Evaluation of Photoreactive and Phenothiazine-based HDAC Inhibitors

Increased histone deacetylase (HDAC) activity and decreased acetylation status have been implicated in a wide array of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s disease. For this reason, developing HDAC inhibitors has risen as a promising therapeutic strategy to treat these ailments. While several HDAC inhibitors have proven quite effective in clinical and pre-clinical studies, there are still some critical barriers for developing successful inhibitors, including lack of HDAC isoform selectivity and potential drug resistance in cells. We hypothesize that we can improve the therapeutic potential of HDAC inhibitors for neurological diseases by (1) evaluating isoform selectivity in a disease-relevant system to aid in the design of selective inhibitors and (2) developing multifunctional HDAC inhibitors to reduce potential drug resistance. The first half of this thesis addresses the first goal, and focuses on optimizing the Binding Ensemble Profiling with Photoaffinity Labeling (BEProFL) approach, developed in our lab, to evaluate the selectivity of photoreactive HDAC inhibitors in a cell-based system. We chose the novel, biologically active HDAC probe, HD-55, as our model compound to optimize this methodology. We report that HD-55 crosslinks to HDAC8 in both a recombinant and cell-based system. Interestingly, this probe also binds the other Class I HDACs and additional proteins, which are currently unidentified. The next half of this thesis covers how we sought to evaluate novel phenothiazine-containing compounds as potential polypharmacological neuroprotective agents that inhibit HDAC function and counter oxidative stress. First, we evaluated these compounds for HDAC activity, radical scavenging ability, and DNA intercalation. Then, we evaluated whether these compounds could induce a neuroprotective effect in differentiated SH-SY5Y cells exposed to oxidative stress; a known model for neurodegenerative disease states. We report that the novel phenothiazine-containing compounds can inhibit HDACs with varying selectivity and scavenge radicals with similar efficacy to known antioxidants without intercalating DNA. We also found that these compounds induce differential increases in the viability of stressed neuronal-like cells. Due to these findings, we conclude that these novel compounds may be potential neuroprotective drugs.