The Disney group is centrally focused on developing a set of rules to allow for the rational exploitation of RNA drug targets present in genomic sequence. RNA is an important but a vastly under exploited drug or chemical probe target. For example: (i) for every protein drug target there is an RNA that could be potentially targeted; (ii) many viruses have RNA genomes, such as HIV and HCV, that could be targeted to develop new antivirals; (iii) many toxic RNAs are produced that cause a variety of neurological and neuromuscular disorders, such as Huntington’s disease and Myotonic Dystrophy; (iv) ribosomes are effective drug targets to treat bacterial infections as well as to stimulate read-through translation in diseases that are caused by pre-mature stop codons; and, (v) pre- and pri-microRNAs are disregulated in a variety of diseases.
Despite the critical roles that RNA plays in biology and thus as a therapeutic or chemical genetics probe target, only a very small number of RNA targets have been exploited as such. The central focus of the Disney group is thus to develop rational methods to exploit any RNA drug target in the human, or other, genome. To accomplish this lofty goal, we are developing methods to obtain information on the types of small molecules, or drugs, that bind RNA with high affinity and specificity and the types of RNA motifs, or folds, that bind small molecules with high affinity. These studies culminate in the development of an RNA motif-ligand database that is mined against genomic sequence to rationally design small molecules to target these RNAs. One therapeutic effort that is currently ongoing is in the rational design of small molecules that can be used to treat Orphan diseases such as Huntington’s Disease, Fragile X Syndrome (only known genetic cause of Autism), Myotonic Muscular Dystrophy, and the Sprinocerebellar ataxias, for example. The general scheme of this approach is depicted in Fig. 1
There are five main areas of research in the Disney group that are centrally focused on exploiting expertise in small molecule synthesis, biophysical chemistry, structural biology, and molecular biology. This includes:
Figure 1: Protocol for identifying the RNA structures that bind small molecules and using this information to rationally design ligands targeting RNA. In this experiment, which we call Two-Dimensional Combinatorial Screening (2DCS), both RNA and chemical spaces are probed simultaneously. A library of chemical ligands are spatially immobilized onto an agarose-coated microarray. The ligand library is hybridized to a library of RNA motifs under conditions of high stringency. RNAs that are bound to arrayed ligands are excised from the array surface, amplified, and sequenced. The structures of the selected RNAs are then predicted by free energy minimization. The output of 2DCS is a set of RNA motif-ligand partners that are deposited into a database. The database is then mined against toxic RNAs to enable the rational design of small molecules targeting them. These studies culminated in the rational and modular design of cell-permeable ligands targeting the toxic RNAs that cause Myotonic Dystrophy types 1 and 2. Designed ligands bind these target RNAs with higher affinity and specificity than the natural protein, Muscleblind-like 1. Our future goals are to expand the database and to develop computational tools to mine genomic sequence and identify new RNA targets to which these strategies can be applied.