Tracking Hidden Binding Pockets Along the Molecular Recognition Path of l‐Trp to Indoleamine 2,3‐Dioxygenase 1

Abstract

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the oxidative cleavage of l-Tryptophan (l-Trp) to yield N-formyl-kynurenine in the first and rate limiting step of the kynurenine pathway. Bioactive metabolites, involved in the regulation of important immunological responses and neurological processes, are then produced by downstream enzymes along the pathway. Inhibitors of IDO1 are being designed and developed as therapeutic agents for immuno-oncology. In this work, we investigated the molecular recognition path of l-Trp to IDO1, integrating biophysical methods with supervised molecular dynamics (suMD) and mutagenesis experiments. Results allowed disclosing for the first time high and low dissociation constants of l-Trp to IDO1, and the presence of a metastable interaction site located at the upper part of a channel whose borders are defined by the EF-loop and the C-terminal part of the JK-loop. Collectively, our results provide new clues for the design of next-generation IDO1 ligands.[a] Dr. interaction energy was calculated using NAMD Energy extension available in VMD. [55] The latter was used to render images and prepare movies of trajectories (ModelA.mpg, ModelB.mpg, Mod-elC.mpg; supporting information). 4 5 6 7 8

One Key and Multiple Locks: Substrate Binding in Structures of Tryptophan Dioxygenases and Hydroxylases

“…[61] While positive allosteric modulators of IDO1 such as indoleamine-ethanol (IDE) and NAS, [44,46] or some uncompetitive inhibitors such as mitomycin C, [60] have been observed or proposed to occupy this proximal pocket, recent computational and biophysical studies unveil clues on the existence of additional metastable and putative hidden pockets of l-Trp in the structure of IDO1. [47] Specifically, supervised molecular dynamics (suMD) drew a plausible approaching pathway of l-Trp from the solvent to the catalytic site of IDO1, pinpointing a metastable binding site on Lys238 that promotes a first interaction to the substrate (Figure 2C). The conformational plasticity of the JK-loop imparted by Gly378 was also found crucial for the molecular recognition process of L-Trp.…”

“…A flexible loop (DE‐loop, residues 260–268) connects the large catalytic domain and the small signaling domain, shaping the catalytic pocket above the sixth coordination site of the heme‐iron. The access to this site is governed by a large flexible loop (JK‐loop, residues 360–382), whose distinct closed, intermediate and open conformations regulate substrate recognition and binding of inhibitors and heme cofactor to the catalytic site [47,55–58] . A channel for the shuttling of water and oxygen molecules from/to the catalytic site is shaped by α‐helices E and F, extending into the distal and proximal pockets above and below the plane of the heme group, respectively (Figure 2).…”

“…This binding mode is in agreement with the high dissociation constant reported for l ‐Trp to the inhibitory pocket of ferric IDO1‐CN‐Trp complex, being in the millimolar range of potency (K d =26 mM) [61] . While positive allosteric modulators of IDO1 such as indoleamine‐ethanol (IDE) and NAS, [44,46] or some uncompetitive inhibitors such as mitomycin C, [60] have been observed or proposed to occupy this proximal pocket, recent computational and biophysical studies unveil clues on the existence of additional metastable and putative hidden pockets of l ‐Trp in the structure of IDO1 [47] …”

“…In this framework, another level of balance between SP and KP may rely on the substrate avidity of the rate limiting enzymes in those districts wherein these proteins are co‐expressed. Indeed, TPH1, TPH2, TDO, IDO1 and IDO2 show different substrate's affinity to l ‐Trp, as evidenced by the relative Michaelis‐Menden constants (K M , Table 1), with some of them (TDO and IDO1) possessing also multiple binding sites for the substrate [45–47] …”

New Insights from Crystallographic Data: Diversity of Structural Motifs and Molecular Recognition Properties between Groups of IDO1 Structures

“…Noteworthy, a heme‐free form (holo‐) of IDO1 has been also characterized, wherein the three clefts shape a unique and larger ligand binding pocket in which potent suicide inhibitors bind to the enzyme . More recently, we have uncovered an additional metastable binding site located at the entrance of the catalytic site as well as a second binding interaction of l ‐Trp ( 1 ) to IDO1, whose location is still elusive …”