Identification and characterisation of small molecule Lck inhibitors that modulate the T-cell receptor signalling pathway

Abstract

T-cells are an important element of the body’s immune system, able to initiate responses to antigens through the T-cell receptor (TCR). The protein tyrosine kinase Lck is critical to TCR signalling, as it is recruited to the receptor complex where it phosphorylates other proteins to initiate signal transduction. Despite the potential of Lck inhibition as a treatment for T-cell dysfunctions, none are in clinical use. The aim of this project was to screen new Lck inhibitors for activity in cell signalling assays and investigate new Lck inhibitor leads. Chapter 3 characterises the response to inhibition of Lck with the multi-kinase inhibitor dasatinib and the more restricted-profile inhibitor masitinib on T-cell receptor signalling in Jurkat cells. Dasatinib was highly efficacious at points proximal and distal to Lck in the TCR pathway including pZap70 and pERK respectively. Masitinib showed a weaker effect only in the proximal part of the pathway. Chapter 4 investigates responses in the TCR signalling pathway to a new chemical class of Lck inhibitors, where three of the compounds modulated the signalling pathway. Responses to dasatinib, masitinib and the novel compound 37804 on basal signalling and agonist activated signalling in Jurkat cells was also examined. All of the compounds reduced pLck and pZap70 levels before and after stimulation. Dasatinib was the most efficacious at blocking TCR signalling and is the only compound that reduced pERK levels. These data suggest compounds with different Lck conformational selectivity may have different effects on modulating the TCR signalling pathway. Chapter 5 investigates a pharmacological relationship between Lck and CSF1R. Despite limited chemical similarity between active inhibitor sets derived from the literature, molecular docking calculations using an ensemble of Lck structures enriched for compounds active against CSF1R. This indicates there could be cross reactivity of Lck and CSF1R inhibitors. Chapter 6 identifies potential Lck binders from clinical kinase inhibitors. FDA approved kinase inhibitors docked into the Lck ATP binding site were analysed for their ability to form a hydrogen bond with the Lck linker residue Met319. Comparison of the predicted binding modes for baricitinib and ribociclib in Lck with known orientations in other kinases indicated these are potential Lck inhibitors.