Repositioning of an existing drug for the neglected tropical disease Onchocerciasis

Article abstract of DOI:10.1073/pnas.0915125107

Onchocerciasis, or river blindness, is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus that affects more than 37 million people, mainly in third world countries. Currently, the only approved drug available for mass treatment is ivermectin, however, drug resistance is beginning to emerge, thus, new therapeutic targets and agents are desperately needed to treat and cure this devastating disease. Chitin metabolism plays a central role in invertebrate biology due to the critical structural function of chitin for the organism. Taken together with its absence in mammals, targeting chitin is an appealing therapeutic avenue. Importantly, the chitinase OvCHT1 from O. volvulus was recently discovered, however, its exact role in the worm's metabolism remains unknown. A screening effort against OvCHT1 was conducted using the Johns Hopkins Clinical Compound Library that contains over 1,500 existing drugs. Closantel, a veterinary anthelmintic with known proton ionophore activities, was identified as a potent and specific inhibitor of filarial chitinases, an activity not previously reported for this compound. Notably, closantel was found also to completely inhibit molting of O. volvulus infective L3 stage larvae. Closantel appears to target two important biochemical processes essential to filarial parasites. To begin to unravel closantel's effects, a retro-fragment-based study was used to define structural elements critical for closantel's chitinase inhibitor function. As resources towards the development of new agents that target neglected tropical diseases are scant, the finding of an existing drug with impact against O. volvulus provides promise in the hunt for new therapies against river blindness.

Full text of DOI:10.1073/pnas.0915125107

Repositioning of an existing drug for the neglected
tropical disease Onchocerciasis
Christian Gloecknera,b,c, Amanda L. Garnera,b, Fana Mershad, Yelena Oksove, Nancy Tricochee,
Lisa M. Eubanksa,b,c, Sara Lustigmane, Gunnar F. Kaufmanna,b, and Kim D. Jandaa,b,c,1
c
aDepartments of Chemistry and Immunology, bThe Skaggs Institute for Chemical Biology, and The Worm Institute for Research and Medicine,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; and dNew England Biolabs, 240 County Road, Ipswich,
MA 01938-2723; eLindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065
Communicated by Sydney B. Brenner, Institute of Molecular and Cell Biology, Singapore, Singapore, December 31, 2009 (received for review November
15, 2009)
Onchocerciasis, or river blindness, is a neglected tropical disease
caused by the filarial nematode Onchocerca volvulus that affects
more than 37 million people, mainly in third world countries.
Currently, the only approved drug available for mass treatment
is ivermectin, however, drug resistance is beginning to emerge,
thus, new therapeutic targets and agents are desperately needed
to treat and cure this devastating disease. Chitin metabolism plays
a central role in invertebrate biology due to the critical structural
function of chitin for the organism. Taken together with its absence
in mammals, targeting chitin is an appealing therapeutic avenue.
Importantly, the chitinase OvCHT1 from O. volvulus was recently
discovered, however, its exact role in the worm’s metabolism re-
mains unknown. A screening effort against OvCHT1 was conducted
using the Johns Hopkins Clinical Compound Library that contains
over 1,500 existing drugs. Closantel, a veterinary anthelmintic with
known proton ionophore activities, was identified as a potent and
specific inhibitor of filarial chitinases, an activity not previously
reported for this compound. Notably, closantel was found also
to completely inhibit molting of O. volvulus infective L3 stage
larvae. Closantel appears to target two important biochemical pro-
cesses essential to filarial parasites. To begin to unravel closantel’s
effects, a retro-fragment-based study was used to define structural
elements critical for closantel’s chitinase inhibitor function. As
resources towards the development of new agents that target
neglected tropical diseases are scant, the finding of an existing
drug with impact against O. volvulus provides promise in the hunt
for new therapies against river blindness.
Central Africa. Victims of onchocerciasis present symptoms of
onchodermatitis (severe skin lesions), musculoskeletal pain
and various stages of blindness; however, patients also experience
decreased body mass index, work productivity, and social stigma-
tization. Currently, the only drug available for mass treatment is
ivermectin (Mectizan®, Merck), which was originally marketed
as a veterinary anthelmintic (7). Although this drug has proven
successful in reducing morbidity, the risk of severe skin or ocular
disease and decrease of microfilarial loads (>99%) after 14 d of
treatment, it only has modest effect on adult worms and must be
continuously administered over decades (8, 9); and it now
appears that ivermectin-resistance is emerging (10). Thus, there
is a crucial need to identify new drug targets and agents that can
effectively treat onchocerciasis.
Recently, chitin metabolism has been implicated in the larval
development of O. volvulus and Acanthocheilonema viteae
(11, 12). While knowledge of chitin biosynthesis in nematodes
is limited (13), two classes of enzymes are critical for maintenance
of the pathway, chitin synthases and chitinases. The dynamic
synthesis and degradation of chitin by these enzymes is a prereq-
uisite for organism development and, thus, serves as a target for
growth control. As chitin synthases are typically membrane-
bound and therefore difficult to express recombinantly, chitinases
represent a better suited target for drug discovery. A chitinase
from O. volvulus, OvCHT1, was recently identified, characterized
and shown to belong to the family 18 chitinases, which are
glycoside hydrolases that follow a substrate-assisted hydrolysis
mechanism (11, 14, 15). Although OvCHT1’s exact metabolic
role is not known, it was found to be expressed only in the infec-
tive L3 larvae with potential involvement in host transmission,
molting, and important developmental processes in the parasite
(14). Similarly, the A. vitae chitinase was shown to be essential for
the molting process using RNAi (16). Yet, chitin has so far not
been identified as a component of the cuticle of nematodes, and
even for well-studied nematodes, such as C. elegans, it has only
been described as a component of the pharynx (17). Immunoe-
lectron microscopy analysis has detected chitinase in the pharyn-
geal glands of L3 of A. viteae (16) and O. volvulus (14), structures
that are proposed to contain a wide variety of proteins essential
for the remodeling processes during molting and the final step of
ecdysis, shedding of the old cuticle. In addition, chitinases are
also found in developing eggs in utero and in microfilariae, thus,
these enzymes may be appropriate targets for other developmen-
tal stages and other filarial parasites. As such, inhibition of this
enzyme may represent a new drug target toward the elimination
chitinase ∣ closantel ∣ filarial parasite ∣ molting ∣ river blindness
eglected tropical diseases (NTD) include 13 poverty-
Npromoting parasitic and bacterial infections that affect over
one billion people worldwide, primarily in impoverished regions
(1). Notably, there is strong evidence that affliction with these
ailments increases susceptibility to or worsens the progression
of morbidity from the “big three” infectious diseases, HIV/AIDS,
tuberculosis, and malaria (2). Thus, controlling NTDs may pro-
vide an effective low cost mechanism for reducing morbidity and
mortality associated with these diseases. Unfortunately, many
factors limit the utility of existing therapies, including high cost,
poor patient compliance, emerging or existing drug resistance,
low therapeutic efficacy, and poor safety profiles (2–4). As a
direct result, new antiparasitic drugs are urgently needed to treat
and control NTDs.
One NTD for which there is pressing urgency for a therapeutic
revolution is onchocerciasis, or river blindness, a leading cause of
blindness in the developing world (5, 6). The infection is caused
by the filarial parasitic nematode Onchocerca volvulus that is
transmitted to humans by the blackfly (Simulium spp.) and the
pathology is resultant of the death of the microfilariae in the skin
and eyes. Despite several eradication efforts, the disease affects
more than 37 million people in Africa, Central and South Amer-
ica as well as Yemen, with >99% of those affected from West and
Author contributions: C.G., A.L.G., S.L., G.F.K., and K.D.J. designed research; C.G., A.L.G.,
F.M., Y.O., and N.T. performed research; C.G., A.L.G., S.L., and K.D.J. analyzed data; C.G.,
A.L.G., L.M.E., S.L., G.F.K., and K.D.J. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: kdjanda@scripps.edu
This article contains supporting information online at www.pnas.org/cgi/content/full/
0915125107/DCSupplemental.
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www.pnas.org/cgi/doi/10.1073/pnas.0915125107

of onchocerciasis. In these regards, allosamidin, a natural product
isolated from the mycelial extract of Streptomyces sp. No. 1,713,
has been investigated and found to be a potent chitinase inhibitor
(18). Despite its potency, the de novo synthesis is quite lengthy,
difficult, and expensive, thus, making it a less than desirable ther-
apeutic lead molecule. While other less potent inhibitors have
also been identified in vitro, chitinase inhibition in vivo remains
to be demonstrated and none of these inhibitors have been tested
against filarial chitinases (19).
Herein, we describe screening efforts against O. volvulus
chitinase (OvCHT1) activity in vitro using the Johns Hopkins
Clinical Compound Library (JHCCL) as a source of potential
inhibitors. Through these studies, we have identified a known
veterinary anthelmintic drug, closantel, previously used in the
treatment of sheep and cattle infected with liver fluke (20, 21).
Importantly, closantel’s mechanism of action has not been linked
to chitinase inhibition. Remarkably, closantel was found to exhi-
bit potent inhibition and high specificity for filarial chitinases in
vitro. Additionally, closantel was found to completely inhibit
molting of the L3 larvae. A retro-fragment-based study was uti-
lized to illuminate the important molecular features for chitinase
inhibition. Based on its specificity, potency, and ease of synthesis,
closantel or one of its analogues might represent a promising
alternative or adjunct therapy in combination with ivermectin
for the treatment of onchocerciasis.
Fig. 1. Structure, potency, and inhibition mode of closantel. For experimen-
tal details, please see the Methods section. (A) Structure of closantel. (B) De-
termination of IC50 for closantel. (C) Determination of mode of inhibition.
Results
Identification of Closantel as an OvCHT1 Inhibitor. To identify inhibi-
tors of OvCHT1 with potential impact as antionchocerciasis
agents, the 1,514 drugs contained in the JHCCL were screened
against OvCHT1 using a fluorescence-based assay and positive
hits were selected by observing a decrease in fluorescence signal
that correlates with a decline in the hydrolytic cleavage of the
fluorescently-labeled chitin substrate 4-methylumbelliferyl-
N-N0-N00-β-chitotrioside (see Materials and Methods for details).
From our initial screening efforts, four hits were identified, lev-
floxacin, lomefloxacin, dexketoprofen, and closantel; however,
only closantel (Fig. 1A), a salicylanilide drug, was found to exhibit
potent inhibition (IC50 ¼ 1.6 ꢀ 0.08 μM), warranting further
investigation (Fig. 1B). Additional experiments showed that
the mode of inhibition for this compound is competitive with a
Ki of 468 ꢀ 84 nM (Fig. 1C).
allosamidin for its potency and specificity towards the above-
mentioned filarial and human enzymes. Table 1 shows allosamidin
potently inhibited all assayed chitinases. However, these data also
indicate a lack of specificity by allosamidin, likely due to the fact
that its molecular structure resembles closely the natural chitinase
substrate, chitin.
Effect of Closantel on Molting in O. volvulus L3 Stage Larvae. The
molting process is considered a potential new target for
chemotherapy against onchocerciasis (22–25). As chitinases
may play a key role in molting, we wanted to determine the effect
of closantel on this process. L3 stage larvae were cultured in the
presence of increasing concentrations of closantel, and the numb-
er of molting larvae was determined on day six. As Fig. 2 shows,
closantel almost completely inhibited the molting of O. volvulus
L3 to L4 larvae at 100 μM (97.6% inhibition; 1.4% molting in the
treated group versus 60.5% in the control group). Although a
dose-dependent response was not observed, closantel at this
concentration did not exhibit an effect on viability, and this lack
of dose dependence was likely due to bioavailability or tissue
penetration issues.
Specificity of Closantel for OvCHT1. Chitinases from several other
parasitic species were used to assess the specificity of closantel,
namely from the filarial nematode Brugia malayi (BmCHT1) and
two unicellular protozoans, Entamoeba histolytica (EhCHT1) and
Plasmodium falciparum (PfCHT1). As an additional control, the
39-kDa isoform of human family 18 chitotriosidase (HsCHT1)
was included. Table 1 (left) shows that closantel demonstrated
excellent specificity towards the filarial family 18 chitinases, as
the IC50 for BmCHT1 (9.0 ꢀ 0.35 μM) is in the same range as
the IC50 for OvCHT1 (1.6 ꢀ 0.08 μM), while the protozoan
chitinases EhCHT1 and PfCHT1 exhibited higher IC50 values
(78 ꢀ 34 μM and 132 ꢀ 32 μM, respectively). Notably, even at
high concentration, closantel did not affect HsCHT1, and there-
fore, no IC50 value could be determined.
Ultrastructural Studies of Larvae That Did Not Molt in the Presence of
Closantel. O. volvulus L3 larvae molt within 4–6 d in culture.
During molting when the cuticles of the L3 and L4 larvae are
Interestingly, the amino acid sequence alignment of different
chitinases indicates a high conservation among the filarial family
18 chitinases OvCHT1 and BmCHT1 with a sequence identity of
67% (Table 1, right). On the other hand, the chitinases from E.
histolytica and P. falciparum show almost no identity (both 13%)
and that from H. sapiens shows slightly higher amino acid identity
compared to OvCHT1 (35%). These findings seem to be in ac-
cord with the IC50 values determined indicating that closantel
may be specific towards the filarial type of family 18 chitinases.
Allosamidin is considered the “gold standard” of chitinases
inhibitors (19). Thus, for comparison, we set forth to investigate
Table 1. IC50 values and sequence identity using chitinases from
different species.
IC50 (μM)
IC50 (μM)
%
Enzyme
(Closantel)
(Allosamidin)
Identity
OvCHT1
BmCHT1
EhCHT1
PfCHT1
HsCHT1
1.6 ꢀ 0.08
9.0 ꢀ 0.35
78 ꢀ 34
132 ꢀ 32
n.d.
0.0013 ꢀ 0.0003
0.0018 ꢀ 0.00004
0.122 ꢀ 0.0037
0.0254 ꢀ 0.0004
0.0119 ꢀ 0.0007
100
67
13
13
35
n:d: ¼ not determined
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Fig. 2. Inhibition of molting in the presence of closantel. O. volvulus L3 were
cultured with closantel at 10, 25, 50, and 100 μM and 1.5 × 105 normal human
peripheral blood mononuclear cells for 6 d as described in the “Methods”
section. The data are presented as percent molting in a total of 10 wells
containing on average 5–10 larvae per well.
being separated, the cuticular material in between the old and
new cuticles is completely degraded before ecdysis, the final stage
of molting when the L4 larvae emerges from the old cuticular cast
of L3 (26). In larvae that were cultured in the presence of 100 μM
closantel, the molting in vitro was almost completely inhibited
(Fig. 2). When the ultrastructural changes of these larvae were
examined, it appeared that in comparison to the normal morpho-
logical changes that occurred in the molting larvae of the control
group (Fig. 3A), the separation between the L3 cuticle and the
newly synthesized L4 cuticle in the closantel-treated worms
was inhibited and the cuticular material in between the cuticles
was not fully degraded (Fig. 3B). Similar phenotypes have also
been observed when L3 larvae were cultured in the presence
of cysteine protease inhibitors or when the transcripts corre-
sponding to O. volvulus cysteine proteases (25) or the serine
protease inhibitor were knocked down using RNAi (27). Thus,
the presented data demonstrate that closantel has a profound
effect on filarial molting. It, however, remains unclear whether
this effect is due to inhibition of the O. volvulus chitinase or other
effects caused by closantel, e.g., uncoupling proton ionophore
activity.
Fig. 3. Ultrastructure of O. volvulus molting in 100 μM closantel-treated L3.
To determine what stage of the molting process was affected by 100 μM
closantel, the O. volvulus treated L3 were collected on day six and fixed
for 2 h at 4 °C with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer
(pH 7.4) and processed for electron microscopy. In the four panels of A,
the normal molting process of L3s is shown. (A) Normal separation between
the L3 cuticle (arrows) and the epicuticle of the newly developed L4 (arrow-
heads) during the normal molting process of larvae cultured in DMSO control
culture media. Please pay attention that the area between the cuticles clears
during the molting process until the two cuticles are completely separated
and before ecdysis; (B) incomplete separation between the L3 and L4 cuticles
in 100 μM closantel-treated worms. Notably, although the cuticle of L4 was
present the separation between the newly synthesized cuticle and the old L3
cuticle was never completely degraded; cuticular material is still present be-
tween the L3 cuticle and the L4 epicuticle. The regions where the cuticles of
L4 and L3 separate are marked by an arrowhead and an arrow, respectively.
Each bar is 500 nm.
Retro-Fragment-Based Studies. Closantel is an approved drug for
veterinary use, thus by definition should not require further
optimization. Yet, with its newly discovered dual biochemical
roles of a chitinase inhibitor as well as proton ionophore, such
activities must not only be accounted for but also dissected. To
begin to define the critical chemical features of closantel
that are essential for chitinase inhibition, we deconstructed the
molecule into simple molecular fragments (Fig. 4).
Closantel can be viewed as a series of three functionalized
aromatic rings (A, B, and C in Fig. 4): 3,5-diiodosalicylic acid
(2, A-ring), 5-chloro-2-methylaniline (3, B-ring) and 4-(chloro-
phenyl)acetonitrile (5, C-ring). 4-Chloroaniline (4) was also
considered due to the para-chloro substituent of the C-ring. Thus,
compounds 6–8 were synthesized, and each contain portions of
the closantel scaffold. Compounds 6 and 7 contain salicylanilide
moieties: 6 is composed of rings A and B, while 7 is composed of
rings A and C. The third fragment, 8, contains rings B and C.
Compounds 2–8 were tested for inhibition potential against
OvCHT1 using the fluorescence assay as previously described.
As shown in Table 2, fragment 7 exhibited potency near to that
of the parent closantel (1.6 μM), and ring A and part of ring C
seem to be the important regions for potent inhibition.
disease, and de novo drug discovery (2, 3). As these approaches
rely more on the screening of compounds that are not already
FDA-approved and in clinical use for other indications, an
exorbitant amount of time and financial resources will still be
needed before drug approval can be obtained, hence, depriving
patients that desperately need treatment.
A more time- and cost-effective approach lies in drug reposi-
tioning, or identifying and developing new uses for existing drugs
(28, 29). In fact, both ivermectin (Fig. 1A) (7) and its analogue,
moxidectin (30), were originally marketed as veterinary anthel-
mintics (20). Ivermectin targets the O. volvulus microfilariae
and has an embryostatic effect on the adult female worms
(8, 31). Although issues exist with this drug, including resistance
development (10, 32–34), its success validates the importance of
drug repositioning toward the discovery of therapeutics against
NTDs. One untapped pathway for the discovery of new therapeu-
tic avenues is chitin metabolism. Although the precise function of
chitinase in filarial nematodes has not been fully deciphered, in
all filarial parasites studied, they appear to be important during
Discussion
Although NTDs, such as onchocerciasis, affect a greater number
of individuals than nearly every other disease known to man,
drug discovery efforts against these ailments have been slow to
develop, principally due to the fact that NTDs are often diseases
of the poor and underprivileged. Current strategies towards the
discovery of unique therapeutics for tropical diseases, include
piggy-back discovery, i.e. the screening of libraries that are
already being assayed for a similar molecular target in another
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Gloeckner et al.

Fig. 4. Fragment analogues of closantel.
egg and microfilariae development, and are probably required to
degrade the chitinous oolema surrounding the developing eggs in
utero (35) or for exsheathment of the microfilariae within the ar-
thropod vector (36). Several natural product chitinase inhibitors
have been previously described, including allosamidin (18), argi-
fin, and argadin (37); however, the complex chemical structures
require lengthy and expensive de novo syntheses as they exist only
in limited supply in Nature.
In the hope of finding “the next ivermectin”, or an existing
drug with a new target, we screened the JHCCL against the
O. volvulus chitinase OvCHT1 (11, 14), which is expressed only
in the infective L3 larvae. The enzyme is stored within the gran-
ules of the esophageal glands until postinfective development
after which it is secreted and found mostly in the cuticle (14).
Although its exact function is not clear, it has been hypothesized
that it is likely involved in host transmission with a potential func-
tion during ecdysis; remodeling of the L4 cuticle and casting of
the L3 cuticle (14). Thus, an inhibitor against this poorly studied
enzyme could help in defining larval development and assist in
uncovering whether chitin metabolism also plays a role during
the L3 to L4 molt.
From our screening efforts, we discovered one hit with potent
inhibition against OvCHT1, closantel, with an IC50 of
1.6 ꢀ 0.08 μM and a competitive inhibition constant (Ki) of
468 ꢀ 84 nM (Fig. 3). Similar to ivermectin and moxidectin, clo-
santel is marketed as a veterinary anthelmintic, and is effective
against bloodsucking nematodes, trematodes (20), filarial M. des-
setae (38) as well as liver fluke (39) in sheep and cattle; addition-
ally it has also shown activity against various insect larval stages
(20). Notably, combinations of ivermectin and closantel have
been used in cattle as a double-dose of antiparasitic with great
success (40). Distinct from ivermectin, previously, closantel’s
mode of action was believed to rely on its role as proton iono-
phore, an uncoupler of oxidative phosphorylation, as similar
salicylanilide drugs are known to possess such activity (20).
However, closantel’s function as a chitinase inhibitor has not
been previously reported, and, thus, this is a new use discovered
for this veterinary anthelmintic drug.
unknown function have been discovered (14), it was important to
first determine the specificity of closantel for OvCHT1. Closantel
was evaluated against chitinases from Brugia malayi, a related
filarial nematode, and Entamoeba histolytica and, Plasmodium
falciparum, protozoans, in addition to the human chitotriosidase.
To our delight, the compound exhibited high specificity for the
filarial chitinases, OvCHT1 and BmCHT1 (Table 1). Brugia
malayi is the parasitic nematode responsible for human lymphatic
filariasis, another devastating NTD (1). BmCHT1 is expressed
only in microfilariae and not in L3s (36). On the other hand,
closantel was approximately 50-fold and 80-fold less active
against the protozoan chitinases EhCHT1 and PfCHT1, respec-
tively; and showed no activity against the human chitotriosidase.
Thus, closantel’s chitinase inhibition is sequence specific as the
IC50 values tracked well with the amino acid sequence identities
(Table 1).
These results were subsequently compared to chitinase inhibi-
tion with allosamidin, the best studied inhibitor to date. Allosa-
midin is a competitive chitinase inhibitor that affects cell
separation in fungi and exerts insect larvae toxicity (19). Impor-
tantly, this compound also blocks malaria parasite transmission
into the mosquito midgut by inhibiting the chitinase of P. falcipar-
um that is essential for the penetration of the host’s peritrophic
matrix (41). Despite the potent activity and thus, its promising
therapeutic implications, allosamidin’s complex structure makes
it difficult to pursue as a lead structure for drug discovery.
Crystallographic studies have revealed that allosamidin binds
to the active site of chitinase in a similar manner as the oxazo-
linium ion reaction intermediate of chitin hydrolysis due to their
structural similarity (15). Most likely because of its transition-
state likeness and the conserved catalytic machinery of chitinases,
allosamidin was found to inhibit all enzymes tested (Table 1).
Thus, unlike closantel, allosamidin does not appear to specifically
inhibit any genus of chitinase.
Previous studies examining the impact of allosamidin on inhi-
bition of L3 larvae molting in vitro were unsuccessful (14); how-
ever, allosamidin’s activity is also known to be pH-dependent,
which might account for the observed lack of activity. Hence,
the chitinolytic activity of OvCHT1 during molting is still un-
known. As closantel was able to inhibit the activity of OvCHT1
in vitro, it was also evaluated for an in vivo effect on the molting
process of O. volvulus L3, a process for which OvCHT1 was
shown to be part of (14). Our experiments demonstrate that,
in fact, closantel at 100 μM concentration almost fully inhibited
molting (Fig. 2). Based on the ultrastructural studies (Fig. 3), it
appears that the process of ecdysis was affected. In the treated
worms, the cuticles did not separate fully as the cuticular material
was not degraded fully. Interestingly, similar phenotypes were
also observed when larvae were RNAi treated targeting cysteine
proteases (27) and a serine protease inhibitor (25), localized also
to glandular esophagus as OvCHT1, thus pointing to a potential
synergy between digestive enzymes and chitinase in this vital
process of development.
Because chitinases are widely expressed in insects, plants,
nematodes, fungi, and bacteria (13), and human chitinases with
Table 2. IC50 values for the inhibition of OvCHT1 by closantel
fragments.
Compound
IC50 (μM)
2
3
4
5
6
7
8
47 ꢀ 8.7
>200
143 ꢀ 30
106 ꢀ 9.1
33 ꢀ 8.4
5.8 ꢀ 0.3
67 ꢀ 15
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Finally, OvCHT1 has been implicated in the degradation of
the cuticular material in between the old and new cuticles during
molting as OvCHT1 expression was shown to be associated with
the glandular esophagus in L3 and molting L3 (14), a structure
built specifically by developing larvae within the arthropod vec-
tor. Yet, the precise role of OvCHT1 may only become apparent
when considered alongside the actions of other gene products ex-
pressed and secreted by the glandular esophagus. However, the
exquisite specificity should lend to the use of closantel not only as
a potential drug, but also as a chemical biology tool for under-
standing the role that OvCHT1 plays in the O. volvulus during
molting.
metabolism of the eggshell or if another O. volvulus chitinase ex-
ists; however, closantel may be used as a molecular tool to probe
this important pathway. Additional studies concerning the gen-
omes of O. volvulus should provide more information concerning
the exact roles that the chitin metabolism plays in the develop-
ment and lifecycle of the parasite. Finally, we highlight that
closantel is an approved drug, albeit veterinary, its application
in patients affected by onchocerciasis in the near future might
be a worthwhile undertaking and offer a desperately needed
new armitarium in the fight against NTDs.
Materials and Methods
Library Screening and Kinetic Analysis. The JHCCL v1.0 is a commercially avail-
able collection of FDA and foreign approved drugs (1,514 compounds).
OvCHT1, BmCHT1, EhCHT1 and PfCHT1 were supplied by New England Bio-
labs (NEB) and human chitotriosidase was supplied by D.v.A. (University of
Dundee, Scotland). Enzyme activity was determined using a 96-well fluores-
cent assay with the substrate 4-methylumbelliferyl-N-N0-N00-β-chitotrioside
purchased from Calbiochem analogous to similar published chitinase assays
(45). Detailed assay conditions are presented in SI Text.
Closantel is an approved veterinary drug; however, knowledge
of its use in humans is limited. Further modifications of its struc-
ture could be necessary before FDA approval for human use can
be petitioned. Drug discovery is an exceedingly complex and de-
manding enterprise. In recent years, there has been considerable
focus on optimizing the absorption, distribution, metabolism, ex-
cretion, and toxicity properties of molecules in addition to their
pharmacology. While small fragments have typically been used to
build lead molecules for drug discovery, using what we will term a
“retro-fragment” based approach, the key elements within clo-
santel that are important for its chitinase inhibition were inves-
tigated. Function-oriented synthesis (42) was used to begin to
unravel the relevant fragments. From these studies, fragment 7
(Fig. 4) was identified with potency similar to closantel, IC50
of 5.8 μM and 1.6 μM, respectively. It will be interesting to inves-
tigate the exact required molecular features underlying the iono-
phore activity as well as the chitinase inhibitory activity in an
effort to identify molecular fragments exhibiting both activities.
In conclusion, we have identified a unique biological activity
for the known drug closantel, a veterinary anthelmintic used to
treat sheep and cattle. Importantly, this drug exhibits potent
and specific inhibition against OvCHT1 in vitro, a chitinase found
in the infective L3 larvae of O. volvulus. Although the exact role
of OvCHT1 is not yet known and knowledge of the chitin meta-
bolism in this parasite is limited, closantel completely prevents
molting from the L3 to L4 stage, a critical development process
that occurs within the human host. Allosamidin, the most widely
studied chitinase inhibitor, was found in previous studies to have
no effect on this molt, and in our hands, exhibits limited speci-
ficity for filarial chitinases. Thus, we posit that this observed in-
hibition of molting might be due to the dual action of closantel
acting as proton ionophore as well as chitinase inhibitor. Aside
from a potential role of chitinase in ecdysis, chitin has also been
found as a major component of the eggshells of O. volvulus (35).
In a related filarial nematode, A. vitaea, which forms its eggshells
in a similar manner, interference with chitinase activity was found
to impact the L3 to L4 molt and led to the death of 50% of female
worms (43). Affecting the female worms is a particularly advan-
tageous approach as the female worm can live for up to 15 years,
and continues to propagate its eggs and offspring, thus keeping
the vicious lifecycle intact. Doxycycline, which targets the endo-
symbiontic Wolbachia bacteria, is currently the only known
macrofilaricidal agent used in the treatment of onchocerciasis
(44). It remains to be seen if OvCHT1 is indeed involved in chitin
Experiments Using O. volvulus L3 Larvae in Molting Assay. Cryopreserved L3
stage larvae were rapidly thawed in a 37 °C water bath and washed in wash
medium (NCTC∶IMDM (1∶1) with 1 × GPS (glutamine, penicillin, streptomy-
cin)). The number of worms was set to 5–10 worms per 50 μL in complete
medium (CM) containing 20% heat inactivated FCS. Worms were distributed
to 10 wells of a 96-well plate per treatment group and 1.5 × 105 normal
PBMCs were added per well in 50 μL. 2X dilutions of compound were then
added to each well, 100 μL per well. Controls using DMSO in complete med-
ium and complete medium were included (CM/DMSO and CM, respectively).
The 96-well plates were then incubated at 37 °C in a 5% CO2 incubator until
day six when molting was recorded under inverted microscope; the presence
of the fourth-stage larvae (L4) and the empty cast of the L3.
Ultrastructural Localization Studies. Worms cultured for 6 d in vitro in the pre-
sence of 100 μM Closantel or in CM/DMSO control group were fixed with 3%
glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 for 2 h at room
temperature. They were then rinsed in 0.1 M sodium cacodylate buffer sev-
eral times, postfixed in 1% osmium tetroxide and then grouped within a
3.5% Sea Plaque agar pad. The worms were then dehydrated in graded etha-
nol solutions (50%–100%), embedded in EMbed-812 media, and cured for
24 h at 56 °C. Ultrathin sections (65–70 nanometer (nm)) were cut on an
MT-XL ultramicrotome, and stained with a uranyl acetate solution followed
by Reynold’s Lead Citrate Stain solution. All reagents were from the EMS
Company. Samples were investigated using a Tecnai G2 Spirit BioTWIN
Transmission Electron Microscope (Phillips/FEI Corporation) at an accelerating
voltage of 80 kV.
Synthesis of Closantel Derivatives for Structure-Activity Relationship Studies.
Synthetic procedures for preparation of closantel fragment analogs are pre-
sented in SI Text.
ACKNOWLEDGMENTS. This work was supported by the Worm Institute for
Research and Medicine (WIRM). C.G. thanks the Deutsche Forschungsge-
meinschaft (DFG) for support. A.L.G. thanks the National Institutes of Health
for support from a postdoctoral fellowship (F32 DK083179-01). The authors
wish to thank Dr. Daan M. F. van Aalten and Mrs. Marianne Schimpl (Univer-
sity of Dundee, United Kingdom) for the kind gift of human chitotriosidase
enzyme, Dr. Mark Hixon for assistance with enzyme kinetics, and Dr. Clotilde
K.S. Carlow, Head, Division of Parasitology (New England Biolabs) as well as
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