Abstract 382: Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP
Authors: Higgins Catherine A. , Fox Jennifer , Majkut Joanna , Fiedler Greti E. , Roberts Jamie , Humphreys Luke , Boffey Ray J. , Perrior Trevor R. , Harrison Timothy , Longley Daniel B. 
Published: August 2019

Abstract
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 and leukemias and has been shown to be a major mediator of resistance to chemo- and radio-therapies and to cell death induced by immune effector cells. 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 regulates induction of apoptosis and necroptosis - two major cell death mechanisms. Herein, we report the development and pre-clinical characterization of first-in-class inhibitors of FLIP.
Molecular modelling 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 protein-protein interaction assays 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 TRAIL-R2 DISC and display nanomolar activity in NSCLC cell-based assays 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 standard-of-care chemotherapeutics, ionizing radiation and targeted anti-cancer agents. FLIP overexpression and CRISPR-mediated deletion of procaspase-8 and FADD abrogated the effects of these novel inhibitors consistent with their expected mechanism-of-action. Recent studies have also demonstrated that lead FLIP inhibitors target Treg cells, which are key immuno-suppressive, tumor-promoting immune cells. Lead molecules have also demonstrated in vivo efficacy in NSCLC cancer models both as a single agent and in combination with a multivalent TRAIL agonist.
The first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of several cancers, including solid tumors and haematological malignancies, either as monotherapy or in combination with other agents.
Acknowledgements: This work was supported by a Seeding Drug Discovery award from the Wellcome Trust.
Citation Format: Catherine A. Higgins, Jennifer Fox, Joanna Majkut, Greti E. Fiedler, Jamie Roberts, Luke Humphreys, Ray J. Boffey, Trevor R. Perrior, 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 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 382.