Highly Sensitive Lysine Deacetylase Assay Based on Acetylated

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Highly sensitive lysine deacetylase assay based on acetylated Firefly luciferase Martin Spinck, Maria Ecke, Sonja Sievers, and Heinz Neumann Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00483 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

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Biochemistry

Highly sensitive lysine deacetylase assay based on acetylated Firefly luciferase Martin Spinck, Maria Ecke, Sonja Sievers and Heinz Neumann Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany Supporting Information Placeholder ABSTRACT: Lysine deacetylases (KDACs) play important roles in many physiological processes and are implicated in many human diseases. Hence, the search for modulators of KDACs is very active and reliable assays to monitor their activity are key to success. Here, we describe a new KDAC assay based on Firefly luciferase harboring an acetylation on an essential active site lysine. We show that several KDACs are able to revert this modification and hence activate luciferase. This new assay is extremely sensitive, reliable as well as fast and can be performed in a continuous format. We used this assay to screen a small library of compounds and identified several novel effectors of SirT2 with low micromolar activity.

Lysine acylations are widespread post-translational modifications of proteins found in all kingdoms of life. Various types of lysine acylation besides acetylation have been identified, including propionylation, butyrylation, crotonylation, malonylation, succinylation, glutarylation, 2-hydroxyisobutyrylation, and myristoylation1. The extent of this plethora of modifications is dynamically controlled by a comparably small number lysine deacetylases (KDACs). KDACs are classified in four enzyme families. Classes 1, 2 and 4 activate a zinccoordinated water molecule to cleave the amide bond, while class 3 enzymes (sirtuins) couple the consumption of NAD+ in nicotinamide (NAM) and O-acyl-ADPribose to the deacylation reaction2. These enzymes are in the limelight of pharmaceutical research because of their role in multiple widespread diseases such as cancer, diabetes, neurodegeneration and aging3. So far, few compounds are available to specifically activate or inhibit individual KDACs, a fact that can be largely attributed to a lack in reliable assays suitable for high-throughput screening4. Robust KDAC assays based on mass spectrometry are time-consuming and of low throughput5. Other discontinuous assays rely

on the separation by HPLC/CE or on the spectrophotometric detection of reaction products4, 6. Continuous assays, which are usually more reliable, couple the KDAC reaction to further chemical or enzymatic reaction steps7, 8 . However, these downstream reactions limit the linear range of the assay and render it more susceptible to side effects by the test compounds. Fluor-de-Lys assays couple the deacylation of the lysine residue to subsequent proteolytic cleavage Cterminally to this residue by trypsin9. This liberates the highly fluorescent 7-amino-4-methyl-coumarin from the C-terminus of the peptide thereby leading to an increase in fluorescence. While this assay is very sensitive, the substrates have suboptimal KM values and the format is usually discontinuous because the protease also degrades the KDAC. This necessitates the use of artificially high enzyme concentrations, which limits the applicability of the Michaelis-Menten equation and the estimation of IC50 and Ki values. Here, we describe a new KDAC assay based on the deacylation of an essential active site lysine residue of Firefly luciferase (FLuc) (Figure 1).

Figure 1. Acetylated Firefly luciferase is activated upon deacetylation by a KDAC.

The active site of FLuc contains a lysine residue that when replaced by any other canonical amino acid renders the enzyme completely inactive10, 11. We investigated whether substitution of this residue with AcK by genetically encoding the modified amino acid would also inactivate the enzyme. Therefore, we expressed FLuc K529ac with a C-terminal His6-tag in E. coli using an

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AcK-specific variant of pyrrolysyl-tRNA synthetase/tRNACUA pair12 and purified the enzyme on Ni-NTA agarose beads (Supplementary Figure 1). Prior to treatment with a KDAC the enzyme produced very little bioluminescence. After incubation with various KDACs the luminescence increased up to 130 fold (Figure 2).

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monitoring the activated luciferase during the KDAC reaction. At increased FLuc K529ac concentration (1 µM) we could quantify SirT2 activity as low as 0.8 nM in the continuous assay format (Supplementary Figure 3). Next, we used our assay to estimate the Ki of NAM for SirT2 and SirT3 (Figure 4). NAM is known to be a non-competitive inhibitor of sirtuins by attacking the bound O-alkylimidate in the active site and reforming NAD+ 13, 14. Inhibition of SirT2 and SirT3 by NAM was measured at four different NAD+ concentrations in the continuous FLuc assay. The Dixon plots15 show a very high linear correlation of the data. The intersection point of the inhibitor lines coincides with the x-axis, which indicates non-competitive inhibition as expected16. The Ki(NAD+) value of NAM obtained for SirT2 (51 µM) is in very good agreement with published values using peptide substrates17.

Figure 2. FLuc K529ac activation by different KDACs. KDACs were used at 20 µg/ml final concentration. All error bars are standard deviations of triplicates using the endpoint assay format.

Hence, FLuc K529ac is a substrate for KDACs and a highly sensitive reporter enzyme for KDAC activity. We titrated SirT2 and CobB to identify the suitable concentration range and compared the performance of the new assay to the established Fluor-de-Lys assay (Figure 3 and Supplementary Figure 2). Optimal working concentrations for the Fluor-de-Lys assay are 10 µg/ml SirT2 and 10 µM substrate peptide. The FLuc-based assay required tenfold less SirT2 and nanomolar concentrations of FLuc K529ac.

Figure 3. Quantification of SirT2 activity using Fluor-deLys (blue) and FLuc K529ac (orange) assays. Shaded areas indicate suitable working concentration of SirT2 in different assays. The gray curve was obtained using the continuous format of the FLuc K529ac assay.

The new assay is very convenient and fast because it can be performed in less than 30 min and does not involve a subsequent proteolysis step. The FLuc-based assay can also be performed in a continuous format,

Figure 4. Dixon plots showing non-competitive inhibition by NAM towards NAD+ in the SirT2 (Ki=51 µM) (A) and SirT3 (Ki=85 µM) (B) catalyzed FLuc K529ac reaction. Data was acquired using the continuous assay format. Error bars are standard deviations of triplicate measurements.

For SirT3 we obtained a Ki(NAD+) value of 85 µM, which is twofold higher than published values18. This may be a result of the difference in the chemical nature and concentration of the deacetylation substrate. Unfortunately, it is impractical to perform the assays under substrate saturation because this would require FLuc K529ac concentrations several fold higher than KM,

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Biochemistry

which we expect to be in the micromolar range. Such a high concentration of FLuc K529ac is difficult to achieve and would immediately consume the substrates of the luciferase reaction. We analyzed NAM inhibition of SirT3 at tenfold higher FLuc:SirT3 ratio, which shifted the Ki(NAD+) to approximately 40 µM (Supplementary Figure 4). Finally, we tested whether the FLuc-based KDAC assay is suitable for screening KDAC inhibitors. Therefore, we composed a set of 351 compounds with similarity to known sirtuin inhibitors. Among these we identified eight compounds that inhibited and one that activated the SirT2 reaction at 10 µM using the FLuc K529ac assay in endpoint format (Figure 5). The inhibitors displayed IC50 values against SirT2 of 3-30 µM, whereas compound 8 enhanced the reaction 1.6 fold at 30 µM (Supplementary Figure 5). Testing these compounds for their direct impact on FLuc revealed compound 9 as a FLuc inhibitor, while the other compounds did not influence FLuc activity directly (Supplementary Figure 6).

The compounds with the highest potency are resveratrol (1) and piceatannol (2). Piceatannol had previously been shown to inhibit SirT219. The structurally very similar resveratrol is a known activator of yeast Sir2 and SirT19. The effect on other sirtuins strongly depends on the combination of KDAC and substrate and can be either activating or inhibitory20. In sum, we present a highly reliable KDAC assay with exceptional sensitivity. The assay is convenient and fast and can be performed in a continuous format. By producing the accordingly modified FLuc, it would be straightforward to adapt the assay to measure the removal of crotonyl, butyryl, propionyl21-23 or 2hydroxyisobutyryl24, 25 groups from lysine residues. We identified seven compounds which inhibit FLuc K529ac deacetylation by SirT2 at low micromolar concentrations. Compound 5 is structurally similar to AGK2, which inhibits SirT2 with an IC50 of 3.5 µM26. Compounds 6 and 7 are similar to SRT1720, which was initially reported as a potent activator of SirT127 and later shown to specifically enhance its interaction with fluorophore-labeled peptides28. Piceatannol (2) and particularly resveratrol (1) are familiar sirtuin activators9. It is therefore surprising that resveratrol and structurally similar compounds are the most active inhibitors of SirT2 identified in our screen. However, resveratrol specifically activates fluorophore-labeled peptide substrates by stabilizing the enzyme-substrate complex29, 30. Resveratrol’s effect on sirtuin activity is highly dependent on the sirtuin-substrate combination and has indeed been shown to be inhibitory for SirT3 by enforcing an unproductive conformation of the enzyme-substrate complex20. The 2-mercapto-quinazoline derivative 8 is structurally similar to thiobarbiturates, which have been reported to inhibit SirT2 at low micromolar concentration31. It will be interesting to further explore the activating potential of this compound class. This work demonstrates the importance of using orthogonal assays to screen small molecule libraries. Hence, the FLuc-based assay described here will be a valuable addition to the arsenal for screening KDAC effectors. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. Experiment details and Supplementary Figures 1-6 (PDF).

AUTHOR INFORMATION Corresponding Author

Figure 5. Effectors identified in FLuc K529ac-based screen. A) Inhibitors of SirT2 (IC50 of 1-7 (µM): 3.2; 5.5; 17; >30; 13; 15; 11. n=2) B) SirT2 activator (1.6 fold at 30 µM) C) Firefly luciferase inhibitor (IC50: 3 µM).

Heinz Neumann, [email protected].

Author Contributions

M.S, M.E. and H.N. performed experiments. S.S. and H.N. analyzed data. H.N. wrote the manuscript.

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Funding Sources

This project was supported financially by the Heisenberg Program of the German Research Foundation (DFG) [NE1589/5-1 to H.N.] and the Max-Planck-Institute of Molecular Physiology.

[10] ACKNOWLEDGMENT We thank Dr. Michael Lammers for KDAC plasmids.

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