Biophysical Characterization and Identification of Small Molecule Inhibitors For the Epigenetic Reader Protein MBD2

Epigenetics;MBD2;methyl binding domain;cancer;cancer therapeutics;sequencing;biomarker;whole genome methyl sequencing;Mi-2;Nurd;Biochemistry

Wyhs, Nicolas AlanYegnasubramanian, Srinivasan ;

Johns Hopkins University

关键词: Epigenetics;    MBD2;    methyl binding domain;    cancer;    cancer therapeutics;    sequencing;    biomarker;    whole genome methyl sequencing;    Mi-2;    Nurd;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/59386/WYHS-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Significant evidence credentialing epigenetic changes as important drivers of cancer has surfaced. As a consequence, there is growing interest to find inhibitors to modulate the epigenome. Current FDA approved medications target either the DNA methylation writer protein DNMT1, or histone acetylation eraser protein family (HDAC). A longstanding interest in our lab is characterization of the 5-methylcytosine DNA methylation (meDNA) reader protein Methyl Binding Domain 2 (MBD2), a member of the MBD family and Mi-2 NuRD repression complex. Previous reports credential MBD2 as a viable cancer therapeutic target in animal and cell line models. In this thesis I describe efforts to find small molecule inhibitors of this epigenetic target. We first characterized the biochemical parameters that can affect MBD2 binding and selectivity for methylated DNA in vitro, determining salt and pH substantially contribute to the affinity and selectivity of the interaction. To prepare for structure aided design we crystallized the native and selenomethionine modified methyl binding domain polypeptides of MBD2 interacting with DNA. Crystals provided data to ~2.4Å resolution. Work is underway to complete refinement and solve the MBD2-MBD structure. With optimal conditions we tested TR-FRET and fluorescence polarization high throughput screening (HTS) assays to find inhibitors of the MBD2-meDNA interaction. Ultimately we chose TR-FRET, with Z’ factors >0.7 in 384 well plates. Pilot screening with the LOPAC1280™ library yielded an unexpected hit, NF449, which bound MBD2 in a dose dependent manner by isothermal calorimetry (ITC), with Ki in the nanomolar range.In collaboration with Scripps Florida investigators, we applied our HTS TR-FRET assay to the NCI-MLPCN library comprising 376,276 compounds. Follow-up and counter-screens identified 18 MBD2 specific small molecules with IC50’s from 1-17µM. We applied these to a TR FRET assay with multiple DNA methylation reader proteins to assess specificity. RNA expression of cancer cell lines treated with compound identified two which relieve DNA methylation mediated epigenetic promoter repression. An intercalator competition assay and ITC experiment confirmed that both bind DNA, independent of methylation status. ChIP experiments suggested that they alter chromatin to form a more open state at epigenetically repressed promoters. In summary we will soon solve a high resolution structure of MBD2-MBD bound to DNA. We also identified at least one small molecule which binds to MBD2 and disrupts the MBD2-meDNA interaction. Two other compounds interact with DNA and somewhat selectively disrupt DNA methylation mediated epigenetic repression.