Evasion of cell death is a major cause of resistance to anti-cancer therapies, making proteins that regulate cell death clinically relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and has been shown by us and others to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors, such as TRAIL-R1 and R2, and treatment with chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We have found that there are important differences between FLIP and procaspase-8 in terms of their preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. Herein, we report the development and pre-clinical characterization of first-in-class inhibitors of FLIP.

Methods Molecular modelling of the FLIP-FADD interaction; cell-free screening assays; cell-based activity assays; biophysical binding assays; on-target mechanism-of-action studies, in vivo studies.

Results Molecular modelling of the FLIP-FADD interaction identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using a FLIP-FADD protein-protein interaction assay identified hits with on-target activity. Medicinal chemistry optimization of these hits identified a series of compounds that are able to disrupt FLIP’s interaction with the DISC and display nanomolar activity in NSCLC and CRC cell-based assays (i.e. caspase activation, cell death and cell survival), which is in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). The pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL, and lead-molecules have been shown to potentiate the effects of the standard-of-care chemotherapeutics such as e.g. cisplatin (NSCLC) or 5FU (CRC). FLIP overexpression and CRISPR-mediated procaspase-8 deletion abrogated the effects of these novel inhibitors consistent with the expected mechanism-of-action. In addition, using peripheral blood mononuclear cells (PBMCs), we demonstrate that FLIP inhibitors have selectivity against cancer cells. Finally, we identified lead molecules with ADME profiles suitable for in vivo evaluation and using these compounds, single-agent anti-tumor effects have been demonstrated in xenograft models.


The first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of cancer, either as monotherapy or in combination with other agents.


This work was supported by a Seeding Drug Discovery award from the Wellcome Trust.

1. Majkut, J., et al., Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly. Nat Commun, 2014. 5: p. 3350.

Citation Format: Joanna Majkut, Catherine Higgins, Jennifer Fox, Greti E. Fiedler, Timothy Harrison, Daniel B. Longley. Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-305. doi:10.1158/1538-7445.AM2017-LB-305