Toxic RNA inhibitors self-assembled in situ

Potent modulators of RNA function can be assembled in cellulo by using the cell as a reaction vessel and a disease-causing RNA as a catalyst. When designing small molecule effectors of function, a balance between permeability and potency must be struck. Low molecular weight compounds are more permeable while higher molecular weight compounds are more potent. The advantages of both types of compounds could be synergized if low molecular weight molecules could be transformed into potent, multivalent ligands via a reaction catalyzed by binding to a target in cells expressing a genetic defect. We demonstrate that this approach is indeed viable in cellulo. Small molecule modules with precisely positioned alkyne and azide moieties bind adjacent internal loops in r(CCUG).sup.exp, the causative agent of myotonic dystrophy type 2 (DM2), and are transformed into oligomeric, potent inhibitors of DM2 RNA dysfunction via a 1,3 Huisgen dipolar cycloaddition reaction, a variant of click chemistry. Additionally, we show that this approach is applicable to the r(CUG) repeating RNA that causes myotonic dystrophy type 1 (DM1). The click chemistry approach also allows for FRET sensors to be synthesized on-site by using r(CUG) repeats as a catalyst. Furthermore it is shown that small molecule binding sites in patient-derived cells can be identified by using reactive approaches termed Chem-CLIP and Chem-CLIP-Map. Lastly, it is shown that small molecules that target r(CUG) expansions can be designed to cleave this RNA by appending a small molecule with a nucleic acid cleaving module.

BACKGROUND(1) RNA dysfunction causes disease through various mechanisms, including microRNA silencing of pro-apoptotic proteins,.sup.[1] translation of aberrant protein,.sup.[2] and gain-of-function..sup.[3] It has been difficult, however, to design small molecule chemical probes of RNA function or lead therapeutics. If broadly applicable methods were developed to drug non-ribosomal RNAs with small molecules, it could have important applications in chemical biology and medicinal chemistry..sup.[4] One class of RNA-mediated diseases is caused by expanded repeating RNAs, or microsatellite disorders. There are >20 known microsatellite disorders, including myotonic dystrophy (DM) and amyotrophic lateral sclerosis (Lou Gehrig's Disease; ALS)..sup.[5] The cellular consequences of repeats are varied and can include alterations at the protein, RNA, and DNA levels. Myotonic dystrophy type 2 (DM2) is caused by a toxic gain-of-function by a r(CCUG) repeat expansion (r(CCUG).sup.exp. Myotonic dystrophy type 1 (DM1) is caused by a toxic gain-of-function by a r(CUG) repeat expansion (r(CUG).sup.exp).SUMMARY(2) We have designed inhibitors or modulators of the RNA that causes myotonic dystrophy type 2 (DM2) based on RNA motif-small molecule interactions..sup.[6] DM2 is caused by a toxic gain-of-function by a r(CCUG) repeat expansion (r(CCUG).sup.exp) located in intron 1 of the zinc finger protein 9 (ZNF9) pre-mRNA..sup.[7] The RNA folds into a hairpin structure that contains repeating units