Our research is aimed at the design and synthesis of small molecule modulators of Histone Acetyltransferase (HAT) enzymes. The paucity of potent and selective small molecule HAT modulators has limited our understanding of the biological functions of this protein family. We are focused specifically on members of the GNAT and MYST families, as there is a wealth of high-resolution structural information available for several important members. Additionally, genetic studies suggest that selective small molecule modulators of these HATs may have potential therapeutic applications in the treatment of a variety of cancers and metabolic disorders. We have synthesized a structurally diverse library of molecules and developed a robust biochemical assay for HAT activity. The structures of the GNAT and MYST active sites demonstrated that each contains a cysteine residue, although the position of the cysteine is distinct in the two families. We have therefore initially focused our synthetic efforts on electrophilic small molecules, which have the potential to interact with the active site cysteine. We have synthesized libraries of reversible and irreversible electrophilic small molecules, and are currently screening these in vitro for modulation of HAT activity. Initial hits will be further developed to increase potency and specificity. We are developing a series of "clickable" HAT probes based on the electrophilic core structure and using these in human cells and lysates to better understand their modes and mechanisms of action. A recent study revealed a novel regulatory region of a human HAT protein outside of the canonical catalytic domain. We are currently extending these studies to understand this regulatory mechanism and further optimize our small molecule modulators. We are also developing derprobes for specific human HAT activities. These probes are constructed from modular protein domains that undergo a conformational switch in response to lysine acetylation. This change in conformation is measured indirectly using Frster Resonance Energy Transfer (FRET). These probes are suitable for in vitro and cell-based studies of HAT activity. The domain structure of the probes will be varied to provide simultaneous read-out of multiple HAT activities within the same cell. Fluorescence imaging techniques are used to visualize and quantify relative HAT activity under a variety of cellular conditions and treatments. These are being developed for the eventual application to cell-based screening for novel molecular modulators of HAT and HDAC pathways.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$403,782
Indirect Cost
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State
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Cheng, Jie; Chen, Chi; Kristopher, Krausz W et al. (2013) Identification of 2-Piperidone as a Biomarker of CYP2E1 Activity Through Metabolomic Phenotyping. Toxicol Sci 135:37-47
Sakakibara, Nozomi; Chen, Dan; Jang, Moon Kyoo et al. (2013) Brd4 is displaced from HPV replication factories as they expand and amplify viral DNA. PLoS Pathog 9:e1003777
Johnson, Caroline H; Bonzo, Jessica A; Cheng, Jie et al. (2013) Cytochrome P450 regulation by α-tocopherol in Pxr-null and PXR-humanized mice. Drug Metab Dispos 41:406-13
Johnson, Caroline H; Slanar, Ondrej; Krausz, Kristopher W et al. (2012) Novel metabolites and roles for ýý-tocopherol in humans and mice discovered by mass spectrometry-based metabolomics. Am J Clin Nutr 96:818-30
Matsubara, Tsutomu; Tanaka, Naoki; Krausz, Kristopher W et al. (2012) Metabolomics identifies an inflammatory cascade involved in dioxin- and diet-induced steatohepatitis. Cell Metab 16:634-44
Matsubara, Tsutomu; Tanaka, Naoki; Sato, Misako et al. (2012) TGF-β-SMAD3 signaling mediates hepatic bile acid and phospholipid metabolism following lithocholic acid-induced liver injury. J Lipid Res 53:2698-707
Johnson, Caroline H; Patterson, Andrew D; Krausz, Kristopher W et al. (2012) Radiation metabolomics. 5. Identification of urinary biomarkers of ionizing radiation exposure in nonhuman primates by mass spectrometry-based metabolomics. Radiat Res 178:328-40
Johnson, Caroline H; Patterson, Andrew D; Krausz, Kristopher W et al. (2011) Radiation metabolomics. 4. UPLC-ESI-QTOFMS-Based metabolomics for urinary biomarker discovery in gamma-irradiated rats. Radiat Res 175:473-84
Cho, Joo-Youn; Matsubara, Tsutomu; Kang, Dong Wook et al. (2010) Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge. J Lipid Res 51:1063-74
Tosh, Dilip K; Chinn, Moshe; Yoo, Lena S et al. (2010) 2-Dialkynyl derivatives of (N)-methanocarba nucleosides: 'Clickable' A(3) adenosine receptor-selective agonists. Bioorg Med Chem 18:508-17