Design, synthesis and evaluation of tryptophan analogues as tool compounds to study IDO1 activity

Abstract

The metabolism of l-tryptophan to N-formyl-l-kynurenine by indoleamine-2,3-dioxygenase 1 (IDO1) is thought to play a critical role in tumour-mediated immune suppression. Whilst there has been significant progress in elucidating the overall enzymatic mechanism of IDO1 and related enzymes, key aspects of the catalytic cycle remain poorly understood. Here we report the design, synthesis and biological evaluation of a series of tryptophan analogues which have the potential to intercept putative intermediates in the metabolism of 1 by IDO1. Functionally-relevant binding to IDO1 was demonstrated through enzymatic inhibition, however no IDO1-mediated metabolism of these compounds was observed. Subsequent T _m-shift analysis shows the most active compound, 17, exhibits a distinct profile from known competitive IDO1 inhibitors, with docking studies supporting the hypothesis that 17 may bind at the recently-discovered S_i site. These findings provide a start-point for development of further mechanistic probes and more potent tryptophan-based IDO1 inhibitors.

Scheme 1. Dioxygenation of l-tryptophan 1 to N-formyl-l-kynurenine 2.

Fig. 1. IDO inhibitor 1-MT 3 and clinically evaluated compounds indoximod 4, epacadostat 5 and navoximod 6.

Scheme 2. Hayashi's 7 and Hamilton's 8 proposed metabolic intermediates.

Scheme 3. Proposed new mechanistic pathways of (A) radical and (B) electrophilic IDO1 mediated dioxygenation of l-tryptophan.

Fig. 2. Proposed mechanistic probes of IDO1 16–18.

Fig. 3. (A) Radical ring opening of cyclopropane 19 giving covalently bound inhibitor complex 20 and forming a captodative radical; (B) radical ring opening of cyclopropane 21 giving covalently bound inhibitor complex 22 and a reactive radical to interact further with the protein; (C) ring opening of epoxide 23via neighbouring group participation to give thionium 24 and a redox inactive protein; (D) loss of a thiolate anion over C–C bond cleavage of hemi-thioacetal 25 giving covalently bound inhibitor-probe complex 26.

Fig. 4. Representation of the IDO1 structure 5WMV, with IDO protein residues depicted in white cartoon/lines and active site surface in white, cut to reveal the CN-coordinated heme (yellow) and substrate binding sites; (A) experimental (cyan) and lowest energy docked (blue) pose of TRP. (B) Lowest energy docked pose of 16 (orange); (C) lowest energy docked pose of 17 (green); (D) projection of lowest energy docked poses of 16 and 17, showing the relationship between the indole ring and cyclopropane bonds; (E) gold docking scores for lowest energy pose and depicted poses with indole in a reactive conformation. Lower numbers are indicative of a more stable bound state.

Scheme 4. Synthesis of α,β-cyclopropyl tryptophan-mimic 16. (a) POCl₃, DMF, 0–50 °C, 2.5 h, 91% (27); (b) (Boc)₂O, Et₃N, DMAP, CH₂Cl₂, 23 °C, 18 h, 99% (28); (c) TsNHNH₂, MeOH, 50 °C, 4 h, 99% (29); (d) (i) 29, Cs₂CO₃, BTAC, Dry PhMe 23 °C, 1.5 h, (ii) 34, 90 °C, 16 h, 91% (92 : 8, E : Z) (30); (e) 10% Pd/C, MeOH, 23 °C, 0.75 h, 94% (31); (f) 4 M HCl in 1,4-dioxane, 23 °C, 23 h, 99% (16); (g) 32, Cs₂CO₃, DMF, 23 °C, 0.3 h, (ii) BnBr, 23 °C, 17.5 h, 78% (33); (h) (i) MsCl, DMAP, 0 °C, 0.25 h, (ii) Et₃N, 0–23 °C, 16.5 h, 83% (34).

Scheme 5. Synthesis of β,β′-cyclopropyl tryptophan-mimic 17.

Scheme 6. Synthesis of sulfenylindole 18.

Scheme 7. Plausible metabolic by-products of IDO1 mediated dioxygenation of tryptophan-mimics (A) 16 and (B) 17.

Scheme 8. Plausible metabolic by-products of IDO1 mediated dioxygenation of sulfenylindole 18 metabolism.

Fig. 5. UV-based inhibition assay data for substrate mimics (A) 16, (B) 17 and (C) 18. (D) Out-competition and (E) co-incubation data for substrate mimic 17.

Fig. 6. Controls – [IDO] μM/SYPRO dye dilution factor.

Fig. 7. T _m analysis in the presence of SYPRO dye (1/2000).

Fig. 8. T _m analysis in the absence of SYPRO dye.

Fig. 9. Microscale thermophoresis (MST) analysis of the dose-dependent interaction between 17 and IDO1, K_d 35.0 μM ± 6.96 μM.

Fig. 10. Representation of the IDO1 structure 5MWV, with IDO protein residues depicted in white cartoon/lines and active site surface in white, cut to reveal the CN-coordinated heme (yellow) and substrate binding sites. Docked pose of tryptophan at the S site (blue) and S_i site (cyan) are overlaid with 17 (green), with F270 which moves to form the Si site depicted in magenta.