Small molecules directly target Tau Pre-mRNA and affect splicing events

As 95% of human genes are alternatively spliced, abnormal splicing events are the common causes of many pathological conditions, including most neurodegenerative diseases. Therapeutic interventions have been developed to target RNAs, such as antisense oligonucleotides, one of which was recently approved by FDA to treat spinal muscular atrophy. Despite high potency and selectivity, current applications of antisense oligonucleotides are impeded by their limited tissue distribution. Directly targeting RNA with small molecules can serve as a promising alternative approach that can achieve systemic distribution.

Figure 1. An Inforna-based search of a database of RNA−small-molecule interactions that target the SRE led to the identification of compounds 1 to 4.

Here in this study, the Disney group developed a series of small molecules directly targeting tau pre-mRNA based on its sequence. Tau protein is one of the most popular targets for neurodegenerative diseases such as Alzheimer's disease. A mutation on tau mRNA can lead to aberrant alternative splicing, 

which results in overexpression and aggregation of an isoform of tau protein (also referred as 4R).  Unlike antisense oligonucleotides targeting unstructured RNA sequences, the small molecule compounds reported in this work target the 3-D structures of tau mRNA.

It is known that overexpression of tau is due to the alternative splicing at exon 10 within tau pre-mRNA. A hairpin element called SRE on the exon 10-intron 10 junction was destabilized due to a mutation, which leads to the over production of 4R. The 3D folds of SRE are searched against the Inforna database (a database developed by the Disney group) to identify compounds that can potentially bind to it.

A series of lead compounds that can potentially bind to SRE and stabilize the hairpin structure were selected by Inforna. Compound 1 from a previous study was used as a query compound in chemical similarity searching for more candidates (Fig. 1). Twenty compounds were identified, and the bioactivities were measured with a luciferase assay (Fig. 2), within which a low luciferase activity indicates the skipped splicing of exon 10. Among the compounds tested, compound 4 demonstrated higher potency at reducing the luciferase activity compared with compound 1.

Figure 2. Schematic diagram of a mini-gene used in cell-based assays with HeLa cells transfected with a DDPAC or WT construct and treated with compounds. Ligand binding at the A-bulge site stabilizes the hairpin and shifts the equilibrium away from unfolded RNA. The firefly luciferase reporter gene is in frame with exon 10 and expressed when exon 10 is included.

Three fluorescent assays were developed to screen compounds that bind to tau mRNA, emphasizing on different aspects of binding properties based on compound 4. The first one was designed based on the stacking of 4 and A-bulge within the RNA (Fig. 3A). The adenine was replaced with 2-aminopurine (2-AP) and binding of 4 would affect the emission of 2-AP. They used this assay for high-throughput screening of other compounds that bind to SRE. The hit compounds were validated with the second competitive binding assay for further evaluation (Fig. 3B). A quencher was labeled at the 5' end and FAM-labeled neomycin was used to bind to A-bulge in the hairpin. The emission intensity decreases when FAM is close to the quencher. When competitive compounds bind to mRNA and displace FAM labeled neomycin, the emission intensity increases.

The third one was designated to mimic the spliceosome recognition of tau pre-mRNA (Fig. 3C). FRET was used in this assay with Cy3 and Cy5 labeled at the 5' end and 3' end, respectively. Binding of U1 will unfold the RNA and decrease the FRET signal, while binding of potential compounds will stabilize the folding and thus the signal will increase. The results showed that the binding of compound 4 impacts the recognition of tau mRNA by the spliceosomal components, thus downregulated the expression of 4R.

Figure 3. In vitro assays to assess compound binding to tau RNA. (A) 2-AP assay. Addition of compound quenches emission. No data are available for 9 due to spectral overlap with 2-AP. (B) Fluorescence resonance energy transfer (FRET) FRET-based quenching (Q-)assay. (C) FRET-based U1 snRNA assay. To the right of the schemes are plots showing correlation between each in vitro binding assay and exon 10 skipping determined at 10 μM for all compounds except 6 and 7, which were determined at 12.5 μM.

With the structures of compound 1-4 as pharmacophores, they did further computation mining based on the properties like H-bonding and hydrophobic interactions. Over three hundred compounds were then selected and tested in the fluorescent assays and cell-based luciferase assay mentioned above (Fig. 3). Compounds 6-8 were selected with improved drug-like properties and used for in silico screening again in a set of 3 million compounds, leading to the identification of compound 9 with improved biochemical potency and therapeutic window (IC₅₀ toxicity >100 µM and IC₅₀ = 10 µM in a cell-based luciferase assay).

Melting points of WT and mutated tau pre-mRNA were also tested in the presence and absence of compounds 4 to 9, validating the higher thermal stability after the addition of the compounds.

Figure 4. The compounds used in Chem-CLIP and competitive Chem-CLIP (C-Chem-CLIP). The Chem-CLIP probe of bioactive ligand 9 (compound 11) reacts with tau pre-mRNA in LAN5 cells, whereas control probe 12 does not.

Compound 11 was designed based on compound 9 with the addition of photo-crosslinking module. Compound 12 was used as a control. The results showed that 11 reacted with tau mRNA with IC₅₀ at 10 µM, while 12 did not label any RNA (Fig. 4). Selectivity was also tested by treating other A-bulge containing RNAs with compound 9. It turned out that treatment of 9 did not affect the expression level of those RNAs in cell environment, indicating the good selectivity of compound 9 on tau RNA over other mRNAs.

Figure 5. Effect of 9 on the pre-mRNA splicing outcome of tau exon 10 in primary neurons harvested from human tau transgenic mice (htau mice). (Top) Primary neurons were extracted and cultured from htau mice brains. (Bottom) Compound 9 downregulated 4R/3R ratio dose-dependently, up to 50%. The 4R-to-3R ASO decreased 4R/3R ratio by 80%, whereas the scrambled ASO had no effect.

For further evaluation, 9 was tested in cultured primary neurons extracted from htau transgenic mice. 4R-to-3R ASO was used as positive control to inhibit the expression 4R and scrambled ASO as negative control (Fig. 5). Consisting with the results measured in above assays, 9 demonstrated dose-dependent effect on the expression level of 4R. The RNA-small molecule complexes were also extensively characterized by NMR.

Overall, the Disney group displayed a comprehensive study on evaluation of compounds binding to tau mRNA by extensive screening and testing. Compound 9 in this study demonstrated optimal properties and can be used for further optimization with conventional medicinal chemistry approaches. The strategies described in this paper can be applied to other interesting RNA targets as well. Even though it's still early to judge whether small molecules can achieve enough selectivity and potency for RNA targets, this emerging research area has gained many attentions both in industry and academia.

References

1. Chen, J. L., et al. (2020). "Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing." Journal of the American Chemical Society.  https://doi.org/10.1021/jacs.0c00768