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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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Related in: MedlinePlus

Evaluation of mitochondrial-uncoupling activity usinga microplatefluorescence assay. HEK 293T/17 cells were incubated with compound(50 μM) and subsequently stained with TMRE. Data shown as meanfluorescence intensity ± sd (n = 3). Unstainedcells (no TMRE) and DMSO were used as negative (−) and positive(+) controls, respectively. RFU = relative fluorescence units (λex = 488 nm, λem = 575 nm).
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fig2: Evaluation of mitochondrial-uncoupling activity usinga microplatefluorescence assay. HEK 293T/17 cells were incubated with compound(50 μM) and subsequently stained with TMRE. Data shown as meanfluorescence intensity ± sd (n = 3). Unstainedcells (no TMRE) and DMSO were used as negative (−) and positive(+) controls, respectively. RFU = relative fluorescence units (λex = 488 nm, λem = 575 nm).

Mentions: To examine mitochondrial-uncouplingactivity, compounds 1–5 were testedusing tetramethylrhodamine ethyl ester (TMRE), a positively charged,mitochondrion-selective dye that serves as a membrane potential sensor.In the presence of a protonophore (e.g., carbonyl cyanide m-chlorophenyl hydrazone or CCCP), the mitochondrion getsdepolarized and thus fails to sequester TMRE, resulting in decreasedfluorescence intensity. A microplate fluorescence assay was developedto monitor mitochondrial uncoupling of HEK-293T/17 cells in the presenceof the inhibitors. Following incubation with the inhibitors at 37°C, the cells were stained with TMRE and subsequently analyzedby fluorescence spectrometry. As depicted in Figure 2, a dissociable proton (i.e., phenolic hydrogen as in 1, 3a, 3h, and 4a)for the salicylanilide class of compounds is critical for mitochondrial-uncouplingactivity. As expected, removal of the hydroxyl moiety (as in compounds 3e, 3j, and 4c) or masking it witha methyl group (compounds 3b, 3i, 3d, and 4b) resulted to a loss of uncouplingactivity. We note, however, that the amide proton is equally significant,as substituting it with a methyl group while retaining the hydroxylmoiety (like in the case of compound 3c) eradicated protonophoricactivity. Results of mitochondrial-uncoupling activity for the othercompounds listed in Table 1 are given in Supporting Information Figure S2. Separate experimentsusing flow cytometry to analyze membrane polarization were also conductedand the results (see Figure S3 in the SupportingInformation) were in agreement to those obtained from the microplatefluorescence assay.


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

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

Evaluation of mitochondrial-uncoupling activity usinga microplatefluorescence assay. HEK 293T/17 cells were incubated with compound(50 μM) and subsequently stained with TMRE. Data shown as meanfluorescence intensity ± sd (n = 3). Unstainedcells (no TMRE) and DMSO were used as negative (−) and positive(+) controls, respectively. RFU = relative fluorescence units (λex = 488 nm, λem = 575 nm).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216208&req=5

fig2: Evaluation of mitochondrial-uncoupling activity usinga microplatefluorescence assay. HEK 293T/17 cells were incubated with compound(50 μM) and subsequently stained with TMRE. Data shown as meanfluorescence intensity ± sd (n = 3). Unstainedcells (no TMRE) and DMSO were used as negative (−) and positive(+) controls, respectively. RFU = relative fluorescence units (λex = 488 nm, λem = 575 nm).
Mentions: To examine mitochondrial-uncouplingactivity, compounds 1–5 were testedusing tetramethylrhodamine ethyl ester (TMRE), a positively charged,mitochondrion-selective dye that serves as a membrane potential sensor.In the presence of a protonophore (e.g., carbonyl cyanide m-chlorophenyl hydrazone or CCCP), the mitochondrion getsdepolarized and thus fails to sequester TMRE, resulting in decreasedfluorescence intensity. A microplate fluorescence assay was developedto monitor mitochondrial uncoupling of HEK-293T/17 cells in the presenceof the inhibitors. Following incubation with the inhibitors at 37°C, the cells were stained with TMRE and subsequently analyzedby fluorescence spectrometry. As depicted in Figure 2, a dissociable proton (i.e., phenolic hydrogen as in 1, 3a, 3h, and 4a)for the salicylanilide class of compounds is critical for mitochondrial-uncouplingactivity. As expected, removal of the hydroxyl moiety (as in compounds 3e, 3j, and 4c) or masking it witha methyl group (compounds 3b, 3i, 3d, and 4b) resulted to a loss of uncouplingactivity. We note, however, that the amide proton is equally significant,as substituting it with a methyl group while retaining the hydroxylmoiety (like in the case of compound 3c) eradicated protonophoricactivity. Results of mitochondrial-uncoupling activity for the othercompounds listed in Table 1 are given in Supporting Information Figure S2. Separate experimentsusing flow cytometry to analyze membrane polarization were also conductedand the results (see Figure S3 in the SupportingInformation) were in agreement to those obtained from the microplatefluorescence assay.

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