الفهرس | Only 14 pages are availabe for public view |
Abstract Histone deacetylases (HDACs) constitute an epigenetic enzyme family that was implicated in cancer and other diseases. Increasing evidence shows that by inhibiting a single isoform, one could regulate a focused subset of genes and achieve the desired therapeutic effect with lower toxicity. The isoform selectivity of HDACi is typically measured in biochemical assays using purified isolated recombinant enzymes and artificial substrate mimics, which doesn’t recapitulate the factors modulating HDAC activity in live settings. Therefore, development of novel chemical tools and assays in live cells, live tissues and in vivo is needed. Photoaffinity labeling uses small molecule trifunctional photoreactive probes (PRPs) that contain: (1) an affinity group which bind to the target protein active site, (2) a photoreactive group (PRG) that, upon UV irradiation, covalently crosslink to the proteins in proximity, and (3) a tagging group that can be conjugated to a reporter tag using bioorthogonal reactions for visualization, enrichment, and identification. In this study, a synthetic strategy to access chemically diverse PRPs based on both FDA approved and investigational, both non-selective and isoform selective HDACi is described, and used for synthesis of a set of chemically diverse HDAC PRPs and control compounds. Tetrafluorophenylazide was the most efficient among the studied PRG. The synthetic strategy was optimized to be applicable to the diverse chemical scaffolds. Molecular modeling guided the placement of the added moieties not to interfere with enzyme-PRP binding. The PRPs and their parent HDACi were tested for in vitro enzyme inhibitory activity, using a common HDAC biochemical assay against recombinant HDAC isoforms. The assay utilizes artificial substrate mimics with a fluorogenic moiety that produces fluorescence signal after being deacetylated by the HDAC. Rational incorporation of the PRGs in the structure of PRPs was found to modestly affect their potency and selectivity determined biochemically. Abstract xxiii S. M. Aboukhatwa, PhD thesis Pharmaceutical Sciences (Pharm. Chemistry), Tanta University 2019 Class I HDACs were identified as targets of the PRPs in MCF-7 cells, using mass-spectrometry based protein profiling. In triple negative breast cancer cells, the cell-based HDAC selectivity profiles of the PRPs were skewed toward HDAC3 irrespective of PRP chemotype and biochemical selectivity, or cellular HDAC isoform abundance. Unlike the HDAC protein abundance and the biochemical activity of PRPs, the PRPs chemotype and the cell type were key in defining HDAC isoform engagement in live SET-2, HepG2, HuH7 and HEK293T cells. Further, photolabeling studies were done in mice liver tissue (ex vivo) as a more realistic model of live cells in vivo. The druglike properties and liver microsomal metabolic stability profile of PRPs indicate their applicability for in vivo studies. The findings of the current study stress the importance of revisiting HDACi selectivity accounting for the multiple roles of HDACs and their regulation by post-translational modifications and multiprotein complex formation. The study reinforces that target engagement of HDAC isoforms by HDACi in vivo is significantly modulated in a cell- and tissue- type dependent manner. The study also highlights the importance of testing HDACi within the cellular context, in the presence of multiprotein complex components and posttranslational modification status. Ultimately, this knowledge can be used to develop and apply novel rational approaches and experimental tools to understand the mechanisms underlying the beneficial and adverse effects of HDACi as a basis for design of more potent and safer therapeutics for cancer. |