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The Binding Mode of N-Hydroxyamidines to Indoleamine 2,3-Dioxygenase 1 (IDO1) Ute Friederike Röhrig, Vincent Zoete, and Olivier Michielin Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00586 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 22, 2017
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The Binding Mode of N-Hydroxyamidines to Indoleamine 2,3Dioxygenase 1 (IDO1) Ute F. Röhrig,a Vincent Zoete,*,a Olivier Michielin*,a,b a Molecular Modeling Group, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland. b Department of Oncology, University of Lausanne and Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland KEYWORDS: IDO1, epacadostat, INCB24360, immune-oncology, density functional theory, QM/MM, docking, inhibitors Supporting Information Placeholder ABSTRACT: Indoleamine 2,3-dioxygenase 1 (IDO1) is
an important target in cancer immunotherapy. The most advanced clinical compound, epacadostat (INCB024360), binds to the heme cofactor of IDO1 through a N-hydroxyamidine function. Conflicting binding modes have recently been proposed, reporting iron binding either through the hydroxyamidine oxygen or nitrogen atoms. Here, we use quantum chemical calculations, docking, and QM/MM calculations based on available X-ray data to resolve this issue and to propose a physically meaningful binding mode. Our findings will aid the design of novel IDO1 ligands based on this pharmacophore.
Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first and rate-limiting step in the kynurenine pathway of tryptophan metabolism, leading to the depletion of tryptophan and to the accumulation of different bioactive metabolites. This pathway has been shown to cause local immunosuppression and to be involved in immune escape by tumors.1-3 Therefore, intense efforts to develop IDO1 inhibitors are ongoing in academia and in the pharmaceutical industry.4
Figure 1. Active site structure of IDO1 (PDB ID 2D0T, 5 ligands not shown).
X-ray structures of IDO1 show one binding pocket in the distal heme site (Pocket A, Figure 1), connected to a second pocket towards the entrance of the active site (Pocket B).6 The size and shape of both pockets differ depending on the bound ligands. In 2009, Incyte Corporation reported Nhydroxyamidines as potent, reversible, competitive IDO1 inhibitors.7 The most potent compound of this series, originally called 5l (1, Figure 2), displayed enzymatic and cellular IC50 values of 67 and 19 nM, suppressed kynurenine generation in vivo, and inhibited melanoma growth in a mouse model. Modeling of the binding of 1 to IDO1 by Incyte predicted that the deprotonated oxygen (O7, Figure 2) of the N-hydroxyamidine binds to the heme iron.8 A cis conformation of the amidine group was assumed. Two intramolecular hydrogen bonds were shown (Figure 2), one between the aniline NH group (N4) and the deprotonated oxygen atom (O7), and a second one between the terminal amino group (N13) and the nitrogen of the hydroxyamidine (N6). The phenyl ring was placed inside the A pocket, while the amino substituent on the furazan ring projected towards the B pocket. Recently, Incyte reported the chemical structure of its clinical compound epacadostat (2, Figure 2), which belongs to a further optimized series of Nhydroxyamidines with an ethyl-sulfamide substituent on the amino group.9 The X-ray structure of isolated epacadostat showed that the two previously postulated intramolecular hydrogen bonds in fact stabilize the cis conformation of the amidine. The planes of the two aromatic rings formed an angle of 63°. Modeling of the binding of epacadostat to IDO1 by the authors, using Xray structure 4PK5,10 consistently showed a covalent bond between the heme iron and the deprotonated oxygen (O7) of the N-hydroxyamidine, the same two intramolecular hydrogen bonds, the substituted phenyl ring in the A pocket, and the sulfamide making a bidentate hydrogen bonding interaction with the heme propionate.9
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On the other hand, an X-ray structure with a resolution of 3.2 Å of compound 1 in complex with IDO1 has recently been reported (PDB ID 5XE1, Figure 3A).11 The N-hydroxyamidine was shown to bind primarily through the nitrogen atom (N6) of the hydroxyamidine to the heme iron. The phenyl ring was placed in the A pocket, while the furazan ring pointed towards the entrance of the pocket. The C3-N4 bond showed a large dihedral twist of -81°. The hydroxyamidine oxygen (O7) formed a hydrogen bond with the terminal amino group (N13), which also made a hydrogen bond to the Ser263 side chain (Figure 3A and Supporting Information).11 Here, we used density functional theory (DFT) calculations, docking, and QM/MM calculations to resolve this discrepancy and to establish the most favorable binding conformation of the important class of Nhydroxyamidine inhibitors to IDO1 in order to aid the design of novel IDO1 ligands based on this pharmacophore.
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at the DFT level. Molecular graphics were produced with the UCSF Chimera package.21 MarvinSketch 16.06.20, 2016, ChemAxon (http://www.chemaxon.com), was used for drawing chemical structures. In vacuo, compound 1 preferentially adopted a cis configuration of the amidine group, with the chloride substituent pointing away from the furazan, the negatively charged oxygen (O7) pointing towards N4, and the planes of the aromatic rings forming an angle of 52° because of the steric hindrance between H1 and N9 (see Supporting Information). The lowest energy conformation with the chloride pointing towards the furazan was +0.49 kcal/mol higher in energy, the most favorable trans amidine had an energy of +2.75 kcal/mol, and the most favorable conformation with O7 pointing towards N13 had an energy of +4.11 kcal/mol. Compound 1 preferentially bound through its deprotonated oxygen (O7) to the iron of ferrous heme (Figure 3C), adopting a conformation very similar to its isolated structure (RMSD 0.1 Å). The iron-oxygen bond length was 1.98 Å, and the binding energy amounted to -7.2 kcal/mol.
Figure 2. Chemical structures of compounds 1 and 2. Dashed lines indicate favorable electrostatic interactions, while red arks indicate steric hindrance.
For the isolated ligand, we generated 100 conformations of the anionic form of compound 1 each in the cis and the trans configurations using Open Babel,12 pre-optimized them with PM6,13 and optimized distinguishable conformations with PBE0/TZVP14-16 in Gaussian09.17 For the complex with the ferrous heme model system (imidazole, porphin2-, and iron II), we used as starting conformations either X-ray coordinates, or coordinates based on previously published models. For the complex between compound 1 and IDO1, we used as starting structures either X-ray structure 5XE111 or the structure of 1 docked into IDO1. Docking was done with Attracting Cavities,18 in combination with Morse-like metal binding potentials19 based on X-ray structure 4PK510 in analogy to the modeling by Incyte. QM/MM calculations were carried out with the interface to Gaussian09 provided in CHARMM20 by Cui and coworkers, treating ligand, heme, and His346 side chain
Figure 3. Structure of compound 1 bound to heme/IDO1. (A) X-ray structure 5XE1. (B) DFT-optimized structure of 1 bound to heme through N6. (C) DFT-optimized structure of 1 bound to heme through O7. (D) QM/MM optimized structure of 1 bound to IDO1 through O7 (for detailed structural parameters see Supporting Information).
Heme binding through N6, based on X-ray structure 5XE1, yielded only a metastable structure (binding energy +2.1 kcal/mol) and an iron-nitrogen bond of 2.05 Å (Figure 3B). The optimized structure differed significantly from the X-ray structure (ligand RMSD 3.4 Å),
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the phenyl ring adopting an almost coplanar conformation to the porphin (22° angle between planes) and an orthogonal conformation to the furazan (73°). This conformation would produce steric clashes with Tyr126, Ser167, Ser163, Ala264, and Gly265 in the IDO1 active site. When optimizing the X-ray structure 5XE1 within a QM/MM approach, the ligand moved away from the heme iron (see Supporting Information). On the other hand, docking of 1 to IDO1 yielded a structure similar to the heme-bound DFT structure (Figure 3C), but with the phenyl ring flipped, so that the chloride substituent could be accommodated in the A pocket (Figure 3D). The structure did not yield steric clashes with the protein and remained stable during QM/MM optimization. The QM/MM optimized structure was overall similar to the structure proposed by Incyte.
Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Computational details, structural parameters, and figures of modeled structures (PDF).
AUTHOR INFORMATION Corresponding Authors
*Tel: +41-21-6924082 E-mail:
[email protected] (V. Zoete) *Tel: +41-21-6924053. E-mail:
[email protected] (O. Michielin) Funding Sources
The authors declare no competing financial interests. We would like to acknowledge financial support from the foundation “Research for Life” (http://researchforlife.ch) by the Reyl Group, Geneva.
ACKNOWLEDGMENT We would like to thank Vital-IT from the SIB Swiss Institute of Bioinformatics for computational resources.
ABBREVIATIONS DFT, density functional theory; IDO1, indoleamine 2,3dioxygenase 1. Figure 4. Comparison of IDO1-bound structure of 1 and known triazole inhibitor 3. (A) Compound 3 docked into IDO1. (B) Superposition of compounds 1 and 3 in IDO1.
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Comparison of the QM/MM-optimized structure of compound 1 with the docked structure of the known IDO1 inhibitor 4-(3-chlorophenyl)-3H-1,2,3-triazole (3, Figure 4A)22 showed a very good structural overlap (see Supporting Material for comparison to 4-phenylimidazole). The triazole heterocycle replaced the Nhydroxyamidine group for heme binding, the phenyl rings overlapped, and the chloride substituents filled the same hydrophobic subpocket (Figure 4B). This overlap may explain the about 50-fold increase in IDO1 inhibitory activity due to the chloride substituent for both scaffolds.7,22 In summary, quantum mechanical calculations, docking studies, and hybrid QM/MM calculations suggest that compounds of the N-hydroxyamidine family including the clinical candidate epacadostat bind through their deprotonated oxygen atom to heme, in contrast to a recently published X-ray structure. These findings will aid the design of novel IDO1 ligands based on this pharmacophore.
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Active site structure of IDO1 (PDB ID 2D0T, ligands not shown). 381x305mm (72 x 72 DPI)
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Chemical structures of compounds 1 and 2. Dashed lines indicate favorable electrostatic interactions, while red arks indicate steric hindrance. 381x268mm (72 x 72 DPI)
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Structure of compound 1 bound to heme/IDO1. (A) X-ray structure 5XE1. (B) DFT-optimized structure of 1 bound to heme through N6. (C) DFT-optimized structure of 1 bound to heme through O7. (D) QM/MM optimized structure of 1 bound to IDO1 through O7 (for detailed structural parameters see Supporting Information). 381x381mm (72 x 72 DPI)
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Comparison of IDO1-bound structure of 1 and known triazole inhibitor 3. (A) Compound 3 docked into IDO1. (B) Superposition of compounds 1 and 3 in IDO1. 381x188mm (72 x 72 DPI)
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