Fluctuations in the brain levels of the neuromodulator kynurenic acid may control cognitive processes and play a causative role in several catastrophic brain diseases. Elimination of the pyridoxal 5′-phosphate dependent enzyme kynurenine aminotransferase II reduces cerebral kynurenic acid synthesis and has procognitive effects. The present description of the crystal structure of human kynurenine aminotransferase II in complex with its potent and specific primary amine-bearing fluoroquinolone inhibitor (S)-(−)-9-(4-aminopiperazin-1-yl)-8-fluoro-3-methyl-6-oxo-2,3-dihydro-6H-1-oxa-3a-azaphenalene-5-carboxylic acid (BFF-122) should facilitate the structure-based development of cognition-enhancing drugs. From a medicinal chemistry perspective our results demonstrate that the issue of inhibitor specificity for highly conserved PLP-dependent enzymes could be successfully addressed.

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Kynurenine aminotransferase (KAT) is a pyridoxal 5′‐phosphate‐dependent enzyme that catalyzes the conversion of kynurenine, an intermediate of the tryptophan degradation pathway, into kynurenic acid, an endogenous antagonist of ionotropic excitatory amino acid receptors in the central nervous system. KATII is the prevalent isoform in mammalian brain and a drug target for the treatment of schizophrenia. We have carried out a spectroscopic and functional characterization of both the human wild‐type KATII and a variant carrying the active site mutation Tyr142→Phe. The transamination and the β‐lytic activity of KATII towards the substrates kynurenine and α‐aminoadipate, the substrate analog β‐chloroalanine and the inhibitors (R)‐2‐amino‐4‐(4‐(ethylsulfonyl))‐4‐oxobutanoic acid and cysteine sulfinate were investigated with both conventional assays and a novel continuous spectrophotometric assay. Furthermore, for high‐throughput KATII inhibitor screenings, an endpoint assay suitable for 96‐well plates was also developed and tested. The availability of these assays and spectroscopic analyses demonstrated that (R)‐2‐amino‐4‐(4‐(ethylsulfonyl))‐4‐oxobutanoic acid and cysteine sulfinate, reported to be KATII inhibitors, are poor substrates that undergo slow transamination.

Kynurenic acid (KYNA), one of the metabolites belonging to the kynurenine pathway, has been described as an important neuroprotective compound, its unbalancing being associated with several pathological conditions. In human brain, the majority of KYNA production is sustained by kynurenine aminotransferase II (KAT II). A selective KAT II inhibitor would be an important pharmacological tool, since it would reduce KYNA formation without causing complete depletion of this neuroprotector. (S)-(4)-(ethylsulfonyl)benzoylalanine (S-ESBA), described as a potent and selective inhibitor of rat KAT II, is unfortunately ineffective towards the human enzyme although the two orthologs share a remarkably high degree of sequence identity. We investigated the molecular basis for this intriguing species-specificity by adopting a site-directed mutagenesis and structural approach. We propose that the source of the inhibitor specificity toward the rat enzyme could reside on S-ESBA interaction/interference with a flexible loop that controls ligand admission to the active site by a classical induced-fit mechanism. Our data further highlights that even in case of highly conserved molecular targets, the flexibility of catalytically important structural elements can have a significant impact on the selectivity of inhibitor action.

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