High Throughput Screening Cascade To Identify Human Aspartate N-Acetyltransferase (ANAT) Inhibitors for Canavan Disease.

Canavan disease (CD) is a progressive, fatal neurological disorder that begins in infancy resulting from a mutation in aspartoacyclase (ASPA), an enzyme that catalyzes the deacetylation of N-acetyl aspartate (NAA) into acetate and aspartate. Increased NAA levels in the brains of affected children are one of the hallmarks of CD. Interestingly, genetic deletion of N-acetyltransferase-8-like (NAT8L), which encodes aspartate N-aceyltransferase (ANAT), an enzyme responsible for the synthesis of NAA from l-aspartate and acetyl-CoA, leads to normalization of NAA levels and improvement of symptoms in several genetically engineered mouse models of CD. Therefore, pharmacological inhibition of ANAT presents a promising therapeutic strategy for treating CD. Currently, however, there are no clinically viable ANAT inhibitors. Herein we describe the development of fluorescence-based high throughput screening (HTS) and radioactive-based orthogonal assays using recombinant human ANAT expressed in E. coli. In the fluorescence-based assay, ANAT activity was linear with respect to time of incubation up to 30 min and protein concentration up to 97.5 ng/μL with Km values for l-aspartate and acetyl-CoA of 237 μM and 11 μM, respectively. Using this optimized assay, we conducted a pilot screening of a 10 000-compound library. Hits from the fluorescence-based assay were subjected to an orthogonal radioactive-based assay using L-[U-14C] aspartate as a substrate. Two compounds were confirmed to have dose-dependent inhibition in both assays. Inhibitory kinetics studies of the most potent compound revealed an uncompetitive inhibitory mechanism with respect to l-aspartate and a noncompetitive inhibitory mechanism against acetyl-CoA. The screening cascade developed herein will enable large-scale compound library screening to identify novel ANAT inhibitors as leads for further medicinal chemistry optimization.