Indoleamine 2,3-dioxygenase 1 (IDO1) is a major enzyme of the kynurenine pathway, where it catalyzes the conversion of L-tryptophan to N-formylkynurenine. Higher expression of the enzyme is observed in the majority of cancer related diseases, which is closely associated with bad prognosis and lower survival rates. IDO1 suppresses effector function of T and natural killer cells, differentiation and activation of immunosuppressive regulatory T lymphocytes and myeloid derived suppressor cells, which promotes spread of tumour cells, neovascularization and diminishes effectiveness of immunotherapies. Results of the first and the second phases of clinical trials showed promising antitumour activity; however, up to date no IDO1 inhibitors have passed all clinical phases.
As a part of master's thesis we wished to express in Escherichia coli, isolate and purify IDO1 that would show equivalent activity to the commercially available enzyme; synthesize novel IDO1 inhibitors and evaluate their inhibitory activity. With optimization of growth medium composition and culture condition we tried to secure higher expression. Cells were provided with heme precursors for its synthesis and we compared expression yields after 24 and 48 hours of cultivation in the presence/omittance of an inductor. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed to confirm identity of the product and its presence in soluble form. In addition, the protein was isolated and purified with immobilized metal affinity chromatography and gel filtration chromatography. Concentration of a purified enzyme was determined spectrophotometrically at 280 nm, but of greater importance was the absorbance ratio A404/A280, which correlates with proportion of cofactorial heme occupation, which influences enzyme activity. Highest IDO1 expression was achieved when the growth medium was supplemented with 5-aminolevulinic acid and bacteria were grown for 24 hours upon recombinant protein induction. The activity of produced enzyme was 5-fold lower compared to the commercial one, most likely due to the fact that the reducent was not added after the last purification step. We synthesized three final compounds based on 2-(3-(4-fluorophenil)-4-oxoisoxasolo[5,4-d]pyrimidine-7(4H)-yl)-N-phenylacetamide scaffold, where we added different substituents on the meta position of the phenyl ring, attached to the N-atom of acetamide group. Inhibitory activity of final compounds was determined with a fluorescence-based assay, where the compound 10 with trifluoromethyl substituent exhibited the most potent inhibitory activity, with the IC50 value of 55.1 µM. Although we failed to produce the enzyme with equivalent activity to the commercial counterpart, our contribution for future research cannot be neglected as it might soon be possible to obtain new crystal structures and design novel more potent inhibitors on the basis of binding site characteristics.
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