A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway.
J Biomol Screen
; 21(6): 626-33, 2016 Jul.
Article
en En
| MEDLINE
| ID: mdl-26962873
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Ensayos de Selección de Medicamentos Antitumorales
/
Complejos Multiproteicos
/
Anemia de Fanconi
/
Ensayos Analíticos de Alto Rendimiento
/
Antineoplásicos
Tipo de estudio:
Diagnostic_studies
/
Guideline
/
Screening_studies
Límite:
Humans
Idioma:
En
Revista:
J Biomol Screen
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2016
Tipo del documento:
Article
País de afiliación:
Estados Unidos