Targeting cancer stem cells via small molecule inhibition of c-FLIP

Lee, Kok 2021. Targeting cancer stem cells via small molecule inhibition of c-FLIP. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The ability of cancer stem cells to self-renew indefinitely and differentiate into multiple tumour cell types has made their elimination critical to completely eradicate tumours. TRAIL (TNF-related apoptosis inducing ligand) is a death ligand that selectively induces apoptosis in cancer cells, but clinical trials utilising recombinant TRAIL or TRAIL agonists eventually failed due to the development of TRAIL resistance post treatment. c-FLIP inhibits TRAIL-mediated apoptosis and its overexpression has since been identified as one of the mechanisms of TRAIL resistance present in cancer stem cells. As a strategy to overcome TRAIL resistance, this project aims to develop new small molecule inhibitors of c-FLIP that re-sensitises cancer stem cells to TRAIL-mediated apoptosis. Twenty different compounds were identified through in silico screening as potential lead compounds targeting DED1 of c-FLIP. TKCC-05 pancreatic cancer cells and HeLa cervical cancer cells were treated with these compounds to test for their ability to sensitise cells to TRAIL-mediated apoptosis. The pan-caspase inhibitor Z-VAD-FMK was used to confirm apoptosis induced by the tested compounds was caspase-mediated. These compounds were also tested for their ability to inhibit the formation of MCF-7 colonies. Three out of twenty compounds (A7, A12 and A13) in combination with TRAIL successfully induced apoptosis in HeLa cells rescuable by caspase inhibitor. A7 was the best candidate out of the three successful candidates, with an EC50 of 72.23 μM. Three out of twenty compounds (A5, A7, and A12) in combination with TRAIL reduced MCF-7 colony formation via sensitisation to TRAIL. A7 was the best performing candidate overall and was selected to be further optimised. Seventeen analogues of A7 were synthesised and tested, yielding the analogue KYL5 that showed an improved EC50 of 40.85 μM. Preliminary modifications to the drug candidate A7 had shown that lipophillic functional groups were favored for improved c-FLIP inhibitory activity. Further cycles of modifications and optimization will be done in the future to establish structure-activity relationships. Several amino acid residues in the DED1 pocket of c-FLIP were mutated to identify key residues important for c-FLIP’s ability to bind onto procaspase-8 and FADD. HeLa cells expressing the 18-45 double mutant c-FLIP lost their resistance to TRAIL-mediated apoptosis, whilst cells expressing the R38A mutant c-FLIP did not respond to OH14 sensitisation. This had provided more information on the structure-function relationships of c-FLIP, and further evidence on DED1-mediated activity of c-FLIP. Overall, the project had successfully synthesised novel small molecules that showed c-FLIP inhibitory activity, and serves as an early proof of concept where pharmocological c-FLIP inhibition can be achieved. Whilst the analogue KYL5 is not ready for clinical testing, several opportunities for improvements are available for the candidate and will become the basis of future work.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Pharmacy
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	6 December 2021
Last Modified:	22 Dec 2022 10:53
URI:	https://orca.cardiff.ac.uk/id/eprint/145953

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