Inhibitors of human indoleamine 2,3‐dioxygenase identified with a target‐based screen in yeast

Abstract

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 to help them escape immune detection, and it has been implicated in depression and in the formation of senile nuclear cataracts. There is a need for potent and selective IDO inhibitors for use in research and as lead compounds for drug development. We show that expression of human IDO in a Saccharomyces cerevisiae tryptophan auxotroph restricts yeast growth in the presence of low tryptophan concentrations and that inhibition of IDO activity can restore growth. We use this assay to screen for IDO inhibitors in collections of pure chemicals and crude natural extracts. We identify NSC 401366 (imidodicarbonimidic diamide, N-methyl-N'-9-phenanthrenyl-, monohydrochloride) as a potent nonindolic IDO inhibitor (Ki=1.5 +/- 0.2 microM) that is competitive with respect to tryptophan. We also use this assay to identify the active compound caulerpin from a crude algal extract. 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.

A Powerful Yeast-Based Screening Assay for the Identification of Inhibitors of Indoleamine 2,3-Dioxygenase

“…We generated a humanized yeast strain-BLOCKADE-where both pathways that lead to NAD + production were abolished by co-deletion of BNA2 and NPT1 genes, making cell survival dependent on the activity of human IDO. Although previous studies suggested that expression of human IDO in yeast was able to promote tryptophan degradation, 21 our work showed for the first time that IDO activity is able to functionally complement disruption of the kynurenine pathway caused by BNA2 deletion. Growth of the BLOCKADE strain was responsive to the level of IDO activity, providing a readout for a life/death assay for the identification of IDO inhibitors.…”

“…24,25 Therefore, most yeast assays are performed using modified strains where pumps are deleted. 21,26 On the contrary, the BLOCKADE strain kept all the transporter systems intact, as we proposed to detect only compounds with high specificity. We deliberately expected to lose some potential hits, to ensure we were selecting the most potent compounds.…”

Tryptophan degradation in autoimmune diseases

“…to treat autoimmune diseases [10] or transplant rejection [66][67][68] or to augment antitumour immune responses [69][70][71][72][73]. In this light, the development of synthetic TCs [10] or pharmacologic inhibitors of IDO [74][75][76][77][78][79] represent promising immu- HAO beef liver [216] rat brain [217] rat liver [217] immortalized murine macrophages MT2 [215] immortalized murine microglial cells N11…”

Natural Product-Inspired Pyranonaphthoquinone Inhibitors of Indoleamine 2,3-Dioxygenase-1 (IDO-1)

“…[13,17] An investigation concerning the inhibitory properties of 4-phenylimidazole (4-PI) [25] has witnessed the crystal structure of IDO-1 being determined, [28] along with the production of nanomolar inhibitors of IDO-1 based on the 4-PI scaffold. [29] Numerous synthetic IDO-1 inhibitors with novel pharmacophores have emerged, including several based on the indole heterocycle, [30][31][32][33] S-benzyl isothioureas, [34] 4-amino-1,2,5-oxadiazole-3-carboximidamides, [35,36] the imidodicarbonimidic diamide NSC 401366, [37] ebselen, [38] and several triazole-based IDO inhibitors. [39] Potent inhibitors have also been discovered using a combination of docking-based pharmacophore model development and fragment-based drug design (FBDD), [40a] along with the identification of potential inhibitors by virtual screening [40b] (Fig.…”