title: A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway.
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.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Voter
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Manthei
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA jlkeck@wisc.edu.
authors.forename: James L
authors.initials: JL
authors.lastname: Keck
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry* / Q000627:therapeutic use; D004249:DNA Damage / Q000187:drug effects; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000235:genetics; D004260:DNA Repair / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D005199:Fanconi Anemia / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D046912:Multiprotein Complexes / Q000037:antagonists & inhibitors*; D060066:Protein Interaction Maps / Q000187:drug effects; D053484:RecQ Helicases / Q000037:antagonists & inhibitors / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D046912:Multiprotein Complexes; C518295:FANCM protein, human; D004265:DNA Helicases; D053484:RecQ Helicases
keywords: DNA repair; Fanconi anemia; protein interaction inhibitor screen