Proteolysis-targeting chimaeras (PROTACs) have recently emerged as a novel paradigm that can hijack existing biological machinery to selectively degrade proteins of interest (POIs), in a catalytic fashion. This thesis explores the application of the PROTAC technology to members of the Janus kinase (JAK) family (JAK1 and JAK2), which have been implicated in several diseases, from inflammatory diseases to cancer. The thesis discloses the first account of PROTAC-mediated proximal membrane protein degradation. Initial efforts focused around selecting JAK warheads (JAK inhibitors) from which to generate PROTACs. The physicochemical properties of each template were optimised to enhance cell permeation, and following the identification of two JAK warheads, the PROTACs’ physicochemical properties were profiled in silico, prior to synthesis, as to identify a physicochemical property space optimal for JAK PROTAC cell penetration. The existing routes to the warheads were scrutinised, and more recent synthetic methodology was exploited to significantly reduce the number of steps and increase the overall yield of key intermediates. Following PROTAC library generation, the PROTACs were evaluated within in vitro biochemical and cellular assays to ascertain target engagement and cell penetration. It was confirmed that the JAK PROTACs were capable of engaging JAK and accessing intracellular compartments. A subset of PROTACs was selected based on chemical diversity of the warhead, linker and ligase binder. This subset was progressed to initial western blot assays within THP-1 cells, and it was identified that only PROTACs possessing an inhibitor of apoptosis (IAP) binder were able to induce JAK1/JAK2 degradation. As such, the IAPcontaining JAK PROTACs were further evaluated and six PROTACs were capable of inducing significant (up to 86 %) JAK1 and/or JAK2 degradation. To confirm the mechanism of JAK PROTAC-mediated JAK degradation to be both IAP- and proteasome-dependent, IAP competition assays were developed, which illustrated the IAP-dependent nature of JAK PROTAC-mediated degradation. Furthermore, proteasome inhibitor dosing, in combination with an active JAK PROTAC, prevented a decrease in intracellular JAK protein levels. Consequently, JAK PROTAC-mediated degradation was determined to be both IAP- and proteasome-dependent. In summary, reported herein is the design of cell penetrant JAK PROTACs from two distinct JAK chemotypes; these novel PROTACs induce degradation of JAK1 and JAK2 at varying levels, demonstrating an extension of the cutting edge PROTAC methodology to an unprecedented class of protein targets. The JAK PROTACs could find utility as biological tool molecules or be further optimised to develop therapeutics for a variety of diseases. [See thesis for illustration]
Date of Award | 1 Aug 2020 |
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Original language | English |
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Awarding Institution | - University Of Strathclyde
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Sponsors | University of Strathclyde |
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Supervisor | John Murphy (Supervisor) & David Nelson (Supervisor) |
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