Cell-Based Identification of New IDO1 Modulator Chemotypes

Abstract

The immunoregulatory enzyme indoleamine-2,3-dioxygenase (IDO1) strengthens cancer immune escape, and inhibition of IDO1 by means of new chemotypes and mechanisms of action is considered a promising opportunity for IDO1 inhibitor discovery. IDO1 is a cofactor-binding, redox-sensitive protein, which calls for monitoring of IDO1 activity in its native cellular environment. We developed a new, robust fluorescence-based assay amenable to high throughput, which detects kynurenine in cells. Screening of a ca. 150 000-member compound library discovered unprecedented, potent IDO1 modulators with different mechanisms of action, including direct IDO1 inhibitors, regulators of IDO1 expression, and inhibitors of heme synthesis. Three IDO1-modulator chemotypes were identified that bind to apo-IDO1 and compete with the heme cofactor. Our new cell-based technology opens up novel opportunities for medicinal chemistry programs in immuno-oncology.

Keywords: cancer; fluorescence; heme proteins; high throughput screening; immunology.

Figure 1

Validation of a coumarin‐based sensor for detection of cellular kynurenine (1, Kyn) levels. A) Cellular Kyn assay. Cells are treated with IFNγ, Trp, and compounds. After 48 h, Kyn levels are detected using the Kyn sensor (2). B) Absorbance (left) and fluorescence (right) scan of 2 in the absence or presence of Kyn in cell culture medium. C) Detection of 3 by means of fluorescence (ex: 555 nm, em: 600 nm) in the presence of Kyn (0–500 μm) in cell culture medium. D) Induction of IDO1 expression in BxPC3 cells with IFNγ for 8, 24, and 48 h. Representative immunoblots for IDO1 and vinculin as a loading control. See Figure S2 for uncropped blots. E) Chemical structures of the IDO1 inhibitors epacadostat (4) and BMS‐986205 (5). F) Kyn assay of epacadostat utilizing p‐DMAB or sensor 2 for Kyn‐level detection. Data are mean values±S.D., n=3. G) Automated Kyn assay for 4 or 5. Kyn levels were detected using 2.

Figure 2

Decrease of cellular Kyn production by identified hit compounds. A) Structures of compound 6, 7, and 8 and IC₅₀±S.D. values in Kyn assay. B) Kyn assay using 2 in BxPC3 cells treated with 6, 7 or 8 for 48 h. C) Detection of Kyn levels using LC‐MS/MS. Cells were treated as described in (B). Data are mean values±S.D., n≥3.

Figure 3

IDO1 is the target of compound 6, 7, and 8. A) Influence of the pre‐incubation temperature on IDO1 activity. IDO1 was incubated with 6, 7 or 8 at 20 °C or 37 °C for 30 min prior to detection of Kyn levels using 2. B) IDO1 enzymatic assay. IDO1 was pre‐incubated with the compounds at 37 °C for 40 min prior to the detection of Kyn levels using p‐DMAB. Data are mean values±S.D., n≥3. C and D) Influence on the melting behavior of IDO1. IDO1 was preincubated with 30 μm of 6, 7 or 8 (C) or 30 μm BMS‐986205 or epacadostat (D) at 37 °C for 30 min prior to SYPRO orange addition. Representative melting curves of IDO1 are shown (n=3, see also Figure S6).

Figure 4

Hit compounds are heme‐competitive IDO1 inhibitors. A) UV/Vis spectrum of IDO1. Human IDO1 was preincubated with the compounds at 37 °C for 120 min prior detection of the UV/Vis spectrum. Representative spectrum (n=3). B) Detection of heme competition. Human IDO1 was preincubated with the compounds or the compounds and 14 μm hemin at 37 °C for 40 min prior to detection of Kyn levels using p‐DMAB. All data are mean values±S.D., n≥3.