1. Epigenetic Processes from a Molecular Perspective INBRE Meeting 2/16/10

2. Outline Analytical tools to measure epigenetic transformations –HPLC –MS –X-Ray crystallography Targeting epigenetic processes with small molecule therapeutics

3. HPLC High performance (or high pressure) liquid chromatography Main components: pump, column, detector

4. HPLC Solid Phase Materials Can vary column size, surface area, organic surface material as needed

5. Separation with HPLC A solvent polarity gradient is used to elute compounds based on polarity Using a “standard” C18 column, highly polar compounds will have little affinity for the greasy surface and pass through quickly relative to nonpolar compounds

6. Tandem HPLC/MS By passing HPLC eluent into a mass spectrometer, masses can be assigned to peaks as they elute For non-tandem systems, peaks can be collected separately as they elute and then inserted into a separate MS

7. MS Techniques “Soft” ionization techniques produce only whole molecular ions, simplifying data analysis “Hard” ionization techniques will fragment whole molecular ions into smaller molecular ions, giving additional structural detail

8. MS/MS Peptide Sequencing Polypeptide backbones fragment in a predictable manner. Fragment data can be used to reconstruct order of attachment within peptide/protein

9. MS/MS Peptide Sequencing “Ladder” sequencing used to construct primary structure

10. MALDI-TOF MS Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry A “soft” ionization method Laser energy is used to vaporize molecules with the use of an intermediary matrix molecule Useful for large biomolecules otherwise difficult to vaporize

11. MALDI-TOF MS Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

12. Examples of MALDI-TOF Data

13. X-Ray Diffraction Analysis

14. Epigenetic Regulatory Reactions Histone acetylation/deacetylation DNA methylation/demethylation

15. Histone Acetylation Promoted by histone acetyltransferase (HAT) Acetylation causes opening of histone protein arms, making DNA in chromatin more accessible Acetylated histones are a mark of gene activity

16. Histone Deacetylation Promoted by histone deacetylase (HDAC) Used to silence gene activity There are 11 known types of HDAC enzymes Targeting these HDACs specifically is a major goal of developing a drug therapy

17. HDAC Inhibitors How are HDAC inhibitors used as potential cancer treatment tools? Inhibiting HDAC will result in an increase of histone acetylation Highly acetylated histone tails will induce genes that suppress the cancer phenotype HDAC inhibitors were observed to block progression of cancer cells in culture

18. HDAC Inhibitors Histone acetylation is suggested to play a role in memory formation HDAC inhibitors are being explored as treatments for neurodegeneration and memory loss

19. MS to Analyze Histone Methylation Parent ion mass can be used to determine quantity of acetylated sites (COCH3 mass is +43) Ladder sequencing techniques can be used to determine where the acetylated lysine residues are located within the primary structure

20. X-Ray Diffraction to Study HDACs Crystal structure of protein allows active site to be defined in 3D Goal of small molecule inhibitor: block active site of enzyme so natural substrate (Lysine side chain) can not bind Co-crystallization with a bound inhibitor give direct insight into mode of action of therapeutic molecule

21. HDAC Mechanism A zinc metalloenzyme Activates the hydrolysis of the amide functional group using a bound water molecule

22. HDAC Protein Structure Cartoon depiction Surface depiction

23. HDAC Active Site Geometry Binding channel mapping Active site residues

24. HDAC Inhibitor Examples

26. DNA Methylation Methylation patterns are unique in different tissues Active genes are less methylated than inactive genes Methylated regions silence gene expression by interacting with proteins and preventing access to DNA

27. DNA Methylase Enzyme substitutes a methyl (CH3) group to cysteine units of DNA CG islands (this can be targeted for inhibition) Inhibiting this process would cause global decrease in methylation level of DNA In conjunction with other enzymatic tools, might DNA methylase inhibition be used as a tool to re-program cells? Revert to progenetor or stem cell state?

28. DNA Demethylation DNA believed not to be directly demethylated but rather methylated cysteine unit is removed entirely and replaced with unmethylated cysteine unit (hence no single enzyme to target for inhibition to perturb this process)

29. Conclusion Epigenetic modifications can be measured using molecular analysis tools such as HPLC, MS and X-ray diffraction Epigenetic modifications can be targeted therapeutically with the use of small molecule enzyme inhibitors