Synthesis and Investigation of Covalent IDO1 Inhibitors as Potential Immunotherapeutic Agents in Cancer Treatment

Abstract

Immunotherapy, which involves harnessing the individual’s immune response against cancer cells, is seen as a new era of cancer treatment. Although effectiveness is patient-dependent, there are mechanisms which can be modulated to sensitize the immune system for therapy. The kynurenine pathway, which produces immunosuppressive metabolites such as kynurenine, is a key example. The rate limiting step, production of kynurenine from tryptophan, is controlled by two enzymes, indoleamine-2,3-dioxygenase 1 (IDO1) and tryptophan-2,3-dioxygenase 2 (TDO), that are elevated in most clinical cancers. Inhibition of this pathway, at the IDO1/TDO step, can reduce the levels of these metabolites resulting in reactivation of the immune system. Since the discovery of these enzymes, hundreds of small molecule IDO1/TDO scaffolds and various dual inhibitors have been reported, but none has yet to reach the market. This is attributed to poor treatment outcomes due to the incomplete arrest of the kynurenine pathway, as production of immunosuppressive metabolites lingers. Protein active-site modelling studies revealed the presence of the cysteine residue Cys129 in the active site of IDO1, implying potential for covalent inhibition through Michael addition onto the cysteine thiol. Covalent inhibition has potential to address issues attributing to the poor outcomes, such as elevated potency, half-life, and reduced frequency for dosing. As a proof of concept, a series of 9 compounds analogous to LY3381916, a clinical IDO1 inhibitor candidate, were synthesized utilizing the 2,3-dihydroindole scaffold to investigate the feasibility of covalent binding. Alterations of the heterocycles and the warheads for binding to Cys129 were made to establish the moieties and positional changes to exert the target effects. Biological testing demonstrated the m-phenyl analogue with an acrylamide warhead to be most promising, exhibiting sub-micromolar IC50 concentrations and activity retention. Further studies revealed additional promising compounds, namely the 4-formylphenyl analogue, which displayed high potency, and the 4-mercaptophenyl analogue, which expressed activity retention when tested for covalent binding. These findings highlighted the potential for covalent IDO1 inhibitors, leading to the possibility of complete arrest of the kynurenine pathway.