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A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway.
- Source :
-
Journal of biomolecular screening [J Biomol Screen] 2016 Jul; Vol. 21 (6), pp. 626-33. Date of Electronic Publication: 2016 Mar 08. - Publication Year :
- 2016
-
Abstract
- Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol.<br /> (© 2016 Society for Laboratory Automation and Screening.)
- Subjects :
- Antineoplastic Agents therapeutic use
DNA Damage drug effects
DNA Helicases antagonists & inhibitors
DNA Helicases genetics
DNA Repair drug effects
Fanconi Anemia genetics
Fanconi Anemia pathology
Humans
Protein Interaction Maps drug effects
RecQ Helicases antagonists & inhibitors
RecQ Helicases genetics
Antineoplastic Agents chemistry
Drug Screening Assays, Antitumor methods
Fanconi Anemia drug therapy
High-Throughput Screening Assays methods
Multiprotein Complexes antagonists & inhibitors
Subjects
Details
- Language :
- English
- ISSN :
- 1552-454X
- Volume :
- 21
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Journal of biomolecular screening
- Publication Type :
- Academic Journal
- Accession number :
- 26962873
- Full Text :
- https://doi.org/10.1177/1087057116635503