Substrate stereo-specificity in tryptophan dioxygenase and indoleamine 2,3-dioxygenase

Abstract

The first and rate-limiting step of the kynurenine pathway, in which tryptophan (Trp) is converted to N-formylkynurenine is catalyzed by two heme-containing proteins, Indoleamine 2,3-dioxygenase (IDO), and Tryptophan 2,3-dioxygenase (TDO). In mammals, TDO is found exclusively in liver tissue, IDO is found ubiquitously in all tissues. IDO has become increasingly popular in pharmaceutical research as it was found to be involved in many physiological situations, including immune escape of cancer. More importantly, small-molecule inhibitors of IDO are currently utilized in cancer therapy. One of the main concerns for the design of human IDO (hIDO) inhibitors is that they should be selective enough to avoid inhibition of TDO. In this work, we have used a combination of classical molecular dynamics (MD) and hybrid quantum-classical (QM/MM) methodologies to establish the structural basis that determine the differences in (a) the interactions of TDO and IDO with small ligands (CO/O(2)) and (b) the substrate stereo-specificity in hIDO and TDO. Our results indicate that the differences in small ligand bound structures of IDO and TDO arise from slight differences in the structure of the bound substrate complex. The results also show that substrate stereo-specificity of TDO is achieved by the perfect fit of L-Trp, but not D-Trp, which exhibits weaker interactions with the protein matrix. For hIDO, the presence of multiple stable binding conformations for L/D-Trp reveal the existence of a large and dynamic active site. Taken together, our data allow determination of key interactions useful for the future design of more potent hIDO-selective inhibitors.

Figure 1

QM-MM optimized structures of xcTDO active site. Top pannel corresponds to the three conformations found in SF TDO-O₂ complex. Lower panel corresponds to the L-Trp-bound xcTDO-O₂ (left) and -CO complex (right), respectively.

Figure 2

The averaged structures of xcTDO-O₂ with D- and L-Trp obtained from the MD simulation. Panel a corresponds to the L-Trp-bound xcTDO-O₂, panel b shows superimposed structures of L and D-Trp complexes, depicted in black and green, respectively, with only one heme displayed for simplicity.

Figure 3

The average structures of D- and L-Trp-bound hIDO-O₂ obtained from the MD simulation. Panels a and b correspond to the L-Trp complexes and show the Cf1 and Cf2 conformations, respectively. Panels c and d correspond to the D-Trp complexes in the Cf1 and Cf2 conformations, respectively.

Figure 4

Free energy profile for the interconversion between Cf1 and Cf2. The results for L and D-Trp are depicted in solid and dashed lines, respectively.

Scheme 1

Schematic illustration of the hIDO reaction. ES₂ and ES₁ represent the protein-L-Trp complexes in Cf2 and Cf1 conformations, respectively. K₂ is the L-Trp dissociation equilibrium constant of ES2, K₂₁ is the equilibrium constant between ES2 and ES1 and k_p is the rate constant of the chemical reaction.