Exploiting the polypharmacology of ?-carbolines to disrupt o. volvulus molting

Article abstract of DOI:10.1021/ml500516r

Onchocerciasis is an infection caused by the filarial worm Onchocerca volvulus, which can eventually result in blindness. The lack of an effective macrofilaricide and the possible development of ivermectin-resistant strains of O. volvulus necessitate the need for alternative treatment strategies. We have shown that targeting the L3-stage-specific chitinase OvCHT1 impairs the shedding of the filarial cuticle. In our continued efforts to discover OvCHT1 inhibitors, we identified the β-carboline alkaloid scaffolding as a chitinase inhibitor that is capable of penetrating the worm cuticle. Herein, we disclose the rich polypharmacology of the β-carboline class of compounds as an approach to abrogate the molting of the parasite and thus the initiation of infection in the human host.

Full text of DOI:10.1021/ml500516r

Letter
pubs.acs.org/acsmedchemlett
Exploiting the Polypharmacology of ß‑Carbolines to Disrupt O.
volvulus Molting
Major Gooyit,† Nancy Tricoche,‡ Sacha Javor,† Sara Lustigman,‡ and Kim D. Janda*,†
†Departments of Chemistry and Immunology and Microbial Science, The Skaggs Institute for Chemical Biology, and The Worm
Institute of Research and Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,
United States
‡Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10065, United States
S
* Supporting Information
ABSTRACT: Onchocerciasis is an infection caused by the filarial worm Onchocerca
volvulus, which can eventually result in blindness. The lack of an effective macrofilaricide
and the possible development of ivermectin-resistant strains of O. volvulus necessitate the
need for alternative treatment strategies. We have shown that targeting the L3-stage-
specific chitinase OvCHT1 impairs the shedding of the filarial cuticle. In our continued
efforts to discover OvCHT1 inhibitors, we identified the β-carboline alkaloid scaffolding
as a chitinase inhibitor that is capable of penetrating the worm cuticle. Herein, we disclose
the rich polypharmacology of the β-carboline class of compounds as an approach to
abrogate the molting of the parasite and thus the initiation of infection in the human host.
KEYWORDS: Onchocerciasis, β-carbolines, chitinase inhibitor, polypharmacology
complete inhibition of OvCHT1 at 25 μM. Compounds 1-4 are
nchocerciasis, or river blindness, is caused by the parasitic
Onematode Onchocerca volvulus and is the second leading β-carboline alkaloids that are known to display a wide range of
biological and pharmacological properties, including antitumor,
antiviral, and antimicrobial activities.9 In addition, β-carbolines
have been reported to display antiparasitic activities. Harmine
inhibited the growth of Leishmania infantum promastigotes as
well as the intracellular amastigote forms of the parasite.10 β-
carboline derivatives were shown to significantly reduce the
growth of Trypanosoma cruzi epimastigotes11 and were also
active against the parasites Plasmodium falciparum and
Trypanosoma brucei rhodesiense.12 In rodents infected with
Acanthoeilonema viteae, Brugia malayi, and Litomosoides carinii,
treatment with β-carbolines led to death of the filariae or
sterilization of female worms.13 The antiparasitic activity of β-
carbolines was thought to be due to respiratory chain inhibition
or inverse agonism of the benzodiazepine receptor; however, it
was not previously linked to chitinase inhibition. Herein, we
present yet another biological activity of the β-carboline class of
compounds and further exploit its polypharmacology as a
means to effectively inhibit O. volvulus L3 molting. We also
show that the β-carboline derivatives are able to penetrate the
highly resistant cuticle of nematodes (using C. elegans as a
model organism of bioaccumulation), which predetermines
their efficacy ex vivo.
infectious cause of blindness (affecting over 37 million people
worldwide).1 Of crucial significance to the survival and
development of O. volvulus in the human host is the shedding
of the L3 cuticle.2 The molting of L3 to L4 larvae is particularly
important for active infection of the human host, and thus,
targeting this transitional stage may help reduce parasite
infection and transmission.
OvCHT1, a chitinase expressed predominantly in the
infective L3 larvae, has been implicated in the development
of O. volvulus.2 We have previously demonstrated that
inhibition of OvCHT1 activity impedes the L3-to-L4 molt3,4
and that a dual-targeting strategy (involving mitochondrial
uncoupling and chitinase inhibition) results in improved
efficacy.4 Multitarget treatments offer several advantages
including increased therapeutic effects and prevention of drug
resistance. As such, the polypharmacological strategy has
emerged as a new paradigm in the discovery of anticancer5,6
and anti-infective7 medications, among others. The dearth of an
efficacious macrofilaricide and the possible emergence of drug-
resistant O. volvulus,8 call for a need to identify alternative
therapeutics for onchocerciasis.
In our continuing search for anti-onchocerciasis agents, we
screened a commercial library of over 500 natural products
toward OvCHT1 inhibition, as described previously.3 Our
screening initiative led to the identification of norharmane (1),
harmane (2), harmine (3), and harmol (4), which all showed
Received: December 11, 2014
Accepted: January 20, 2015
© XXXX American Chemical Society
A
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On the basis of the initial hits 1−4, a small library of β-
carboline analogues was prepared to identify key features that
could impart potency toward OvCHT1 inhibition. An initial
structure−activity relationship was gleaned from the library
screening as methyl and hydroxy/methoxy substitutions are
tolerated at positions 1 and 7, respectively. As derivatization of
β-carbolines is already well established in the literature, we
focused on simple elaborations of the β-carboline framework by
diversification at positions 2, 6, 7, and 9. The synthetic routes
for the preparation of β-carboline derivatives 5−12 are outlined
in Scheme 1. Reaction of 1 with chlorosulfonic acid furnished
2. However, disubstitution of 2 at positions 2 and 7 (analogues
9 and 11) led to increased potency against OvCHT1 (Figure
1B). N9-Alkylation of 3 (compounds 10a−b) diminished its
activity, whereas simultaneous N9- and 7-O-benzylation (12)
displayed complete inhibition of OvCHT1 at 5 μM. The IC50
values of the initial hits 1−4 and the more potent derivatives
are listed in Table 1. Compound 3 appeared to exhibit a
competitive mode of inhibition as determined using Dixon plot,
with an inhibition constant (Ki) of 3.78
0.79 μM. Our
straightforward derivatization of the β-carboline scaffold
resulted in up to 5-fold increase in potency; analogue 11 had
an IC50 of 1.43
constant Ki of 0.98 0.15 μM.
0.09 μM and a competitive inhibitory
a
Scheme 1. Synthesis of β-Carboline Derivatives
With regard to chitinase selectivity, representative com-
pounds (3, 4, 9a, 11, and 12) were tested for inhibition of
chitinases from other species including Brugia malayi
(BmCHT1) and Entamoeba histolytica (EhCHT1). All
compounds preferentially inhibited OvCHT1 over BmCHT1
and EhCHT1 except for compound 11, which showed
complete inhibition of all three chitinases at 10 μM
(Supporting Information Figure S1).
Next, we conducted docking of compound 11 into the active
site of OvCHT1 using the homology model described by
Segura-Cabrera et al.17 Analysis of the docked poses showed
that the tricyclic indole ring fills the binding pocket by
interacting with residues Tyr268, Trp361, and Thr362, while
the N9-(p-chloro)benzyl system is involved in π-stacking
interaction with Phe365 (Figure 2). The binding mode is
consistent with that of closantel,17 where the terminal
chlorobenzene and the central aromatic system occupy the
same respective binding pockets. In the case of 11, the more
extended structure predisposes its 7-O-(p-chloro)benzyl moiety
to interaction via van der Waals contacts with residues Tyr27,
Phe58, Asp145, Ala184, and Trp361 (Figure 2), exploiting thus
an additional binding pocket that is not accessible with the
closantel ligand (Figure S2 in the Supporting Information).
N2-Methylated β-carbolines were previously reported to
inhibit mitochondrial respiration.18 This activity was rational-
ized on their ability to form the neutral anhydronium base
(through indole N9-deprotonation), which could passively
diffuse across the mitochondrial membrane.18 To evaluate the
mitochondrial uncoupling activity of the β-carboline derivatives,
we performed a microplate fluorescence assay using the
mitochondrion-selective probe TMRE to detect membrane
depolarization.4 Quarternary alkaloids 9a, 9b, and 11 proved to
be good uncouplers of oxidative phosphorylation relative to
CCCP, a known protonophore (Figure 3). It would seem that
the uncoupling behavior is dependent on the availability of the
indole N−H proton as N9-alkylated 12 was completely devoid
of protonophoric activity. Compounds 1 and 2 were found to
be inactive, whereas the 7-OH/OMe-substituted counterparts
(compounds 3 and 4) displayed comparable uncoupling activity
as 9a/9b.
a
Reagents and conditions: (a) ClSO3H, 0 °C, 1 h; (b) RNH2, DMF,
rt, 1 h; (c) RBr or RI, EtOAc/iPrOH, reflux, 16 h; (d) NaH, RBr,
DMF, 0 °C to rt, 1 h; (e) HBr/HOAc, reflux, 16 h; (f) Cs2CO3, RBr,
DMF, rt, 12 h.
the 6-sulfonyl chloride intermediate,14 which was subsequently
reacted with alkyl/aryl amines to produce 6-sulfonamides 5a−e.
N2-, N9-, and 7-O-alkylated derivatives 6−12 were prepared
according to published methods.15,16 Next, we tested
compounds 1−12 for OvCHT1 inhibition at 10 and 5 μM
(Figure 1). Functionalization with a sulfonamide moiety at
position 6 (derivatives 5a−e) did not cause a significant effect
on potency, relative to 1 (Figure 1A). The same could be said
for compounds 6−8; N2-, N9-, or 7-O-alkylation showed no
significant improvement on inhibitory activity as compared to
The foregoing studies reveal compounds 3, 4, 9a, 9b, and 11
to possess both protonophoric and chitinase inhibitory
activities, whereas analogues 1, 2, and 12 act as chitinase
inhibitors only. With these data in hand, we went ahead and
tested the compounds for their ability to inhibit O. volvulus
molting. At 10 μM, the dual protonophore-chitinase inhibitors
9a, 9b, and 11 eradicated L3 molting, while compounds 1, 2,
and 12 (OvCHT1 inhibitors only) had no effect on the
parasite’s developmental process (Figure 4A). When dosed at
100 μM, compounds 1, 2, and 12 prevented the shedding of
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Figure 1. Evaluation of β-carboline derivatives in a fluorescence-based OvCHT1 inhibition assay. Analogues were examined at (A) 10 and (B) 5 μM.
Data shown as % OvCHT1 activity, relative to control (0.5% DMSO).
Table 1. IC50 of O. volvulus Chitinase Inhibition
cmpd
IC50 (μM)
cmpd
IC50 (μM)
1
2
3
4
8.46 1.36
7.33 0.06
7.06 0.42
7.08 0.20
9a
9b
11
12
3.57 0.79
3.49 0.17
1.43 0.09
2.08 0.03
Figure 4. Molting of O. volvulus L3 larvae in the presence of β-
carboline derivatives. Percent molting at (A) 10 and (B) 1 μM
inhibitor concentration. Data presented as percent molting in a total of
2−3 wells containing on average 5−10 larvae per well. (C)
Bioaccumulation in C. elegans. Late-stage L4 worms were incubated
with 10 μM inhibitor (equivalent to 2 nmol/mg worm) for 6 h. Data
shown as mean concentration SD (n = 3), expressed in nmol/mg
worm.
Figure 2. Lowest energy pose of compound 11 docked into O. volvulus
chitinase (OvCHT1) using AutoDock Vina. Color scheme: oxygens
are in red, nitrogens in blue, chlorines in green, and carbons in yellow
(compounds 11) or gray (OvCHT1).
molting, respectively (Figure 4A). These results demonstrate
that the presence of both mitochondrial uncoupling and
chitinase inhibitory activities in a single molecule more
effectively inhibits the L3-to-L4 molt, as we have earlier
shown for the closantel analogues.4
Derivatives 9a, 9b, and 11 were further evaluated for their
effect on L3 molting at 1 μM (Figure 4B). Treatment with 9b
and 11 led to 45% and 27% inhibition, respectively, whereas 9a
had no impact on molting at 1 μM. Considering that
compounds 9a, 9b, and 11 have similar inhibitory profiles,
we reasoned that the difference in efficacy might be due to
bioaccumulation issues. O. volvulus, as with other nematodes, is
equipped with a thick cuticle composed primarily of cross-
linked collagen, giving rise to a highly compact structure
resistant to exogenous perturbation.19 Thus, cuticle penetration
is a pharmacological determinant of the drug sensitivity of O.
volvulus and other filarial worms. To shed light on this matter,
we used C. elegans as a predictive model of nematode
penetrability.20 We have previously employed this model
system to determine the accumulation of closantel derivatives
within the worm.4 Late-stage L4 C. elegans were incubated with
analogues 9a, 9b, or 11 at 2 nmol/mg worm for 6 h, and worm
homogenates were then analyzed by LC−MS for quantitation.
As depicted in Figure 4C, all three compounds penetrate the
Figure 3. Evaluation of the mitochondrial-uncoupling activity of β-
carboline derivatives. HEK 293T/17 cells were incubated with
compound (50 μM) and subsequently stained with TMRE. Data
shown as mean fluorescence intensity SD (n = 3). Unstained cells
(no TMRE) and 0.5% DMSO were used as negative (−) and positive
(+) control, respectively. RFU = relative fluorescence units (λex = 488
nm, λem = 575 nm).
the L3 cuticle. Compared to 9a/9b, the 2-fold less active
chitinase inhibitors 3 and 4 showed 57% and 61% inhibition of
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cuticle, albeit, at low concentrations. Levels of 9b were 0.22
0.10 nmol/mg worm, 6-fold higher than those of 9a (0.033
0.001 nmol/mg worm). Compound 9a was also found to
undergo N2-dealkylation to 3, which in turn had worm
treatments of onchocerchiasis. From our screening efforts, we
have identified the β-carbolines as novel inhibitors of the O.
volvulus chitinase OvCHT1. We took advantage of the rich
polypharmacology of the β-carboline class of compounds and
investigated its ability to abrogate the shedding of the L3
cuticle. The multitarget derivatives (9a, 9b, and 11) with
protonophoric and chitinase inhibitory activities displayed more
potent inhibition of molting. As penetration of the filarial
cuticle is a requisite for an effective anthelmintic drug, we also
demonstrated that the β-carboline analogues accumulate within
the worm using C. elegans as the model nematode. Future
efforts will focus on the optimization of our multitarget leads
and exploitation of their beneficial polypharmacology as an
alternative approach to combat onchocerchiasis.
concentrations of 0.052
0.006 nmol/mg. Note that other
possible metabolism pathways, including 7-O-dealkylation,
aromatic ring, and N2-oxidation, were also expressly looked
for but were not identified in the worm homogenates. We
hasten to add that the bioaccumulation of 9a, 9b, and 11
correlated well with the bioactivity observed in O. volvulus.
It is intriguing that despite the low accumulation levels (up to
11% of the initial dose for 9b) and modest chitinase inhibitory
activities (IC50 values in the low micromolar concentration) of
the β-carbolines, the casting of the L3 cuticle was still
significantly inhibited. The efficacy observed in O. volvulus is
likely the result of the dynamic modulation of a pharmaco-
logical network of relevant targets. Early electron microscopic
studies of O. volvulus revealed that the infective L3 has a
compact morphology, with the glandular esophagus making up
over two-thirds of the larval body.21 The glandular tissue is
packed with secretory structures and rough endoplasmic
reticulum,21 signifying an enhanced rate of protein synthesis
crucial for the development of the L3 larvae. Enzymes such as
cysteine proteases,22 serine proteases,23 transglutaminases,24
and chitinases2 have earlier been implicated in the L3-to-L4
molt of O. volvulus. Thus, the uncoupling of mitochondrial
activity (as demonstrated by compounds 9a, 9b, and 11, Figure
3) in the esophageal glands may impact the production of
essential proteins (including OvCHT1) that are necessary for
molting. In addition, β-carbolines can interact with DNA
through groove binding or intercalation,25 thereby interfering
with DNA replication/transcription and hence the overall
protein biosynthesis in the granules of the glandular esophagus.
Interestingly, no significant effects on the viability of human
HEK 293T/17 cells were observed upon incubation with 9a,
9b, or 11 at a concentration (5 μM) above their IC50 values
(Figure S3 in the Supporting Information), thus indicating a
level of confidence about their safety at the effective
concentration used.
ASSOCIATED CONTENT
* Supporting Information
■
S
General syntheses and characterization of compounds, assays
on chitinase inhibition, molecular docking, bioaccumulation in
C. elegans, L3 molting assay, mitochondrial uncoupling, and cell
viability. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (858) 785-2515. Fax: (858) 784-2595. E-mail:
kdjanda@scripps.edu.
■
Funding
This work was supported by NIH Grant AI092076 (to K.D.J.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Aldo Segura-Cabrera for providing the PDB file
of the homology model of OvCHT1 structure used for
molecular docking. This is manuscript No. 29015 from The
Scripps Research Institute.
β-carboline alkaloids were also reported as inverse agonists at
ABBREVIATIONS
■
the benzodiazepine allosteric site of the GABAA receptor,26
a
O. volvulus, Onchocerca volvulus; OvCHT1, Onchocerca volvulus
chitinase; L3, third larval stage; L4, fourth larval stage; CCCP,
carbonyl cyanide m-chlorophenyl hydrazone; C. elegans,
Caenorhabditis elegans; LC, liquid chromatography; MS, mass
spectrometry
biochemical target for filaricidal agents. Incidentally, expressed
sequence tag (EST) analysis of cDNA libraries from O. volvulus
showed upregulation of an ionotropic GABA receptor in the
molting L3 larvae of O. volvulus.27 Compounds 9a, 9b, and 11
could possibly interact with this uncharacterized GABA
receptor to modulate the neurotransmitter-mediated signaling
pathways. A next logical step would be to isolate and
characterize the molting L3 GABA receptor (or the homologue
in C. elegans unc-49)27,28 and investigate the inverse agonistic
effects of the β-carboline analogues.
Most drug discovery efforts are directed toward selective
regulation of a therapeutic target in an attempt to maximize
efficacy and minimize harmful side effects. However, single-
targeted compounds are often ineffective in treating complex
diseases involving a wide array of cellular signaling networks.
The use of polypharmacologicals renders the benefits of
superior efficacy (via modulation of multiple disease pathways)
while reducing the risk of drug resistance. Although it raises
safety concerns, multitarget therapy does not necessarily equate
to toxicity, as was shown for the analgesic tapentadol29 and
antidepressants.30 The possible occurrence of ivermectin-
resistant strains of O. volvulus8 warrants the need for effective
REFERENCES
■
(1) Allen, J. E.; Adjei, O.; Bain, O.; Hoerauf, A.; Hoffmann, W. H.;
Makepeace, B. L.; Schulz-Key, H.; Tanya, V. N.; Trees, A. J.; Wanji, S.;
Taylor, D. W. Of mice, cattle, and humans: The immunology and
treatment of river blindness. PLoS Negl. Trop. Dis. 2008, 2, e217.
(2) Wu, Y.; Egerton, G.; Underwood, A. P.; Sakuda, S.; Bianco, A. E.
Expression and secretion of a larval-specific chitinase (family 18
glycosyl hydrolase) by the infective stages of the parasitic nematode
Onchocerca volvulus. J. Biol. Chem. 2001, 276, 42557−42564.
(3) Gloeckner, C.; Garner, A. L.; Mersha, F.; Oksov, Y.; Tricoche, N.;
Eubanks, L. M.; Lustigman, S.; Kaufmann, G. F.; Janda, K. D.
Repositioning of an existing drug for the neglected tropical disease
Onchocerciasis. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 3424−3429.
(4) Gooyit, M.; Tricoche, N.; Lustigman, S.; Janda, K. D. Dual
protonophore-chitinase inhibitors dramatically affect O. volvulus
molting. J. Med. Chem. 2014, 57, 5792−5799.
(5) Azmi, A. S. Network pharmacology for cancer drug discovery:
Are we there yet? Future Med. Chem. 2012, 4, 939−941.
D
DOI: 10.1021/ml500516r
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(6) Knight, Z. A.; Lin, H.; Shokat, K. M. Targeting the cancer kinome
through polypharmacology. Nat. Rev. Cancer 2010, 10, 130−137.
(7) East, S. P.; Silver, L. L. Multitarget ligands in antibacterial
research: progress and opportunities. Expert Opin. Drug Discovery
2013, 8, 143−156.
(8) Lustigman, S.; McCarter, J. P. Ivermectin resistance in Onchocerca
volvulus: Toward a genetic basis. PLoS Negl. Trop. Dis. 2007, 1, e76.
(9) Cao, R.; Peng, W.; Wang, Z.; Xu, A. Beta-carboline alkaloids:
Biochemical and pharmacological functions. Curr. Med. Chem. 2007,
14, 479−500.
(10) Di Giorgio, C.; Delmas, F.; Ollivier, E.; Elias, R.; Balansard, G.;
Timon-David, P. In vitro activity of the beta-carboline alkaloids
harmane, harmine, and harmaline toward parasites of the species
Leishmania infantum. Exp. Parasitol. 2004, 106, 67−74.
(11) Rivas, P.; Cassels, B. K.; Morello, A.; Repetto, Y. Effects of some
beta-carboline alkaloids on intact Trypanosoma cruzi epimastigotes.
Comp. Biochem. Physiol., Part C: Pharmacol. Toxicol. Endocrinol. 1999,
122, 27−31.
(12) Franca, P. H.; Barbosa, D. P.; da Silva, D. L.; Ribeiro, E. A.;
Santana, A. E.; Santos, B. V.; Barbosa-Filho, J. M.; Quintans, J. S.;
Barreto, R. S.; Quintans-Junior, L. J.; de Araujo-Junior, J. X. Indole
alkaloids from marine sources as potential leads against infectious
diseases. Biomed. Res. Int. 2014, 2014, 375423.
(13) Srivastava, S. K.; Agarwal, A.; Chauhan, P. M.; Agarwal, S. K.;
Bhaduri, A. P.; Singh, S. N.; Fatima, N.; Chatterjee, R. K. Potent 1,3-
disubstituted-9H-pyrido[3,4-b]indoles as new lead compounds in
antifilarial chemotherapy. Bioorg. Med. Chem. 1999, 7, 1223−1236.
(14) Xin, B.; Tang, W.; Wang, Y.; Lin, G.; Liu, H.; Jiao, Y.; Zhu, Y.;
Yuan, H.; Chen, Y.; Lu, T. Design, synthesis and biological evaluation
of beta-carboline derivatives as novel inhibitors targeting B-Raf kinase.
Bioorg. Med. Chem. Lett. 2012, 22, 4783−4786.
(24) Lustigman, S.; Brotman, B.; Huima, T.; Castelhano, A. L.; Singh,
R. N.; Mehta, K.; Prince, A. M. Transglutaminase-catalyzed reaction is
important for molting of Onchocerca volvulus third-stage larvae.
Antimicrob. Agents Chemother. 1995, 39, 1913−1919.
(25) Nafisi, S.; Bonsaii, M.; Maali, P.; Khalilzadeh, M. A.;
Manouchehri, F. Beta-carboline alkaloids bind DNA. J. Photochem.
Photobiol., B 2010, 100, 84−91.
(26) Malatynska, E.; Knapp, R.; Ikeda, M.; Yamamura, H. I. Beta-
carboline interactions at the BZ-GABA receptor chloride-ionophore
complex in the rat cerebral cortex. Brain Res. Bull. 1989, 22, 845−848.
(27) Lizotte-Waniewski, M.; Tawe, W.; Guiliano, D. B.; Lu, W.; Liu,
J.; Williams, S. A.; Lustigman, S. Identification of potential vaccine and
drug target candidates by expressed sequence tag analysis and
immunoscreening of Onchocerca volvulus larval cDNA libraries. Infect.
Immun. 2000, 68, 3491−3501.
(28) Bamber, B. A.; Beg, A. A.; Twyman, R. E.; Jorgensen, E. M. The
Caenorhabditis elegans unc-49 locus encodes multiple subunits of a
heteromultimeric GABA receptor. J. Neurosci. 1999, 19, 5348−5359.
(29) Tzschentke, T. M.; Christoph, T.; Kogel, B.; Schiene, K.;
Hennies, H. H.; Englberger, W.; Haurand, M.; Jahnel, U.; Cremers, T.;
Friderichs, E.; De Vry, J. (−)-(1R,2R)-3-(3-Dimethylamino-1-ethyl-2-
methyl-propyl)-phenol hydrochloride (tapentadol HCl): A novel mu-
opioid receptor agonist/norepinephrine reuptake inhibitor with broad-
spectrum analgesic properties. J. Pharmacol. Exp. Ther. 2007, 323,
265−276.
(30) Millan, M. J. Dual- and triple-acting agents for treating core and
co-morbid symptoms of major depression: Novel concepts, new drugs.
Neurotherapeutics 2009, 6, 53−77.
(15) Chen, Z.; Cao, R.; Shi, B.; Guo, L.; Sun, J.; Ma, Q.; Fan, W.;
Song, H. Synthesis and biological evaluation of 1,9-disubstituted beta-
carbolines as potent DNA intercalating and cytotoxic agents. Eur. J.
Med. Chem. 2011, 46, 5127−5137.
(16) Cao, R.; Guan, X.; Shi, B.; Chen, Z.; Ren, Z.; Peng, W.; Song, H.
Design, synthesis and 3D-QSAR of beta-carboline derivatives as potent
antitumor agents. Eur. J. Med. Chem. 2010, 45, 2503−2515.
(17) Segura-Cabrera, A.; Bocanegra-Garcia, V.; Lizarazo-Ortega, C.;
Guo, X.; Correa-Basurto, J.; Rodriguez-Perez, M. A. A computational
analysis of the binding mode of closantel as inhibitor of the Onchocerca
volvulus chitinase: Insights on macrofilaricidal drug design. J. Comput.-
Aided Mol. Des. 2011, 25, 1107−1119.
(18) Albores, R.; Neafsey, E. J.; Drucker, G.; Fields, J. Z.; Collins, M.
A. Mitochondrial respiratory inhibition by N-methylated beta-
carboline derivatives structurally resembling N-methyl-4-phenylpyr-
idine. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9368−9372.
(19) Lustigman, S.; Huima, T.; Brotman, B.; Miller, K.; Prince, A. M.
Onchocerca volvulus: Biochemical and morphological characteristics of
the surface of third- and fourth-stage larvae. Exp. Parasitol. 1990, 71,
489−495.
(20) Burns, A. R.; Wallace, I. M.; Wildenhain, J.; Tyers, M.; Giaever,
G.; Bader, G. D.; Nislow, C.; Cutler, S. R.; Roy, P. J. A predictive
model for drug bioaccumulation and bioactivity in Caenorhabditis
elegans. Nat. Chem. Biol. 2010, 6, 549−557.
(21) Strote, G.; Bonow, I. Morphological demonstration of essential
functional changes after in vitro and in vivo transition of infective
Onchocerca volvulus to the post-infective stage. Parasitol. Res. 1991, 77,
526−535.
(22) Lustigman, S.; Zhang, J.; Liu, J.; Oksov, Y.; Hashmi, S. RNA
interference targeting cathepsin L and Z-like cysteine proteases of
Onchocerca volvulus confirmed their essential function during L3
molting. Mol. Biochem. Parasitol. 2004, 138, 165−170.
(23) Ford, L.; Guiliano, D. B.; Oksov, Y.; Debnath, A. K.; Liu, J.;
Williams, S. A.; Blaxter, M. L.; Lustigman, S. Characterization of a
novel filarial serine protease inhibitor, Ov-SPI-1, from Onchocerca
volvulus, with potential multifunctional roles during development of
the parasite. J. Biol. Chem. 2005, 280, 40845−40856.
E
DOI: 10.1021/ml500516r
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX