UBC Theses and Dissertations
Inhibitors of human indoleamine 2,3-dioxygenas Vottero, Eduardo Ricardo
Indoleamine 2,3-dioxygenase (IDO) is a tryptophan degradation enzyme that is emerging as an important drug target. IDO is expressed by many human tumors and results in suppression of the T-cell based immune response. IDO has also been implicated in depression and in the formation of senile nuclear cataracts. Recognition of these vital roles played by IDO, has led to considerable interest and resulted in the development of potent and selective IDO inhibitors for use in research and as lead compounds for drug development. In the present work, a bacterial expression system for production of recombinant human IDO has been constructed, and the purified enzyme has been used to develop a high-throughput activity assay for IDO that has enabled assay-guided fractionation of extracts prepared from marine invertebrates by the Andersen group at this university. Screening of this library of ~4500 extracts led to the identification of potent inhibitors from two marine organisms. The polyketides annulin C, annulin A and annulin B were identified as the most potent IDO inhibitors from the hydroid Garveia annulata, and three quinones, adociaquinones A and B and xestoquinone were identified as potent IDO inhibitors from a sponge of the genus Xestospongia. Structure analogues of these natural compounds that are commercially available have also been studied. Most of these inhibitors exhibit K[sub i] values greater than any reported previously, none are substrate analogs, and those that have been evaluated exhibit uncompetitive inhibition with respect to tryptophan. The expression of human IDO in a Saccharomyces cerevisiae tryptophan auxotroph restricts yeast growth in the presence of low tryptophan concentrations, and inhibition of this IDO activity can restore growth. This growth response was used as an assay for IDO activity in vivo to screen crude extracts and the compounds present in the NCI Diversity Set for IDO inhibitors. In this manner, NSC 401366 (imidodicarbonimidic diamide, N -methyl-N'-9-phenanthrenyl-, monohydrochloride) was identified as a potent non-indolic IDO inhibitor (K[sub i] = 1.5 ± 0.2 μM) that is competitive with respect to tryptophan. The yeast growth restoration assay is simple and inexpensive. It combines desirable attributes of cell- and target-based screens and is an attractive tool for chemical biology and drug screening. The evolutionary relationship of IDO to some gastropod myoglobins suggests that IDO may undergo autoxidation in vivo such that one or more currently unidentified electron donors are required to maintain IDO heme iron in the active, ferrous state. To evaluate this hypothesis, yeast knockout mutants were used in combination with the yeast growth assay for IDO activity in vivo to demonstrate a role for cytochrome b₅ and cytochrome b₅ reductase in maintaining IDO activity in vivo. The availability of high throughput assays for IDO activity both in vivo and in vitro permitted the design of a variety of informative experiments that identify a number of new directions for study in which physical (kinetic, thermodynamic and spectroscopic) methods can be employed to understand the structural and mechanistic basis for the catalytic and physiological activities of this enzyme.
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