Abstract
BackgroundInhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins.MethodsWe have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen.ResultsOur primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding.ConclusionsThe assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.
Highlights
Inhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins
By applying our screening method we have identified that the HDAC inhibitor anti-cancer agent [42,43,44,45,46,47] Givinostat inhibits CGGBP1-DNA binding
An assay combining Dot-Blot and ImmunoDetection (DBID) detects in vitro DNA-protein interactions To develop a method for finding out a chemical inhibitor of DNA binding and testing it on CGGBP1, we needed to conform to the following preconditions: (i) detecting sequence-independent DNA-binding and its inhibition, (ii) the DNA-binding shall include binding to repeats, and (iii) no requirement of any prior knowledge about the structure of the target protein
Summary
Inhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. The advent of large scale screening technologies has allowed researchers to screen libraries of small molecule chemicals for their inhibitory activities against chosen target enzymes [6,7,8] The latter is a blind hit and trial approach, which can help us identify sub-optimal inhibitors of a target enzyme from a library that would need further optimization. Such optimization could be driven by the structural information of the target protein or for reducing the concentration of the chemical required for efficient inhibition. The knowledge and strategies of enzyme inhibitions by chemicals can not be extended for inhibition of DNA-protein interactions
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