Development of Fluorescent Probes for the Real-time Detection of Histone Deacetylase Activity

摘要

Significant research efforts have advanced our understanding of lysine acetylation in biology, but many questions still remain unanswered. In this thesis, we discuss the development of fluorescent probes as a general platform for the real-time detection of activity of enzymes responsible for cleaving lysine post-translational modifications.;Chapter 1 introduces lysine acetylation in biology and histone deacetylases (HDACs). The chapter discusses the limitations of transposing the current deacetylase activity assay designs into cellular assays and focuses on the benefits of developing a general platform to monitor cellular deacetylase activity.;Chapter 2 describes the design and synthesis of a small-molecule deacetylase probe that acts as both a histone deacetylase substrate and optical reporter. Real-time detection of deacetylase activity with recombinant HDACs as well as in a diluted HeLa nuclear extract was demonstrated. Furthermore, the IC 50 of an HDAC inhibitor was determined in the presence of HDAC3/NCOR1, proving the utility of our small-molecule probe. Lastly, the probe was employed to detect overall deacetylase activity in live cells. In A549 cells overexpressing HDAC6, a significant increase in average fluorescence per cell was observed relative to wild-type cell expressing endogenous levels of HDACs, demonstrating that the probe can detect changes in cellular deacetylase activity.;Chapter 3 describes the development of peptide-based deacetylase activity probes with a similar amine-reactive optical reporter moiety. These probes expanded the scope of our deacetylase assay to study the NAD+-dependent sirtuins, showing that the probes with the natural peptide backbone are better substrates for these isozymes. Deacetylase activity with recombinant sirtuins as well as metal-dependent HDACs was monitored in real time. With the ability to monitor sirtuin activity, the role of Fe3+-sirtinol complex as an inhibitor of SIRT2 activity was investigated. Lastly, deacylase activity of HDAC3/NCOR1 was monitored using a decrontonylase variation of our peptide-based activity probe, showing the broader appeal of our design for studying other deacylation processes.;Chapter 4 summarizes a side project highlighting the development of a probe aimed at selectively coordinating free magnesium in biological systems for fluorescence-based detection. The probe described requires optimization and possible design suggestions are provided in the chapter.