Original article
Identification of benzofuran-3-yl(phenyl)methanones as novel SIRT1 inhibitors: Binding mode, inhibitory mechanism and biological action

https://doi.org/10.1016/j.ejmech.2012.12.026Get rights and content

Abstract

SIRT1 is a NAD+-dependent deacetylase. Here we described new SIRT1 inhibitors with the scaffold of benzofuran-3-yl(phenyl)methanone. The inhibitors were predicted to bind in C-pocket of SIRT1, forming hydrophobic interactions with Phe273, Phe312 and Ile347. Introducing hydroxyl to meta position of phenyl may form H-bond with Asn346. Indeed, (2,5-dihydroxyphenyl)(5-hydroxy-1-benzofuran-3-yl)methanone (16), an analogue with hydroxyls at ortho and meta positions, showed greater inhibition. The binding mode was validated by structural modifications and kinetic studies. Since C-pocket is the site where the nicotinamide moiety of NAD+ binds and the hydrolysis takes place, binding of 16 in C-pocket would block the transformation of NAD+ to productive conformation and hence inhibit the deacetylase activity. Consistently, 16 inhibited SIRT1 through up-regulating p53 acetylation on cellular level.

Graphical abstract

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New SIRT1 inhibitors with scaffold of benzofuran-3-yl(phenyl) methanone, as represented by (2,5 dihydroxyphenyl) (5-hydroxy- 1-benzofuran-3-yl)methanone (16), were designed and determined to bind in the C-pocket of SIRT1.

Highlights

► Benzofuran-3-yl(phenyl)methanone derivatives were identified as SIRT1 inhibitors. ► The inhibition pattern of the inhibitor was determined against the substrates. ► The inhibitor binds in the C-pocket of SIRT1. ► The binding mode was validated by structural modification and mutagenesis studies. ► The inhibitor up-regulates the acetylation of p53 on cellular level.

Introduction

SIRT1 is a nuclear nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that deacetylates histones, tumour suppressor protein p53 and several other transcription factors [1], [2]. It plays important roles in many biological functions such as fat mobilization [3], life span regulation [4], [5], cellular stress responses [5], [6], inflammation [7] and apoptosis [8], [9], which renders it a potential target for the design of drugs against cancer, diabetes and age-related diseases. Up to present, numbers of SIRT1 inhibitors, such as nicotinamide [10], Ex527 [11], sirtinol [12] and splitomicin derivatives [13] have been reported. These inhibitors had been used to explore the biological functions of SIRT1 [14], [15], [16], and been demonstrated to increase p53 acetylation and thus induce apoptosis of cancer cells [17], [18], [19]. Here we describe a series of new SIRT1 inhibitors with the scaffold of benzofuran-3-yl(phenyl)methanone, which were discovered from our in-house compound library. Through the structure–activity relationship (SAR) analysis and docking simulation, we predicted that the inhibitors bind in the C-pocket of SIRT1. Based on the interaction model, another more potent SIRT1 inhibitor, (2, 5-dihydroxyphenyl) (5-hydroxy-1-benzofuran-3-yl) methanone (16), was designed with an IC50 of 2.8 μM. The interactions between the inhibitor and SIRT1 were further supported by structural modifications of 16 and enzyme kinetics studies. Moreover, we also demonstrated that compound 16 inhibits SIRT1 deacetylase activity and up-regulates the acetylation of p53 on cellular level, suggesting that benzofuran-3-yl(phenyl)methanone may serve as a novel structural scaffold for developing potent SIRT1 inhibitors that have potential application in cancer treatment.

Section snippets

Identification, SAR analysis and binding mode prediction of new SIRT1 inhibitor

We employed a 4-amino-7-methylcoumarin (AMC)-based fluorescent assay to identify the compounds that inhibit SIRT1 enzyme activity [20]. The acetyl peptide substrate, ac-RHKKac-AMC, is derived from the p53 sequence and its C-terminus was conjugated with AMC. The AMC fluorophore can be cleaved from the peptide by trypsin only when the ɛ-lysine of the peptide is deacetylated. The fluorescence from the free AMC can be quantified at 490 nm with the excitation wavelength of 340 nm. A known SIRT1

Conclusions

In this paper we identified a new class of SIRT1 inhibitors with the benzofuran-3-yl(phenyl)methanone scaffold by high through-put screening. The binding mode of the inhibitors was predicted through the SAR analysis and docking simulations, based on which another more potent SIRT1 inhibitor, (2, 5-dihydroxyphenyl) (5-hydroxy-1-benzofuran-3-yl) methanone (16), was found and confirmed to be a noncompetitive inhibitor for acetyl peptide and a mixed-type competitive inhibitor for NAD+. The SAR

Protein expression and purification

The coding region of SIRT1(156-664) was inserted between the NdeI and XhoI restriction sites in pET28a vector together with an N-terminal hexa-histidine tag. The plasmid was transformed into Escherichia coli BL21 (DE3) competent cells. A single colony was inoculated in LB medium containing 50 μg/mL kanamysin at 37 °C. When OD600 reached 0.8–1.0, the culture was induced with 0.5 mM IPTG at 16 °C overnight. The protein was purified on a Ni-NTA column and stored at −80 °C. SIRT1 mutations SIRT1

Acknowledgement

This work was supported by the CAS “Introducing Outstanding Oversea Scientists Project”, the National Science Fund for Creative Research Groups (Grant 21021063), and the Science and Technology Commission of Shanghai Municipality (Grant 08JC1422100).

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