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The synthesis of inhibitors of TGF-β cytokine signalling and identification of their protein binding partners

posted on 06.02.2017, 02:56 by Ciayadi, Rudy
The transforming growth factor-β (TGF-β) protein superfamily is a set of signalling proteins (cytokines) which control many physiological processes such as cell proliferation and differentiation, immune responses, wound healing and various endocrine activities. Over-expression of specific TGF-β cytokines is associated with various disease states such as: cancer, inflammatory disorders, fibrosis, endometriosis, and also complications in female reproduction and pregnancy. Consequently, it has been proposed that inhibiting the signalling of specific TGF-β cytokines may be beneficial in diseases such as cancer, kidney disorders and pre-eclampsia. This could possibly be achieved using compounds that inhibit the kinase activity of the relevant TGF-β cytokine receptor. The initial aim of this project was to synthesize low molecular weight inhibitors of the kinase activity of TGF-β cytokine receptors and thus the signalling functions of TGF-β cytokines. An additional aim was to measure the selectivity of their inhibition of the medicinally important activin A and TGF-β1 signalling pathways. A further aim was to determine what other proteins the pharmacophore class bind, in order to confirm the mechanism of TGF-β signalling inhibition and to identify novel biological targets in the context of usage of the inhibitors as pharmaceuticals. A variety of inhibitors based on a 3,4-bis-pyridinylated pyrazole scaffold containing different phenyl or nitrogen heterocycle substituents were synthesized. Compounds containing phenyl or aromatic nitrogen heterocycle substituents inhibited both types of signalling in HEK-293 cell culture, with a selectivity preference for TGF-β1. Further specific modifications of phenyl or 3-pyridinyl-based substituents led to improved activities. Compounds containing 5-membered imidazole or pyrazole substituents were found to be comparable or more active inhibitors than those containing a phenyl, 2-pyridinyl or 3-pyridinyl substituent, and constitute a new lead class for further inhibitor development. Protein binding partners of the pharmacophore class were identified by immobilizing an exemplary inhibitor structure onto a chromatography resin and then using this affinity resin to capture proteins from whole cell lysates. Specifically-bound proteins were isolated and subjected to mass spectrometry peptide mass fingerprinting (MSPMF) to identify them. This process identified a number of different proteins, including medicinally important heat shock 70 kDa protein (HSP70), as potential binding partners of the pharmacophore class. Overall, these results suggest that the inhibitor class under investigation may have novel biological activities related to binding interactions with HSP70 and other proteins.


Campus location


Principal supervisor

Milton Hearn

Additional supervisor 1

Geoffrey Kelso, Simon Harris

Year of Award


Department, School or Centre

Centre for Green Chemistry


Doctor of Philosophy

Degree Type



Faculty of Science