Merits and pitfalls of conventional and covalent docking in identifying new hydroxyl aryl aldehyde like compounds as human IRE1 inhibitors

Abstract

IRE1 is an endoplasmic reticulum (ER) bound transmembrane bifunctional kinase and endoribonuclease protein crucial for the unfolded protein response (UPR) signaling pathway. Upon ER stress, IRE1 homodimerizes, oligomerizes and autophosphorylates resulting in endoribonuclease activity responsible for excision of a 26 nucleotide intron from the X-box binding protein 1 (XBP1) mRNA. This unique splicing mechanism results in activation of the XBP1s transcription factor to specifically restore ER stress. Small molecules targeting the reactive lysine residue (Lys907) in IRE1α's RNase domain have been shown to inhibit the cleavage of XBP1 mRNA. Crystal structures of murine IRE1 in complex with covalently bound hydroxyl aryl aldehyde (HAA) inhibitors show that these molecules form hydrophobic interactions with His910 and Phe889, a hydrogen bond with Tyr892 and an indispensable Schiff-base with Lys907. The availability of such data prompted interest in exploring structure-based drug design as a strategy to develop new covalently binding ligands. We extensively evaluated conventional and covalent docking for drug discovery targeting the catalytic site of the RNase domain. The results indicate that neither computational approach is fully successful in the current case, and we highlight herein the potential and limitations of the methods for the design of novel IRE1 RNase binders.

Figure 1

(A) Ligands co-crystallized in murine IRE1 RNase active site. Reactive aldehyde groups are highlighted with circles. (B) Superposition of the 3D structures of murine IRE1; PDB code 4PL3 (green), 4PL4 (blue), and 4PL5 (red). Key residues involved in binding of HAA are shown in stick model. (C,D) Per amino-acid interaction energy map for co-crystallized inhibitors in the murine IRE1-HAA binding site. Lys907 is not depicted in the heat maps as it is involved in the covalent bond with the substrates. (C) Electrostatic energy values (kcal mol⁻¹); (D) Hydrophobic score (arbitrary units).

Figure 2

Per amino-acid interaction energy map for a dataset of hydroxy aryl aldehyde (HAA) class of IRE1 endoribonuclease inhibitors docked inside the binding site of MKC9989 using conventional docking studies (PDB code: 4PL3). (A) Electrostatic energy values (kcal mol⁻¹); (B) Hydrophobic score (arbitrary units).

Figure 3

(A) Crystal structure of murine IRE1 with MKC9989 bound to the binding pocket of the RNase active site (PDB 4PL3). Comparison of (B) conventional docked pose, (C) Covalent Docking pose using VS workflow, and (D) crystal pose of MKC9989.

Figure 4

Superposition of the best-scoring docked poses from covalent docking (grey) onto the native crystal one (green). Lys907 and key residues in the binding site are highlighted and all other residues are hidden for clarity (A) MKC9989 (PDB code: 4PL3) (B) OICR464 (PDB code: 4PL4) (C) OICR573 (PDB code: 4PL5).