Abnormal levels of kynurenic acid (KA) has been thought to accompany several neuropsychiatric diseases. This is consistent with the role of KA as an antagonist acting at the glutamate-binding site of the N-methyl D-aspartic acid receptor. Overproduction of this metabolite by kynurenine aminotransferases (hKATs), could contribute to pathophysiology of psychosis, making the kynurenine pathway a valuable target for the treatment of such disease. Novel inhibitors will be valuable, and on this basis we have been pursuing the design of inhibitors.
Both hKAT-I and hKAT-2 catalyse the production of kynurenic acid from kynurenine. We have prepared recombinant hKAT-I and reported a series of phenyl hydrazone compounds and seven candidates were found to inhibit hKAT-I representing the best reported inhibitors1. To assist our study, we have now embarked upon the heterologous expression of hKAT-2 from which we will be able to pursue the design of selective inhibitors. These proteins are required in a fully functional state and in quantities suitable to allow us to advance our structure-base drug design program, and our strategies have required combinations of codon optimisation, E. coli and insect cell (Sf9) expression systems, as well as optimised purification strategies sometimes including his-tagging. Most recently we have successfully expressed hKAT-2 in Rosetta E.coli cells using an optimized hKAT-2 codon sequence. Purification has proceeded with a his-tagged hKAT-2 using immobilized nickel affinity chromatography and a concentration step, and the recombinant hKAT-2 was shown to be active in our activity assay. The hKAT-2 expression and purification will be further optimized for its supply in inhibitor design, including crystallographic studies.