Fig 1.
Tryptophan metabolism through the kynurenine pathway.
Tryptophan metabolism proceeds through kynurenine in multiple branches, which includes the formation of KYNA by the KAT enzymes.
Fig 2.
Kynurenine in the KAT-II active site.
The amino acids within 5.0 Å of kynurenine (green) were chosen for display, with PLP in magenta and residue Tyr-142 removed for clarity. Depicted is the aromatic ring forming pi-pi interactions (green dashes) with Tyr-74 and pi-cation interactions (blue dashes) with Arg-20. A hydrogen bond (yellow dashes) between the amine group and Asn-202, and a salt bridge (yellow dashes) formed between the carboxyl and Arg-399 helps anchor kynurenine in the correct conformation for transamination. Image generated with PyMOL [29].
Fig 3.
NS-1502 docked into the KAT-II active site.
The amino acids within 5.0 Å of NS-1502 (green) were chosen for display. Residues Ser-143, Gly-144, and Gln-289 were removed for clarity. The carboxyl group forms hydrogen bonds (yellow dashes) with Asn-202 and Arg-399, and the aromatic ring has pi-pi interactions (green dashes) with Tyr-142 and pi-cation interactions (blue dashes) with Lys-263. Image generated with PyMOL [29].
Fig 4.
Comparative lengths of the structures when fully extended.
Distances measured in the 3D conformer of the compounds, generated from LigPrep at the time of preparation for docking, from the first carbon atom (A) in each compound to the carbon from which the carboxyl branches off (B), the furthest carbon atom (C), or the sulfur atom in the sulfate/sulfonamide groups (D).
Fig 5.
Dose-dependent inhibition of KAT-II.
Inhibition assays with 0.5 μg KAT-II incubated for 10 min at 37 °C in a 50 μL reaction mixture containing 50 μM PLP, 5 mM α-ketoglutarate, 5 mM l-kynurenine and the inhibitor studied. The reaction was terminated with 0.8 M formic acid and analysed using HPLC. Experiments were performed as independent replicates, and figures were produced using GraphPad Prism v7.02 [40] (A) JN-01 inhibition (IC50: 73.8 (95% CI: 66.3–81.8) μM, R2: 0.91). (B) JN-02 inhibition (IC50: 112.8 (95% CI: 100.8–126.1) μM, R2: 0.92).
Fig 6.
JN-01 docked into the KAT-II active site.
The amino acids within 5.0 Å of JN-01 (green) were chosen for display. Residues Ser-143, Gly-144, and Gln-289 were removed for clarity. The oxygen atoms in the sulfonamide form hydrogen bonds (yellow dashes) with Asn-202 and Lys-263, and the aromatic ring has pi-pi interactions (green dashes) with Tyr-74. Image generated with PyMOL [29].
Fig 7.
JN-02 docked into the KAT-II active site.
The amino acids within 5.0 Å of JN-02 (green) were chosen for display. Residues Ser-143, Gly-144, and Gln-289 were removed for clarity. The amine in the sulfonamide forms hydrogen bonds (yellow dashes) with Gln-118, and the carbonyl in the phthalimide rings forms a hydrogen bond with Tyr-142. The aromatic ring has pi-pi interactions (green dashes) with Tyr-142 and pi-cation interactions (blue dashes) with Lys-263. Image generated with PyMOL [29].
Fig 8.
JN-03 docked into the KAT-II active site.
The amino acids within 5.0 Å of JN-03 (green) were chosen for display. Residues Ser-143, Gly-144, and Gln-289 were removed for clarity. The sulfate forms hydrogen bonds with Tyr-74, Gln-118, Lys-263, and Arg-270, and the carboxyl moiety forms hydrogen bonds with Asn-202 and Arg-399. The aromatic ring has pi-cation interactions (blue dashes) with Lys-263. Image generated with PyMOL [29].