Title: Synthesis and screening of spiroepoxy polycyclic small molecules
Description: Small molecules can exert specific biological effects through binding and altering functions of macromolecules. In the field of chemical genetics, small molecules are used to systematically perturb and study biological processes. The challenge of finding the appropriate small molecule binders largely requires two collaborative efforts: the assembly of small molecule "libraries" and the high-throughput screening of such libraries to identify biologically active molecules. This thesis concerns both of these efforts. A collection of structurally complex spiroepoxy polycyclic molecules with four distinct molecular skeletons were synthesized via tandem Becker-Adler and Diels-Alder reactions. Enantiomeric separation methods for these compounds were also optimized. Evaluation of representative spiroepoxy polycyclic molecules in both protein-binding and phenotypic assays identified some spiroepoxy[2.2.2]octenones as inhibitors of Hepatitis C viral RNA replication. Several structural analogues were synthesized as part of a continuing structure-activity relationship study. A preliminary analysis suggests the epoxide moiety is an important element of the antiviral effect.

Towards an interconnected effort in chemical genetics, a high-throughput cell-based assay was developed to identify small molecules that restored phosphorylation levels of S6 protein, an effector of mTOR, in the presence of rapamycin or amino acid starvation. An anthraquinone sulfonamide was identified from the screen and was found to regulate S6 phosphorylation independently of S6K1. In parallel, the same molecule was also identified as a small molecule that restored growth of yeast cells arrested by treatment with rapamycin. The characterization of this anthraquinone sulfonamide activity in mammalian and yeast cells suggested the existence of a conserved and uncharacterized S6 regulatory pathway. Preliminary transcriptional profiling of the compound in yeast suggested that it was a specific activator of the stress-responsive transcription factors, Msn2p/Msn4p.