TY - JOUR
T1 - High Throughput Screening Cascade To Identify Human Aspartate N-Acetyltransferase (ANAT) Inhibitors for Canavan Disease
AU - Nešuta, Ondřej
AU - Thomas, Ajit G.
AU - Alt, Jesse
AU - Hin, Niyada
AU - Neužilová, Anna
AU - Long, Shunyou
AU - Tsukamoto, Takashi
AU - Rojas, Camilo
AU - Wei, Huijun
AU - Slusher, Barbara S.
N1 - Funding Information:
This work was fund by 1R61NS119659 from NIH/NINDS.
Publisher Copyright:
© 2021 American Chemical Society.
PY - 2021/9/15
Y1 - 2021/9/15
N2 - 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.
AB - 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.
KW - Canavan disease
KW - N-acetyl aspartate (NAA)
KW - N-acetyltransferase-8-like (NAT8L)
KW - acetyl-CoA
KW - aspartate N-aceyltransferase (ANAT)
KW - aspartoacyclase (ASPA)
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U2 - 10.1021/acschemneuro.1c00455
DO - 10.1021/acschemneuro.1c00455
M3 - Article
C2 - 34477360
AN - SCOPUS:85115119152
SN - 1948-7193
VL - 12
SP - 3445
EP - 3455
JO - ACS Chemical Neuroscience
JF - ACS Chemical Neuroscience
IS - 18
ER -