Inhibition of human kynurenine aminotransferase isozymes by estrogen and its derivatives

Abstract

The kynurenine aminotransferase (KAT) enzymes are pyridoxal 5'-phosphate-dependent homodimers that catalyse the irreversible transamination of kynurenine into kynurenic acid (KYNA) in the tryptophan metabolic pathway. Kynurenic acid is implicated in cognitive diseases such as schizophrenia, and several inhibitors have been reported that selectively target KAT-II as it is primarily responsible for kynurenic acid production in the human brain. Not only is schizophrenia a sexually dimorphic condition, but women that have schizophrenia have reduced estrogen levels in their serum. Estrogens are also known to interact in the kynurenine pathway therefore exploring these interactions can yield a better understanding of the condition and improve approaches in ameliorating its effects. Enzyme inhibitory assays and binding studies showed that estradiol disulfate is a strong inhibitor of KAT-I and KAT-II (IC₅₀: 291.5 μM and 26.3 μM, respectively), with estradiol, estradiol 3-sulfate and estrone sulfate being much weaker (IC₅₀ > 2 mM). Therefore it is possible that estrogen levels can dictate the balance of kynurenic acid in the brain. Inhibition assay results and modelling suggests that the 17-sulfate moiety in estradiol disulfate is very important in improving its potency as an inhibitor, increasing the inhibition by approximately 10-100 fold compared to estradiol.

Figure 1

The kynurenine pathway. The first step is rate-limiting, involving tryptophan getting cleaved by indoleamine 2,3-dioxygenase (IDO1/IDO2; EC 1.13.11.52) or tryptophan 2,3-dioxygenase 2 (TDO2; EC 1.13.11.11) to form N-formylkynurenine. Kynurenine formamidase (EC 3.5.1.9) metabolises this further into L-kynurenine, where it is converted into either kynurenic acid by kynurenine aminotransferases (KAT; EC 2.6.1.7), 3-hydroxykynurenine (3-HK) by kynurenine 3-monooxygenase (EC 1.14.13.9), or anthranilic acid by kynureninase (EC 3.7.1.3). 3-HK can be metabolised into xanthurenic acid by KAT, or 3-hydroxyanthranilic acid (3-HANA) by kynureninase. The latter is also a product that is formed by anthranilate 3-monooxygenase (EC 1.14.16.3) acting on anthranilic acid. Downstream of 3-HANA, quinolinic acid is formed and this progresses into nicotinamide metabolism which produces nicotinamide adenosine dinucleotide (NAD). The transamination of kynurenine to kynurenic acid by the KAT enzymes is denoted in red. Figure adapted with permission from Jayawickrama, et al..

Figure 2

PLP-dependent transamination reaction. (a) The active site is formed from PLP (red) and Lys-263 (blue). (b) The transamination process which irreversibly converts kynurenine (brown) into kynurenic acid (brown); PLP is regenerated in this process via an α-ketoacid acceptor. Figure adapted with permission from Jayawickrama, et al..

Figure 3

Kynurenine in the active site of KAT-II. The amino acids with an atom within 5.0 Å of kynurenine (yellow), and PLP (magenta), were chosen for display. Residue Tyr-142 was removed for clarity. The aromatic ring of kynurenine forms pi-pi interactions (green dashes) with Tyr-74, and pi-cation (blue dashes) interactions with Arg-20. The carboxyl moiety forms a salt bridge (yellow dashes) with Arg-399, and the neighbouring amine group forms hydrogen bonds with Asn-202 (yellow dashes). Image generated with PyMOL.

Figure 4

Chemical structures of KAT-II inhibitors S-ESBA, BFF-122, PF-04859989, BFF-816, and NS-1502. The reversible inhibitors are drawn in blue; the irreversible inhibitors are in red.

Figure 5

Scaffold of estrogen compounds. X is off the 3-carbon position. Y is off the 17-carbon position. The four rings are labelled A, B, C, D, respectively.

Figure 6

Inhibition assays of estradiol disulfate on KAT. (a) Inhibitory activity of estradiol disulfate in a dose-dependent format on KAT-I (IC₅₀: 291.5 (±19.6) μM, R² = 0.95). (b) Inhibitory activity of estradiol disulfate in a dose-dependent format on KAT-II (IC₅₀: 26.3 (±1.4) μM, R² = 0.98). Both plots used data from at least 3 sets of tests. Diagrams were produced using GraphPad Prism v7.02.

Figure 7

Estradiol disulfate docked into the active site of KAT-II. The amino acids with an atom within 5.0 Å of estradiol (yellow) were chosen for display. Residues Tyr-142 and Gly-144 were removed for clarity. The 3-sulfate group of estradiol disulfate forms hydrogen bonds (yellow dashes) with Arg-20, and the 17-sulfate group forms hydrogen bonds (yellow dashes) with Asn-202 and Lys-263. Image generated with PyMOL.