Design and optimization of aspartate N -acetyltransferase inhibitors for the potential treatment of Canavan disease

Thangavelu, Bharani; Mutthamsetty, Vinay; Wang, Qinzhe; Viola, Ronald E.

doi:10.1016/j.bmc.2016.11.060

Cited by 14 publications

(18 citation statements)

References 20 publications

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“…Structures of the compounds ( 1 – 5 ) identified by AtomNet and validated in vitro to be inhibitors of ANAT. P59, a previously identified inhibitor from a fragment library screen, is included for the purpose of structural comparison.…”

Section: Resultsmentioning

confidence: 99%

“…The experimental assay is based on a custom protocol developed by the Viola lab described previously. 43 Because most of the compounds that were examined have relatively low water solubility, the stock solutions were prepared in DMSO, leading to a final concentration of 10−20% DMSO in the activity assays. Control experiments run in the absence of inhibitors found that DMSO levels as high as 20% had a negligible effect on the enzyme activity.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…ANAT activity was measured by monitoring the production of coenzyme A using 5,5′-dithio-bis[2-nitrobenzoic acid] (DTNB) as described previously . The assay buffer contained 20 mM HEPES, pH 7.5, 150 mM NaCl, 5% glycerol, 40 μM DTNB, 40 μM acetyl-CoA ( K m = 3.1 μM), 2 mM l -aspartate ( K m = 0.16 mM), 10 μg of enzyme ( k cat = 0.071 U/mg), and varying concentrations of inhibitors.…”

Section: Methodsmentioning

confidence: 99%

“…Despite these advancements in the etiology of CD, there are no FDA-approved ANAT inhibitors on the market today. The current set of known inhibitors was identified through a fragment library screen, which was subsequently iteratively optimized to produce the first potent inhibitors of this enzyme . While further potency and selectivity optimization of these core structures is possible, the compounds contain multiple carboxylic acid functional groups, which make them clinically undesirable due to cell permeability and potential toxicity concerns. , New, drug-like scaffolds are needed to interrogate the clinical hypothesis of ANAT inhibition as a therapeutic approach for CD in vivo .…”

Section: Introductionmentioning

confidence: 99%

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Discovery of Novel Inhibitors of a Critical Brain Enzyme Using a Homology Model and a Deep Convolutional Neural Network

2020

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Rare neglected diseases may be neglected but are hardly rare, affecting hundreds of millions of people around the world. Here, we present a hit identification approach using AtomNet, the world's first deep convolutional neural network for structure-based drug discovery, to identify inhibitors targeting aspartate N-acetyltransferase (ANAT), a promising target for the treatment of patients suffering from Canavan disease. Despite the lack of a protein structure or high sequence identity homologous templates, the approach successfully identified five low-micromolar inhibitors with drug-like properties.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Discovery of Novel Inhibitors of a Critical Brain Enzyme Using a Homology Model and a Deep Convolutional Neural Network

2020

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“…14,20 Constitutive knockout of a single Nat8l allele, which reduced [NAA B ] in these mice toward but not below the normal range, reduced leukodystrophy severity. 20,21 Though initial progress has been made in developing specific Nat8l antagonists, 27 no druggable Nat8l inhibitor is yet available. We therefore tested the therapeutic efficacy in Aspa Nur7/Nur7 mice of in vivo brain Nat8l knockdown via neonatal combined intracerebroventricular/intracisternal administration of an AAV vector driving transduction of an Nat8l inhibitory shRNA construct.…”

Section: Discussionmentioning

confidence: 99%

Brain Nat8l Knockdown Suppresses Spongiform Leukodystrophy in an Aspartoacylase-Deficient Canavan Disease Mouse Model

et al. 2018

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Canavan disease, a leukodystrophy caused by loss-of-function ASPA mutations, is characterized by brain dysmyelination, vacuolation, and astrogliosis ("spongiform leukodystrophy"). ASPA encodes aspartoacylase, an oligodendroglial enzyme that cleaves the abundant brain amino acid N-acetyl-L-aspartate (NAA) to L-aspartate and acetate. Aspartoacylase deficiency results in a 50% or greater elevation in brain NAA concentration ([NAA]). Prior studies showed that homozygous constitutive knockout of Nat8l, the gene encoding the neuronal NAA synthesizing enzyme N-acetyltransferase 8-like, prevents aspartoacylase-deficient mice from developing spongiform leukodystrophy. We now report that brain Nat8l knockdown elicited by intracerebroventricular/intracisternal administration of an adeno-associated viral vector carrying a short hairpin Nat8l inhibitory RNA to neonatal aspartoacylase-deficient Aspa mice lowers [NAA] and suppresses development of spongiform leukodystrophy.

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NAAG synthetase deficiency has only low influence on pathogenesis in a Canavan disease mouse model

Becker,

Wang‐Eckhardt,

Eckhardt

2023

J of Inher Metab Disea

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Canavan disease (CD) is a leukodystrophy caused by mutations in the N‐acetylaspartate (NAA) hydrolase aspartoacylase (ASPA). Inability to degrade NAA and its accumulation in the brain results in spongiform myelin degeneration. NAA is mainly synthesized by neurons, where it is also a precursor of the neuropeptide N‐acetylaspartylglutamate (NAAG). Hydrolysis of this peptide by glutamate carboxypeptidases is an additional source of extracellular NAA besides the instant neuronal release of NAA. This study examines to what extent NAA released from NAAG contributes to NAA accumulation and pathogenesis in the brain of Aspanur7/nur7 mutant mice, an established model of CD. Towards this aim, Aspanur7/nur7 mice with additional deficiencies in NAAG synthetase genes Rimklb and/or Rimkla were generated. Loss of myelin in Aspanur7/nur7 mice was not significantly affected by Rimkla and Rimklb deficiency and there was also no obvious change in the extent of brain vacuolation. Astrogliosis was slightly reduced in the forebrain of Rimkla and Rimklb double deficient Aspanur7/nur7 mice. However, only minor improvements at the behavioral level were found. The brain NAA accumulation in CD mice was, however, not significantly reduced in the absence of NAAG synthesis. In summary, there was only a weak tendency towards reduced pathogenic symptoms in Aspanur7/nur7 mice deficient in NAAG synthesis. Therefore, we conclude that NAAG metabolism has little influence on NAA accumulation in Aspanur7/nur7 mice and development of pathological symptoms in CD.

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Design and optimization of aspartate N -acetyltransferase inhibitors for the potential treatment of Canavan disease

Cited by 14 publications

References 20 publications

Discovery of Novel Inhibitors of a Critical Brain Enzyme Using a Homology Model and a Deep Convolutional Neural Network

Discovery of Novel Inhibitors of a Critical Brain Enzyme Using a Homology Model and a Deep Convolutional Neural Network

Brain Nat8l Knockdown Suppresses Spongiform Leukodystrophy in an Aspartoacylase-Deficient Canavan Disease Mouse Model

NAAG synthetase deficiency has only low influence on pathogenesis in a Canavan disease mouse model

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