A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening

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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 372 (2008) 72–81 www.elsevier.com/locate/yabio

A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening Thomas Ciossek 1, Heiko Julius, Heike Wieland, Thomas Maier, Thomas Beckers

2,*

Therapeutic Area Oncology, ALTANA Pharma—a member of the Nycomed Group, Byk-Gulden Str. 2, 78467 Konstanz, Germany Received 13 June 2007 Available online 2 August 2007

Abstract Most cellular assays that quantify the efficacy of histone deacetylase (HDAC) inhibitors measure hyperacetylation of core histone proteins H3 and H4. Here we describe a new approach, directly measuring cellular HDAC enzymatic activity using the substrate Boc-K(Ac)-7-amino-4-methylcoumarin (AMC). After penetration into HeLa cervical carcinoma or K562 chronic myeloid leukemia cells, the deacetylated product Boc-K-AMC is formed which, after cell lysis, is cleaved by trypsin, finally releasing the fluorophor AMC. The cellular potency of suberoylanilide hydroxamic acid, LBH589, trichostatin A, and MS275 as well-known HDAC inhibitors was determined using this assay. IC50 values derived from concentration–effect curves correlated well with EC50 values derived from a cellomics array scan histone H3 hyperacetylation assay. The cellular HDAC activity assay was adapted to a homogeneous format, fully compatible with robotic screening. Concentration–effect curves generated on a Tecan Genesis Freedom workstation were highly reproducible with a signal-to-noise ratio of 5.7 and a Z’ factor of 0.88, indicating a very robust assay. Finally, a HDAC-inhibitor focused library was profiled in a medium-throughput screening campaign. Inhibition of cellular HDAC activity correlated well with cytotoxicity and histone H3 hyperacetylation in HeLa cells and with inhibition of human recombinant HDAC1 in a biochemical assay. Thus, by using BocK(Ac)-AMC as a cell-permeable HDAC substrate, the activity of various protein lysine-specific deacetylases including HDAC1-containing complexes is measurable in intact cells in a simple and homogeneous manner.  2007 Elsevier Inc. All rights reserved. Keywords: HDAC inhibitor; Cellular HDAC activity; Medium-throughput screening; Assay development

Posttranslational protein modification by reversible acetylation of lysine residues is now recognized as an important modification modulating protein function [1,2]. The modification of core histone proteins by acetylation of N-terminal lysine residues is well described and part of the so-called histone code [3]. Nevertheless, an increasing number of nonhistone proteins modified by acetylation at lysine residues, including the signal transducer and activa*

tor of transcription 1 and 3 (STAT1, STAT3)3, heat shock protein 90 (Hsp90), the tumor suppressor protein p53, and a-tubulin, have been reported [2,4]. As an example, in melanoma cells the interaction of NFjB p65 with STAT1 is dependent on CRE binding protein and HDAC-regulated STAT1 acetylation, controlling the nuclear translocation and antiapoptotic function of NFjB [5]. The status of core histone H3/H4 acetylation correlates with the transcrip-

Corresponding author. Fax: +49 761 5155955. E-mail address: [email protected] (T. Beckers). 1 Present address: Boehringer Ingelheim Pharma, Biberach an der Riss, Germany. 2 Present address: Oncotest GmbH, Am Flughafen 12-14, 79106 Freiburg, Germany. 3 Abbreviations used: AMC, 7-amino-4-methylcoumarin; CML, chronic myeloid leukemia; DMSO, dimethyl-sulfoxide; FCS, fetal calf serum; HAT, histone acetyltransferase; HCS, high-content screening; HDAC, histone deacetylase; HDI, histone deacetylase inhibitor; MTS, medium throughput screening; SAHA, suberoylanilide hydroxamic acid; TFA, trifluoracetate; TSA, trichostatin A; STAT, signal transducer and activator of transcription; aa, amino acids. 0003-2697/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.07.024

Homogeneous cellular histone deacetylase assay / T. Ciossek et al. / Anal. Biochem. 372 (2008) 72–81

tional activity of chromatin with histone acetyltransferases (HATs) and histone deacetylases as key players regulating reversible histone acetylation [2]. Up to now, 11 different HDAC isoenzymes belonging to the class I (HDAC1, 2, 3, 8), class II (HDAC4–7, 9, 10), and class IV (HDAC11) families have been described [6,7]. HDAC class III enzymes, also named Sirtuins/Sir2 homologs with seven human isoenzymes (SIRT1–7), are NAD+-dependent enzymes and insensitive to inhibition by HDAC class I/II inhibitors such as trichostatin A (TSA) [8]. Inhibition of HDAC class I/II enzymes is well established as a new approach for solid and hematological tumor therapy [7,9–11]. HDAC inhibitors (HDIs) affect the transcriptional regulation and induce or repress genes involved in differentiation, proliferation, cell cycle regulation, protein turnover, and apoptosis [7]. As such, they are considered as drugs for targeted cancer therapy and HDIs of divergent chemical structure are currently in clinical development [12]. These include the hydroxamate analogs LBH589 [13] and suberoylanilid hydroxamic acid (SAHA; Zolinza), which was recently approved for treatment of therapy-refractory cutaneous T-cell lymphoma [14]. Different strategies to measure the target-related pharmacodynamic effects of HDIs in cells or tissue have been published [15]. Predominantly, cellular histone hyperacetylation in HDI-treated tumor or normal cells/tissue is quantified directly by immunohistochemistry [16], cytoblot assay, or Western blotting [17,18], enzyme-linked immunosorbent assay [19], flow cytometry [20], single-cell imaging [21], or indirectly by reporter gene assays [18]. An alternative approach is based on the trifluoroacetate analog Boc-K(TFA)-OH as an indicator for cellular HDAC activity by using magnetic resonance spectroscopy [22]. Recently it has been shown that the promiscuous HDAC substrate Boc-K(Ac)-AMC permeates into intact, vital cells. The formation of the deacetylated product BocK-AMC can be used as a direct measure for cellular HDAC activity [23–25]. Here we show the validation of this cellular HDAC activity assay using adherent HeLa cervical carcinoma and nonadherent K562 chronic myeloid leukemia (CML) cancer cell lines. The assay was simplified and adapted to a homogeneous format compatible with a fully automated, robotic screening. Finally, a focused HDI library was evaluated in a medium-throughput screening (MTS) campaign. The inhibition of cellular HDAC activity was correlated with cellular cytotoxicity and induction of histone H3 hyperacetylation and with inhibition of nuclear extract HDAC and recombinant HDAC1 activity in biochemical assays. The cellular HDAC assay proved to be robust, resulting in highly reproducible IC50 determinations. Inhibition of cellular HDAC activity correlated best with HDAC1 inhibition but also with cytotoxicity and histone H3 hyperacetylation but not significantly with inhibition of nuclear extract HDAC activity.

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Materials and methods Materials The HDAC inhibitors suberoylanilide hydroxamic acid (SAHA), MS275, and LBH589 were synthesized internally; TSA was supplied by Calbiochem (Darmstadt, Germany). The focused library was synthesized internally and consists of about 1000 compounds with different HDAC inhibitor head groups, mainly hydroxamate and benzamide analogs (for details see published patent applications WO200739404 and WO2007039403). In general, the library and reference HDAC inhibitors were dissolved as 10 mM stock solutions in dimethyl sulfoxide (DMSO) and subsequently diluted for respective biochemical and cellular assays. The HDAC substrates Boc-K(TFA)-AMC, Ac-NH-GGK(Ac)-AMC, and Ac-SDK(Ac)-AMC were supplied by Thermo Biosciences (Ulm, Germany), BOCK(Ac)-AMC was supplied by Bachem (Weil am Rhein, Germany), and (Ac)RHK(Ac)K(Ac)-AMC was supplied by BioMol (Hamburg, Germany). Cell lines and cell culture The human tumor cell lines HeLa (cervical carcinoma, ATCC CCL-2) and K562 (chronic myeloid leukemia, ATCC ACC-10) were from Promochem (Wesel, Germany). All cell lines were cultivated in Dulbecco’s modified Eagle’s medium high glucose or RPMI 1640 cell culture medium supplemented with 10 vol% fetal calf serum (FCS). Cell culture was done under standard conditions at 37 C and 5% CO2. Cytotoxicity assays The antiproliferative cytotoxic activity of HDIs on HeLa cervical carcinoma cells was evaluated using the Alamar blue (Resazurin) cell viability assay as described [26]. HeLa cells were seeded into 96-well cell culture microtiter plates at 1 · 103 cells/well and cultivated for 24 h before addition of compound dissolved in DMSO (200-fold dilution). After incubation for 72 h at 37 C under standard cell culture conditions, metabolic activity was determined by addition of Resazurin solution (final concentration 9 mg/ml Resazurin; Sigma, Munich, Germany) and measurement of fluorescence at excitation of k = 544 nm and emission of k = 590 nm. For microscopic analysis of individual cells, HeLa cells were treated for 6 and 24 h with 16 lM SAHA, TSA, or LBH589. Instead of cell lysis, cells were washed and stained with calcein AM and ethidium bromide homodimer using the LIVE/DEAD assay kit (Molecular Probes, Karlsruhe, Germany). Stained cells were analyzed by fluorescence microscopy, discriminating between red and green fluorescence for apoptotic/necrotic cells (ethidiumbromide homodimer positive) and vital, metabolicly active (esterase positive) cells, respectively.

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Biochemical HDAC assays

Data analysis

HDAC activity was isolated from HeLa nuclear extracts according to a method originally described by Dignam et al. [27]. Recombinant HDAC1 was expressed in insect cells and purified as described [21]. The HDAC enzyme activity assay was essentially done as described [24,25].

Data were normalized using DMSO-treated controls (100% activity) and blank controls (0% activity) in the Alamar blue assay (wells without HeLa cells) and cellular HDAC assay (10 lM TSA added before substrate treatment). Data from manually performed experiments were analyzed using the GraphPad Prism (San Diego, USA) software. For analysis of fully automated MTS-based assays, the Screener software package was utilized (Genedata, Basel, Switzerland). For visualization of large data sets, Spotfire Decision Site was used (Spotfire, Somerville, USA). Z’ values were calculated using the method described by Zhang et al. [28].

Robotic, homogeneous cellular HDAC activity assay The manual assay was established based on information using Fluor-deLys as substrate (BioMol http:// www.biomol.com/SiteData/docs/ProductData/ak503.pdf). HeLa cells were seeded into white 96-well tissue culture plates at a density of 5 · 103 cells/well (total volume 81 ll culture medium) and cultivated for 24 h under standard cell culture conditions. Next, 5 ll of the test compound dissolved in DMSO at varied concentrations was diluted into a sterile 96-well microtiter plate containing 95 ll/well culture medium using the Tecan Genesis Freedom workstation with the 96-channel pipetting head (Tecan, Ma¨nnedorf, Switzerland). From this dilution, 9 ll/well was added to the HeLa cells and incubation was continued for 3 h under cell culture conditions (Heraeus Cytomat). As negative and positive controls, DMSO and 10 lM TSA were added in separate wells. After this treatment period, 10 ll of a 2 mM stock solution of the substrate Boc-K(Ac)-AMC in culture medium was added by using the 96-channel pipetting head (final concentration 0.85 vol% DMSO and 200 lM Boc-K(Ac)AMC). Microtiter plates were incubated under cell culture conditions for an additional 3 h before addition of 100 ll/ well lysis/developer buffer mix (50 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 vol% Nonidet-P40, 2.0 mg/ml trypsin, 10 lM TSA) using the EasySpense-2 Multidrop (Tecan). After final incubation for 3 h under cell culture conditions, fluorescence was measured at excitation of k = 355 nm and emission of k = 460 nm on the Perkin–Elmer Wallac Victor V multilabel plate reader (Perkin–Elmer, Wellesley, USA), integrated into the Tecan robotic system. Cellular histone H3 hyperacetylation assay Histone H3 hyperacetylation was quantified using a single-cell-based high-content screening (HCS) format on the Cellomics ArrayScan II platform (Thermo Fisher Scientific, Waltham, USA) as described [21]. Briefly, HeLa cells in 96-well microtiter plates were treated with test compound for 24 h, fixed, permeabilized, and stained with a polyclonal rabbit antibody specific for histone H3K(Ac)1-20 (Calbiochem, Darmstadt, Germany). Data analysis was done with the Cellomics algorithm mitotic index, calculating normalized nuclear fluorescence intensity and correlating with the level of histone H3 hyperacetylation.

Results Basic concept and selection of suitable substrates In general, the cellular HDAC activity assay is based on an assay concept published by Wegener et al. [24,25]. Short tri- or tetrapeptides acetylated on defined lysine residues and conjugated via a peptide bond to 7-amino-4-methylcoumarin are substrates of HDAC isoenzmyes. The deacetylated peptide is cleaved proteolytically by a trypsin– containing developer solution, thus releasing AMC as fluorophore with emission at k = 460 nm. The lysine mimetic Boc-K(Ac)-AMC is also a substrate for certain HDACs, including HDAC1 [24,25] (data not shown). To select a substrate suitable for a cellular HDAC activity assay, the peptides and lysine mimetics as shown in Fig. 1 were evaluated. The peptidic HDAC substrates were derived from histone H4/aa 7–9 (Ac-GGK(Ac)-AMC), p53/aa 379–382 ((Ac)RHK(Ac)K(Ac)-AMC), a-tubulin/aa 38–40 ((Ac)SDK(Ac)-AMC), or lysine mimetics Boc-K(Ac)-AMC [24,25] and Boc-K(TFA)-AMC [29]. As shown in Fig. 1, only the lysine mimetics are cellular HDAC substrates, permeating into HeLa cells. Boc-K(Ac)-AMC showed a superior absolute signal and signal-to-noise ratio and was selected for all further experiments. Kinetics of substrate cleavage in HeLa and K562 cell lines The adherent HeLa cervical carcinoma and the nonadherent K562 CML cell lines were selected to study the kinetics of cellular substrate cleavage. As shown in Fig. 2A for HeLa cells and respective culture medium, a linear increase in Boc-K-AMC generation over 6 h occured. The cellular signal can be largely antagonized by 10 lM TSA, proving a HDAC mediated deacetylation of Boc-K(Ac)-AMC in HeLa cells. Only a small percentage of the weak fluorescent signal in the culture medium is inhibited by TSA. In K562 cells, the formation of cellular Boc-K-AMC is faster and declining after 2 h, which is explained by release of the deacetylated product into the culture medium (Fig. 2B). This is clearly seen by a linear increase of a strong, TSA-sensitive fluorescent signal in

Homogeneous cellular histone deacetylase assay / T. Ciossek et al. / Anal. Biochem. 372 (2008) 72–81

Cellomics Array Scan HCS assay for SAHA and TSA. In contrast, a six- to sevenfold higher activity for LBH589 and MS275 in the cellular HDAC activity assay was seen, a trend also evident in the MTS campaign (see Fig. 7A).

40000

30000

Ac-SDK(Ac)-AMC

DMSO

DMSO

DMSO

Ac-GGK(Ac)-AMC

Boc-K(TFA)-AMC

(Ac)RHK(Ac)K(Ac)-AMC

DMSO

DMSO Boc-K(Ac)-AMC

0

10µM TSA

For adaptation to a homogeneous MTS-compatible assay format, adherent HeLa cells were selected. This cell line is also the source of the nuclear HDAC mixture and is used in the histone H3 hyperacetylation HCS assay. Nevertheless, a homogeneous MTS assay is possible for any adherent or nonadherent cell line after selection of optimal treatment schedules. Treatment of HeLa cells with BocK(Ac)-AMC was routinely done for 3 h and the wash steps of the manual assay after treatment with HDI and substrate were omitted in this context. This adaptation to a homogeneous assay format needed various alterations, namely (i) a volume reduction of the cell culture medium, (ii) the addition of prediluted test compound to reduce total DMSO concentration, and (iii) the combining of cell lysis and developer steps by using a twofold concentrated lysis/developer buffer with a higher trypsin concentration. The stability of the fluorescent signal in the cell lysate after formation of AMC was initially studied, showing no decline up to 6 h and stable IC50 values for TSA (Fig. 3A). For scheduling reasons, an incubation time of 3 h was selected for all further experiments. Next, the effect of variable FCS concentrations in the culture medium was determined. Increasing concentrations of FCS in the culture medium strongly decreased the signal intensity by inhibition of trypsin in the lysis buffer (Fig. 3B). This effect could be circumvented by increasing the amount of trypsin in the lysis buffer as shown in Fig. 3C. A trypsin concentration of 2 mg/ml was finally selected, fully compensating the antagonizing effect of 10 vol% serum in the homogeneous assay format.

10µM TSA

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10µM TSA

Adaptation of the cellular HDAC assay for robotic screening

10µM TSA

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10µM TSA

fluorescence units

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Fig. 1. Analysis of different HDAC substrates. HeLa cells were treated for 24 h with either 0.5 vol% DMSO or 10 lM TSA. After changing the culture medium, the cells were treated with the HDAC substrates as shown (AMC-coupled peptides and lysine mimetics) at 200 lM final concentration for 6 h. Substrate cleavage was measured in cell lysates as described under Materials and methods and is expressed as fluorescence units (emission at k = 460 nm).

the culture medium over 6 h. Using a manual assay including two wash steps to measure Boc-K-AMC formation in HeLa and K562 cell lysates, IC50 values were determined for the hydroxamate analogs TSA, SAHA, and LBH589 and the benzamide analog MS275 as reference HDIs. Data from respective concentration–effect curves are summarized in Table 1 showing a clear correlation with histone H3 hyperacetylation in HeLa cells as determined in the

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time [h] Fig. 2. Kinetics of substrate deacetylation. HeLa (A) and K562 cells (B) were treated for 24 h with 0.5% DMSO or 10 lM TSA. Next, the culture medium was changed and incubation continued with 200 lM substrate Boc-K(Ac)-AMC for the time as indicated. The amount of AMC after cleavage of the deacetylated product Boc-K-AMC in lysed cells and culture supernatant was quantified and is expressed as fluorescence units (emission at k = 460 nm).

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Table 1 Inhibition of cellular HDAC activity ID

Structure

HeLa IC50 [lM]

K562 IC50 [lM]

Histone H3 hyperacetylation EC50 [lM]

0.0022 ± 0.0004

0.00086 ± 0.00024

0.013 ± 0.018

5.49 ± 1,39

5.40 ± 1,13

6.7 ± 4,69

0.124 ± 0.009

0.51 ± 0.013

0.27

0.33 ± 0.005

0.21 ± 0.04

2.35 ± 0.35

O OH N H

H N

LBH589 N H

O H N

SAHA

NH OH

O

TSA

O O

MS275

NH2

N H

H N

N O

Inhibition of HDAC activity in HeLa cervical carcinoma and K562 CML cell lines by the hydroxamate analogs LBH589, SAHA, and TSA and the benzamide analog MS275 is summarized and compared to histone H3 hyperacetylation measured using a HCS assay. Mean values ± SD of at least two independent manual experiments are shown.

Assay validation on a robotic scale To estimate the assay reproducibility in a fully automated MTS run, the HDIs SAHA, LBH589, and TSA were used as tool compounds for IC50 determinations on the Tecan Genesis Freedom workstation. The same compound dilution plate was reapplied 10 times for assaying two cellular assay plates in each repetition. The first MTS run was done with HeLa cells seeded in culture medium without FCS. By comparing individual IC50 concentration–effect curves from separate assay plates, an increase of potency in assay plates analyzed at later time points became evident (Fig. 4A). This could be explained by an up to 2.5 h longer incubation of cells in serum-free medium between assay plate 1 and plate 10, which presumably affected cell viability and sensitivity toward HDIs (total incubation time between 16 and 18.5 h). Thus, in the second MTS run cells were seeded in culture medium with 10 vol% FCS, making a higher tyrpsin concentration in the lysis buffer necessary (see Fig. 3). As shown in Fig. 4B, individual concentration– effect curves no longer displayed a shift in potency between early and late assay plates as seen in the first MTS run. A third MTS run decreasing the DMSO concentration from 0.85 to 0.43 vol% by adding a reduced volume of test compound solution was performed. Both DMSO concentrations gave similar results and did not affect the concentration–effect curves (Fig. 4C). Mean IC50 values of these three MTS test runs are summarized in Table 2. The conditions of the second run, showing a signal-to-noise

ratio of 5.7 and a Z’ factor of 0.88, indicating a very robust cellular assay [28], were selected for all further screens. To exclude any cytotoxic effect of the HDIs during the assay, HeLa cells were treated with high concentrations of LBH589, SAHA, and TSA for 6 and 24 h. After 6 h treatment, the percentage of apoptotic/necrotic cells was not enhanced as visualized by LIVE/DEAD staining of HeLa cells (Fig. 5A). After 24 h treatment, only a very high concentration of 16 lM LBH589 displayed weak cytotoxicity (Fig. 5B). Since the overall time of HDI treatment is 6 h in the cellular HDAC assay, any effect of cell viability on cellular HDAC activity is unlikely. Fully automated screening of a focused library The cellular HDAC activity assay is of high value for primary screening and for hit profiling to quantify cellular, target-related activity. To evaluate the usefulness in primary screening, the MTS assay procedure was used for screening of a focused library consisting of about 1000 compounds derived from an internal medicinal chemistry optimization program. In parallel MTS campaigns, cytotoxicity (HeLa cells, 72 h treatment) and inhibition of HeLa nuclear extract HDAC and rHDAC1 activity in biochemical assays were determined. Most cytotoxic and HDAC inhibitory compounds were also tested using the HCS assay quantifying histone H3 hyperacetylation (HeLa cells, 24 h treatment). The final analysis using the Spotfire decision site software showed that the inhibitory potency

A IC50 TSA [nM]

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Fluorescence units (xE4)

Homogeneous cellular histone deacetylase assay / T. Ciossek et al. / Anal. Biochem. 372 (2008) 72–81

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10 μ M TSA 0.5 % DMSO LogIC50 TSA

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trypsin [mg/ml] Fig. 3. Optimization of assay parameters. (A) Stability of the final product and fluorophor AMC in the HeLa cell lysate and inhibition by TSA (IC50 values) as a function of time after cell lysis. HeLa cells in serum-free culture were treated with 0.5 vol% DMSO and TSA (10 lM or different concentrations for IC50 determination) for 3 h before addition of 200 lM Boc-K(Ac)-AMC. After an additional 3 h, cells were lysed (lysis/developer buffer with 0.5 mg/ml trypsin) and fluorescence was measured at k = 460 nm up to 6 h after lysis. (B) Effect of different FCS concentrations (0 to 10 vol%) on substrate cleavage and flourescent signal intensity. (C) Effect of increased trypsin concentrations on substrate cleavage in HeLa cells cultivated in medium with 2 or 10 vol% FCS. Cells were treated with either 0.5 vol% DMSO or 10 lM TSA for 24 h before substrate treatment and for an additional 3 h and cell lysis.

in the cellular HDAC assay correlated well with histone H3 hyperacetylation and cytotoxicity (Figs. 6A and 6B). Nevertheless, a trend toward lower IC50 values/higher potency in the cellular HDAC assay is seen. Surprisingly, no significant correlation with inhibition of HeLa nuclear extract HDACs is seen (Fig. 6C). Finally, the inhibitory potency in the cellular HDAC assay correlated very well with rHDAC1 inhibition (Fig. 6D), supporting the concept that the assay also measures cellular HDAC1 activity. Discussion Reversible protein acetylation is increasingly recognized as an important posttranslational modification regulating diverse biological processes including gene transcription [1,2]. Modification of core histone proteins by acetylation via HDACs and HATs as part of transcriptional regulation is well elucidated, with a dysregulated function of HDAC recruiting fusion proteins in leukemia shown by different

groups [30]. Nevertheless, the relevance of nonhistone substrates is far less clear, but recent studies provided evidence for lysine acetylation regulating, e.g., p53 and STAT1 function [5,31]. Different HDAC inhibitors are currently in clinical trials with SAHA recently approved for treatment of advanced, therapy-refractory cutaneous T-cell lymphoma [7,14]. In many pharmaceutical companies, programs are aiming at the identification of HDIs with pharmacological and safety profiles superior to those of second generation HDIs such as SAHA, LBH589, or MS275. Robust and reliable primary and secondary profiling assays are needed. Simple biochemical HDAC activity assays using recombinant HDAC isoenzymes or mixtures derived from cancer cell lines or tissues are well established and widely used for primary screening and isoenzyme selectivity profiling [21,24,25,32]. For profiling of HTS hits or analogs derived from medicinal chemistry programs, reliable and robust cellular assays measuring target inhibition in a cellular con-

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activity [%]

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100 80 60 40 20 0

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100 80 60 40 20 0 -10

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100 80 60 40 20 0 -10

Concentration [logM]

activity [%]

C 100

100

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0

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Concentration [logM] Fig. 4. Concentration–effect curves in a homogeneous, robotic assay format. Concentration–effect curves for LBH589, TSA, and SAHA were determined under different assay conditions on the Tecan Genesis Freedom workstation. HeLa cells were treated with the HDI for 3 h in medium without FCS/ 0.85 vol% DMSO (A), with 10 vol% FCS/0.85 vol% DMSO (B), and with 10 vol% FCS/0.43 vol% DMSO (C). The lysis buffer contained 0.5 and 2 mg/ml trypsin for cells cultivated without and with 10 vol% serum, respectively. Concentration–effect curves for compound plates 2, 7, and 10 in a robotic screening run with 10 identical compound plates are shown. Mean IC50 values are shown in Table 2.

Table 2 Assay parameters derived from different MTS runs

LBH589 SAHA TSA Z’ Factor Signal-to-noise ratio

0% FCS(1st MTS run, n=10)

10% FCS(2nd MTS run, n=18)

reduced DMSO (3rd MTS run, n=10)

2.6 nM 510 nM 14 nM 0.86 4.2

1.5 nM 210 nM 20 nM 0.88 5.7

2.0 nM 250 nM 16 nM 0.84 10.6

Mean IC50 values for selected HDIs and assay quality for different assay conditions are summarized. Selected concentration–effect curves are shown in Figs. 4A–4C.

text are needed. Most available cellular HDAC assays quantify induction of histone hyperacetylation by different means [16–21]. In general these assays are complicated, with low signal-to-noise ratios. Also, reagents such as antibodies are quite expensive and the non-homogeneous assay format makes easy adaptation to screening robotics questionable. Finally, these assays are restricted by not measuring HDAC-mediated effects distinct to core histone modifications. Therefore, we evaluated a new concept using the cell-permeable, promiscous HDAC substrate

Boc-K(Ac)-AMC. The advantages of this assay concept are the direct measure of cellular HDAC activity with simple and cheap assay components, the similarity to widely used biochemical assays, the opportunity to use HDAC isoenzyme-specific substrates, and the simplicity of a homogeneous assay format. The assay has been described recently for use with peripheral blood mononuclear cells [23] and cancer cells (BioMol homepage) and is based on acetylated lysine analogs conjugated with AMC. Only the deacetylated lysine analog is cleaved by trypsin, liberating the fluorophor AMC [24,25]. So far only the lysine analogs Boc-K(Ac)-AMC and Boc-K(TFA)-AMC permeated cells and functioned as cellular HDAC substrates. The tri- and tetrapeptides as studied gave no signal and presumably did not permeate into intact, vital cells (Fig. 1). A new concept would be to use HDAC substrate peptides fused with a cell-penetrating peptide sequence allowing efficient translocation across the plasma and nuclear membranes as recently described for the synthetic peptides C105Y and PFVYLI derived from a1 antitrypsin [33]. This concept would be transferable to HDAC isotype-selective substrates as described [34]. In the present study, the substrate Boc-K(Ac)-AMC behaved in a superior fashion and was used for all further experiments. Interestingly, the kinetics

Homogeneous cellular histone deacetylase assay / T. Ciossek et al. / Anal. Biochem. 372 (2008) 72–81

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Fig. 5. Cytotoxicity of HDIs. Cytotoxicity of LBH589, TSA, and SAHA at 16 lM concentration after 6 h (A) and 24 h (B) treatment as analysed by fluorescence microscopy. HeLa cells were stained with the LIVE/DEAD assay kit with vital cells stained in green and apoptotic/necrotic cells stained in red. Representative pictures are shown with apoptotic/necrotic cells after 24 h treatment with 16 lM LBH589 marked by arrow.

Cellular HDAC assay

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Fig. 6. Profiling of a focused HDI library. Focused HDI library was screened using the cellular HDAC assay format. In addition, robotic MTS assays were also run for inhibition of HeLa nuclear extract activity and recombinant HDAC1, cytotoxicity toward HeLa cells, and a manual HCS assay for induction of histone H3 hyperacetylation in HeLa cells. For selected compounds (compounds with inhibition > lgIC50 –4 were excluded), the correlation of cellular HDAC activity inhibition with histone H3 hyperacetylation (A), cytotoxicity (B), HeLa nuclear extract HDAC activity (C), and recombinant HDAC1 (D) are shown. All data are shown as IC50 values in log[M].

of substrate deacetylation differed between cell types. Substrate deacetylation and release of Boc-K-AMC into the cell culture supernatant was faster in K562 CML than in HeLa cervical carcinoma cells (Fig. 2). Thus, the duration of substrate treatment has to be adapted for each cell line,

but this does not restrict the general applicability of the assay. The assay as initially established in a manual format was validated by IC50 determinations using SAHA, LBH589, TSA, and MS275 as reference HDIs. The results obtained

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in K562 and HeLa cells were similar with a clear correlation to cellular histone H3 hyperacetylation. Trends toward higher potency in the cellular HDAC activity assay were seen with MS275, LBH589, and the focused library screen. Since the cellular HDAC activity assay also quantifies HDAC isoenzymes/complexes with substrates distinct to histone proteins, differences between these two assay are not unexpected. To have a compatibility with a homogeneous format suitable for use on the Tecan Genesis robot, the manual assay needed various adaptations, namely (i) a single cell lysis/developer step, (ii) a higher trypsin concentration of 2 mg/ml as a consequence of 10 vol% FCS needed in the culture medium, and (iii) prediluted compound solutions to reduce total DMSO concentration. Although reasonable signal-to-noise ratios were obtained in serum-free culture medium, IC50 values were affected by handling times most likely due to enhanced cytotoxicity of test compounds in serum-free medium (Fig. 4). Therefore, cells were routinely treated with test compound in culture medium containing 10 vol% FCS. Under these experimental conditions, any cytotoxic effect of the HDIs that might interfere with the assay could be excluded (see Fig. 5). Since final DMSO concentrations of 0.85 and 0.43 vol% gave similar results, the assay was routinely done with 0.85 vol% DMSO. Using the adaptations mentioned, the assay proved to be robust with an excellent Z’ factor of 0.88 and a signal-to-noise ratio of 5.7. The IC50 values were slightly lower than those in the manual assay. This might be due to the 6 h continous HDI treatment in the homogeneous assay compared to 4 h HDI treatment followed by incubation with substrate without HDI for an additional 3 h in the manual assay. The overall throughput on the Tecan Genesis Freedom workstation was 1600 data points/day based on 80 IC50 determinations with 10 half-log compound dilutions performed in duplicate. An increased incubation time with lysis buffer of 3 h allowed for an easy scheduling on the workstation. Finally, a focused HDI library consisting of diverse structures mainly of hydroxamate and benzamide analogs derived from medicinal chemistry was screened using the robotic cellular HDAC activity assay. A correlation analysis was done including biochemical and cellular data derived from parallel manual and robotic screening (see Fig. 6). A good correlation between cellular HDAC inhibition and histone H3 hyperacetylation and cytotoxicity in HeLa cells was seen with a trend towards higher activity in the cellular HDAC assay. As discussed before, this might be explained by measuring HDAC- or, more generally, lysine-specific deacetylase activity in cells. The specificity is mainly affected by the substrate Boc-K(Ac)-AMC, which is a good substrate for at least HDAC1, 3, and 6 but not for HDAC8 [35]. However, there was no significant correlation with the inhibition of HDAC activity purified from HeLa nuclei consisting of at least HDAC1, 2, 3, 5, and 8. This might be due to the test compounds displaying variable inhibition profiles in the assay using

nuclear extract HDACs, namely flat concentration–effect curves and/or partial maximal inhibition even at highest concentration (data not shown). Curve fitting and IC50 calculations therefore might be misleading, in particular if only a minor subpopulation of HDAC complexes is inhibited. Also, we do not know the efficiency of substrate permeation into the nucleus, which certainly would affect the correlation analysis. Finally, a clear correlation is seen for inhibition of HDAC1 reflecting in part the strategy of medicinal chemistry efforts aiming at optimizing HDAC1 inhibition and cellular activity. This perfect correlation would not be expected when screening a random compound library, where compounds not penetrating into cells will be active only in the biochemical HDAC1 assay. We conclude that inhibition of HDAC1-containing complexes in HeLa cells is one read-out of the cellular HDAC activity assay. To summarize, the cellular HDAC activity assay as described is robust and compatible with a homogeneous robotic screening format. It can be used in primary screening as well as hit or analog profiling to exact measurement of target-specific cellular activity. By using BocK(Ac)-AMC as a lysine-mimetic HDAC substrate, the activity of various protein lysine-specific deacetylases including HDAC1-containing complexes in intact, vital cells is reproducibly measurable. Further studies with new cell-permeable and isoenzyme-selective substrates offer the opportunity to quantify the enzymatic activity of single, pathophysiologically relevant HDAC isoenzymes in a relevant cellular background. A general applicability is also supported by new results, showing a compatibility with various cancer cell lines, normal cells such as peripheral blood mononuclear cells, and even whole human blood. Acknowledgments The HDAC1-expressing HEK293 cell line was kindly provided by E. Verdin, Gladstone Institute for Virology and Immunology, San Francisco, USA. We also thank colleagues at ALTANA Discovery Research, in particular U. Bosch and G. Quintini for providing HDAC preparations, C. Burkhardt for excellent technical assistance, and A. Schwienhorst for critical reading of the manuscript. References [1] T. Cohen, T.P. Yao, AcK-Knowledge reversible acetylation, Sci. STKE. 3 (2004) pe42. [2] K. Zhang, S.Y. Dent, Histone modifying enzymes and cancer: going beyond histones, J. Cell Biochem. 96 (2005) 1137–1148. [3] B.D. Strahl, C.D. Allis, The language of covalent histone modifications, Nature 403 (2000) 41–45. [4] R.W. Johnstone, J.D. Licht, Histone deacetylase inhibitors in cancer therapy: is transcription the primary target? Cancer Cell 4 (2003) 13–18. [5] O.H. Kramer, D. Baus, S.K. Knauer, S. Stein, E. Jager, R.H. Stauber, M. Grez, E. Pfitzner, T. Heinzel, Acetylation of Stat1 modulates NF-kappaB activity, Genes Dev. 20 (2006) 473–485.

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