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Dual protonophore-chitinase inhibitors dramatically affect O. volvulus molting.

Gooyit M, Tricoche N, Lustigman S, Janda KD - J. Med. Chem. (2014)

Bottom Line: The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis.Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor.As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis. Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor. As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles. Furthermore, we present that either OvCHT1 inhibition or protonophoric activity was capable of affecting O. volvulus L3 molting and that the presence of both activities in a single molecule yielded more potent inhibition of the nematode's developmental process.

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Molting of O. volvulus L3 larvaein the presence of inhibitors. Percent molting at (A) 10 μMand (B) 1 μM inhibitor concentration, and (C) in the presenceof 1:1 combination of 4b and CCCP each at concentrationsshown. Data presented as percent molting in a total of 10 wells containingon average 5–10 larvae per well.
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fig4: Molting of O. volvulus L3 larvaein the presence of inhibitors. Percent molting at (A) 10 μMand (B) 1 μM inhibitor concentration, and (C) in the presenceof 1:1 combination of 4b and CCCP each at concentrationsshown. Data presented as percent molting in a total of 10 wells containingon average 5–10 larvae per well.

Mentions: The target proteinOvCHT1 is expressed predominantly in the infectiveL3 larvae and may be critical in the molting and development of thenematode.11 Filarial molting of O. volvulus L3 larvae is typically assayed by incubationof the larvae in complete growth medium in the presence of peripheralblood mononuclear cells (PBMCs). To evaluate the efficacy of the inhibitorsbased solely on their inhibitory properties, the L3 larvae were initiallyincubated with select compounds (10 μM) for 24 h (to allow sufficienttime for accumulation) prior to addition of the growth medium andPBMCs and monitored until day 6 when molting was assessed. Under thisassay condition, compounds 3h, 3m, and 4a (all have dual protonophoric and chitinase inhibitory activities)completely abolished molting at 10 μM, as indicated in Figure 4A. Significant effects on molting (up to 84% inhibition)were also observed at a lower concentration of 1 μM for compounds 3h, 3m, and 4a (Figure 4B). Of note, no considerable effect on cell viabilitywas observed when HEK 293T/17 cells were treated with 1 and 10 μMof 3h, 3m, or 4a, for 24 h(see Figure S5 in the Supporting Information). Treatment of L3 larvae with compound 6b (neitheran OvCHT1 inhibitor nor a protonophore), showed no inhibition on molting(Figure 4A). To ascertain which activity isrelevant for inhibition, we also evaluated compounds 3i and 4b (chitinase inhibitors only) and CCCP (a protonophoreonly) for their impact on L3 molting. Compounds 3i and 4b have differing effects; while 4b inhibitedmolting at 10 μM, only marginal inhibition was observed with 3i at the same inhibitor concentration (Figure 4A). The difference in efficacy between the two compounds at10 μM could not be readily explained as both display similarIC50 values against OvCHT1; however, compound 4b could be accumulating within O. volvulus at higher levels, as was observed in the C. elegans model (vide supra), which might account for its better activity.At a higher concentration of 100 μM, compound 3i completely hindered L3 molting. CCCP also fully inhibited the moltingof L3 larvae at 10 μM, suggesting that mitochondrial-uncoupling,by itself, affects this developmental process. Many of the proteinsthat are necessary for O. volvulus L3molting are stored within the granules of the esophageal glands.24 Mitochondrial-uncoupling in the glandular esophagusmay, therefore, affect the synthesis of key proteins (including OvCHT1)that are essential for larval development. At a final concentrationof 1 μM, both 4b and CCCP did not show any impacton molting (Figure 4B). As both chitinase inhibitoryand protonophoric activities are likely required for more potent inhibition,we investigated the effect of incubating L3 larvae with a 1:1 mixtureof 4b and CCCP. Whereas 3h, 3m, and 4a were effective at 1 μM, no effect onmolting was observed using a combination of 4b + CCCPat 0.5 or 1 μM of each compound (Figure 4C). Treatment with 2.5 μM each of 4b and CCCPwas sufficient to completely suppress the L3-to-L4 molt.


Dual protonophore-chitinase inhibitors dramatically affect O. volvulus molting.

Gooyit M, Tricoche N, Lustigman S, Janda KD - J. Med. Chem. (2014)

Molting of O. volvulus L3 larvaein the presence of inhibitors. Percent molting at (A) 10 μMand (B) 1 μM inhibitor concentration, and (C) in the presenceof 1:1 combination of 4b and CCCP each at concentrationsshown. Data presented as percent molting in a total of 10 wells containingon average 5–10 larvae per well.
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fig4: Molting of O. volvulus L3 larvaein the presence of inhibitors. Percent molting at (A) 10 μMand (B) 1 μM inhibitor concentration, and (C) in the presenceof 1:1 combination of 4b and CCCP each at concentrationsshown. Data presented as percent molting in a total of 10 wells containingon average 5–10 larvae per well.
Mentions: The target proteinOvCHT1 is expressed predominantly in the infectiveL3 larvae and may be critical in the molting and development of thenematode.11 Filarial molting of O. volvulus L3 larvae is typically assayed by incubationof the larvae in complete growth medium in the presence of peripheralblood mononuclear cells (PBMCs). To evaluate the efficacy of the inhibitorsbased solely on their inhibitory properties, the L3 larvae were initiallyincubated with select compounds (10 μM) for 24 h (to allow sufficienttime for accumulation) prior to addition of the growth medium andPBMCs and monitored until day 6 when molting was assessed. Under thisassay condition, compounds 3h, 3m, and 4a (all have dual protonophoric and chitinase inhibitory activities)completely abolished molting at 10 μM, as indicated in Figure 4A. Significant effects on molting (up to 84% inhibition)were also observed at a lower concentration of 1 μM for compounds 3h, 3m, and 4a (Figure 4B). Of note, no considerable effect on cell viabilitywas observed when HEK 293T/17 cells were treated with 1 and 10 μMof 3h, 3m, or 4a, for 24 h(see Figure S5 in the Supporting Information). Treatment of L3 larvae with compound 6b (neitheran OvCHT1 inhibitor nor a protonophore), showed no inhibition on molting(Figure 4A). To ascertain which activity isrelevant for inhibition, we also evaluated compounds 3i and 4b (chitinase inhibitors only) and CCCP (a protonophoreonly) for their impact on L3 molting. Compounds 3i and 4b have differing effects; while 4b inhibitedmolting at 10 μM, only marginal inhibition was observed with 3i at the same inhibitor concentration (Figure 4A). The difference in efficacy between the two compounds at10 μM could not be readily explained as both display similarIC50 values against OvCHT1; however, compound 4b could be accumulating within O. volvulus at higher levels, as was observed in the C. elegans model (vide supra), which might account for its better activity.At a higher concentration of 100 μM, compound 3i completely hindered L3 molting. CCCP also fully inhibited the moltingof L3 larvae at 10 μM, suggesting that mitochondrial-uncoupling,by itself, affects this developmental process. Many of the proteinsthat are necessary for O. volvulus L3molting are stored within the granules of the esophageal glands.24 Mitochondrial-uncoupling in the glandular esophagusmay, therefore, affect the synthesis of key proteins (including OvCHT1)that are essential for larval development. At a final concentrationof 1 μM, both 4b and CCCP did not show any impacton molting (Figure 4B). As both chitinase inhibitoryand protonophoric activities are likely required for more potent inhibition,we investigated the effect of incubating L3 larvae with a 1:1 mixtureof 4b and CCCP. Whereas 3h, 3m, and 4a were effective at 1 μM, no effect onmolting was observed using a combination of 4b + CCCPat 0.5 or 1 μM of each compound (Figure 4C). Treatment with 2.5 μM each of 4b and CCCPwas sufficient to completely suppress the L3-to-L4 molt.

Bottom Line: The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis.Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor.As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis. Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor. As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles. Furthermore, we present that either OvCHT1 inhibition or protonophoric activity was capable of affecting O. volvulus L3 molting and that the presence of both activities in a single molecule yielded more potent inhibition of the nematode's developmental process.

Show MeSH
Related in: MedlinePlus