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

Abstract

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 K_m 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.

Keywords: Canavan disease; N-acetyl aspartate (NAA); N-acetyltransferase-8-like (NAT8L); acetyl-CoA; aspartate N-aceyltransferase (ANAT); aspartoacyclase (ASPA); l-aspartate.

Fig. 1 -

Evaluation of 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM) as chemical sensor of CoASH production. (A) Emission spectra of CPM-SCoA adduct after excitation at 388 nm. Maximal fluorescence intensity was seen at 478 nm. Fluorescence intensity is negligible when CPM and acetyl-CoA or CPM only are used. (B) Time course of reaction between CPM (10 μM) and various concentrations of CoASH. (C) Dependence of fluorescence at various concentrations of CoASH and excess CPM at 10 μM. (D) Background fluorescence generated from reaction of CPM (10 μM) and acetyl-CoA that had been pre-treated with acetic anhydride.

Fig 2 -

Dependence of rate of ANAT-catalyzed reaction on (A) time of incubation when using 24.4 ng/μL ANAT, (B) protein concentration after 30 min incubation, (C) L-aspartate concentration when using 100 μM acetyl-CoA, 24.4 ng/μL ANAT and 30 min incubation, and (D) acetyl-CoA concentration. Experimental conditions were the same as in (C) except acetyl-CoA concentration was varied and l-aspartate concentration was kept constant at 1 mM. The amount of NAA production was calculated based on the Relative Fluorescent Units (RFUs). Analysis of results in (C) and (D) was performed using GraphPad Prism 8 software (Microsoft) using a non-linear least square fit to the Michaelis-Menten model to determine V_max and K_m.

Fig 3-

Results of a pilot HTS of 10,000 compound library - (A) Coefficient of variance (CV), (B) Z’ factor, and (C) percent inhibition (average of 16 determinations ± S.E.M.) by the prototype inhibitor N-carbobenzyloxy-l-aspartate (Cbz-l-asp) at 100 μM were calculated for each of 32 screening plates. (D) Scatter chart plot for percentage inhibition of all compounds.

Fig 4.. Separation of NAA and L-aspartate using cation exchange resin in an orthogonal radiochemical assay.

(A) Scheme of the orthogonal assay. (B) Analysis of NAA and L-aspartate by mass spectrometry before applying to the cation exchange column. Equal amount of NAA and L-aspartate (25 μM) were mix together and separated by HPLC and detected by mass spectrometry. The NAA was detected between 2.8 to 3 min (blue peak) whereas L-aspartate was detected between 3.6 to 3.8 min (green peak). (C) Analysis of NAA and L-aspartate by mass spectrometry in the eluant passing the cation exchange column. 90 μL of mixture containing 25 μM NAA and 250 μM L-aspartate were loaded onto a spin column packed with strong cation exchange resin (AG^® 50W-X8, 200–400 mesh), followed by elution with 100 μL of 0.01 N HCl twice. The eluants were concentrated and loaded and analyzed by mass spectrometry for the NAA and L-aspartate.

Fig 5.. kinetics studies of V002–2064 and Cbz-L-asp on ANAT inhibition.

Lineweaver-Burk plot of ANAT inhibition by V002–2064 (A) or Cbz-L-asp (B) in related to L-aspartate, and Lineweaver-Burk plot of ANAT inhibition by V002–2064 (C) or Cbz-L-asp (D) in related to Acetyl-CoA.

Fig 6.

Flow chart for ANAT inhibitor screening cascade

Scheme 1 -. Metabolism of N-acetyl aspartate (NAA) in the brain -

NAA biosynthesis from l-aspartate and acetyl-coenzyme A is catalyzed by aspartate-N-aceyltransferase (ANAT) in neurons. NAA is transported from neurons to oligodendrocytes where it is hydrolyzed to L-aspartate and acetate in a reaction catalyzed by aspartoacylase (ASPA). Pharmacological inhibition of neuronal ANAT would lead to decreased production of NAA and thus prevent toxic cumulation of NAA-water complex in surrounding extracellular fluid.

Scheme 2 -

Reaction diagram of fluorescence-based ANAT assay using 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM) as a chemical sensor of CoASH production.