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-
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
John J. Moores (WIRM) and Hannah Park (WIRM) for helpful discussions.
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