Development of a NanoBRET assay to validate inhibitors of Sirt2-mediated lysine deacetylation and defatty-acylation that block prostate cancer cell migration

Vogelmann, Anja; Schiedel, Matthias; Wössner, Nathalie; Merz, Annika; Herp, Daniel; Hammelmann, Sören; Colcerasa, Arianna; Komaniecki, Garrison; Hong, Jun Young; Sum, Manuela; Metzger, Eric; Neuwirt, Emilia; Zhang, L; Einsle, Oliver; Groß, Olaf; Schüle, Roland; Lin, Hening; Sippl, Wolfgang; Jung, Manfred

doi:10.1039/d1cb00244a

Cited by 17 publications

(67 citation statements)

References 83 publications

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“…This finding is in line with the proposed binding mode of the inhibitor. Additionally, the calculated K i value of 13 nM ( Figure 5 C) for cmp12 was lower than any other K i value reported for small molecule SIRT2 inhibitors [ 66 ]. In order to validate the substrate F4 , we compared IC 50 values of known SIRT2 inhibitors (including AGK2 [ 69 ], KK-22 [ 70 ], S2iL5 [ 71 ], and SMyr [ 55 ]) using different substrates described in continuous and discontinuous activity assays and with both acetylated and myristoylated lysine residues ( Table 2 ).…”

Section: Resultsmentioning

confidence: 55%

“…Recently, novel derivatives of SirReal have been described (i.e., cmp12 ; the structure is shown in Figure S13 ) which potently inhibit SIRT2-mediated demyristoylation reactions in vitro and in cells [ 66 ]. We determined the respective K i value for cmp12 using the substrate F4 and SIRT2.…”

Section: Resultsmentioning

confidence: 99%

“…Fmoc-protected β-(7-methoxy-coumarin-4-yl)-alanine (Mcm) was purchased from Bachem (Bubendorf, Switzerland). KK22 , cmp12, S2iL5, Fmoc-Lys(Ns)-OH, and SMyr were synthesized as described previously [ 50 , 66 , 70 ]. SirReal2 was purchased from Biomol (Hamburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…The values in the table represent the mean of three independent replicates. ( B ) v/[S] plot of SIRT2 (1 nM) with different concentrations of F4 , 500 µM of NAD + , and different concentrations of cmp12 [ 66 ]. A nonlinear regression analysis was done according to the Michaelis-Menten equation.…”

Section: Figurementioning

confidence: 99%

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Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Zessin

Meleshin

Hilscher

et al. 2023

IJMS

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Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M−1s−1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.

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Section: Resultsmentioning

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Zessin

Meleshin

Hilscher

et al. 2023

IJMS

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“…47 Thus, we were interested to see whether our dual Sirt2/HDAC6 inhibitors also prevent Sirt2-catalyzed defatty acylation. Using a previously reported biochemical in vitro demyristoylation assay that is based on the small molecule myristoylated substrate ZMML, 47 several of our triazole-based dual Sirt2/HDAC6 inhibitors were identified as low micromolar inhibitors of Sirt2-catalyzed demyristoylation (Table 2). Consistent with the data for Sirt2-mediated deacetylation (Table 1), the inhibitors with a merged Sirt2/ HDAC6 pharmacophore (21,22) showed the weakest inhibition of Sirt2-mediated demyristoylation.…”

Section: Synthesis Of Hdac6 Inhibitors As Control Compoundsmentioning

confidence: 99%

Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation

Sinatra,

Vogelmann,

Friedrich

et al. 2023

J. Med. Chem.

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Dysregulation of both tubulin deacetylases sirtuin 2 (Sirt2) and the histone deacetylase 6 (HDAC6) has been associated with the pathogenesis of cancer and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. Herein, we report the design, synthesis, and biological characterization of the first-in-class dual Sirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulin deacetylation. Using biochemical in vitro assays and cell-based methods for target engagement, we identified Mz325 (33) as a potent and selective inhibitor of both target enzymes. Inhibition of both targets was further confirmed by X-ray crystal structures of Sirt2 and HDAC6 in complex with building blocks of 33. In ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to single or combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus, our dual Sirt2/HDAC6 inhibitors are important new tools to study the consequences and the therapeutic potential of dual inhibition of tubulin deacetylation.

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Symmetrical 2,7‐disubstituted 9H‐fluoren‐9‐one as a novel and promising scaffold for selective targeting of SIRT2

Kaya,

Eren,

Massarotti

et al. 2024

Archiv der Pharmazie

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Sirtuin 2 (SIRT2) belongs to the family of silent information regulators (sirtuins), which comprises nicotinamide adenine dinucleotide (NAD+)‐dependent protein lysine deacetylases. With a distribution across numerous tissues and organs of the human body, SIRT2 is involved in a wide range of physiological and pathological processes, such as regulating the cell cycle, energy metabolism, DNA repair, and tumorigenesis. Aberrant expression of SIRT2 has been closely associated with particular etiologies of human diseases, positioning SIRT2 as a promising therapeutic target. Herein, we detail the design overview and findings of novel symmetrical 2,7‐disubstituted 9H‐fluoren‐9‐one derivatives targeting SIRT2. SG3 displayed the most potent SIRT2‐selective inhibitory profile, with an IC50 value of 1.95 , and reduced the cell viability of human breast cancer MCF‐7 cells accompanied by hyperacetylation of α‐tubulin. Finally, molecular docking, molecular dynamics simulations, and binding free energy calculations using molecular mechanics/generalized born surface area method were performed to verify the binding ability of SG3 to SIRT2. Taken together, these results could enhance our understanding of the structural elements necessary for inhibiting SIRT2 and shed light on the mechanism of inhibition.

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Development of a NanoBRET assay to validate inhibitors of Sirt2-mediated lysine deacetylation and defatty-acylation that block prostate cancer cell migration

Abstract: Sirtuin2 (Sirt2) with its NAD+-dependent deacetylase and defatty-acylase activities plays a central role in the regulation of specific cellular functions. Dysregulation of Sirt2 activity has been associated with the pathogenesis...

Cited by 17 publications

References 83 publications

Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation

Symmetrical 2,7‐disubstituted 9H‐fluoren‐9‐one as a novel and promising scaffold for selective targeting of SIRT2

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