The Rational Design and Synthesis of novel PPARγ Phosphorylation Inhibiting Agonism-Free Ligands and The Library Construction of Small Molecule Autophagy Modulators

Abstract

Chapter 1: The transcription factors peroxisome proliferator-activated receptors (PPAR) are well known for their key roles in the regulation of glucose and lipid metabolism. Naturally, the PPAR family has become an attractive target for researchers seeking to create compounds that can interact with the receptors and in turn modulate their downstream gene expression levels to instigate a desired biological response. With over-activation of PPARγ being linked to a cascade of serious side effects and relatively recent developments attributing cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation inhibition to the dysregulation of several insulin sensitizing genes, a rationally designed set of isoxazole-based compounds which exhibit moderate to no agonism towards PPARγ while simultaneously inhibiting phosphorylation of PPARγ at Ser273 have been synthesized and will be presented in this thesis. The compound set has been split into 3 families (carbamate, urea and amide), each differing slightly from the last in terms of rigidity and their electronic environment adjacent to the isoxazole core. The compound set was designed around a convergence principle in which functional group modifications that generated desired biological outcomes in one family were then incorporated into the other family sets for more accurate cross-comparisons along with converging our compound set into a smaller more active subset comprising the most biologically active compounds of all three of the family sets. The compounds were all tested for their effectiveness in inhibiting CDK5-mediated phosphorylation through western blotting tests and select compounds also underwent transcriptional activity assays to determine the extent of their agonism towards PPARγ. Afterwards, docking studies were carried out to first, gain insight into what possible interactions between our ligand and PPARγs binding pocket could be generating the observed bio-activity and second, provide us with some rationale regarding future functional group modifications. Of the 3 family sets, the urea set proved to be the most consistently active with regards to phosphorylation inhibition as well as having moderate to no agonism towards PPARγ, next was the carbamate set and last was the amide set. In Chapter 2, the isoxazole-based ligand core from Chapter 1 was incorporated into the library construction of small molecule autophagy modulators. Autophagy is the major intercellular degradation system by which unnecessary or dysfunctional cytoplasmic material is delivered and degraded within the lysosome. Intra and extracellular Amyloid Beta (Aβ) plaque accumulation within the brain, a signature pathological biomarker for Alzheimers disease, has been shown to leave neurons in an autophagy-dependent manner, and suggests that aggregation of intracellular Aβ plaque contributes to Alzheimers pathology. With this in mind, a small molecule library based off of a hit compound (P41H06) from our previous image-based phenotypic HTS for autophagy modulation was constructed and tested for its relative effect on autophagy modulation. The goal of this compound set was to synthesize compounds that have a moderate autophagy inducing effect, such as to avoid the potential side effects that may be associated from autophagy over-activation.