Inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) for cancer therapy

Abstract

Immune escape is a central hallmark of cancer. A tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase-1 (IDO1) is a dominant immune escape mechanism in a broad range of human tumours and its expression is associated with a poor prognosis in cancer. Blockade of IDO1 by small-molecule inhibitors is a validated cancer therapy and two IDO1 inhibitors are showing promise in human clinical trials. The work in this thesis contributed to an overall goal at the Auckland Cancer Society Research Centre to identify and investigate the mode of action of novel IDO1 inhibitors as potential anti-cancer agents. It entailed the establishment of a sensitive and automated IDO1 enzyme assay to screen compound libraries for potential drug development leads. The new fluorescence IDO1 enzyme assay developed in this work is 30-fold more sensitive (limit of detection 153 nM N-formylkynurenine) than pre-existing assays. It is economical and features low interference from test compounds. The assay utilises a fluorescing tetrahydroquinolone adduct formed in a chemical reaction between Nformylkynurenine and cyclic amines involving transamidation and amine rearrangement, not previously described. This assay was automated and used to screen the National Cancer Institute Diversity Set III library (1,597 compounds) and to validate the thirty hits obtained from the screening of a commercial library of 40,000 molecules. This afforded eight IDO1 inhibitors from which pyrimidinone, indolonoxide, and isoxazole classes emerged as suitable drug development leads. All three showed excellent cell permeability and good potency (IC50 0.066 - 8 μM) in cell-based assays with negligible cell toxicity. Three of the top eight hits, including pyrimidinone, blocked IDO1 activity reversibly, identical to a well-studied IDO1 inhibitor, 4-phenyl-1H-imidazole. The other five hits, including isoxazole, elicited essentially irreversible IDO1 inactivation, an inhibitory mechanism not previously documented for IDO1 inhibitors. Testing the NCI library against serine-167 and cysteine-129 alanine replacement IDO1 mutants, established that serine-167 but not cysteine-129 in the IDO1 active site, is important for the binding of a broad range of inhibitors. This project discovered three novel IDO1 inhibitors suitable for drug development. The isoxazole lead is currently being optimised by rational medicinal chemistry for development as a potential anti-cancer drug for the future.