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. 2019 Nov 14;9(1):16872.
doi: 10.1038/s41598-019-53410-z.

Discovery of a Potent Small Molecule Inhibiting Huntington's Disease (HD) Pathogenesis via Targeting CAG Repeats RNA and Poly Q Protein

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Free PMC article

Discovery of a Potent Small Molecule Inhibiting Huntington's Disease (HD) Pathogenesis via Targeting CAG Repeats RNA and Poly Q Protein

Eshan Khan et al. Sci Rep. .
Free PMC article

Abstract

CAG repeats RNA causes various fatal neurodegenerative diseases exemplified by Huntington's disease (HD) and several spinocerebellar ataxias (SCAs). Although there are differences in the pathogenic mechanisms, these diseases share the common cause, i.e., expansion of CAG repeats. The shared cause of these diseases raises the possibility for the exploiting the common target as a potential therapeutic approach. Oligonucleotide-based therapeutics are designed earlier with the help of the base pairing rule but are not very promiscuous, considering the nonspecific stimulation of the immune system and the poor cellular delivery. Therefore, small molecules-based therapeutics are preferred for targeting the repeats expansion disorders. Here, we have used the chemical similarity search approach to discern the small molecules that selectively target toxic CAG RNA. The lead compounds showed the specificity towards AA mismatch in biophysical studies including CD, ITC, and NMR spectroscopy and thus aided to forestall the polyQ mediated pathogenicity. Furthermore, the lead compounds also explicitly alleviate the polyQ mediated toxicity in HD cell models and patient-derived cells. These findings suggest that the lead compound could act as a chemical probe for AA mismatch containing RNA as well as plays a neuroprotective role in fatal neurodegenerative diseases like HD and SCAs.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Screening of small molecules targeting CAG repeat RNA. (a) A plot of binding constant values of compounds against 1 × 1 single loop 5′CAG/3′GAC RNA with control duplex 5′CAG/3′GUC RNA (b) A plot of binding constant values of shortlisted compounds with multiple loop 5′CAG/3′GAC RNA (c) A comparative Plot of binding constant values of potential lead compounds with Myricetin (d) Structural analysis of lead compound in comparison to query molecule (Myricetin).
Figure 2
Figure 2
Isothermal titration calorimetry assay of (5′CAG/3′GAC)x6 RNA with compounds (a) CP2 (b) CP3 (c) CP4 (d) CP6 (e) CP8 (f) CP13 (g) CP14 (h) Myricetin.
Figure 3
Figure 3
Circular Dichroism spectra of free RNA (20.0 µM) and in the presence of compounds CP2, CP6 & CP13 (left to right) with (a–c) (5′CAG/3′GAC)x20 (d–f) (5′CAG/3′GAC)x6 (g–i) (5′CAG/3′GUC)x6.
Figure 4
Figure 4
Thermal denaturation curve of free RNA and in the presence of compounds CP2, CP6 & CP13 (left to right) with (a–c) (5′CAG/3′GAC)x20 (d–f) (5′CAG/3′GAC)x6 (g–i) (5′CAG/3′GUC)x6.
Figure 5
Figure 5
Structural insight into the interaction of 5′r(CCGCAGCGG)3′ with CP13 using NMR spectroscopy. One dimensional proton spectra of RNA as a function of increasing concentration of CP13 showing (a) base region (b) NMR titration of CP13 with increasing concentration of RNA (c–d) Two dimensional NMR spectroscopy of 5′r(CCGCAGCGG)3′ with CP13 at 298 K, at D/N ratios of (c) D/N = 0.0 (d) D/N = 2.0 (e–f) Docked structure of RNA with compounds (e) CP2 (f) CP6 (g) CP13
Figure 6
Figure 6
Effect of compounds on expanded polyglutamine aggregates. (a) Micrographs of EGFP-HD23Q, EGFP-74Q transiently transfected COS-7 cells, treated with control as well as compounds (100.0 µM) for 12 hours. The arrowheads represent aggregates. (b) Cells were transiently transfected with HDQ74 and AtaxinQ84 constructs; after transfection cells were treated with different compounds in a concentration-dependent manner, as shown in the figure. Aggregate positive cells with or without compounds were observed under a fluorescence microscope, and quantification of aggregates was performed with the help micrographs. * p < 0.05 as compared with the control group.
Figure 7
Figure 7
Effect of compounds on polyQ mediated aggregation reduction and Thioflavin assay. (a–f) Few same sets of cells expressing expanded polyglutamine proteins were used for quantitative fluorescence analysis of the aggregates. (g–i) Plasmid expression analysis of the cells was confirmed by immunoblot, using anti-GFP antibodies (For full blot image, see Supplementary Figure S6b-g) (j–l) polyQ aggregation inhibition assay in HD patient-derived cells. Total protein was extracted from HD cells (GM04281) in the absence and presence of compounds and emission spectra of Thioflavin T was recorded. (j) CP2 (k) CP6 (l) CP13
Figure 8
Figure 8
Cell viability assay in HD patient-derived cells treated with compounds (a–c) GM04281 cells expressing 74 CAG repeats treated with (a) CP2 (b) CP6 (c) CP13 (d–f) GM07492 cells expressing 17 CAG repeats (d) CP2 (e) CP6 (f) CP13 (g–h) Bright field micrograph of patient-derived cells treated with compounds (g) HD Cells (GM04281) (h) Normal fibroblast cells (GM07492). Blue line showing the curve for vehicle (control) while black lines represents cell viability of cells treated with compounds.

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References

    1. Connelly CM, Moon MH, Schneekloth JS., Jr. The Emerging Role of RNA as a Therapeutic Target for Small Molecules. Cell Chem Biol. 2016;23:1077–1090. doi: 10.1016/j.chembiol.2016.05.021. - DOI - PMC - PubMed
    1. Mirkin SM. Expandable DNA repeats and human disease. Nature. 2007;447:932. doi: 10.1038/nature05977. - DOI - PubMed
    1. Orr HT, Zoghbi HY. Trinucleotide repeat disorders. Annu Rev Neurosci. 2007;30:575–621. doi: 10.1146/annurev.neuro.29.051605.113042. - DOI - PubMed
    1. Zoghbi HY, Orr HT. Glutamine repeats and neurodegeneration. Annu Rev Neurosci. 2000;23:217–247. doi: 10.1146/annurev.neuro.23.1.217. - DOI - PubMed
    1. Banez-Coronel M, et al. RAN Translation in Huntington Disease. Neuron. 2015;88:667–677. doi: 10.1016/j.neuron.2015.10.038. - DOI - PMC - PubMed

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