Original articleIdentification of benzofuran-3-yl(phenyl)methanones as novel SIRT1 inhibitors: Binding mode, inhibitory mechanism and biological action
Graphical abstract
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).
References (35)
- et al.
SIRT1 modulating compounds from high-throughput screening as anti-inflammatory and insulin-sensitizing agents
J. Biomol. Screen.
(2006) - et al.
Mammalian SIRT1 represses forkhead transcription factors
Cell
(2004) - et al.
hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase
Cell
(2001) - et al.
Synthesis and biological activity of splitomicin analogs targeted at human NAD(+)-dependent histone deacetylases (sirtuins)
Bioorg. Med. Chem.
(2011) Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of Yeast Sir2 and human SIRT1
J. Biol. Chem.
(2002)- et al.
Structural basis for nicotinamide inhibition and base exchange in Sir2 enzymes
Mol. Cell
(2007) - et al.
Mechanism of sirtuin inhibition by nicotinamide: altering the NAD+ Cosubstrate Specificity of a Sir2 enzyme
Mol. Cell
(2005) - et al.
Crystal structures of human SIRT3 displaying substrate-induced conformational changes
J. Biol. Chem.
(2009) - et al.
SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1
J. Biol. Chem.
(2010) - et al.
A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide
Anal. Biochem.
(2009)
Structural insights into intermediate steps in the Sir2 deacetylation reaction
Structure
SirT1 is an inhibitor of proliferation and tumor formation in colon cancer
J. Biol. Chem.
Negative control of p53 by Sir2alpha promotes cell survival under stress
Cell
Discovery, in vivo activity, and mechanism of action of a small-molecule p53 activator
Cancer Cell
Crystal structure of a SIR2 homolog-NAD complex
Cell
Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity
Proc. Natl. Acad. Sci. U.S.A.
Sirtuin activators mimic caloric restriction and delay ageing in metazoans
Nature
Cited by (22)
Molecular modeling of alkaloids bouchardatine and orirenierine binding to sirtuin-1 (SIRT1)
2022, Digital Chinese MedicineCitation Excerpt :This site Ile347 was defined as the potential binding site for the other ligands tested in our docking analysis, and was juxtaposed to the C-pocket, where the nicotinamide moiety of NAD+ binds and the hydrolysis takes place. Drug binding to this site is known to block the transformation of NAD+ to productive conformation and to inhibit the deacetylase activity 23,24. The secondary weak site (Ile316) has never been mentioned in other studies; it is considered too weak and irrelevant.
Polycyclic heterocycles by condensation of 1,4-benzoquinone analogs and nucleophiles
2021, Advances in Heterocyclic ChemistryAntiproliferative activity of (R)-4′-methylklavuzon on hepatocellular carcinoma cells and EpCAM<sup>+</sup>/CD133<sup>+</sup> cancer stem cells via SIRT1 and Exportin-1 (CRM1) inhibition
2019, European Journal of Medicinal ChemistryCitation Excerpt :Moreover, structural similarity of (R)-4′-methylklavuzon and TK126 to goniothalamin which was shown to be CRM1 inhibitor, allowed us to test its CRM1 inhibitory properties in HCC cells for the first time in this study. There have been several studies including antiproliferative activities of small molecules in various types of cancer cell lines via SIRT1 inhibition [42,43]. Up to date, there is no reported covalent inhibitor of SIRT1.
Design, synthesis and biological evaluation of benzofuran appended benzothiazepine derivatives as inhibitors of butyrylcholinesterase and antimicrobial agents
2018, Bioorganic and Medicinal ChemistrySynthesis and biological evaluation of new benzofuran carboxamide derivatives
2017, Journal of Saudi Chemical SocietyCitation Excerpt :In skeleton modifications of the benzofurans are possible in six different positions. For example, Marriott et al. proved that benzofuran-2-carboxamide derivatives act as sigma receptors [24] and Wu et al. synthesized modified benzofuran as SIRT1 inhibitor [10]. Similarly Kirilmis et al. reported the antimicrobial activity of benzofuran-2-ethanone derivatives [14] and Xie et al. reported the anti-inflammatory activity of benzofuran-2-carboxalamide derivatives [25].
Biological and medicinal significance of benzofuran
2015, European Journal of Medicinal ChemistryCitation Excerpt :21 derivatives of benzofuran-3-yl(phenyl)methanones were synthesized and evaluated for their inhibitory activity on SIRT1. Compound 33 was found be more active derivative among this series (IC50 = 2.8 μM) [28]. Mitogen-activated protein kinase phosphatase-1 (MKP-1) enzyme is highly expressed in prostate, breast, gastric and renal cancers and is considered as a good target for these cancers.