thapsigargin and Fanconi-Anemia

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.

Topics: Antineoplastic Agents; DNA Damage; DNA Helicases; DNA Repair; Drug Screening Assays, Antitumor; Fanconi Anemia; High-Throughput Screening Assays; Humans; Multiprotein Complexes; Protein Interaction Maps; RecQ Helicases

Fanconi Anemia (FA) is a rare and complex inherited blood disorder associated with bone marrow failure and malignancies. Many alterations in FA physiology appear linked to red-ox unbalance including alterations in the morphology and structure of nuclei, intermediate filaments and mitochondria, defective respiration, reduced ATP production and altered ATP/AMP ratio. These defects are consistently associated with impaired oxygen metabolism indeed treatment with antioxidants N-acetylcysteine (NAC) and resveratrol (RV) does rescue FA physiology. Due to the importance of the intracellular calcium signaling and its key function in the control of intracellular functions we were interested to study calcium homeostasis in FA. We found that FANCA cells display a dramatically low intracellular calcium concentration ([Ca(2+)]i) in resting conditions. This condition affects cellular responses to stress. The flux of Ca(2+) mobilized by H2O2 from internal stores is significantly lower in FANCA cells in comparison to controls. The low basal [Ca(2+)]i in FANCA appears to be an actively maintained process controlled by a finely tuned interplay between different intracellular Ca(2+) stores. The defects associated with the altered Ca(2+) homeostasis appear consistently overlapping those related to the unbalanced oxidative metabolism in FA cells underlining a contiguity between oxidative stress and calcium homeostasis.

Topics: Acetylcysteine; Antioxidants; Calcium; Calcium-Transporting ATPases; Carbocyanines; Cell Line; Fanconi Anemia; Fanconi Anemia Complementation Group Proteins; Fibroblasts; Heterocyclic Compounds, 3-Ring; Homeostasis; Humans; Hydrogen Peroxide; Kinetics; Microscopy, Confocal; Mitochondria; Models, Biological; Resveratrol; Stilbenes; Thapsigargin