A spectrophotometric assay for histone deacetylase 8

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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 372 (2008) 82–88 www.elsevier.com/locate/yabio

A spectrophotometric assay for histone deacetylase 8 David G. Fatkins, Weiping Zheng

*

Department of Chemistry, University of Akron, 190 E. Buchtel Commons, Akron, OH 44325, USA Received 14 June 2007 Available online 1 September 2007

Abstract Inhibitors for the classical protein deacetylase enzymes have been actively pursued to develop the next generation of cancer therapy. Developing a novel convenient assay platform for the classical enzyme-catalyzed reactions could thus facilitate the drug discovery process. Based on our previous studies demonstrating the functional mimicry of Ne-thioacetyl-lysine for Ne-acetyl-lysine in the reaction catalyzed by the classical enzyme histone deacetylase 8 (HDAC8) on a peptide template derived from the C terminus of the human p53 tumor suppressor protein, we have developed a spectrophotometric HDAC8 assay via quantifying thioacetate produced from the enzymatic dethioacetylation with Ellman’s reagent 5,5 0 -dithiobis(2-nitrobenzoate). We further demonstrated that this novel assay was selective for HDAC8 versus HDAC1 and 2 and for other classical protein deacetylase enzymes present in the HeLa nuclear extracts, thus making it potentially suitable not only for screening HDAC8-selective inhibitors but also for selectively assessing HDAC8 activity under (patho)physiological conditions.  2007 Elsevier Inc. All rights reserved. Keywords: HDAC8; Spectrophotometric assay; DTNB

Protein acetyltransferases and protein deacetylases are the two families of enzymes that, respectively, catalyze the specific lysine Ne-acetylation and Ne-deacetylation on proteins such as the core histone proteins, various transcription factors, a-tubulin, acetyl-coenzyme A synthetases, and human immunodeficiency virus (HIV) Tat protein that are, respectively, involved in gene transcriptional, cytoskeletal, metabolic control, and HIV infection (Fig. 1) [1–7]. Based on homology with yeast transcriptional repressors, phylogenetic analysis, and different cofactor requirements, human protein deacetylase enzymes have been categorized into class I (HDAC1,2,3,8; HDAC1 is the abbreviation for histone deacetylase that is named after the first discovered protein substrate histone), class II (HDAC4,5,6,7,9,10), class III (SIRT1,2,3,4,5,6,7), and *

class IV (HDAC11 and its related enzymes) subfamilies [8,9]. Class I, II, and IV (also collectively known as classical) enzymes require a catalytic zinc (Zn2+), whereas class III (also known as sirtuins) enzymes require coenzyme nicotinamide adenine dinucleotide (NAD+) for activity. The classical protein deacetylase-catalyzed reaction has been targeted for developing novel therapies for cancer [10,11]. Suberoylanilide hydroxamic acid (SAHA) has recently been approved (as the first classical protein deacetylase inhibitor) by the U.S. Food and Drug Administration for treating cutaneous T cell lymphoma. Several other small-molecule inhibitors for the classical enzymes have also moved onto different stages of clinical trials primarily to evaluate their anticancer profiles. These developments have fueled ongoing efforts in both industry and

Corresponding author. Fax: +1 (330) 972 7370. E-mail address: [email protected] (W. Zheng). 1 Abbreviations used: HDAC, histone deacetylase; NAD+, nicotinamide adenine dinucleotide; SAHA, suberoylanilide hydroxamic acid; SIRT1, human sirtuin type 1; DTNB, 5,5 0 -dithiobis(2-nitrobenzoic acid); TNB, 2-nitro-5-thiobenzoate; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate; HIV, human immunodeficiency virus; HOBt, N-hydroxybenzotriazole; DMF, N,N-dimethylformamide; NMM, 4-methylmorpholine; EDTA, ethylenediaminetetracetic acid; BSA, bovine serum albumin; RT, room temperature; RP-HPLC, reversed-phase high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; ESI, electrospray ionization; UV, ultraviolet. 0003-2697/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.08.031

Spectrophotometric assay for HDAC8 / D.G. Fatkins, W. Zheng / Anal. Biochem. 372 (2008) 82–88

lyzed dethioacetylation reaction by its reaction with the Ellman’s reagent 5,5 0 -dithiobis(2-nitrobenzoate) (DTNB) [13], with the reaction product 2-nitro-5-thiobenzoate (TNB) being monitored at 412 nm (Fig. 2).

H3C O H3N

HN Protein deacetylases H N H

H

Protein acetyltransferases

Materials and methods

N H

O Deacetylated protein

O Acetylated protein

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Materials Peptide synthesis and purification. All Fmoc-protected amino acids (except Na-Fmoc-Ne-thioacetyl-lysine), resins, the coupling reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3tetramethylaminium hexafluorophosphate (HBTU), and the additive N-hydroxybenzotriazole (HOBt) were purchased from Novabiochem (La Jolla, CA, U.S.A). Na-Fmoc-Ne-thioacetyl-lysine was synthesized from NaFmoc-lysine and ethyl dithioacetate as described previously [12] and used to incorporate Ne-thioacetyl-lysine into peptides. Trifluoroacetic acid, N,N-dimethylformamide (DMF), and acetonitrile were purchased from EMD Biosciences (San Diego, CA, U.S.A). Anhydrous diethyl ether was purchased from Fisher (Pittsburgh, PA, U.S.A). 4-Methylmorpholine (NMM), piperidine, phenol, thioanisole, and ethanedithiol were purchased from Aldrich (Milwaukee, WI, U.S.A). Assays. DTNB, Trizma, guanidinium chloride, ethylenediaminetetracetic acid (EDTA) disodium salt, and a 1 M solution of MgCl2 (molecular-biology grade) were purchased from Sigma (St. Louis, MO, U.S.A). Thioacetic acid was purchased from Aldrich. The bovine serum albu-

Fig. 1. Lysine Ne-acetylation and Ne-deacetylation reactions catalyzed, respectively, by protein acetyltransferases and protein deacetylases.

academia to pursue further inhibitors, especially the isoform-selective inhibitors for the classical protein deacetylase enzymes. With regard to this, developing novel convenient enzyme assay platforms could facilitate the drug discovery process. By evaluating human p53 tumor suppressor protein Cterminal peptides (amino acid residues 372–389) containing Ne-thioacetyl-lysine or Ne-acetyl-lysine at the 382 position, we previously demonstrated [12] that, whereas the former peptide potently inhibited human sirtuin type 1 (SIRT1) with an IC50  2 lM, the two peptides were comparably de(thio)acetylated by human HDAC8, suggesting that, when placed within an appropriate amino acid sequence, the thioacetyl group can serve as a functional mimic for the acetyl group for the enzymatic deacetylation reaction catalyzed by HDAC8. We thus hypothesized that a spectrophotometric HDAC8 assay could be developed via quantifying thioacetate released from the HDAC8-cataH3C S

H3N

HN HDAC8

O

+

H

H

S

HN

HN

CH3

thioacetate

O p53 peptide

O thioacetyl-lysine p53 peptide NO2

COO NO2

NO2

O

COO S

CH3

thioacetate

+

S S

COO

+ S

S O OOC NO2 5,5'-dithiobis(2-nitrobenzoate) (DTNB)

2-nitro-5-thiobenzoate (TNB) monitored at 412 nm

S

H3C

Fig. 2. Reaction scheme for the DTNB-based spectrophotometric assay developed in this study. Peptide sequence: H2N-KKGQSTSRHKXLMFKTEGCOOH with X = Lys (p53 peptide), Ne-thioacetyl-lysine (thioacetyl-lysine p53 peptide).

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min (BSA) with reduced fatty acid content was also purchased from Sigma (Cat. No. A3803), and was used for all the assays. NaCl, KCl, and NaH2PO4 were purchased from Fisher. The human recombinant HDAC8 and HDAC1 and the HeLa nuclear extracts (enriched in HDAC1 and 2) were purchased from Biomol International, L.P. (Plymouth Meeting, PA, U.S.A.) (Cat. Nos. SE145-0100, SE456-0050, and KI140-0100, respectively). The human recombinant HDAC2 was purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.) (Cat. No. 10009377). Peptide synthesis and purification The following peptides used in this study were synthesized based on the Fmoc chemistry strategy [14] on a PS3 peptide synthesizer (Protein Technologies Inc., Tucson, AZ, U.S.A.): (i) H2N-KKGQSTSRHK(K)LMFKTEGCOOH (the p53 peptide shown in Fig. 2), (ii) H2NKKGQSTSRHK(ThAcK)LMFKTEG-COOH (ThAcK = Ne-thioacetyl-lysine, the thioacetyl-lysine p53 peptide shown in Fig. 2), (iii) H2N-KKGQSTSRHK(AcK)LMFKTEG-COOH (AcK = Ne-acetyl-lysine, the acetyl-lysine p53 peptide), (iv) AcNH-RH(AcK)(AcK)-CONH2, and (v) AcNH-RH(ThAcK)(ThAcK)-CONH2. Whereas peptides i–iii were synthesized from the Wang resin preloaded with Fmoc-Gly, peptides iv and v were synthesized from the Rink amide resin. Of note, peptides i–iii were also previously used by us [12,15] and others [16–18] for protein deacetylase and bromodomain binding assays and/or Xray crystallographical studies. For each peptide coupling reaction, 4 equivalents of a Fmoc-protected amino acid, 3.8–4.0 equivalents of the coupling reagent HBTU, and the additive HOBt were used in the presence of 0.4 M NMM/DMF, and the coupling reaction was allowed to proceed at room temperature (RT) for 1 h. A 20% (v/v) piperidine/DMF solution was used for Fmoc removal. All peptides were cleaved from the resins by reagent K (83.6% (v/v) trifluoroacetic acid, 5.9% (v/v) phenol, 4.2% (v/v) ddH2O, 4.2% (v/v) thioanisole, 2.1% (v/v) ethanedithiol), precipitated in cold diethyl ether, and purified by reversed-phase high pressure liquid chromatography (RP-HPLC) on a preparative ˚ , 2.14 · 25 cm). The column was eluted C18 column (100 A with a gradient of double-deionized water containing 0.05% (v/v) trifluoroacetic acid and acetonitrile containing 0.05% (v/v) trifluoroacetic acid at 10 mL/min and monitored at 214 nm. The pooled HPLC fractions were stripped of acetonitrile and lyophilized to give all peptides as puffy white solids. Peptide purity (>95%) was verified by RP-HPLC on ˚ , 0.46 · 25 cm). The column an analytical C18 column (100 A was eluted with a gradient of double-deionized water containing 0.05% (v/v) trifluoroacetic acid and acetonitrile containing 0.05% (v/v) trifluoroacetic acid at 1 mL/min and monitored at 214 nm. The molecular weights of all purified peptides were confirmed by either matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)

or electrospray ionization (ESI) mass spectrometric analysis. Peptide i: MS (MALDI-TOF) m/e 2091 [M+H]+; peptide ii: MS (MALDI-TOF) m/e 2149 [M+H]+; peptide iii: MS (MALDI-TOF) m/e 2133 [M+H]+; peptide iv: MS (ESI) m/e 693 [M+H]+; peptide v: MS (ESI) m/e 725 [M+H]+. Kinetic analysis of the reaction between DTNB and commercially available thioacetic acid A HDAC8 assay solution (300 lL) (without HDAC8 and its substrate but with added thioacetic acid (0, 10, 25, 50, 75, or 100 lM, final concentrations)) that contained 25 mM Tris–HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 1 mg/mL BSA was diluted by three-fold with the addition of 600 lL of the following quench buffer: 3.2 M guanidinium chloride in 100 mM sodium phosphate (pH 6.8). Of note, the final concentration range (0 to 100 lM) for thioacetic acid in these experiments was chosen to approximately cover the concentration range of thioacetic acid that could possibly be generated in actual enzymatic dethioacetylation reactions. To each of the above solutions with different final thioacetic acid concentrations was added 100 lL DTNB solution (400 mM sodium phosphate (pH 6.8), 20 mM DTNB, and 100 mM EDTA), the mixture was further incubated at RT for 0, 5, 10, 15, 20, 30, 60, or 90 min, and A412 was recorded at each time point with a Cary 100 UV-Vis spectrophotometer (Varian, Inc., Walnut Creek, CA, U.S.A.). The results are depicted in Fig. 3. HPLC-based enzymatic assay The HDAC8-catalyzed dethioacetylation reaction was performed in the same HDAC8 assay solution as described above, except that no thioacetic acid was added and human recombinant HDAC8 and its substrate 0.4 0.35 0.3

A412, AU

84

0.25 0.2 0.15 0.1 0.05 0

0

10

20

30

40

50

60

70

80

90

100

Time, minutes

Fig. 3. Kinetic analysis of the reaction between DTNB and commercially available thioacetic acid. Each data point represented an average from duplicate measurements that agreed with each other within 10%. Concentrations of thioacetic acid used are as follows: s, 10 lM; d, 25 lM; j, 50 lM; , 75 lM; m, 100 lM.

Spectrophotometric assay for HDAC8 / D.G. Fatkins, W. Zheng / Anal. Biochem. 372 (2008) 82–88

(i.e., the thioacetyl-lysine p53 peptide shown in Fig. 2) were added to final concentrations of 375 nM and 0.3 mM, respectively. An enzymatic reaction was initiated by the addition of HDAC8 at RT and incubated at RT until quenched at different time points with the following stop solution: 3.2 M guanidinium chloride in 100 mM sodium phosphate (pH 6.8). One portion (40 lL) of the HDAC8 assay mixture was quenched with 80 lL of the above stop solution and analyzed essentially as before [12] by RP-HPLC with a C18 analytical col˚ , 0.46 · 25 cm), eluting with the following umn (100 A gradient of double-deionized water containing 0.05% (v/v) trifluoroacetic acid (mobile phase A) and acetonitrile containing 0.05% (v/v) trifluoroacetic acid (mobile phase B): linear increase from 0% B to 35% B from 0 to 40 min (1 mL/min) with ultraviolet (UV) monitoring at 214 nm. The enzymatically formed dethioacetylated product (i.e., the p53 peptide shown in Fig. 2) was confirmed by its comigration with the chemically synthesized authentic sample and by mass spectrometric analysis with MALDI-TOF and quantified by HPLC peak integration and comparison with that of synthetic authentic sample. The same assay protocol was used when the HeLa nuclear extracts were used instead of human HDAC8. Nearly the same assay protocols were used when human HDAC1 and 2 were employed instead of human HDAC8, with the following exceptions: (i) for HDAC1 assays, 50 mM Tris–HCl (pH 8.0) was used instead of 25 mM Tris–HCl (pH 8.0), (ii) HDAC2 assays were performed at 30 C instead of RT, and (iii) HDAC1 and 2 assay solutions were quenched with the stop solution 1.0 M HCl and 0.16 M acetic acid. DTNB-based enzymatic assay Another portion (300 lL) of the above HDAC8 assay mixture was quenched with 600 lL of the stop solution 3.2 M guanidinium chloride in 100 mM sodium phosphate (pH 6.8) and analyzed by the DTNB-based assay procedure established in the current study (see ‘‘Results and discussion’’); i.e., 60 min after the quench and DTNB (100 lL) addition, A412 was recorded for each sample derived at different time points of the same assay reaction. Again, the same assay protocol was used when the HeLa nuclear extracts were used instead of human HDAC8. To measure the kinetic parameters for the thioacetyl-lysine p53 peptide as an in vitro HDAC8 substrate, all assay reactions contained 375 nM HDAC8, a range of substrate concentrations varied around the Km value, and were allowed to proceed at RT for 1 h before quenching. Reaction velocities were determined under initial condition (substrate turnover <10%). This experiment was performed twice, and each duplicate measurement agreed within 20%. By using a nonlinear least squares approach with the computer program Kaleidagraph (Reading, PA, U.S.A.), kinetic parameters (kcat and Km) were derived from the Michaelis–Menten plot based on the experimental data.

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Results and discussion DTNB-based assay condition establishment DTNB can selectively react with thiol-containing substances including free thiols (e.g., free cysteine or the cysteine residues on a denatured protein molecule) and thioacids (e.g., thioacetic acid, thiobenzoic acid, or a peptidic thioacid), with the released TNB having maximum absorbance at 412 nm [13,19,20]. However, the reaction with free thiols is much faster than that with thioacids [13,19,20]. The reaction of DTNB with a free thiol can be complete within 1 min at RT, as judged by the achievement of maximum absorbance at 412 nm. However, the reaction of DTNB with a free thioacid may take 50–60 min at RT to achieve maximum absorbance at 412 nm, as documented for the reaction of DTNB with thiobenzoic acid and with a peptidic thioacid. This likely explains why DTNB has been primarily used to quantify free thiols via quantifying TNB from its absorbance at 412 nm. But the selective reaction of DTNB toward thioacids such as those mentioned above should not preclude its general use in quantifying thioacids. However, to employ DTNB to quantify a thioacid released from an enzymatic reaction, it is essential to ensure a complete conversion of the thioacid to TNB in the presence of an excess amount of DTNB. We thus first performed a detailed kinetic analysis for the reaction between DTNB and commercially available thioacetic acid under our enzymatic assay conditions in the absence of an enzyme and its substrate. Following the procedure detailed under Materials and methods, the obtained A412 values were plotted against times for each individual thioacetic acid concentration. As indicated in Fig. 3, maximum A412 was obtained or closely approached starting at 60 min following DTNB addition for all thioacetic acid concentrations examined. We thus used 60 min as the fixed post-DTNB time point for the actual enzymatic assays. Assay with recombinant HDAC8 Using the thioacetyl-lysine p53 peptide as an in vitro substrate for HDAC8, both the HPLC-based and the DTNB-based assays were performed to evaluate the HDAC8-catalyzed dethioacetylation reaction, following the procedure detailed under Materials and methods. For the DTNB-based assay, the extinction coefficient (e) value for TNB reported in literature (13,700 M1 cm1) [13] was used for our calculations even though a very close average value (13,505 M1 cm1) was also obtained from two standard curves (not shown) for thioacetic acid that we generated according to the DTNB-based assay procedure established in the current study. Fig. 4 shows the DTNB-based and HPLC-based assay results and it is clear that these two assay formats gave rise to mutually agreeable measurements (within 8%) for HDAC8 activity when the thioacetyl-lysine p53 peptide was employed as a substrate, even though the well-established HPLC-based assay

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Spectrophotometric assay for HDAC8 / D.G. Fatkins, W. Zheng / Anal. Biochem. 372 (2008) 82–88

Fig. 4. Comparative analyses of enzymatic dethioacetylation of the thioacetyl-lysine p53 peptide to form the p53 peptide (product). HPLC: HPLC-based assay quantifying the released p53 peptide; UV-Vis: DTNB-based spectrophotometric assay quantifying the released thioacetate.

measured the peptide product whereas our newly developed DTNB-based assay measured another product, i.e., thioacetic acid (or thioacetate under assay pH). The kinetic parameters for the in vitro HDAC8 substrate, i.e. the thioacetyllysine p53 peptide, were determined by the DTNB-based assay with kcat = 0.26 ± 0.02 min1 and Km = 33.7 ± 7.2 lM. From these kinetic parameters, it is clear that our newly developed DTNB-based assay offers another advantage in that reliable HDAC8 activity measurements could be made with low substrate concentrations, because this Km value is smaller than any of those for the currently available HDAC8 in vitro substrates [21–23].

nuclear extracts (9 mg protein/mL) were, respectively, used, with kobs = 0.23 ± 0.01 lM/(min Æ 4 lL of the nuclear extracts), as judged by HPLC analysis. This same phenomenon was also observed when AcNH-RH(AcK)-(AcK)CONH2 and AcNH-RH(ThAcK)(ThAcK)-CONH2 (analogs of H2N-RH(AcK)(AcK)-COOH [24]) were used, with the former being deacetylated significantly (kobs = 0.29 ± 0.02 lM/(min Æ 4 lL of the nuclear extracts) at 30 min), but no detectable dethioacetylation was observed for the latter. Again, intact AcNHRH(ThAcK)(ThAcK)-CONH2 was quantitatively recovered from the assay mixture as revealed by the HPLCbased assay (data not shown).

Assay with HeLa nuclear extracts Assay with recombinant HDAC1 and HDAC2 To examine the selectivity of our newly developed DTNB-based spectrophotometric assay among different classical HDAC enzymes, we initially performed both the HPLC-based and the DTNB-based assays with the HeLa nuclear extracts enriched in HDAC1 and 2. The procedures for the HDAC8 assay were followed. It is apparent from Fig. 4 that no detectable dethioacetylation of the thioacetyl-lysine p53 peptide was observed with both the HPLCbased and the DTNB-based assay formats, when HDAC8 was replaced with the HeLa nuclear extracts. That the intact thioacetyl-lysine p53 peptide was quantitatively recovered from the assay mixture as revealed by the HPLC-based assay (data not shown) argued against the possibility that the degradation of the thioacetyl-lysine p53 peptide under assay conditions contributed to the lack of detectable dethioacetylation. However, under the same assay conditions, the corresponding acetyl-lysine p53 peptide was able to be deacetylated significantly by the HeLa nuclear extracts, in that 2.3 and 6.5% of substrate turnover were observed at 30 min when 4 and 12 lL of the HeLa

The above-described assay results with HeLa nuclear extracts suggested that our newly developed spectrophotometric assay was selective for HDAC8 versus HDAC1 and 2 and for other classical protein deacetylase enzymes present in the HeLa nuclear extracts, due to the incapability of HDACs other than HDAC8 to catalyze the dethioacetylation of the thioacetyl-lysine p53 peptide. To provide further evidence for the selectivity of our new assay format, we performed analogous assays with purified human recombinant HDAC1 and 2 instead of the HeLa nuclear extracts. Under the assay conditions detailed under Materials and methods, no detectable dethioacetylation of the thioacetyl-lysine p53 peptide was observed in both HDAC1 and HDAC2 assays, when the HPLC-based assay format was followed. However, under the same assay conditions, both HDAC1 and 2 were able to deacetylate the corresponding acetyl-lysine p53 peptide with the following observed reaction rates: kobs = 0.41 ± 0.23 min1 at 2 h for HDAC1 and kobs = 6.78 ± 2.39 min1 at 30 min for HDAC2. These assay results

Spectrophotometric assay for HDAC8 / D.G. Fatkins, W. Zheng / Anal. Biochem. 372 (2008) 82–88

clearly reinforced the notion that HDAC1 and 2 are unable to catalyze the dethioacetylation of the thioacetyl-lysine p53 peptide. They also suggested that the lack of detectable dethioacetylation of the thioacetyl-lysine p53 peptide in the assays with the HeLa nuclear extracts could not have been due to the possible interference from materials other than HDACs in the HeLa nuclear extracts with the recognition between the HDACs and the thioacetyl-lysine p53 peptide. Taken together, our assay results with both the HeLa nuclear extracts and the purified HDAC1 and 2 strongly suggested that our newly developed DTNB-based spectrophotometric assay is selective for HDAC8 versus HDAC1 and 2 and other classical protein deacetylase enzymes present in the HeLa nuclear extracts. Based on the currently available theoretical and experimental studies [7,8,25–31], while the catalytic domain is highly conserved among the classical HDAC enzymes, the acetyl-lysine binding pocket in HDAC8 appears to be more malleable than those in other classical HDACs. This could explain our observed capability and incapability, respectively, for HDAC8 and HDACs present in HeLa nuclear extracts to catalyze dethioacetylation reaction, due to the larger Van der Waals radius of S versus O. Conclusion In this study, we have developed and validated the first spectrophotometric HDAC8 assay via quantifying with DTNB the thioacetate product released from the enzymatic dethioacetylation of the thioacetyl-lysine p53 peptide. Our results further suggested that this novel assay was selective for HDAC8 versus HDAC1 and 2 and other classical HDAC enzymes. In addition to furnishing a fast and environment-friendly assay platform, compared to the currently existing HDAC8 assays, i.e., radioactive assay, HPLC assay, and fluorescence assay [23,32–35], our newly developed spectrophotometric assay also represents the most cost-effective format in that a readily available UV-Vis spectrophotomer was used and the substrate was readily obtained through facile organic/peptide synthesis. This novel spectrophotometric assay may thus find applications for highthroughput screening of HDAC8-selective inhibitors and for selective reporting of HDAC8 activity under (patho)physiological conditions. Acknowledgments The financial support from the James L. and Martha J. Foght Endowment, the University of Akron Research Foundation, and the University of Akron Faculty Research Fellowship is highly appreciated. We are also grateful for Professor Chrys Wesdemiotis and his research group at the University of Akron for the assistance with mass spectrometric analyses of the peptides involved in the current study.

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