Development of a single-step fluorogenic sirtuin assay and its applications for high-throughput screening

Wang, Pan; Chen, Di; An, Jian-Xiong; Lin, Shuxian; Liu, Ting; Li, Yan; Chen, Lei; He, Bin

doi:10.1039/d1ob02347k

Cited by 6 publications

(4 citation statements)

References 41 publications

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“…As discussed in the previous chapter, the interest in sirtuins has its origin in the impact on life extension. The aim is to find the effective and selective modulators of their activity (Wang et al 2022 ). Sirtuins are involved in various metabolic pathways and alterations in their activity may affect different targets differently.…”

Section: Oxidative Stress Diseases Aging and Antioxidant Interventionsmentioning

confidence: 99%

Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging

Jomova,

Raptova,

Alomar

et al. 2023

Arch Toxicol

520

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A physiological level of oxygen/nitrogen free radicals and non-radical reactive species (collectively known as ROS/RNS) is termed oxidative eustress or “good stress” and is characterized by low to mild levels of oxidants involved in the regulation of various biochemical transformations such as carboxylation, hydroxylation, peroxidation, or modulation of signal transduction pathways such as Nuclear factor-κB (NF-κB), Mitogen-activated protein kinase (MAPK) cascade, phosphoinositide-3-kinase, nuclear factor erythroid 2–related factor 2 (Nrf2) and other processes. Increased levels of ROS/RNS, generated from both endogenous (mitochondria, NADPH oxidases) and/or exogenous sources (radiation, certain drugs, foods, cigarette smoking, pollution) result in a harmful condition termed oxidative stress (“bad stress”). Although it is widely accepted, that many chronic diseases are multifactorial in origin, they share oxidative stress as a common denominator. Here we review the importance of oxidative stress and the mechanisms through which oxidative stress contributes to the pathological states of an organism. Attention is focused on the chemistry of ROS and RNS (e.g. superoxide radical, hydrogen peroxide, hydroxyl radicals, peroxyl radicals, nitric oxide, peroxynitrite), and their role in oxidative damage of DNA, proteins, and membrane lipids. Quantitative and qualitative assessment of oxidative stress biomarkers is also discussed. Oxidative stress contributes to the pathology of cancer, cardiovascular diseases, diabetes, neurological disorders (Alzheimer’s and Parkinson’s diseases, Down syndrome), psychiatric diseases (depression, schizophrenia, bipolar disorder), renal disease, lung disease (chronic pulmonary obstruction, lung cancer), and aging. The concerted action of antioxidants to ameliorate the harmful effect of oxidative stress is achieved by antioxidant enzymes (Superoxide dismutases-SODs, catalase, glutathione peroxidase-GPx), and small molecular weight antioxidants (vitamins C and E, flavonoids, carotenoids, melatonin, ergothioneine, and others). Perhaps one of the most effective low molecular weight antioxidants is vitamin E, the first line of defense against the peroxidation of lipids. A promising approach appears to be the use of certain antioxidants (e.g. flavonoids), showing weak prooxidant properties that may boost cellular antioxidant systems and thus act as preventive anticancer agents. Redox metal-based enzyme mimetic compounds as potential pharmaceutical interventions and sirtuins as promising therapeutic targets for age-related diseases and anti-aging strategies are discussed.

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Section: Oxidative Stress Diseases Aging and Antioxidant Interventionsmentioning

confidence: 99%

Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging

Jomova,

Raptova,

Alomar

et al. 2023

Arch Toxicol

520

View full text Add to dashboard Cite

show abstract

“…Previously reported high-throughput screens targeting SIRT2 focused on inhibiting its deacetylase activity [28][29][30] or identifying ligands that bind to a SIRT2-decanoyl-peptide complex [25]. New assays that are compatible with screening have been developed to detect inhibition of SIRT2's defatty-acylase activity [24,26,27,31]. Assays developed to study other defatty-acylases including other sirtuin isoforms and classical histone deacetylases (HDACs) may also be adapted to identify SIRT2 inhibitors [32].…”

Section: Introductionmentioning

confidence: 99%

A homogeneous time-resolved fluorescence screen to identify SIRT2 deacetylase and defatty-acylase inhibitors

Yang,

Cassel,

Boyle

et al. 2024

PLoS ONE

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Human sirtuin-2 (SIRT2) has emerged as an attractive drug target for a variety of diseases. The enzyme is a deacylase that can remove chemically different acyl modifications from protein lysine residues. Here, we developed a high-throughput screen based on a homogeneous time-resolved fluorescence (HTRF) binding assay to identify inhibitors of SIRT2’s demyristoylase activity, which is uncommon among many ligands that only affect its deacetylase activity. From a test screen of 9600 compounds, we identified a small molecule that inhibited SIRT2’s deacetylase activity (IC50 = 7 μM) as well as its demyristoylase activity (IC50 = 37 μM). The inhibitor was composed of two small fragments that independently inhibited SIRT2: a halogenated phenol fragment inhibited its deacetylase activity, and a tricyclic thiazolobenzimidazole fragment inhibited its demyristoylase activity. The high-throughput screen also detected multiple deacetylase-specific SIRT2 inhibitors.

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Section: Introductionmentioning

confidence: 99%

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Development of a single-step fluorogenic sirtuin assay and its applications for high-throughput screening

Abstract: Sirtuins (SIRTs) are a class of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases. Since SIRTs have different subcellular locations and different preferences for deacylation activity, SIRTs are not only highly gaining...

Cited by 6 publications

References 41 publications

Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging

Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging

A homogeneous time-resolved fluorescence screen to identify SIRT2 deacetylase and defatty-acylase inhibitors

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