Burns, EM; (2015) Structural and functional aspects of RET receptor tyrosine kinase maturation, signalling and chemical inhibition. Doctoral thesis , UCL (University College London).

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Abstract

The RET receptor tyrosine kinase (RTK) is crucial for embryonic and adult development of multiple organs, tissues and neurons. Gain-of-function mutations in the RET gene are found in human cancer, while loss-of-function mutations are associated with congenital anomalies of the kidney and urinary tract (CAGUT) and Hirschsprung's Disease (HSCR). Previous work has identified that some HSCR RET mutations result in a bottleneck in RET folding and a subsequent loss of RET export. This thesis presents work examining the characteristics of wild type (WT) and HSCR RET maturation, export and signalling in stably transfected mammalian cell lines. High throughput siRNA screening was used to identify components involved in WT and HSCR RET maturation and export; preliminary validation has implicated Endoplasmic Reticulum associated degradation (ERAD), autophagy and the N-glycosylation pathway. RET is also a validated cancer target, as a driver of cancers including multiple endocrine neoplasia (MEN) 2A and B. While there are several FDA-approved RET inhibitors available, their lack of specificity and potency has resulted in high levels of off-target toxicity and low life expectancy extensions. As such, a new generation of more optimal inhibitors is required. This thesis presents the investigation of the molecular basis of RET kinase inhibition, through the elucidation of the RET kinase domain (KD) structure bound to several ATP-competitive chemical inhibitors that are known to inhibit RET in vitro. Preliminary development of an updated RET pharmacophore is described, defining key residue interactions and combining observations with biochemical and thermal stability data.

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