College of Science and Health Theses and Dissertations

Date of Award

6-2012

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Abstract

Inhibiting histone deacetylases (HDACs) can suppress tumor cell growth eventually leading to their death, makeing HDACs an important drug target. Therefore, understanding the mechanism behind inhibiting HDACs is imperative. To determine the thermodynamic parameters for the interaction of a known competitive HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), with zinc(II), the metal ion in the HDAC active site, and cobalt(II), isothermal titration calorimetry (ITC) was used. The results showed that SAHA binds to zinc(II) more strongly than it does to cobalt(II) as the equilibrium binding constants were 4.3 ± 0.5 x 102 M-1 and 2.0 ± 0.5 x 102 M-1 for zinc (II) and cobalt (II), respectively. In addition, the calculated value of ΔG⁰ for SAHA and zinc(II) of -15.0 ± 0.3 kJ/mol compared to -35.6 kJ/mol for SAHA and HDAC suggest that the hydroxamic acid functional group contributes significantly to the overall binding energetics of the interaction. Additional titration experiments using the literature prototype for hydroxamic acid binding, acetohydroxamic acid (AHA), suggested that the binding parameters obtained for SAHA interacting with zinc(II) and cobalt(II) in the non-physiological mixture are likely comparable to what they would be in aqueous conditions. To further characterize HDAC as a drug target, various nitric oxide (NO) donors, known to chemically modify and inhibit HDACs, were screened using an HDAC8 discontinuous fluorogenic assay. These assays allowed the effectiveness of the NO donors to be determined through comparison of HDAC8 percent activity relative to a control. The screened commercially available NO donors displayed no inhibition, whereas SAHA, a different class of NO donors, did decrease HDAC8 activity. Overall these studies suggest that inhibitors which bind in the active site of HDACs are more effective than those that covalently modify cysteine residues.