Fig. 1. From small molecules fragments bound to the doorstop pocket to the inhibitors designed in this study.

a A simplified picture of the enzymatic mechanism of TGR is shown (for a comprehensive TGR mechanism see refs. ^{17, 30}). In the reductive half-reaction 2 eq of NADPH are consumed to produce the EH₄ species, the 4-electron state, the one competent for substrate reduction in the oxidized half-reaction. After the initial reduction of the oxidized enzyme (E_ox) to EH₂, the 2-electron reduced state, TGR oscillates between EH₂ and EH₄ during turn-over. b TGR homodimer is shown in cartoons and each subunit is differently colored. The FAD cofactor is in green sticks. The doorstop pocket adjacent to the NADPH binding site is shown as a pink surface in one subunit. c A magnification of the doorstop pocket with representative bound fragments identified by X-ray crystallography (PDB ID 6FTC – magenta and PDB ID 6FP4 – cyan and yellow models); the molecular surface of the doorstop pocket is colored according to its hydrophobic features (green = hydrophobic; magenta = hydrophilic). Subpockets A-C are outlined in different colors and for each fragment the PDB ID is reported. d The TGR inhibitors designed in this study. e Gameplan non-polar hypotheses (magenta sticks) generated for chimera molecule (green) built using the X-ray fragments found in subpockets A-C in PDB ID 6FP4 and PBD ID 6FTC and connected by a short CH₂CH₂ linker to facilitate calculations and analysis. The binding site surface is colored with VIDA hydrophobicity palette, brown is hydrophobic, and blue is hydrophilic. f SZMAP grid results for TGR-chimera (gray sticks) complex processed with WaterOrientation VIDA extension show the most probable probe positions (polar substituents - yellow bubbles, non-polar substituents - purple bubbles) and the corresponding free energy values and the location of the water molecules (red bubbles) found in the X-ray structures of TGR.