"RNA was long thought to be an 'undruggable' target for small molecules, because most cellular RNAs have extensive secondary structure, but only limited tertiary structure," Matthew Disney told BioWorld Science.
Disney is a professor of Chemistry at Scripps Research Institute, Florida Campus, and an overarching goal of his lab is to change that perception of "RNA as a legitimate drug target and not as an afterthought that it is now," he said.
In work reported in the November 5, 2020 issue of Cell Chemical Biology, Disney and his colleagues described a potential drug molecule targeting a CUG repeat expansion that causes two distinct diseases, myotonic dystrophy type 1 (DM-1) and Fuchs endothelial corneal dystrophy (FECD).
RNA repeat expansion transcripts cause or contribute to more than 40 disorders and are due to an inherited expansion of unstable repetitive elements, so-called microsatellites, in a gene or intron.
Since the 2016 approval of Spinraza (nusinersen; Biogen), a number of antisense oligonucleotides (ASOs) have been approved for the treatment of muscular dystrophies, and pioneer Ionis Pharmaceuticals and others have a broad pipeline of ASOs for multiple diseases.
But since tandem repeats are ubiquitous in the genome, the specificity of these ASOs is severely limited, and ASOs for microsatellite diseases recognize sequences unique to each gene, rather than the repeat expansion itself. Thus, an ASO needs to be customized for each disease, complementary to regions outside of the repeating sequence, even if two or more diseases are caused by the same repeat expansion.
On the other hand, small-molecule inhibitors that can target and bind the repeat expansion with high affinity and are broadly selective would be highly desirable -- for they could be, in principle, able to target any disease harboring the toxic repeat expansion.
The first such RNA-targeting small molecule, Evrysdi (risdiplam; Roche) was approved in September for the treatment of spinal muscular atrophy.
The molecule described by Disney and his team is based on bis-benzimidazole H as a ligand for the nucleotide UU internal loops found in CUG expansion repeat. By conjugating the ligand to bleomycin A5, Disney's group was able to selectively cleave the disease-causing RNA in mice models of DM-1 and FECD and reverse the molecular defects associated with the diseases.
Disney further added that "The CUG repeat itself is the same in both... DM and FECD, but the location in the genome is different. That is why the two diseases are different despite being caused by the same toxic RNA. Different tissues express the gene differently and that is why one is manifested as a muscular dystrophy while another is a front of the eye disease."
Biochemically, too, the two diseases present different mechanisms of RNA toxicity.
In muscular dystrophy, the CUG repeat is in a mature mRNA and the compound does not affect the mRNA levels. Instead, the repeat binds to and sequesters proteins like muscle blind-like 1 (MBNL1) that control pre-mRNA splicing. Sequestration of these proteins causes system-wide defects in patients, such as muscle atrophy and myotonia.
In FECD, the CUG repeat is in in a noncoding part of the gene called an intron. Normally, introns when copied into RNA are cut out of the RNA almost immediately and degraded by disposal systems in the cell. In FECD, the presence of the CUG-repeat expansion prevents the affected intron from being excised. When the small molecule binds to the intronic mRNA it is removed and degraded via the nuclear exosome.
Thus, "the small molecule can cause elimination via native quality control of a toxic CUG repeat. That is unprecedented but we have now found this in several diseases... This is the most important part of the work -- that small molecules binding the same repeats have differing consequences," Disney said.
Small molecule interacting with RNA
In this study, Disney's group used several structural modeling approaches to show that specific types of small molecule structures targeted a particular CUG repeat. The authors also showed that the molecule directly bound to the RNA in patient derived cells, as studied by the target profiling method, competitive chemical cross-linking and isolation by pull-down (C-Chem-CLIP).
Disney said that "the current work highlights not only new approaches to affect RNA biology but also a key platform that we have developed over the past 15 years." Next, the authors tested the therapeutic potential of the compound in DM1 and FECD. The lead compound was found to successfully reduce the depletion of MBNL1 and the loss of its function in cultured cells derived from patients with DM1, as well as in an animal model of the disease.
Disney is a co-founder of Expansion Therapeutics, a startup that specializes in developing therapies for patients with RNA repeat expansion disorders. Disney has also filed a patent (US20190152924A1) related to this work.
The compound reported in this study has been licensed to Expansion Therapeutics, which will take on the drug discovery aspects of the study. Disney said that he "was excited to use the results to deliver medicines to patients. Several of these small molecules interacting with RNA are bioactive in mice. The challenge is to get them to work in humans and that is very difficult... We are excited to see this hypothesis tested and, as always, the real test and value inflection is delivering compounds to patients. We want to get to that point, but these studies are the very beginning on that long road." (Angelbello, A.J. et al. Cell Chem Biol 2020, Advanced publication).