Open Access Dissertation
Date of Award
Cancers are one of the leading causes of death in United States, effecting roughly 20% of the population. To develop effective anti-cancer agents, vast research is in progress that targets various factors leading to cancer. One such area of cancer research is developing drugs that inhibit histone deacetylase (HDAC) proteins. HDACs are histone modifying enzymes that regulate transcription of genes. Aberrant recruitment of HDACs to the transcription factors leads to tumor onset and growth. Because of their potential anti-cancer therapeutic interest, several HDAC inhibitors are in various stages of clinical trials. SAHA and Romidepsin are FDA-approved drugs for the treatment of cutaneous T-cell lymphoma. Human HDAC proteins have high sequence similarity and hence many known inhibitors non-specifically interact with all or most of the eleven HDAC isoforms, leading to possible side-effects. Hence, HDAC inhibitors specific to one HDAC isoforms (Isoform selective HDAC inhibitors) may overcome the potential clinical side effects and be effective anti-cancer drugs. In addition, isoform selective HDAC inhibitors would also be great tools to dissect the individual functions of HDAC isoforms in carcinogenesis. The most common assay to monitor the deacetylase activity of HDAC, is the in vitro fluorescence assay that employs HDAC-Fluor-De-LysTM substrate. To screen small molecule for isoform selectivity using the assay, baclovirus expressed recombinant HDAC proteins are employed. However, inadequate, low throughput and expensive screening strategies using recombinant HDAC proteins have stalled the identification of isoform selective HDAC inhibitors. We report here the development of a high throughput ELISA-based HDAC activity assay to screen the inhibitors against human-derived HDAC isoforms for selectivity in a cost effective manner. A mini pilot screen was performed using the developed assay to determine the selectivity of the SAHA derivatives modified at the linker position with hydrophobic substituents, the C7-SAHA, C2-SAHA, and C3-Ethyl SAHA derivatives. A HDAC6-selective inhibitor has been identified from the SAHA derivatives, which showed similar selectivity compared to the known HDAC6 selective tubastatin. The combined screening data of the SAHA derivatives suggest that modification at the linker region close to hydroxamic acid of SAHA with hydrophobic groups display some HDAC6 selectivity. However, SAHA derivatives modified at the region close to the capping group lead to more potent but less specific for compounds. Overall, the developed ELISA-based HDAC activity assay show immense potential in the identification of selective HDAC inhibitors in cost-effective and high throughput manner using mammalian-cell derived proteins, which contribute to the greater field of anti-cancer drug development and HDAC-related cancer research.
Padige, Geetha, "Development Of Elisa-Based Hdac Activity Assay For Identification Of Isoform Selective Hdac Inhibitors" (2014). Wayne State University Dissertations. 1054.