Small‐Molecule‐Mediated Cleavage of RNA in Living Cells (2012)

Very few small molecule therapeutics bind RNA and modulate its function. The most notable exceptions target the bacterial ribosome or riboswitches. The ribosome comprises ca. 80–90% of the total RNA content of living cells and thus can be effectively targeted with small molecules even if they are not exquisitely selective.[5] Small molecules that modulate riboswitch function can be designed by mimicking the riboswitch’s natural metabolite, mirroring substrate mimicry approaches that have been a mainstay to target enzymes.[6] Modulating the function of other cellular RNAs is therefore difficult due to their low expression levels (compared to ribosomal RNAs) and the lack of lead small molecules.[7]

In an effort to identify lead small molecules to modulate RNA function, we have developed a bottom-up approach to target RNA. That is, the RNA secondary structural elements (motifs) that are preferred to bind small molecules are identified. These RNA motifs are then searched for in RNAs that cause or contribute to disease. In ideal cases, the disease-causing RNA has more than one targetable motif such that a multivalent ligand can be designed to increase ligand affinity and specificity. We have successfully applied this approach to modulate the toxicity of expanded repeating RNAs that cause or contribute to myotonic dystrophy type 1 (DM1) and Huntington’s disease (HD).[4, 8] [9]

DM1 is caused by an expanded r(CUG) repeat (50–1000+ repeats) in the 3’ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. The expanded repeat, or r(CUG)exp, folds into an RNA hairpin that sequesters proteins, including muscleblind-like 1 (MBNL1), a pre-mRNA splicing regulator (Figure 1B).[3, 10] Sequestration of MBNL1 by r(CUG)exp leads to its inactivation, and hence alternative splicing defects in a subset of pre-mRNAs including the cardiac troponin T (cTNT), the insulin receptor, and the muscle-specific chloride ion channel (Figure 1A & B). These splicing defects explain the heart defects, insulin insensitivity, and myotonia that are experienced by DM1 patients. The disease mechanism for DM1 points to a therapeutic strategy in which a compound binds r(CUG)exp and displaces MBNL1, allowing it to properly regulate pre-mRNA alternative splicing (Figure 1C). Several compounds have been used to validate this as a therapeutic strategy in both cellular and animal models of disease.[9a, 9b, 11]



Figure 1

Disease pathology for DM1 and the strategy that is used to target the causative agent, r(CUG)exp. A, in wild type cells, MBNL1 regulates exon inclusion and exclusion by binding to various transcripts including cTNT pre-mRNA. In DM1-affected cells, MBNL1 ...



We previously reported a designed, modularly assembled compound that binds r(CUG)exp and modulates its toxicity in vivo.[9a] The compound consists of a peptoid scaffold that displays two copies of a bis-benzimidazole (H, Figure 2), separated by four spacing modules (2H-4, Figure 2). The H module binds a single 5’CUG/3’GUC motif displayed in r(CUG)exp while 2H-4 binds two adjacent 5’CUG/3’GUC motifs.[9c] We sought to improve the potency of 2H-4 in vivo by by equipping the RNA-binding compound with a moiety that can induce degradation of r(CUG)exp. The RNA-binding portion of the compound displaces MBNL1 from r(CUG)exp and provides specificity while the cleavage-moiety leads to degradation of r(CUG)exp. Both modes of action should lead to improvement of DM1-associated defects.[11d]



Figure 2

The use of hydroxyl pyridine derivatives to photochemically generate hydroxyl radicals near targeted transcripts in living cells. Top, the mechanism by which hydroxyl radicals are generated by hydroxyl pyridines, as shown previously.[11] Bottom, structures ...



We utilized a derivative of N-hydroxypyridine-2(1H)-thione that generates hydroxyl radicals upon irradiation to induce cleavage of r(CUG)exp. Previous studies have employed N-hydroxypyridine- 2(1H)-thione to probe RNA structure in vitro.[12] Thus, we synthesized 1-hydroxy-6-thioxo-1,6-dihydropyridine-2-carboxylic acid (N-HPT) that allows for conjugation of the hydroxyl radical-producing war head onto 2H-4 via an amide bond (2H-4-HPT) (Figure 2).[13] A compound without the RNA-binding (H) module (2P-4-HPT) serves as a negative control.

The compounds were studied for cleaving r(CUG)10 in vitro (Figure 3). These studies show that 2H-4-HPT cleaves r(CUG)10 in the presence of UV light. Some cleavage is also observed when r(CUG)10 is incubated with 2H-4, but it is less than what is observed for 2H-4-HPT. A slower mobility band was observed when r(CUG)10 was incubated with both 2H-4 and 2H-4-HPT, suggesting a cross-link mediated by Hoechst occurs upon irradiation with light. No cleavage is observed for 2P-4-HPT as expected since 2P-4 does not bind r(CUG) repeats.[9c]



Figure 3

Studying the ability of 2H-4, 2H-4-HPT, and 2P-4-HPT to cleave r(CUG)10 in vitro. Top, a plot of the amount of cleavage as a function of nucleotide position at 10 µM compound. As expected, the extent of cleavage by 2H-4-HPT is more than 2H-4 and ...



Next, the in vitro potencies of the compounds for inhibiting the r(CUG)10-MBNL1 complex were determined via a time-resolved fluorescence resonance energy transfer (TR-FRET) assay (Table 1).[9b] Two sets of experiments were completed: (i) the RNA was incubated with the compound of interest and irradiated with light. MBNL1 was then added; and, (ii) the RNA was incubated with the compound of interest and then MBNL1 was added (no irradiation). The IC50 for 2H-4 is similar whether the sample is irradiated or not. In contrast, a 6-fold increase in potency is observed for 2H-4-HPT when the sample is irradiated. The increase in potency of 2H-4- HPT is due to degradation of r(CUG)10, as evidenced by in vitro cleavage (Figure 2). The decrease in potency of 2H-4 in the presence light irradiation is presumably due to the degradation of ligand. (Figure S5). No effect on complex formation is observed with N-HPT, as expected.



Table 1

Potencies of 2H-4, 2H-4-HPT, and N-HPT for the inhibition of the r(CUG)10-MBNL1 complex in vitro in the presence and absence of light.



Since 2H-4-HPT is a more potent in vitro inhibitor of a model r(CUG)exp-MBNL1 complex than 2H-4 and cleaves r(CUG) repeats, it is possible that it has improved potency in vivo. Therefore, 2H-4- HPT was studied in a cell culture model for improving DM1- associated pre-mRNA splicing defects (Figure 4). HeLa cells were co-transfected with a DM1 mini-gene that expresses r(CUG)exp and a mini-gene construct of cTNT alternative splicing. After transfection, the cells were dosed with compound in growth medium. After 6 h, cells were irradiated (+light). Total RNA was harvested 12–14 h later, and the amount of each alternatively spliced isoform of the cTNT mini-gene transcript was determined by RT-PCR. When cells were treated with 5µM of 2H-4-HPT and irradiated with light, the levels of cTNT pre-mRNA splicing shifted towards what is observed in the absence of r(CUG)exp (Figure 4). Furthermore, no toxicity is observed at these conditions as assessed by morphological changes and a MTT viability assay (Figure S7). This suggests that even though highly reactive species are being generated, they are localized to r(CUG)exp and not damaging bystander, essential biomolecules. This specificity is afforded by 2H-4. Under identical conditions, 2H-4 does not improve pre-mRNA splicing defects at 5 µM, indicating that the improved bioactivity of 2H-4-HPT is due to cleavage of r(CUG)exp. Additional control experiments show that 2H-4-HPT has no effect on the alternative splicing of PLEKHH2 pre-mRNA, which is not controlled MBNL1 (Figure S6).[10a]



Figure 4

2H-4-HPT improves a DM1-associated alternative pre-mRNA splicing defect in a cell culture model. In the presence of light, 2H-4-HPT shifts the cTNT alternative splicing pattern towards that observed in cells that do not express r(CUG)exp. No improvement ...



To determine if 2H-4-HPT is cleaving r(CUG)exp in living cells, total RNA was harvested and the amount of r(CUG)exp relative to β-actin was measured by real-time RT-PCR (Figure 5).[11d] These studies show that approximately 50% of r(CUG)exp is cleaved upon irradiation while in the absence of irradiation none of the transcript is cleaved. These data support the hypothesis that the improvement in pre-mRNA splicing defects observed with 2H-4-HPT in the presence of light are due to specific cleavage of r(CUG)exp and subsequent removal of the transcript by RNA degradation pathways. This cleavage frees MBNL1 from r(CUG)exp, allowing it to regulate pre-mRNA splicing.



Figure 5

The results from real-time RT-PCR analysis of r(CUG)exp in the presence of 2H-4-HPT with and without light. Top, schematic of the DMPK mini-gene that was used in these studies. Arrows indicate where primers are positioned.[10d] Data show that 2H-4-HPT ...



In summary, we have shown, for the first time, that small molecules can be designed to cleave a specific RNA in living cells and modulate function. This approach could be used in conjunction with photodynamic therapy to cleave specific cancer- or other disease-associated transcripts, to design chemical probes of RNA function or potentially selective therapeutics, or to probe RNA structure in vitro or in vivo,[14] provided that the RNA-binding preferences of small molecules were sufficiently defined.