Myricetin Reduces Toxic Level of CAG Repeats RNA in Huntington's

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Myricetin reduces toxic level of CAG repeats RNA in Huntington’s Disease (HD) and Spino Cerebellar Ataxia (SCAs) ESHAN KHAN, Arpita tawani, Subodh Kumar Mishra, Arun Kumar Verma, Arun Upadhyay, Mohit Kumar, Rajat Sandhir, Amit Mishra, and Amit Kumar ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00699 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017

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ACS Chemical Biology

Myricetin reduces toxic level of CAG repeats RNA in Huntington’s Disease (HD) and Spino Cerebellar Ataxia (SCAs) Eshan Khan1†, Arpita Tawani1†, Subodh Kumar Mishra1†, Arun Kumar Verma1, Arun Upadhyay2, Mohit 3 3 2 1* Kumar , Rajat Sandhir , Amit Mishra and Amit Kumar 1

Discipline of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Madhya Pradesh 453552, India 2 Cellular and Molecular Neurobiology Unit, Indian Institute of Technology Jodhpur, Rajasthan 342011, India 3 Department of Biochemistry, Panjab University, Chandigarh 160014, India * To whom correspondence should be addressed. Tel: +91-731-2438-771; Fax: +91-731-2438-721; Email: [email protected]. †

Equal Contribution

ABSTRACT Huntington's disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of CAG repeats in HTT gene. The transcribed mutant RNA contains expanded CAG repeats that translate into a mutant huntingtin protein. This expanded CAG repeat also causes mis-splicing of premRNA due to sequestration of muscle blind like-1 splicing factor (MBNL1) and thus both of these elicits the pathogenesis of HD. Targeting the onset as well as progression of HD by small molecules could be a potent therapeutic approach. We have screened a set of small molecules to target this transcript and found Myricetin, a flavonoid, as a lead molecule that interacts with CAG motif and thus prevents the translation of mutant huntingtin protein as well as sequestration of MBNL1. Here, we report the first solution structure of complex formed between Myricetin and RNA containing 5'CAG/3'GAC motif. Myricetin interacts with this RNA via base stacking at AA mismatch. Moreover, Myricetin was also found reducing the proteo-toxicity generated due to the aggregation of polyglutamine and further, its supplementation also improves neurobehavioral deficits in HD mouse model. Our study provides the structural and mechanistic basis of Myricetin as an effective therapeutic candidate for HD and other polyQ related disorders. INTRODUCTION 1

RNA is gaining significant attention as a potential drug target. One such class includes transcripts containing expansion of trinucleotide repeats (TNR) that causes Trinucleotide Repeat Expansion Disorders (TREDs).2 TREDs comprise more than 20 neurodegenerative and neuromuscular disorders

3, 4

5

, amongst which CAG repeat RNAs alone impart a majority of disorders like Huntington's

disease (HD), several spinocerebellar ataxias (SCAs)6, etc. HD is caused by abnormal expansion of CAG repeats in the coding region of HTT gene. Expansion of this triplet repeat beyond a threshold 7

level (usually >37) causes disorder and its severity depends upon increase in number of repeats.

The mutant transcript r(CAG)exp translates into mutant protein (mHTT) that harbors expanded polyglutamine (polyQ) repeat near to its amino terminus. mHTT causes cellular toxicity specifically by 8

formation of protein aggregation and also causes sequestration of biologically important proteins.

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In addition to the polyQ proteins, r(CAG)exp also undergoes the repeat associated non-ATG (RAN) translation and expresses four types of homo-polymeric proteins (polyAla, polySer, polyLeu, and polyCys) out of all possible open reading frames which forms the protein aggregates inside the cells.

13, 14

These events have detrimental effects on number of cellular functions that eventually lead

to neurodegeneration. Earlier studies reported that reduction in the amount of mutant proteins or its aggregation

could 16

oligonucleotide.

be

inhibited

by

using

caspase-6

inhibiting

peptides15

and

antisense

But allergic reactions, poor absorption and blood brain barrier were the major

limiting factors of these therapies.17 Conversely, small molecules based therapeutic strategies could be favored as they can aid in overcoming those limitations. Previously, some of the small molecules were designed

18, 19

and employed for targeting trinucleotide repeats 21-23

disorders including HD.

20

that causes various neurological

However, majority of these molecules were synthetic in nature and impart

various side effects to human health. For treatment of disorders like HD, usually long term continuous consumption of drugs is needed and therefore, drug dose must be endurable to human body. Naturally available small molecules are often appreciated for providing low systemic toxicity. Combining this property with their availability in human diets could make them a potential therapeutic candidate. Flavonoids are one of these kinds of bioactive polyphenolic compounds that are readily available in human diets.24 They have been known for providing health beneficiary effects like anti25

oxidant , anti-cancer disease.

30-32

26, 27

28, 29

, neuro-protection

as well as prevent the onset of neurodegenerative

In 2006, Ehrnhoefer et. al. reported the inhibition of mutant protein aggregation in HD

yeast and fly model by (2)-epigallocatechin-3-gallate (EGCG).

33

Although, targeting protein

aggregation is one of the effective strategy to combat HD, but the root cause of toxicity is due to the presence of r(CAG)exp within exon 1 of HTT mRNA34 that could lead to sequestration of several 12

10

splicing regulators MBNL-1 , CBP , PQBP-1.

11

Indeed for any therapeutic approach, targeting the

actual cause of the disorder rather than its consequences is always preferred. Here in this study, we have identified a lead small molecule as a potent binder of CAG motif among various known DNA binding molecules including dyes.

35

These molecules have been used in

fluorescence based assay with all combination of 1×1 nucleotide internal loops RNA as r(CNG) motif. Out of 36 DNA binding small molecules screened, Myricetin has been obtained as lead molecule. Further, various biophysical studies were performed to get detail insights of interaction between Myricetin and RNA containing r(CAG)exp. Solution structure of Myricetin- 5'CAG/3'GAC motif containing RNA complex revealed that Myricetin intercalates in the AA loop of the RNA. It also inhibits the aggregation of mHTT in cell culture models. Moreover, the applicability of our decrees with respect to HD pathogenesis in mice models was also determined, showing Myricetin ameliorated mitochondrial dysfunctions and motor impairments in HD rats. RESULTS AND DISCUSSION Screening of small molecules for binding to RNA library containing mis-matched internal loops involved in TREDs Small molecule modulator that targets RNA has been promoted as a potential therapeutic approach. The proof of the principle for this strategy has been best exemplified by antibiotic drugs that bind to


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bacterial rRNA. This consideration has powered a curiosity to target unusual cellular RNAs that are associated with diseased conditions. In this connection, trinucleotide repeat expansion disorders (TREDs) provides a striking example for targeting RNA. TREDs majorly involves the CNG repeats (N = A,U,G,C) and is known to be associated with neuro degenerative diseases like fragile X mental retardation syndrome (FXS), Huntington’s disease, myotonic dystrophy (DM), several spinocerebellar ataxias (SCAs), etc. Several DNA-binding agents have been reported with RNAs containing 1x1 36

nucleotide internal loops which are found in expanded triplet repeats.

We therefore screened a set

of 36 nucleic acid binders whose codes were assigned to each of them for the convenience of this study (Table S1). Screening was performed against a library of 11 RNA sequences containing all combination of 5'CNG/3'GNC motif, (where N is A, U, G, C), i.e 1×1 nucleotide internal loops, closed by CG pairs and embedded in a cassette of RNA hairpin to ensure its proper folding (Figure S1).37, 38 Some of these are known to be involved in different neurodegenerative disorders like 5'CAG/3'GAC, 2

5'CGG/3'GGC and 5'CUG/3'GUC are associated with HD and SCA3, FXTAS and DM1, respectively.

5'CAG/3'GUC is canonically paired RNA sequence that will serve as control along with duplex DNA that is calf thymus DNA (CT-DNA). On comparing the fluorescence based binding affinity of 36 molecules with these 11 RNA sequences, it has been found that CP1032 (Thiazole Orange) possesses best affinity for most of RNA sequences. But, it does not show specificity towards non-canonical RNAs over canonically paired duplex RNA (Figure S2, Table S2). However in drug development strategies, specificity for target is an important requisite. Additionally, synthetic molecules may provide toxicity to normal cells and have various side effects. Previously, small molecules have been designed that could bind r(CAG) repeat, but it caused toxicity to normal cells.38 Moreover, considering the toxicity produced by synthetic molecules to normal cells, we have centralized our study on assessing the affinity of natural compounds for these RNA targets. Specific binding preference towards 5'CAG/3'GAC motif RNA was observed for compound CP1005 (Myricetin) amongst all molecules used in this study (Figure 1A). It has more than 5000 fold and 80000 fold higher selectivity for 5'CAG/3'GAC motif over duplex DNA and canonically paired AU-RNA, respectively (Figures 1B and 1C, Table S3, Figure S3H, Figure S3I,). Moreover its affinity for AA multiple loop containing RNA sequence that is (CAG)×2, (CAG)×3, (CAG)×4, (CAG)×5, (CAG)×6 and (CAG)×33 were also determined (Figure 1D, Table S3; Figure S3). The plot of change in fluorescence intensity vs. RNA concentration was best fitted by two mode binding model. The computed binding constant values show that Myricetin has good binding affinity for all of AA multiple loop containing RNA sequences with several fold high specificity over canonically paired RNA sequence (AU-RNA) as well as duplex DNA (CT-DNA). Further insights of Myricetin-RNA interaction were explored by analysing the fluorescence life time decay profile of Myricetin by employing Time Correlated Single Photon Counting (TCSPC) studies. Usually, the life time decay of fluorophore in its excited state is responsive to change in its environment and could be used to probe its interaction with nucleic acid. The lifetime of small molecule increases when it interacts strongly with macromolecule. TCSPC method was used to record life time decay of Myricetin in absence and presence of both RNA sequences having



5'CAG/3'GAC motif and duplex AU-RNA respectively (Figures 1E and 1F). In unbound form, Myricetin shows tri-exponential decay profiles containing three lifetimes (τ1, τ2 and τ3) and three amplitudes (β1, β2 and β3) with average lifetime value of 32 ps (Table S4). With increasing the RNA concentration upto drug/nucleic acid (D/N) = 2.0 ratio, the decay profile of Myricetin in this complex was best fitted with tri-exponential decay function and its average lifetime increases by ~ 6 fold for 5'CAG/3'GAC motif-RNA while ~ 2.5 fold

for duplex AU-RNA. Remarkably, this changes were

pronounced for 5'CAG/3'GAC motif RNA specificity rather for AU-paired RNA. Moreover, we have also performed isothermal titration calorimetry (ITC) study to corroborate the binding affinity of Myricetin with 5'CAG/3'GAC and 5'CAG/3'GUC RNA. The data were fitted using two site binding model for both of RNAs. ITC data showed the exothermic peaks due to increased π-π stacking interaction of Myricetin with RNA (Figure S4).39 The dissociation constants obtained for AA pair and AU pair were 0.25 µM and 202 µM respectively that suggests the higher affinity and specificity of Myricetin with 5'CAG/3'GAC motif-RNA over AU paired duplex RNA control. This ensures strong and tight binding as well as confirms the nanomolar range of binding obtained from fluorescence binding assay with 5'CAG/3'GAC motif-RNA. Assessment of conformational changes and gel mobility of CAG containing RNA upon binding with Myricetin CD spectra for RNA (20 µM) containing 5'CAG/3'GAC and 5'CAG/3'GUC motif showed a large positive peak near 270 nm and a small negative peak near 240 nm, which was consistent with double stranded A-form like structure (Figure 1G and 1H).

40

With the addition of Myricetin upto D/N = 2.0, the

intensity of positive peak at 270 nm was decreased for 5'CAG/3'GAC motif RNA. This reduction in CD signal indicates that Myricetin interacts strongly with RNA. As this band is sensitive to base-stacking, thus, decrease in its intensity could be inferred as alteration of base-stacking in RNA, however overall conformation of RNA remains the same. Moreover, this change takes place only in 5'CAG/3'GAC motif RNA while no changes were observed for paired AU-RNA. We have also recorded UV-melting profiles of RNA and its complex with Myricetin. Figures 1I and 1J portray the melting profiles that showed that 5'CAG/3'GAC motif RNA unfolds with single transition of ~1-4ºC with increasing number of repeats whereas no changes were observed in paired AU-RNA (Figures 1I and 1J; Figure S5; Table S5). The CD and UV-melting results were coherent with the TCSPC results stating the strong and selective binding of Myricetin with the 5'CAG/3'GAC motif RNA. Furthermore, the binding of Myricetin also causes changes in the stacking interactions of 5'CAG/3'GAC motif RNA that leads to changes in melting temperature. Moreover, we have also performed the gel retardation assay to confirm the binding of Myricetin to r(CAG)exp RNA. With the increasing concentration of Myricetin, a retardation in mobility of 5'CAG/3'GAC motif RNA bands was observed, however, retardation in the mobility of 5'CAG/3'GUC was not observed. (Figure S6A and S6B, S6C). Additionally, we have also performed Taq

Polymerase stop assay utilizing (5'CAG/3'GAC)x1, (5'CAG/3'GAC)x6 and

(5'CAG/3'GUC)x6 motif containing DNA. Decrease in the intensity of PCR bands confirms binding of Myricetin to 5'CAG/3'GAC motif that hinders the activity of polymerase enzyme to amplify DNA


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(Figure S6D and S6E). Nonetheless, the intensity of PCR bands of (5'CAG/3'GUC)x6 DNA motif was not altered (Figure S6F) that also affirms the specific binding of Myricetin with CAG motif. Therefore, Retardation in the mobility of various AA internal loops and polymerase stop assay further supports the specific binding of Myricetin to 5'CAG/3'GAC motif. . Understanding the binding of Myricetin to CAG×1 motif containing RNA by NMR spectroscopy Binding of Myricetin to AA- RNA was further monitored by one-dimensional (1D) proton NMR titration experiment. For this experiment we have chosen short sequence of RNA containing 1×AA loop ((CAG)×1) that is 5'- rC1rC2rG3rC4rA5rG6rC7rG8rG9-3'. Previously, we have revealed the solution structure of (CAG)×1 motif containing RNA that helped us in proton assignment.41 Upon gradual addition of Myricetin to RNA, changes in the chemical shifts as well as shape of the proton resonances were observed (Figures 2A, 2B and 2C) that corroborates the binding of Myricetin to (CAG)×1 motif containing RNA. In 1D proton NMR spectra, these changes emanated from AA internal loop and its neighboring nucleotides as seen in Figures 2A, 2B and 2C. Single set of resonances of imino protons demonstrated the intermediate to fast exchange regime between free and bounded RNA on NMR time scale. Information about binding region for Myricetin on (CAG)×1 RNA was gathered by mapping all shifts that are larger than 0.03 ppm. Largest upfield shift of 0.19 ppm was observed for G6NH proton, while resonances of G3NH and G9NH showed upfield shift of 0.06 ppm (Figure 2A). Comparison of chemical shifts in 1D proton NMR spectra of free RNA with Myricetin – RNA complex in 2:1 D/N ratio revealed the following significant changes (positive and negative values in ppm represents downfield and upfield shifts, respectively): A5H8 (-0.05), G6H8 (+0.05), G6H1'(0.06) coupled with broadening of proton resonances of A5H1', C4H1' with increase in concentration of Myricetin (Figures 2B and 2C). Notably, C4H6 resonance got resolved as individual peak with increase in D/N ratio upto 1.0 then gradually broadens upto D/N = 2.0. Remarkably, with further addition of Myricetin after one equivalent to RNA sample, distinct changes in resonances for C1, C2, G8 and G9 protons were observed. As Myricetin was added to RNA, C1H6 starts to emerge as individual peak that significantly broadens after D/N = 1.0 and showed upfield shift of 0.11 ppm. C2H6 resonance showed 0.07 ppm upfield shift and it also gets broadened with increasing concentration of Myricetin. C1H5 showed 0.14 ppm upfield shift that becomes individual resonances after D/N = 1.0 (Figures 2B and 2C). Similarly, G8H8 resonance gets broadened while G9H1' peaks shows an upfield shift of 0.06 ppm. All these distinct changes posit that apart from preferred AA internal loop, Myricetin also binds at second, terminal site on this CAG motif containing RNA. These results were in-line with our biophysical studies which also states more than one binding sites for Myricetin on AA-RNA. Further, to determine the involvement of Myricetin protons in interaction, we have performed NMR titration experiment of Myricetin by gradual addition of AA- RNA. With the successive addition of RNA to Myricetin, there occur changes in its proton resonances (Figure 2D). The resonance for 2', 6', 8 and 6 protons started disappearing at D/N ratio of 100:1 while 5' OH resonance starts to appear and get sharpened at successive increment in D/N ratio. This result suggests the involvement of these protons in the binding of Myricetin with r(CAG) RNA. Thus to get clear picture of this interaction, we



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have performed two dimensional proton NMR studies of this complex in order to understand the structural basis of its interaction. Structure determination and calculation of Myricetin - CAG motif containing RNA complex We have determined the solution structure (CAG)×1 motif RNA bound to Myricetin at D/N = 2.0 ratio. NOESY spectra were collected at various mixing times at different D/N ratios (Figure 3). As demonstrated by changes in chemical shift of various proton resonances, these structural revamping does not change the NOE pattern of nucleotide bases apart from AA internal loop of (CAG)×1 motif. This indicates that no structural changes take place in the rest part of this RNA and sequential H1' H8/H6 walk is possible from C1 through G9 (Figure 3). Further, in NOESY spectra, 9 intermolecular NOEs between Myricetin and RNA protons were observed (Figure 4A, Table S6). These NOEs include cross peaks between A5H2-Myr H8, A5H8-Myr H6 at D/N = 0.6 and 1.0 ratio (Figures S7A and S7B). Upon increasing the Myricetin concentration beyond D/N = 1.0 emergence of NOE for C1 and C2 protons with Myricetin protons was also observed (Figure 4A). For structure calculation of RNA-Myricetin complex, NOE-derived intermolecular distances were used as distance restraints. In a qualitative manner, the intensities of cross peak were employed, in which the distances were approximately 1.8–2.5, 2.5–3.0, 3.0–3.5, 3.5–4.0, and 4.0–5.0 Å for strong intense (ss), strong (s), medium (m), weak (w) and very weak (vw) respectively (Table S6). A 100 ns of restrained molecular dynamic simulation were performed and 100 converged structures with no NOE violations larger than 0.5 Å were obtained. The results favoured the distances obtained from the NOE experiment with energy minimized model for Myricetin – (CAG)×1 RNA obtained after rMD simulations (Figure 4B) has final potential energy of -17012.30 kcal/mol (Table S7) and an ensemble of ten conformations with the lowest potential energy were superimposed (Figure 4C). This lowest potential energy model showed binding of Myricetin between C4-A5 as well as above the C1 base step (Figure 4B). This follows the fact that the obtained NOE-derived distance restraints will be fulfilled only with base stacking mode above C1 and intercalative mode at AA internal loop. Further support for intercalation of Myricetin could be provided by the (0.05 ppm) upfield shift of Myricetin protons with increasing D/N ratio. This structural data reveals that Myricetin binds specifically to CAG motif of the RNA with intercalation mode. In this complex, Myricetin stacks in between C4pA5 and its benzopyran ring is involved in π-π stacking with C4 and G6 (Figure S7C). As a result, one of the adenine residues of AA mismatch gets protrudes out that could also be facilitated by dynamic nature of AA internal loop.

41

Interestingly, the

conventional Watson- Crick base pairing of C4 and G6 allows formation of a continuous helix in the RNA. This supports CD and melting analysis that binding of Myricetin causes no change in the global RNA structure formed by (CAG) motif containing RNA. O21 of Myricetin forms hydrogen bond with amine protons of C4 at this location (Figure S7C). Also, A5 acquires anti-conformation and aids in interaction of Myricetin by forming hydrogen bond with its O4' and H5 of Myricetin (Figure S7D). Apart from this, Myricetin also bounds at terminal bases of CAG motif containing RNA sequence that is C1 and G9. At this location, Myricetin is oriented in such a manner that it forms π-π stacking interactions with C1 and G9 (Figure S7E). However, binding of Myricetin near to C1 base is not significant as in actual condition of pathogenesis due to the CAG repeats, such terminal sequences of C1C2G3 does


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not exist. Furthermore, as mutant protein also contributed to cell function impairment by aggregation8, therefore, we have protracted our studies to understand the potential of Myricetin to plummet toxicity caused by mutant protein. Treatment of Myricetin Induces Expanded Polyglutamine Proteins Degradation and Alleviates Cytotoxicity Cos-7 cells were transiently transfected with normal (EGFP-HDQ23) and expanded polyglutamine (EGFP-HDQ74) constructs. Post-transfected cells were treated with DMSO (control) and Myricetin and used for micrographs analysis as shown in Figure 5A. As shown in Figures 5B and 5C higher magnification fluorescence analysis was performed for Myricetin treated cells expressing normal (EGFP-HDQ23 and ataxin-3 (Q28)) and expanded polyglutamine (EGFP-HDQ74 and ataxin-3 (Q84)) proteins. Arrowheads indicate aggregates formation inside the cells. Cell lysates of control cells were used for immunoblot analysis (Figure 5D) using GFP and β-actin antibodies to further confirm intracellular plasmids expression. Next, we performed aggregates counting, as shown in Figure 5E, which represents quantification of aggregate formation with or without Myricetin in cells expressing normal and expanded polyglutamine proteins. Few of the same sets of cells were also used for immunoblot analysis and blots were incubated with GFP and β-actin antibodies as shown in Figures 5F and 5G. To further ascertain the effects of Myricetin against cytotoxicity, generated by expanded polyglutamine proteins, we performed MTT assay to observe cell viability as represented in Figure 5H and 5I. Our cell viability results showed that Myricetin alleviates proteotoxicity of expanded polyglutamine proteins in cells which were in-line with previous reports affirming the role of Myricetin in cytoprotection against cytokine-induced cell death and oxidative stress-induced apoptosis.

42, 43

Effect of Myricetin supplementation on 3-NP-induced mitochondrial respiratory chain enzymes, oxidative stress and swelling A large number of evidence suggests that 3-Nitropropionic acid (3-NP) induced HD model might offer the suitable platform for therapeutic testing.44 The neuro-protective role of some of the flavonoids like 45

46

47

Naringin , Hesperidine , Quercetin

48

ECGC

etc. has been reported in 3-NP induced HD rats. Our

results also affirms these studies that Myricetin a flavonoid, also has potential to prevent mitochondrial dysfunctions, oxidative stress along with motor deficits and helps in management of HD and several SCAs. High resolution clear native (hrCN-)PAGE analysis followed by in- gel staining for individual complexes was done to evaluate electron transport chain enzymes activities after 3-NP exposure followed by densitometry analysis (Figures S8A). The densitometric quantification of mitochondrial complex activity based on in gel assay staining was performed to evaluate the individual mitochondrial complex activity (Figure S8B) Administration of 3-NP significantly inhibited the levels of mitochondrial complex I (26.43%), complex II (15.65%), complex III+IV (40.74%), complex IV (15.56%), and complex V (11.42%). Myricetin supplementation to 3-NP treated animals significantly increased the activity of complex I (18.56%), complex II (19.07%), complex III+IV (37.5%), complex IV (13.47%), and complex V (9.67%). Further, the administration of 3-NP also inhibited the activities of mitochondrial respiratory chain enzymes. The activity of NADH dehydrogenase was inhibited by



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37.8% (Figure S8C), SDH by 39.05% (Figure S8D), cytochrome oxidase by 40.49% (Figure S8E) and F1F0-ATP synthase by 32.07% (Figure S8F). Upon supplementation of Myricetin to 3-NP treated animals for 17 days, a significant increase in the activities of NADH dehydrogenase (62.75%), SDH (55.38%), cytochrome oxidase (68.63%), F1F0-ATP synthase (45.21%) (Figure S8C-S8F) were observed. Mitochondrial oxidative stress was found to be elevated in terms of increased levels of mitochondrial lipid peroxidation (68.77%), ROS formation (42.9%), nitrite species (216.54%) and mitochondrial swelling (156.71%) in 3-NP treated animals as compared to controls (Figure S8G-S8J). Supplementation of Myricetin significantly decreased the levels of mitochondrial lipid peroxides (35.01%), protein ROS levels (40.96%), nitrite levels (72.27%) and mitochondrial swelling (66.87%). Conversely, the activities of mitochondrial Mn-SOD and catalase were found to be significantly reduced by 45.39% and 52.61% respectively in the 3-NP-treated animals. Myricetin supplementation significantly increased the enzymatic activities of Mn-SOD (43.77%) whereas, no significant change was observed in catalase activity. Moreover, no significant changes were observed in GSH, GSSG and redox ratio in all the groups (Figure S9A-S9E). Effect of Myricetin on neurobehavioral deficits The numbers of counts for 3-NP treated animals fell to an average of 154.8 as compared to controls (235.6),

suggesting

a

significant

impairment

in

locomotor

functions.

However,

Myricetin

supplementation increased the average number of counts to 226.8. Narrow beam walk test was used to assess hind-limb impairment, wherein the time taken by the rats to walk across a narrow beam with progressively decreasing width was recorded (Figure S9F-S9H). The maximum time allowed to each animal for traversing the beam was 120 s. The average time recorded by 3-NP treated animals was 71.4 s as compared to control (20.4 s) indicating a significant impairment in hind-limb function. Myricetin supplemented animals on the other hand took an average of 22.4 s. The results indicate that Myricetin administration was effective in improving 3-NP induced motor impairments (Figure S9FS9H). The effectiveness of Myricetin was also ascertained by providing its oral supplementation to 3NP induced HD rats. CONCLUSION Structural aspects of drug-nucleic acid complex are important as it is likely to provide intimate details of their interaction. Our study provides useful structural information and the first solution structure of Myricetin - 5' r(CCGCAGCGG)3' RNA complex (coordinates of the NMR model of this complex have been deposited in the PDB as 5XI1). We have determined Myricetin as potent and selective binder of 5'CAG/3'GAC motif containing RNA that causes neurological disorders. Myricetin has potential to aids in rescuing polyglutamine protein-induced cell death by inhibiting polyQ aggregation and it is effective in 3-NP induced rat HD model. Our study provides one of the possible ways for action of Myricetin to exert its effects in improving the pathogenicity of CAG repeat disorders and strongly support the potential of Myricetin as potent therapeutic candidate for the CAG repeat causing TREDs.


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ACCESSION NUMBERS The coordinates for NMR model have been deposited in the Protein Data Bank, with accession code 5XI1. SUPPLEMENTARY DATA The Supporting Information is available free of charge via the internet at http://pubs.acs.org All experimental procedure with secondary structure of various RNA, complete biophysical experimental binding data (fluorescence titration curve, ITC, thermal denaturation profile), Gel retardation, NMR data showing intermolecular NOEs, enzyme assays showing effect of myricetin on mitochondrial functions. The authors declare no conflict of interest. ACKNOWLEDGEMENT This work was supported with the funding provided by Department of Science and Technology, Government of India, [DST/FT/LS29/2012]. SKM is thankful to UGC, New Delhi and EK, AKV to DBT, New Delhi for their fellowships. Authors are thankful to Sophisticated Instrumentation Facility at IIT Indore for NMR, CD and TCSPC experiments. We sincerely thank H. L. Paulson (The University of Michigan Health System, Department of Neurology, Ann Arbor, MI) for pEGFP-C1-Ataxin3Q28 and pEGFP-C1Ataxin3Q84 constructs and A. Tunnacliffe (Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK) for EGFP-HDQ23 and EGFP-HDQ74 constructs.

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FIGURES LEGENDS

Figure 1. Screening of small molecules that targets the trinucleotide repeats RNAs. (a) A plot of binding constant values of compounds against AA pair and AU pair RNA.(b) A plot of binding constant values of flavonoids against trinucleotide repeats RNAs (c,d) Fluorescence titration curve of Myricetin as a function of various RNA concentration (e-f) Fluorescence life time decay curve of Myricetin and its complex with RNAs (e) 5'CAG/3'GAC RNA (f) 5'CAG/3'GUC RNA. (g-h) Circular dichroism Spectrum of free RNA and in presence of increasing concentration of Myricetin (g) 5'CAG/3'GAC RNA (h) 5'CAG/3'GUC RNA (i-j) Thermal denaturation curve of free RNA and in presence of Myricetin (i) 5'CAG/3'GAC RNA (j) 5'CAG/3'GUC RNA. Figure 2. Structural insight into the interaction of 5'r(CCGCAGCGG)3' with Myricetin using NMR spectroscopy. One dimensional proton spectra of RNA 5'r(CCGCAGCGG)3' as a function of increasing concentration of Myricetin showing (a) Imino region (b) base region (c) H1' region (d) NMR titration of Myricetin with increasing concentration of 5'r(CCGCAGCGG)3' RNA. Figure 3. Two dimensional NMR spectroscopy of 5'r(CCGCAGCGG)3' with Myricetin at 298 K at D/N ratio (a) D/N=0.0 (b) D/N= 2.0. Figure 4. Myricetin and 5'r(CCGCAGCGG)3' complex at D/N = 2.0. (a) Various regions of NOESY spectrum showing intermolecular cross-peaks between Myricetin and 5' r(CCGCAGCGG)3'RNA (b) Lowest potential energy model of the complex after rMD simulation. Black dashes showing hydrogen bond formed between Myricetin (blue) and r(CAG) RNA. All the nucleotides are shown in dark purple and A-A mismatch is shown in dark cyan colour, RNA backbone is shown in worm-representation (c) Ensemble of ten lowest energy structures after restrained molecular dynamics simulation (PDB code 5XI1). Figure 5. Myricetin contributes in degradation of polyglutamine aggregates. (a) EGFP-HDQ23 and EGFP-HDQ74 transiently transfected Cos-7 cells were treated with DMSO and Myricetin in concentration-dependent manner for 12 hours and quantitative fluorescence analysis was performed; arrowheads indicate aggregates. (b-c) Aggregate formation in cells transfected with normal (EGFP-


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HDQ23 and ataxin-3(Q28)) and expanded polyglutamine constructs (EGFP-HDQ74 and ataxin3(Q84)) were observed under fluorescence microscope, scale bar 20 µM. (d) Total cell lysates, collected from similar sets of cells, were subjected to immunoblotting by anti-GFP and anti-β-actin antibodies. (e) Quantitative analysis was done for aggregates formed in various sets of cells, transfected with normal and expanded polyglutamine constructs, followed by treatment of Myricetin in concentration-dependent manner. (f-g) Expanded polyglutamine-expressing cells, after treatment with DMSO and Myricetin, were subjected to immunoblot analysis by anti-GFP antibody; while anti-β-actin was used as loading control. (h-i) Similar transfected sets of polyglutamine-expressing cells, after treatment of Myricetin were subjected to MTT assay to asse ss cell viability. Values presented are the mean ± SD, from three independent experiments.*p

Myricetin Reduces Toxic Level of CAG Repeats RNA in Huntington's

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