Design, synthesis and evaluation of mechanism-based inhibitors of IDO1 and PROTAC-degraders of JAK2

Cundy, Nicholas John ORCID: https://orcid.org/0000-0001-6783-5422 (2020). Design, synthesis and evaluation of mechanism-based inhibitors of IDO1 and PROTAC-degraders of JAK2. University of Birmingham. Ph.D.

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Abstract

Over-expression of indolamine-2,3-dioxygenase 1 (IDO1) and programmed death-ligand 1 (PD-L1) are common phenotypes of immunoresistant cancers. The expression of IDO1 and PD-L1 is mediated by the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in response to stimulation by interferon-gamma (IFN gamma), with tumoral signalling specifically transduced via JAK2.
IDO1’s role in tumour immunoresistance is mediated by both depletion of its substrate, L-tryptophan, and by metabolites derived from its product, N-formyl-L-kynurenine. The mechanism of IDO1-mediated dioxygenation of L-tryptophan to N-formyl-L-kynurenine proceeds by either a radical or electrophilic-based mechanism. Radicals adjacent to cyclopropane rings undergo ring-opening driven by relief of ring strain, with the rate of ring-opening dependent on the stability of the resulting radical. A series of cyclopropane-containing IDO1 substrate mimics were designed to divert the hypothesised radical dioxygenation mechanism, potentially resulting in enzyme inhibition resulting from either a mechanistic ‘dead-end’ or covalent inactivation of the protein. Based on tryptophan and tryptamine, 1,2-cyclopropanated analogues were synthesised via a 1,3-dipolar cycloaddition of an amino acrylate and a indolo-diazo species or the Rh-catalysed cyclopropanation of a terminal olefin. Complimentary spirocyclic cyclopropane analogues of tryptophan and tryptamine were accessed via cyclodialkylation of 3-indoleacetonitrile. A library of sulfenylindoles was also synthesised to divert late-stage metabolic intermediates in a non-radical dependent manner. The synthesised inhibitors were exposed to IFN gamma-stimulated SCOV-3 cells and demonstrated a broadly poor ability to inhibit IDO1 turnover of tryptophan. Novel cell-free assays were developed to ascertain whether the reduced reactivity was related to compound cell-permeability and to further probe the mechanistic-detail of IDO1. A promising IDO1 inhibitor was identified in the cell-free assays and the further evaluation of the candidate is described.
Study of JAK2’s role in tumour immune escape has been complicated by inhibitors direct effects on immune cells, which is thought to be mediated by inhibition of other JAK family members. Proteolysis targeting chimeras (PROTACs) have the ability to degrade proteins within cells by harnessing the ubiquitin-proteasomal system. Based on FDA-approved JAK2 ligand fedratinib, a series of PROTACs were synthesised. The synthesised PROTACs were evaluated in JAK2-expressing HeLa cells. Degradation of JAK2 in HeLa cells was not observed in the preliminary western-blot analysis – future investigations of JAK2 PROTACs is discussed.

Type of Work:	Thesis (Doctorates > Ph.D.)
Award Type:	Doctorates > Ph.D.
Supervisor(s):	Supervisor(s) Email ORCID Grainger, Richard S. UNSPECIFIED orcid.org/0000-0003-0611-4386 Butterworth, Sam UNSPECIFIED orcid.org/0000-0002-6549-3753 Barnes, Nicholas M. UNSPECIFIED UNSPECIFIED
Licence:	All rights reserved
College/Faculty:	Colleges (2008 onwards) > College of Engineering & Physical Sciences
School or Department:	School of Chemistry
Funders:	Engineering and Physical Sciences Research Council
Subjects:	Q Science > QD Chemistry
URI:	http://etheses.bham.ac.uk/id/eprint/10457

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