Modelling and targeting FLIP's interactions at the DISC

Abstract

FLIP is a well-established regulator of the extrinsic apoptotic pathway which is mediated through death receptors. Following death receptor ligation a protein complex called the DISC is formed and this is where procaspase 8 is gets activated by homodimerisation and autoprocessing. By binding to the DISC and dimerizing with procaspase 8, FLIP can prevent caspase 8 processing and thus inhibit the extrinsic apoptotic signalling cascade. .. " FLIP overexpression is a mechanism by which cancer cells survive apoptotic pressure therefore FLIP is an attractive target for anti-cancer therapies. However, strategies for targeting FLIP are challenging due to its lack of enzymatic activity and a crystal structure. Based on the structure of viral FLIP, a homology model of FLIP was built. Using a combination of molecular modelling, mutagenesis and functional assays, we determined the preferential sites and mode of interaction for FLIP-FADD. Moreover an homology model of procaspase 8 was also developed, again based on the structure of MC159 vFLlP. Using similar molecular modelling, mutagenesis and functional assays, we determined the mode of interaction of all three key apoptotic players at the DISC and a novel model of DISC assembly was developed. In addition, small molecule inhibitors of FLIP were developed. Having identified the critical region of the FLIP-FADD interaction, virtual screening was performed to identify candidate small molecules. The panel of candidate compounds was tested in a high throughput assay and this led to identification and prioritisation of a leading hit series. The selected compounds were further tested in a panel both cell-free and cell-based assays for FLIP-FADD inhibition and apoptosis induction. In summary, these studies provide insight into the modes of interaction between FLIP, FADD and procaspase 8 at the DISC and identify small molecule starting points for the development of inhibitors for therapeutically targeting FLIP for the treatment of cancer.EThOS - Electronic Theses Online ServiceGBUnited Kingdo