Investigating the immunological efficacy and target profiling of a small molecule immunomodulator
Creators: Tabana, Yasser
Publisher: name: University of Alberta Library
Year: 2023

<p>Immunotherapy has emerged as a promising new therapy for treating cancer and viral infections. There has been a growing interest in using small molecules for immunomodulation. Small molecules are compounds with low molecular weights that can be chemically synthesised and modified, making them ideal candidates for drug development. An example of such small molecules is Compound B, the focus of this Thesis. Compound B was initially identified through an earlier high throughput screening campaign by our group to discover immunologically active compounds. Despite its observed outstanding immunological activity and acceptable safety profile, the mode of action of this compound was not clear. Therefore, this thesis has been focused on investigating its immunological efficacy and on its target profiling. Compound B&#39;s physiochemical properties, immunological effects and transcriptomic profiling were first investigated in human peripheral blood mononuclear cells (PBMCs). In a collaboration with West group at the University of Alberta, a trifluoromethyl phenyl diazirine (TPD)-bearing trifunctional Probe 1 and a biotinylated (TPD)-bearing trifunctional Probe 12 were synthesized based on the chemical structure of Compound B. These two probes were used to identify the potential targets for Compound B via proteomic experiments. We then developed a procedure for pull-down purification using bovine serum albumin (BSA) in combination with photoaffinity and click chemistry (PAL) as an approach for target identification using immortalized human T lymphocyte Jurkat cells. Our data showed that Compound B exhibited drug-like physicochemical properties with minimal cellular toxicity. Compound B also showed increased T-cell proliferation and significantly enhanced the production of a variety of cytokines and chemokines in PBMCs with roles in both innate and adaptive immune responses. Additionally, we observed that Compound B upregulated a number of genes in PBMCs which are key upstream regulators associated with immune cell activation. In addition, we successfully developed a proof-of-concept protocol for pull-down purification using BSA and Probe 1 to be utilized as a model to identify the targets in living cells. We confirmed that Compound B and Probe 12 both have immunostimulatory efficacy in Jurkat cells. By performing LC-MS/MS analysis of the pull-down assay samples, we identified a total of 660 proteins. Out of these, 17 proteins demonstrated a significant increase in expression after treatment with Probe 12 compared to the control treated with the solvent (DMSO) (0.2%). Among the upregulated proteins, cyclin-dependent kinase 6 (CDK6) is a protein whose inhibition has been linked to immunomodulatory effects. Compound B was confirmed as a potential target for CDK6 through computer simulations, suggesting that Compound B may have potential to be developed as a CDK6 inhibitor. Taken together, the findings of this thesis provide a valuable starting point for the development of Compound B as a new class of immunotherapeutic agents.</p>