Article

Genome-wide localization of small molecules

Nature Biotechnology (Impact Factor: 39.08). 12/2013; 32(1). DOI: 10.1038/nbt.2776
Source: PubMed

ABSTRACT A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chemical entities that interact with DNA or genome-associated proteins.

Download full-text

Full-text

Available from: Alla A Sigova, Oct 17, 2014
3 Followers
 · 
138 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cat-anionic vesicles were prepared by mixing non stoichiometric amounts of sodium bis-2-ethylhexylsulfosuccinate and dioctyldimethylammonium bromide in water. Depending on the concentration and mole ratios between the surfactants, cat-anionic vesicular aggregates are formed in part of the phase diagram. They have either negative or positive charges in excess and are endowed with significant thermodynamic and kinetic stability. Vesicle characterization was performed by combining dynamic light scattering and electro-phoretic mobility. It was inferred that vesicle size scales in inverse proportion with its surface charge density, diverges as the latter quantity approaches zero, and/or the mole ratio equals unity. Therefore, both variables are controlled by the anionic/cationic mole ratio. Small angle X-ray scattering, in addition, indicates that vesicles are uni-lamellar. Selected anionic vesicular systems were reacted with poly-L-lysine hydrobromide, or lysozyme. Polymer binding continues until an almost complete neutralization of the negatively charged sites on the vesicles surface is attained, as inferred by electro-phoretic mobility. Lipo-plexes are formed as a result of significant electrostatic interactions between cationic poly-electrolytes and negatively charged vesicles.
    Langmuir 02/2014; 30(10). DOI:10.1021/la500199w · 4.38 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The bromodomain and extraterminal (BET) protein Brd4 recruits transcriptional regulatory complexes to acetylated chromatin. While Brd4 is considered to be a general transcriptional regulator, pharmacological inhibition of BET proteins shows therapeutic activity in a variety of different pathologies, particularly in models of cancer and inflammation. Such effects have been attributed to a specific set of downstream target genes whose expression is disproportionately sensitive to pharmacological targeting of BET proteins. Emerging evidence links the transcriptional consequences of BET inhibition to the association of Brd4 with enhancer elements, which tend to be involved in lineage-specific gene regulation. Furthermore, Brd4 engages in direct regulatory interactions with several DNA-binding transcription factors to influence their disease-relevant functions. Here we review the current understanding of molecular mechanisms that underlie the promising therapeutic effects of BET bromodomain inhibition.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Understanding the mechanisms by which compounds discovered using cell-based phenotypic screening strategies might exert their effects would be highly augmented by new approaches exploring their potential interactions with the genome. For example, altered androgen receptor (AR) transcriptional programs, including castration resistance and subsequent chromosomal translocations, play key roles in prostate cancer pathological progression, making the quest for identification of new therapeutic agents and an understanding of their actions a continued priority. Here we report an approach that has permitted us to uncover the sites and mechanisms of action of a drug, referred to as "SD70," initially identified by phenotypic screening for inhibitors of ligand and genotoxic stress-induced translocations in prostate cancer cells. Based on synthesis of a derivatized form of SD70 that permits its application for a ChIP-sequencing-like approach, referred to as "Chem-seq," we were next able to efficiently map the genome-wide binding locations of this small molecule, revealing that it largely colocalized with AR on regulatory enhancers. Based on these observations, we performed the appropriate global analyses to ascertain that SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the Jumonji domain-containing demethylase, KDM4C. Global location of candidate drugs represents a powerful strategy for new drug development by mapping genome-wide location of small molecules, a powerful adjunct to contemporary drug development strategies.
    Proceedings of the National Academy of Sciences 06/2014; 111(25). DOI:10.1073/pnas.1404303111 · 9.81 Impact Factor