting the multi-unit
to be demonstrated in humans. vitamin K epoxide reductase complex, resulting in impair-
ment of the activation of vitamin K-dependent coagulation
Potential novel therapies factors II, VII, IX, and X. Based on microorganism studies
Small molecule-based therapies in vitamin K, warfarin administration in mice showed to
In MLD, no specific treatment has been shown to completely reduce the levels of sulfatides by 33%, along with cerebro-
arrest disease progression. Therapies using small molecules sides and sphingomyelin in the brain.123 Warfarin was then
(in general, ,500 D) have hardly been explored in MLD. hypothesized as a potential substrate-reducing agent to be
Small molecule-based therapies have shown encouraging explored therapeutically for MLD. Recently, a clinical trial
results in the case of some lysosomal disorders like Gaucher in a small cohort of MLD patients tested whether warfarin
disease and NPC. N-butyldeoxynojirimycin (NB-DNJ) would control the progression of MLD.124 During a 45-day
miglustat (Zavesca®, Actelion Pharmaceuticals Ltd, Allschwil, treatment with warfarin at doses of 0.2 mg/kg/day, adjusted
Switzerland) was showed to reduce the biosynthesis of glu- to maintain INR within the range 2–2.5, no beneficial effect
cosylceramide by competitively inhibiting the UDP-glucose- of this small molecule was observed in four enrolled patients
N-acysphingosine D-glucosyltransferase (EC [Link]) or with the late infantile form of MLD (Table 1).124 The urinary
ceramide-specific glucosyltransferase.114 The mechanism of sulfatide levels of these patients remained elevated. Brain
action of miglustat was shown using an in cellulo model of magnetic resonance spectroscopy showed that the levels of
Gaucher disease.115 In studies using cultured human mac- N-acetyl-aspartate and myo-Inositol remained unchanged
rophages pre-exposed with conduritol-β-epoxide (CBE), after the treatment period.124
miglustat was shown to reduce glucosylceramide storage,
which is the main substrate accumulated in Gaucher disease.115 Small molecules as enzyme
CBE is an irreversible inhibitor of glucocerobrosidase, leading enhancement agents
to accumulation of glucosylceramide in cultured cells.116,117 Lysosomal storage disorders can be caused by missense
Ceramide-specific glucosyltransferase is the key enzyme mutations in specific genes that encode misfolded mutant
on the biosynthetic pathway of several glycosphingolipids, lysosomal enzymes (Figure  2). Most of these misfolded
including the ganglio- series (acidic glycosphingolipids), mutant lysosomal enzymes are earlier degraded in the ERAD
lacto(neo)- series (neutral glycosphingolipids), and globo- pathways125 and, consequently, a very small amount of the
series (Figure 1).118 Based on this principle, miglustat was mutant enzyme reaches the lysosomal compartment and is

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Wild type ASA peptide Mutant ASA peptide

Partially folded Misfolded mutant ASA

wild type ASA

ER – folding ER – associate
system degradation (ERAD)

Pharmacological chaperones

Proteostasis regulators

Figure  2 Schematic representation of the mechanism of action of enzyme enhancement agents including pharmacological chaperones (PCs) and proteostasis
regulators (PRs).
Notes: ASA is synthesized in the endoplasmic reticulum (ER) as several other proteins target to different intracellular organelles, plasma membrane, and those destined to be
secreted to the extracellular environment. Concomitant to the translation process, nascent ASA peptide interacts with several components of the ER folding system. Once
folded properly, ASA is targeted to the ER exit pathway and ultimately reaches the lysosomal compartment, where it exerts its physiological function. Missense mutations in the
ARSA gene can affect physicochemical interactions between amino acids, resulting in impairment of the folding process of the mutant ASA. PCs (blue boxes) are small molecules
that physically interact with the target protein, in this case ASA, and assist its folding, preventing it from being targeted to the ERAD pathway and ultimately degraded by the
ubiquitin-proteasomal system. Thus, increased levels of mutant ASA reach the lysosomes to degrade the natural substrate (sulfatides). Eventually, other small molecules function
as PRs (beige/brown boxes), which can improve the cellular folding capacity by interacting with ER folding components. These molecules can also promote physiological cellular
responses, such as unfolded protein response, which can be advantageous to enhance folding of specific misfolded ASA mutants. Other PRs (red boxes) can also potentially inhibit
components of ERAD and favor the interactions of misfolded ASA mutants with the ER folding elements, increasing the probability of reaching a folding-like conformation.
Abbreviations: ASA, arylsulfatase A; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum associate degradation; PC, pharmacological chaperones; PR, proteostasis
regulators.

functionally active. The resultant residual enzymatic activ- (Figure 2).127 Thus, by evading the ERAD pathway, and sub-
ity, in the case of ASA mutants, ranges between 0%–10% sequently enhancing the level of the available mutant ASA
of the normal activity detected from the wild-type ASA.126 to the lysosomal compartment, the residual ASA enzymatic
This ASA enzymatic activity, above which the activity is activity will be increased and potentially overcome the
sufficient to degrade the sulfatides and prevent their accu- 10% critical threshold.126 In this regard, the ER folding
mulation, is known as the critical threshold – which is ∼10% system, with its numerous ER-resident chaperones, plays
of that measured from the wild-type ASA.126 Interestingly, an important physiological role in promoting adequate and
the levels of ASA enzymatic activity of the pseudodeficiency functional folding of several lysosomal enzymes, including
ARSA alleles are just above the critical threshold. Therefore, ASA. The ER-resident chaperones are proteins that assist
in ASA pseudodeficiency, the levels of enzyme activity the folding or unfolding of nascent proteins synthesized in
between 10%–15% of wild-type ASA levels result in no the ER that are not able to do so spontaneously.127 Similarly,
sulfatide accumulation, and consequently no disease-related pharmacological chaperones (PCs) are small molecules
symptoms of MLD.126 that function like chaperones, enhancing the levels of the
Specific small molecules are able to rescue misfolded misfolded-prone mutant enzymes by assisting their ER
proteins. These molecules are able to physically interact folding and preventing their interaction with the ERAD
with a specific misfolded protein, assist its folding process, pathway and subsequent degradation.128–130 Small molecules
and prevent it from being targeted to the ERAD pathway and functioning as PCs were identified for misfolded mutant
ultimately degraded by the ubiquitin-proteasomal system hexosaminidase A, the lysosomal enzyme deficient in

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GM2  gangliosidosis,128 and for misfolded mutant gluco- primary screening stage.140 However, in most cellular HTS
cerebrosidase (Glc-ase), the lysosomal enzyme deficient assays, the cells utilized are usually not disease relevant.
in Gaucher disease.129,131 In addition to PCs, proteostasis Thus, using patient-derived cell lines for HTS campaigns
regulators (PRs) are small molecules that improve the brings considerable advantages, compared to the traditional,
protein folding capacity of cells, resulting in the enhance- single-target biochemical assays (Figure  3).141,142 Cellular
ment of folded mutant proteins.132 PRs work by interacting assays utilizing cells from affected patients provide a unique
with the ER folding components and eventually activating opportunity to assess a target protein and/or pathway in the
physiological cellular response mechanisms, including context of potentially disrupted biochemical and/or signal-
unfolded protein response and heat shock proteins (Fig- ing pathways secondary to the disease process. In addition,
ure 2). PRs can also function by inhibiting the degradation this pathogenic in cellulo setting permits the evaluation of
of the misfolded mutant enzyme, by interacting with the multiple intervention points, which are potentially altered in
ERAD components or HSC molecules (Figure 2).133–135 The the disease, as opposed to the commonly-used cell expres-
effect of PRs can be enhanced by simultaneously using sion systems of a specific protein target, or a predefined step
PCs for assisting the folding of misfolded-prone mutant of a purified or recombinant protein-based assay (Figure 3).
enzymes.127 Interestingly, these two classes of small mol- Therefore, cell-based HTS can identify both PCs and
ecules can work together synergistically to guide the mis- PRs.141,142 A cell-based HTS assay was developed to identify
folded mutant enzyme to a folded state (assisted by PRs) and small molecules for mutant ASAs.141 Using a transformed
maintain its structural stability (assisted by PCs).136 Some fibroblast cell line from a MLD patient with measurable
PRs have been identified for specific lysosomal mutant residual ASA activity, a fluorescence quench absorbance ASA
enzymes.136,137 Inhibitors of histone deacetylase (HDAC) assay was developed. This HTS assay showed robust signals
are showed to reduce the acetylation of heat shock protein in diverse multi-well plates, including 1,536-well plates.141
90 (Hsp90)138. Since Hsp90 plays an important role in the The implementation of the cell-based HTS assay against
degradation pathway of the misfolded N370S and L444P larger and diverse small molecule libraries should allow
Glc-ase mutants, HCAD inhibitors resulted in increased the identification of novel potential drugs for MLD. This
levels of mutant Glc-ase enzymatic activity.138 Thus, these and other cell-based HTS assays open a novel opportunity
inhibitors act as PRs by preventing the degradation of to identify numerous small molecules, as several potential
mutant enzymes while interacting with components of therapeutic targets are accessible in this disease-cellular
the degradation pathway. MG-132 and celastrol are other environment.141,142
examples of small molecules that were shown to function
as PRs for some mutant Glc-ase and Hex A enzymes.136 PRs Small molecules as substrate
can be potentially applied to a range of LSDs, since these reduction agents
small molecules target the cellular enzyme folding pathway Some small molecules can also have their point of action
and their effect can be enhanced using PCs. These specific upstream to the biochemical defect: ie, sulfatide accumu-
small molecule-based therapies can work in conjunction lation in MLD (Figure  1). Miglustat is an example of a
with ERT by interacting with and, consequently, stabilizing substrate reduction therapy (SRT) agent that functions as
the administered recombinant ASA, extending its half-life a competitive inhibitor of ceramide-specific transferase.118
in either extra or intracellular compartments. Similarly, the sulfatide synthesis pathway can be potentially
How can these therapeutic small molecules be identi- targeted for substrate (sulfatide) reduction at multiple points
fied? Small molecules that work as PCs or PRs for specific in the pathway, as seen in Figure 1. Specifically, ceramide
lysosomal mutant enzymes can be identified using high synthase can be targeted using small molecules, which would
throughput screening (HTS) assays. Recently, the use of cell- lead to reduction of the biosynthesis of sulfatides (Figure 1).
based assays in the HTS of chemical compound collections Reducing ceramide synthase catalytic activity can result in
has become increasingly common, as targets are presented the reduction of further accumulation of sulfatides. However,
in a more biologically relevant context than classical in vitro complete arrest of sulfatide biosynthesis may cause dis-
biochemical-based HTS assays.139 In cell-based HTS assays, ruption of myelin stability, as shown in different murine
the identification of small molecules that penetrate cellular models described above.48,51,54 Thus, physiological levels of
membranes and reach specific cellular organelles, along with sulfatides are crucial, as these are main glycosphingolipids
their cytotoxicity information, can already be obtained at the of the myelin sheath. Hence, the optimality of therapeutic

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A 1
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

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B
HO
OH H
HCI
NO2 H
O n
N CH3 OCH3 • HCI
CH3 N CI NH2
HO CI
O N O OH
H O
CI H NH2 N
NH N
NN OH
N
N NH2 H O H2N N N
H HO OH • HCI H
CI
OH

O
NHCH(CH3)2 O
H CH3
O
O N
N N O HO CH CHCH2CH3
H H
N • CH3SO3H H3C CH3
NH N • HCI
H3C CH3
N H
N N O OH
H OH

C D
80 80
6000
Fluorescent signal

6000
Fluorescent signal

% of HMU signal
% of HMU signal
70 70
4.6 µM 23 µM
5000 5000 60 60
50 50
4000 4000 40 40

3000 30 30
3000
20 20
2000 2000 10 10
0 0
1000 1000
−10 −10
−5.5 −5.0 −4.5 −4.0 −3.5 −5.5 −5.0 −4.5 −4.0 −3.5
0 0 O
0 10 20 30 0 10 20 30 NO2
Log [ ]M
Log [ H
N CI ]M O
CH3

Row number Row number O N O OH H3C CH3

N • HCI
H H3C CH3
H

Figure 3 Cell-based high-throughput screening (HTS) assay.

Notes: Here, a cell-based HTS assay for galactocerebrosidase (GALC), which is the lysosomal enzyme deficient in Krabbe disease or globoid cell leukodystrophy (GLD),
is depicted for (A and B). The 1,536-well plate contains GLD patient cells that are cultured and treated with a small molecule collection for a specific period of time. After
the treatment period, the specific enzyme GALC can be measured using a fluorescent substrate, 6-hexadecanoylamino-4-methylumbelliferyl-β-D-galactoside (HMU) for the
enzyme. In each 1,536-well plate, control cells (columns 2 and 3) are seeded and later assayed. These control cells have normal Gal-ase enzymatic activity and are not treated
with the library. The columns 1 and 2 contain only culture medium and no cells, and are used as backgrounds for the assays. Columns 5–48 contain cultured patients cells (B)
– in this case, fibroblasts. Each well (yellow square) corresponds to a specific small molecule from the library, which is tested in different concentrations. (C) Two-plate view
diagrams are shown with the fluorescent signals generated from the HMU against the different rows of a 1,536-well plate. Substantial assay window is observed between
GALC enzymatic activity of controls (triangles) and GLD patient cells (circles). The small molecule candidates are selected based on the enhancement of the residual GALC
enzyme activity. (D) The quantitative nature of the HTS assay, where seven different concentrations of the target library were tested, allows the generation of concentration-
response curves (CRCs) given by the HMU fluorescence signal increase. Based on the characteristics of the CRCs,146 prioritization of “hits” can be done during the validation
process with secondary and tertiary assays.

dose of potential small molecules functioning as SRT agents are characterized by disturbances in molecular signaling
is a prerequisite for the success of this therapeutic strategy. pathways occurring secondary to sulfatide accumulation,
Therefore, SRT agent doses should be determined by the ultimately leading to apoptosis.
molecule concentrations capable of partially inhibiting In a few lysosomal disorders, some small molecules have
the catalytic activity of a target enzyme in the biosynthetic been identified to target specific pathogenic cascades. In stud-
pathway of sulfatides at intracellular levels (ie, in oligo- ies of NPC patient fibroblasts and Npc1−/− mice, sphingosine
dendrocytes and Schwann cells) (Figure  1). This will be storage was shown to precede low calcium levels in the lyso-
crucial to avoid potential adverse events of SRT agents in somal compartment, which explains the profound block in
the treatment of MLD. late endosome to lysosome transport, resulting in the storage
of cholesterol and glycosphingolipids – the cellular hallmark
Small molecules to control of this lysosomal disorder.70 In the presence of curcumin, a
pathogenic cascades small molecule that is a weak antagonist of the sarco (endo)
Small molecules can also be used to manipulate downstream plasmic reticulum ATPase (SERCA), the levels of calcium
pathways that are initiated or disturbed secondary to the in the acidic compartments were normalized and clearance
primary lysosomal enzymatic defect. Diverse pathways of lipid storage was noted, resulting in increased Npc1−/− life
have been described to be impaired in lysosomal storage expectancy.70 SERCA is a pump that transports calcium ions
diseases. These are known as pathogenic cascades, which from the cytoplasm into the sarco(endo)plasmic reticulum.143

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The inflammatory cascades can be targeted using non-steroidal collaborators who donated cell lines including Dr Hirokazu
and anti-inflammatory drugs, which have resulted in clini- Furuya MD, PhD, neurologist from NHO Omuta Hospital,
cal benefits in other lysosomal diseases, including Sandhoff Omuta, Fukuoka, Japan.
disease144 and NPC145. Most of these small molecules have
already been characterized to interact with specific molecular Disclosure
targets. To discover further small molecules, cell-based HTS Shilpa Patil is partially supported by National MPS
assays can be developed using as the readout signal a specific Society. Gustavo Maegawa is currently supported by
relevant target of a pathogenic cascade related to ASA defi- grants NIH-NIMH 1R03MH098689 and NIH-NINDS
ciency. These HTS assays will identify potential therapeutic 1R01NS079655. Dr Maegawa also receives research grants
small molecules to tackle signaling pathways that are disturbed from the National MPS Society and received research grants
secondary to the ASA deficiency. These small molecules may from the National Tay-Sachs Disease and Allied Diseases
produce synergistic effects along with the therapeutic modali- Association. Dr Maegawa receives research funds for clini-
ties that tackle primarily the ASA deficiency, such as ERT, gene cal and educational studies from Genzyme Therapeutics Ltd,
therapy, and cell-based therapies. By using combinations of Biomarin Pharmaceutical Inc, Protalix Biotherapeutics Inc,
therapies to specific targets, it is more likely that the arrest or and Shire HGT. The authors declare no other conflicts of
even reversal of disease progression can be achieved. interest in this work.

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