Simultaneous In Vitro Assay of the First Four Enzymes in the Fungal Aspartate Pathway Identifies a New Class of Aspartate Kinase Inhibitor

The biosynthesis of amino acids derived from Asp (Met, Thr, and Ile) is a target for antifungal agents. We have developed a simultaneous in vitro assay of the first four enzymes of the fungal aspartate pathway: aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, and homoserine O-acetyltransferase. This reconstructed pathway assay was initiated with the readily accessible amino acid L-Asp and thus circumvents the obstacles of substrate availability and stability for aspartate semialdehyde dehydrogenase and homoserine dehydrogenase. The assay was shown to be suitable for high-throughput screening of chemical libraries for the identification of inhibitors of all four component enzymes. A screen of a library of 1000 small molecules identified a novel class of 7-chloro-4(1, 3, 4thiadiazol-2-ylsulfanyl)-quinoline aspartate kinase inhibitors that have the potential to act as leads in the development of new antifungal agents.