High-Throughput Molecular Imaging for the Identification of FADD Kinase Inhibitors

Abstract

Fas-associated protein with death domain (FADD) was originally reported as a proapoptotic adaptor molecule that mediates receptor-induced apoptosis. Recent studies have revealed a potential role of FADD in NF-κB activation, embryogenesis, and cell cycle regulation and proliferation. Overexpression of FADD and its phosphorylation have been associated with the transformed phenotype in many cancers and is therefore a potential target for therapeutic intervention. In an effort to delineate signaling events that lead to FADD phosphorylation and to identify novel compounds that impinge on this pathway, the authors developed a cell-based reporter for FADD kinase activity. The reporter assay, optimized for a high-throughput screen (HTS), measures bioluminescence in response to modulation of FADD kinase activity in live cells. In addition, the potential use of the reporter cell line in the rapid evaluation of pharmacologic properties of HTS hits in mouse models has been demonstrated.

Phosphorylation of FADD by the kinase CK1α promotes KRAS G12D -induced lung cancer

“…3B). To evaluate a requirement for CK1α, the kinase that phosphorylates FADD (14, 27–28), we treated K Luc cells with a CK1α inhibitor, CKI-7., We observed a marked decrease in the proliferative ability of CKI-7-treated K Luc cells, whereas that of CKI-7-treated KF Luc or Luc cells was not substantially affected (Fig. 3C).…”

A Novel Kinase Inhibitor of FADD Phosphorylation Chemosensitizes through the Inhibition of NF-κB

“…We have previously demonstrated that inhibition of FADD phosphorylation results in its ubiquitin-dependent degradation. Using MG132, a proteosome inhibitor, have confirmed that a decrease in total FADD levels in response to FADD dephosphorylation (i.e., in the presence of a FADD kinase inhibitor) can be reversed by inhibiting proteosomal degradation (18). …”

“…Research from our lab has shown these lines to express high levels of phosphorylated FADD protein, thereby providing the impetus toward selecting these models for the identification and evaluation of a potential FADD-kinase inhibitor. Using A549 lung cancer cells expressing our recently described FADD-Kinase Reporter, FKR (18), we performed a high-throughput screen with an NCI diversity compound collection and identified NSC 47147 as a potent inhibitor of FADD phosphorylation. NSC 47147 is a prodigiosin, a family of natural red pigments synthesized by a variety of microorganisms.…”

“…No vial of cells was cultured for more than 1–2 months. A549-FKR and SW620-BGCR cells have been previously described (18–19). A549-FKR findings were validated using freshly obtained A549 cultures from the ATCC.…”

“…The bioluminescent FADD-kinase reporter assay was executed as previously described (18). Briefly, A549 expressing FKR cells were seeded (1×10 5 cells/well) in opaque 96-well plates, 24-hours prior to assaying.…”