Small Molecule Recognition and Tools to Study Modulation of r(CGG)^exp in Fragile X-Associated Tremor Ataxia Syndrome

Abstract

RNA transcripts containing expanded nucleotide repeats cause many incurable diseases via various mechanisms. One such disorder, fragile X-associated tremor ataxia syndrome (FXTAS), is caused by a noncoding r(CGG) repeat expansion (r(CGG)exp) that (i) sequesters proteins involved in RNA metabolism in nuclear foci, causing dysregulation of alternative pre-mRNA splicing, and (ii) undergoes repeat associated non-ATG translation (RANT), which produces toxic homopolymeric proteins without using a start codon. Here, we describe the design of two small molecules that inhibit both modes of toxicity and the implementation of various tools to study perturbation of these cellular events. Competitive Chemical Cross Linking and Isolation by Pull Down (C-Chem-CLIP) established that compounds bind r(CGG)exp and defined small molecule occupancy of r(CGG)exp in cells, the first approach to do so. Using an RNA GFP mimic, r(CGG)exp-Spinach2, we observe that our optimal designed compound binds r(CGG)exp and affects RNA localization by disrupting preformed RNA foci. These events correlate with an improvement of pre-mRNA splicing defects caused by RNA gain of function. In addition, the compounds reduced levels of toxic homopolymeric proteins formed via RANT. Polysome profiling studies showed that small molecules decreased loading of polysomes onto r(CGG)exp, explaining decreased translation.

Approved Anti-cancer Drugs Target Oncogenic Non-coding RNAs

“…Other viral RNAs such as the hepatitis C virus internal ribosomal entry site (HCV IRES) and Influenza A have been focus of several targeting studies (Dibrov, et al, 2014; Lee, et al, 2014). Recently, several small molecules have been designed to target nucleotide repeat expansions including fragile X-associated tremor ataxia syndrome (FXTAS; CGG) (Yang, et al, 2016), myotonic dystrophy type 1 (DM1; CUG) (Gareiss, et al, 2008; Jahromi, et al, 2013; Pushechnikov, et al, 2009), myotonic dystrophy type 2 (DM2; CCUG) (Childs-Disney, et al, 2014), Huntington’s disease (HD; CAG) (Kumar, et al, 2012), and frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALD; G 4 C 2 ) (Su, et al, 2014). A salient question is if small molecules that target non-coding RNAs can be identified and developed into medicines or chemical probes.…”

Curcumin Regulates the r(CGG)exp RNA Hairpin Structure and Ameliorate Defects in Fragile X-Associated Tremor Ataxia Syndrome

“…CGG repeats also produces toxic polyglycine protein, FMRpolyG via repeat-associated non-AUG (RAN) translation (Todd et al, 2013;Sellier et al, 2017). Previous studies have shown that selective targeting of CGG hairpin structures via small molecules to reduce both RNA foci and PolyG aggregates formation is the most successful approach (Yang et al, 2015(Yang et al, , 2016b. Thus, we sought to find out natural small molecule FIGURE 6 | In vitro potency of Curcumin in FXTAS cultured cell line.…”

“…In this study, we focused on the identification of small molecules targeting expanded CGG RNA repeats [r(CGG) exp ] causing FXTAS. Ideally, a potential compound would prevent non-canonical translation of the CGG repeats sequence into the toxic FMRpolyG protein, as well as correct the alternative splicing defects caused by sequestration of RNA regulatory proteins by expanded CGG nucleotide (Yang et al, 2015(Yang et al, , 2016bVerma et al, 2019b). Here, we found that Curcumin, a polyphenol used as a traditional herbal medicine, binds to CGG RNA repeats (Nafisi et al, 2009;Kulkarni and Dhir, 2010).…”

“…To assess the effect of Curcumin on the thermal profile of CGG repeat RNAs, which could affect CGG associated splicing defects and RAN translation (Yang et al, 2016b), we used CD thermal denaturation assays. CGG RNA thermal profile were monitored at 267 nm and the changes in melting temperature ( T m ) were of 1.49, 3.48, 4.19, 5.25, and 6.28 • C for r(CGGx1), r(CGGx6), r(CGGx20), r(CGGx40), and r(CGGx60), respectively ( Figures 4A-E).…”

“…40% and 50% of RAN translation were impaired at 50 and 100 µM, respectively (Figure 7E). Thus, enhancing thermostability of r(CGG) exp through compounds correlates with the reduction of protein aggregates (Yang et al, 2015(Yang et al, , 2016b.…”