Small molecule evolution: dynamic combinatorial selection applied to the discovery of novel RNA-binding small molecules.

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2008.RNA is no longer simply an intermediary between the genetic code of DNA and proteins; we are living in an RNA world. Ribonucleic acids have been shown to be involved in many important cellular processes such as transcriptional and translational regulation, enzymatic catalysis, protein function, and various essential interactions in cellular machinery and disease states. Selective and high affinity small molecule ligands for RNA binding have utility as tools in the understanding of fundamental RNA interactions, and in the development of novel therapeutics. As such, the development of small molecules capable of binding RNA sequences with high affinity and selectivity is currently a large and growing area of bioorganic chemical research. Dynamic combinatorial chemistry (DCC) is an emerging technique that allows both the synthesis and screening of small molecule libraries in situ. Governed by thermodynamic control, dynamic combinatorial chemistry employs reversible assembly of starting materials into a dynamic combinatorial library (DCL). Addition of a target biomolecule to the DCL enhances production of library members with affinity for the target; in essence it drives small molecule evolution. The research described herein developed a novel Resin Bound Dynamic Combinatorial Chemistry (RBDCC) strategy for the development of small molecule ligands for two biomedically important RNA hairpin sequences: a frameshift inducing stemloop involved in HIV-1 translation, and a (CUG)n triplet repeat RNA involved in myotonic dystrophy pathogenesis. An 11,325 member disulfide exchange based Resin Bound Dynamic Combinatorial Library (RBDCL), the largest reported to date, was synthesized and screened against these two important disease related RNA hairpin structures. In the context of HIV, screening the 11,325 member RBDCL selected a small molecule ligand that binds the target HIV-1 frameshift inducing RNA stemloop with high affinity (low μM dissociation constants), and significant selectivity over other related RNA hairpins. Additionally, in the context of myotonic dystrophy, library screening selected a series of ligands that bind the target (CUG)n RNA with high affinity (low μM dissociation constants), and good selectivity over other RNA sequences. Importantly, the (CUG)n ligands were shown to inhibit the interaction of (CUG)n RNA and the splicing factor MBNL1 in vitro. The (CUG)n – MBNL1 interaction causes misregulated splicing in myotonic dystrophy, and as such, compounds that inhibit the interaction hold promising therapeutic utility. Together, this research highlights the utility of the RBDCC library synthesis and screening approach to develop novel small molecule ligands with high affinity and selectivity for two biomedically important RNA sequences