Design, synthesis and evaluation of novel immune checkpoint small molecule inhibitors as potential cancer treatment

Avigliano, Marialuce 2023. Design, synthesis and evaluation of novel immune checkpoint small molecule inhibitors as potential cancer treatment. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Immune checkpoints are receptor-mediated signalling pathways that exert a negative control on immune cells, playing a crucial role in maintaining self-tolerance and facilitating the evasion of tumours from immune surveillance. Over the past few years, monoclonal antibodies (mAbs) have brought about a significant transformation in cancer immunotherapy. However, these biological therapies still have several drawbacks. In the context of immunotherapy, the use of small molecules as inhibitors of negative immunological checkpoints has become a crucial and innovative area of research in drug discovery. This thesis investigates the design, synthesis, and in vitro assessment of novel small molecule inhibitors targeting the PD1/PD-L1 and CD200/CD200R pathways. PD-1 and PD-L1 are upregulated in many immune cells. Furthermore, PD-L1 has been seen to be excessively expressed on the surface of various forms of cancer, such as breast cancer, lung cancer, bladder cancer, lymphoma, glioma, and melanoma. In a previous work, a combination of computer-based screening and laboratory tests successfully identified five compounds (1-5) that can inhibit the interaction between PD1 and PD-L1 at μM concentrations. Out of these five compounds, compounds 4 and 5 were selected as the initial candidates for the optimisation process. Various analogues of both compounds were synthesised to investigate the correlations between their structure and activity. The novel compounds were evaluated in a displacement ELISA assay to test their ability to disrupt the original interaction. Some derivatives retained their hit activity, therefore demonstrating the significance of specific components or introducing new ones (11 and 50). Simultaneously, the toxicity of hits 1-5 was assessed and two cell-based assays were developed. The first assay involved directly measuring the displacement through light signal production, while the second assay involved analysing the changes in IL-2 amounts. P1019.7 was identified through the application of a comparable method used for studying the interaction between CD200R and CD200, a newly discovered immune checkpoint target. Various series of derivatives were synthesised to enhance the biological activity and drug-like properties of the compound, based on its structure. The novel compounds have undergone investigation in an optimised displacement ELISA assay, demonstrating encouraging preliminary results (86 and 91). Additionally, two cell-based assays were developed to study CD200R-CD200 interaction processes. These assays attempt to find additional cell-outputs for further study with the new derivatives. The first assay aims to evaluate MAPK pathway, specifically ERK phosphorylation, by western blot examination. Despite extensive optimisation, the interaction of CD200 peptide with CD200R has not decreased ERK phosphorylation as stated in the literature. The second cell-based experiment examined Fas and FasL gene expression, early apoptotic markers. The goal was to confirm that CD200R-CD200 interaction promotes NK92MI cell death. Both cell-based assays have undergone rigorous optimisation and will require ongoing efforts to become reliable and strong biological tools.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Pharmacy
Subjects:	Q Science > Q Science (General)
Funders:	Cancer Research Wales
Date of First Compliant Deposit:	14 August 2024
Last Modified:	14 Aug 2024 09:17
URI:	https://orca.cardiff.ac.uk/id/eprint/171385

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