See the published version of this preprint at BMC Cancer.
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Umashankar Singh
Indian Institute of Technology Gandhinagar
Corresponding Author
ORCiD: https://orcid.org/0000-0001-8578-8201
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Background: Inhibition 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.
Methods: We 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.
Results: Our 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.
Conclusions: The 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.
Keywords
DNA-protein interaction, library screen, CGGBP1, Givinostat
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CURRENT STATUS: Published
View the published article at BMC Cancer.
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Editorial decision: Accept
On 14 Oct, 2020
Editor assigned
On 13 Oct, 2020
Submission checks complete
On 12 Oct, 2020
Editor invited
On 12 Oct, 2020
Version 3
Posted 02 Sep, 2020
No comments provided
Editorial decision: Minor revision
On 09 Oct, 2020
Review #4 received
Received 02 Oct, 2020
Review #3 received
Received 02 Oct, 2020
Review #2 received
Received 15 Sep, 2020
Review #1 received
Received 09 Sep, 2020
Reviewer #4 agreed
On 07 Sep, 2020
Reviewer #3 agreed
On 04 Sep, 2020
Reviewer #1 agreed
On 02 Sep, 2020
Reviewer #2 agreed
On 02 Sep, 2020
Reviewers invited
Invitations sent on 01 Sep, 2020
Editor assigned
On 30 Aug, 2020
Submission checks complete
On 29 Aug, 2020
Editor invited
On 29 Aug, 2020
Version (no preprint)
Posted 30 Jan, 2020
Review #5 received
Received 28 Jul, 2020
Reviewer #5 agreed
On 08 Jul, 2020
Editorial decision: Major revision
On 19 Mar, 2020
Review #3 received
Received 17 Mar, 2020
Review #4 received
Received 16 Mar, 2020
Reviewer #4 agreed
On 26 Feb, 2020
Review #2 received
Received 25 Feb, 2020
Reviewer #3 agreed
On 24 Feb, 2020
Review #1 received
Received 20 Feb, 2020
Reviewer #2 agreed
On 14 Feb, 2020
Reviewer #1 agreed
On 06 Feb, 2020
Editor assigned
On 03 Feb, 2020
Reviewers invited
Invitations sent on 03 Feb, 2020
Submission checks complete
On 02 Feb, 2020
Editor invited
On 02 Feb, 2020
This version was not preprinted
No comments provided
Editor assigned
On 28 Jan, 2020
Submission checks complete
On 27 Jan, 2020
Editor invited
On 27 Jan, 2020
No comments provided
Editor assigned
On 22 Jan, 2020
Editorial decision: Revise before sending to peer reviewers
On 22 Jan, 2020
Submission checks complete
On 17 Jan, 2020
Editor invited
On 17 Jan, 2020
First submitted
On 14 Jan, 2020
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A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
See the published version of this preprint at BMC Cancer.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Technical advance
Umashankar Singh
Indian Institute of Technology Gandhinagar
Corresponding Author
ORCiD: https://orcid.org/0000-0001-8578-8201
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Cancer.
No community comments so far
Editorial decision: Accept
On 14 Oct, 2020
Editor assigned
On 13 Oct, 2020
Submission checks complete
On 12 Oct, 2020
Editor invited
On 12 Oct, 2020
Version 3
Posted 02 Sep, 2020
No comments provided
Editorial decision: Minor revision
On 09 Oct, 2020
Review #4 received
Received 02 Oct, 2020
Review #3 received
Received 02 Oct, 2020
Review #2 received
Received 15 Sep, 2020
Review #1 received
Received 09 Sep, 2020
Reviewer #4 agreed
On 07 Sep, 2020
Reviewer #3 agreed
On 04 Sep, 2020
Reviewer #1 agreed
On 02 Sep, 2020
Reviewer #2 agreed
On 02 Sep, 2020
Reviewers invited
Invitations sent on 01 Sep, 2020
Editor assigned
On 30 Aug, 2020
Submission checks complete
On 29 Aug, 2020
Editor invited
On 29 Aug, 2020
Version (no preprint)
Posted 30 Jan, 2020
Review #5 received
Received 28 Jul, 2020
Reviewer #5 agreed
On 08 Jul, 2020
Editorial decision: Major revision
On 19 Mar, 2020
Review #3 received
Received 17 Mar, 2020
Review #4 received
Received 16 Mar, 2020
Reviewer #4 agreed
On 26 Feb, 2020
Review #2 received
Received 25 Feb, 2020
Reviewer #3 agreed
On 24 Feb, 2020
Review #1 received
Received 20 Feb, 2020
Reviewer #2 agreed
On 14 Feb, 2020
Reviewer #1 agreed
On 06 Feb, 2020
Editor assigned
On 03 Feb, 2020
Reviewers invited
Invitations sent on 03 Feb, 2020
Submission checks complete
On 02 Feb, 2020
Editor invited
On 02 Feb, 2020
This version was not preprinted
No comments provided
Editor assigned
On 28 Jan, 2020
Submission checks complete
On 27 Jan, 2020
Editor invited
On 27 Jan, 2020
No comments provided
Editor assigned
On 22 Jan, 2020
Editorial decision: Revise before sending to peer reviewers
On 22 Jan, 2020
Submission checks complete
On 17 Jan, 2020
Editor invited
On 17 Jan, 2020
First submitted
On 14 Jan, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Cancer.
Background: Inhibition 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.
Methods: We 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.
Results: Our 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.
Conclusions: The 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.
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
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