Abstract LB-037: Development and preclinical assessment of a first-in-class small molecule inhibitor of the major cell death regulator protein FLIP
Authors: Majkut Joanna , Higgins Catherine , Malik Adnan , Nemeth Zsusannah , Blurton Peter , Boffey Ray , Perrior Trevor R. , Johnston Patrick G. , Haigh David , Harrison Timothy , Longley Daniel B. 
Published: August 2015

Abstract
Background
Evasion of cell death is a major cause of resistance to cancer therapy, making proteins that regulate cell death clinically-relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers 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 cytotoxic chemotherapeutics. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We recently reported that there are important differences between FLIP and procaspase-8 in terms of both their binding affinities and preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. We now report our subsequent work leading to the development and pre-clinical characterisation of first-in-class inhibitors of FLIP.
Methods
Molecular modelling of the FLIP-FADD complex; virtual small molecule library screening; cell-free screening assays; cell-based activity assays; biophysical binding assays; in vivo anti-tumor studies.
Results
Molecular modelling of the FLIP-FADD complex 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 optimisation of these hits afforded lead and back-up series with nanomolar activity in 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). Lead compounds have been shown to block recruitment of FLIP to the TRAIL-R2 death-inducing signalling complex (DISC), confirming their on-target activity. Moreover, 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 and radiotherapy. To further confirm the mechanism of action, FLIP overexpression and procaspase-8 depletion abrogated the effects of these novel inhibitors. Lead molecules have been identified with ADME profiles suitable for in vivo evaluation. Using these compounds, single-agent anti-tumor effects have been demonstrated in xenograft models
Conclusions
The novel, first-in-class inhibitors of FLIP developed in this study have the potential for broad application in a range of cancers, either as monotherapy or in combination with other agents.
Acknowledgements
This work was supported by a grant from the Wellcome Trust's Seeding Drug Discovery Initiative (reference: 099470).
Reference
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, Adnan Malik, Zsusannah Nemeth, Peter Blurton, Ray Boffey, Trevor R. Perrior, Patrick G. Johnston, David Haigh, Timothy Harrison, Daniel B. Longley. Development and preclinical assessment of a first-in-class small molecule inhibitor of the major cell death regulator protein FLIP. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-037. doi:10.1158/1538-7445.AM2015-LB-037