Identification of Novel Small-Molecule Histone Deacetylase Inhibitors By

Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorogenic assay Dennis Wegener, Christian Hildmann, Daniel Riester, Andreas Schober, Franz-Josef Meyer-Almes, Hedwig Deubzer, Ina Oehme, Olaf Witt, Siegmund Lang, Martina Jaensch, et al.

To cite this version:

Dennis Wegener, Christian Hildmann, Daniel Riester, Andreas Schober, Franz-Josef Meyer-Almes, et al.. Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorogenic assay. Biochemical Journal, Portland Press, 2008, 413 (1), pp.143-150. 10.1042/BJ20080536. hal-00478995

HAL Id: hal-00478995 https://hal.archives-ouvertes.fr/hal-00478995 Submitted on 30 Apr 2010

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Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorogenic assay

Dennis Wegener1,2,3, Christian Hildmann1,2*, Daniel Riester1, Andreas Schober2, Franz- Josef Meyer-Almes4, Hedwig E. Deubzer3, Ina Oehme3, Olaf Witt3, Siegmund Lang5, Martina Jaensch6, Vadim Makarov7, Corinna Lange8, Benedikt Busse9 and Andreas Schwienhorst1

1 Department of Molecular Genetics and Preparative Molecular Biology, Institute for Microbiology und Genetics, Grisebachstr. 8, 37077 Goettingen, Germany 2 Institute for Micro- and Nanotechnologies, Technical University of Ilmenau, Gustaf-Kirchhoff-Straße 7, 98693 Ilmenau, Germany 3 CCU Pediatric Oncology G340, German Cancer Research Center (DKFZ) Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany 4 Department of Chemical Engineering and Biotechnology, University of Applied Sciences Darmstadt, Schnittspahnstr. 12, 64287 Darmstadt, Germany 5 TU Braunschweig, Institut für Biochemie und Biotechnologie, Spielmannstr. 7, 38106 Braunschweig, Germany 6 AnalytiCon Discovery GmbH, Hermannswerder Haus 17, 14473 Potsdam, Germany 7 State Research Center for Antibiotics, 3f, Nagatinsksaya str., Moscow 117105 Russia 8 Leibniz-Institut für Naturstoffforschung und Infektionsbiologie,-Hans-Knöll Institut, Beutenbergstr. 11a, 07745 Jena, Germany 9 zell-kontakt GmbH, Industriestrasse 3, 37176 Noerten-Hardenberg, Germany

SHORT TITLE: HDAH inhibitor screening

______*To whom correspondence should be addressed: Christian Hildmann Technical University Ilmenau Institute for Micro- and Nanotechnologies (ZMN) Gustav-Kirchhoff-Str. 7 98693 Ilmenau THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536 Germany Phone: 49-3677-693410; fax: 49-3677-693455; Email: [email protected]

Stage 2(a) POST-PRINT

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The abbreviations used: MCA 7-amino-4-methylcoumarin DMSO dimethylsulfoxide FRET fluorescence resonance energy transfer HDAC histone deacetylase HDAH histone deacetylase-like amidohydrolase (from strain FB188) IC50 Inhibitor concentration at 50 % inhibition MCA 4-methylcoumarin-7-amide PAGE polyacryl amide gel electrophoresis PBS phosphate buffered saline PEG polyethylene glycol RT room temperature SAHA suberoylanilide hydroxamic acid TBS Tris-buffered saline Tos Tosyl TSA trichostatin A VPA valproic acid

KEY WORDS: FB188 HDAH, high-throughput screening, trifluoromethylketone, pyran-4- one, p-benzochinone, chromone

ABSTRACT

Histone deacetylases (HDACs) are considered to be among the most important enzymes regulating gene expression in eukaryotic cells. In general, increased levels of histone acetylation are associated with increased transcriptional activity, whereas decreased levels are linked to repression of gene expression. HDACs associate with a number of cellular oncogenes and tumor-suppressor genes leading to an aberrant recruitment of HDAC activity, which results in changes of gene expression, impaired differentiation and excessive proliferation of tumor cells. Therefore, HDAC-inhibitors are efficient anti-proliferative agents in both in vitro and in vivo preclinical models of cancer making them promising anti-cancer therapeutics. Here, we present the results of a medium-throughput screening program aiming at the identification of novel HDAC-inhibitors using histone deacetylase-like amidohydrolase

from strain FB188 (FB188 HDAH) as model enzyme. Within a library of 3719 compounds, THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536 several new classes of HDAC-inhibitors were identified. Among these hit compounds, there were also potent inhibitors of eukaryotic HDACs, as demonstrated by an increase in histone H4 acetylation accompanied by a decrease in tumor cell metabolism in both SHEP neuroblastoma and T24 bladder carcinoma cells. In conclusion, screening of a compound library using FB188 HDAH as model enzyme identified several promising new lead structures for further development.

INTRODUCTION Histone deacetylases (HDACs) are important enzymes in the regulation of gene expression in eukaryotic cells [1]. Together with histone acetyltransferases (HATs) HDACs maintain the delicate equilibrium between acetylated and deacetylated ε-amino groups of lysines in the aminoStage terminal parts of histone 2(a) proteins. In general,POST-PRINT increased levels of histone acetylation are associated with increased transcriptional activity, whereas decreased levels of acetylation are

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associated with repression of gene expression [2]. Although histone deacetylation appears to have a fundamental role in regulating gene expression, HDAC inhibitors seem to directly affect transcription of only a relatively small number of genes [3, 4]. Most of the genes regulated by HDAC inhibitors participate in the control of cell growth and survival, providing a mechanistic explanation for the anticancer properties of these inhibitors. Particularly, activation of differentiation programs, inhibition of the cell cycle and induction of apoptosis are key antitumor activities that make HDAC inhibitors promising anticancer agents. Several synthetic compounds and natural products have been reported to inhibit HDACs (reviewed in:[5-7]. Furthermore, a small number of drug candidates are currently in phase I – III clinical trials (reviewed in: [8] with the first drug, vorinostat (Zolinza, Merck & Co., Inc.) recently approved by the FDA. However, many currently available HDAC inhibitors are disadvantageous e.g. in regard of their limited metabolic stability [9, 10] or their cross- reactivity against other enzymes such as esterases [11]. Thus, there is still a need for new HDAC inhibitors belonging to yet unexplored structural classes of compounds. Here, we report the identification of novel small-molecule HDAC inhibitors through medium- throughput screening of a compound library using a bacterial histone deacetylase-like amidohydrolase (HDAH; [12-14]) as a model system. Several of the compounds specifically inhibit the bacterial enzyme. Other compounds proved to be pan-inhibitors that also inhibit eukaryotic HDACs and induce hyperacetylation in a neuroblastoma cell line (SHEP) and bladder carcinoma cells (T24). The novel inhibitor structures provide new lead structures for further development of improved anticancer drugs.

EXPERIMENTAL

Reagents FB188-HDAH was expressed in E. coli and purified by affinity chromatography (Zn2+- IMAC) as described [12]. The enzyme was stable for weeks as 150 µg/ml solution in 10 mM potassium phosphate, pH 8.0 at 4°C. Rat liver HDAC was purchased from Merk Biosciences (Bad Soden, Germany), whereas human HDAC8 was expressed in E. coli and purified as described [15]. Trypsin from porcine pancreas (Type IX-S, 13,000–20,000 BAEE units/mg) was purchased from Sigma (Taufkirchen, Germany) and after solvation in trypsin buffer (50 mM Tris-HCl, pH 8.5, 100 mM NaCl, 0.1% PEG8000) to a solution of 10 mg/ml was stable for several days at 4 °C. Boc-Lys(Ac)-MCA (Bachem, Weil am Rhein, Germany) and Tos- GPR-MCA (Sigma, Taufkirchen, Germany) were dissolved to 30 mM in DMSO. Tos- GPK(Ac)-MCA was synthesized as described [16]. Sodium valproate (Sigma, Taufkirchen, Germany) was dissolved to 1 M in phosphate-buffered saline (PBS) and stored at -20 °C. Apart from RPMI 1640 medium and trypsin/EDTA (Cambrex, Nottingham, UK), all cell

culture reagents were purchased from Sigma (Taufkirchen Germany). THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536

Library Screening The compound collection was obtained from the Hans-Knöll-Institute (HKI, Jena) as solutions of approximately 10 µg/ml and stored at -80 °C. The thawed substances were diluted 1:25 in assay buffer (10 mM potassium phosphate, pH 8.0). From each dilution 20 µl were transferred into cavities of black 96-well screening plates (Fluotrac200, Greiner, Frickenhausen, Germany). Screening of the compound library for inhibitors of the target enzyme FB188-HDAH was performed using the 1-step version of the standard fluorogenic assay [16, 17]. A robotic screening device (CyBi-Screen-Machine, CyBio, Jena, Germany) equipped with a Polarstar MTP reader (BMG Labtech, Offenburg, Germany) was used (see also supplementary material). To excludeStage false-positive candidates2(a), control POST-PRINTexperiments were conducted with all hits from primary screening. For detection of compound autofluorescence, reactions without enzymes

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or substrate but with the same concentration of each compound as used for screening were measured. To discriminate real hits from substances predominantly inhibiting the reporter enzyme, trypsin inhibition assays were performed for all primary hits at the same compound concentrations as used in primary screening. 150 µl of the mock reactions used for autofluorescence measurements were transferred in black 96 well MTP and 10 µl trypsin (10 ng/ml diluted in trypsin buffer) were added. After 10 min at 30 °C, the reaction was started with 50 µl trypsin substrate (200 µM Tos-GPR-MCA in trypsin buffer). Trypsin activity was recorded for 10 min as increasing MCA fluorescence at 30°C in the plate reader at 460 nm after excitation at 390 nm.

Determination of Z´-factor For determination of the Z´-factor, a quality parameter used to judge assay performance control enzyme inhibition experiments were carried out using the known HDAH inhibitor CYPX [14]. In principle, the screening protocol was used except that deacetylase activity was measured in the presence (48 wells) or absence (48 wells) of 10 µM model inhibitor instead of library compounds, respectively. The Z´-factor was calculated as described previously [18].

Determination of IC50 values To assess the potency of hits 8 point dose-response curves were measured in triplicates. For HDAH, the same protocol was applied as for primary screening. Reactions were monitored in the presence of 20 µl of hit compounds diluted in assay buffer to final concentrations of 1 nM to 100 µM. Obtained slopes of the activity curves were corrected against blank reactions without HDAH. Dose-response curves for primary hits with rat liver HDAC and human HDAC8 were generated using a two-step HDAC assay protocol as described [15, 16]. IC50 values were calculated with the help of GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, USA).

Cell culture SHEP neuroblastoma cells were cultured in RPMI 1640 medium (Cambrex) supplemented with 10% FCS (Sigma) at 37 °C, 5% CO2 [19]. Primary human fibroblasts (PHFs) derived from a healthy donor were grown in DMEM (10% FCS, 1% NEAA) at 37 °C, 5% CO2 as described previously [19]. Cellular metabolic activity Metabolic activity was assessed with the tetrazolium derivative WST-1, which is reduced to a soluble coloured formazan product by cellular mitochondrial dehydrogenases. The metabolic

activity measured corresponds to the number of living cells and is used as a screening assay THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536 for cell viability [20]. Cells were plated at 2 x 103 cells/ well into 96-well plates and exposed to different concentrations of compounds 46F08 and 50B09 for 48 h. Compound 46F08 was dissolved in MeOH p.a. to give a stock solution of 100 mM, compound 50B09 was dissolved in DMSO to give a stock solution of 10 mM. Compounds were further diluted in cell culture medium to give the final concentrations (Fig. 2). In control experiments, cells were treated with MeOH (0.1 ‰ v/v) and DMSO (0.25% v/v) corresponding to the highest solvent concentration applied in the compound treated cells. WST-1 reagent was added to a final dilution of 1:10 to the wells with those with medium only serving as blank control. After 60 min at 37 °C, absorbance at 450 nm was determined with a plate reader (BMG Labtech, Offenburg, Germany) and corrected for absorbance at 650 nm. Blank control values were subtractedStage from all results and2(a) values for untreated POST-PRINT cells were set as 100%.

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Western Blot Analysis The level of acetylated histone H4 was assessed as previously described [21]. Protein concentrations of the cell lysates were determined using the BCA protein assay (Pierce, Rockford, USA). Briefly, 5µl of cell lysates were incubated for 1h at 37°C with 200 µl of BCA-Mix. Absorbance was measured with a plate reader (BMG Labtech, Offenburg, Germany). The following primary antibodies were used: anti-β-actin mouse monoclonal (clone AC-15, Sigma) and anti-acetyl-histone 4 rabbit polyclonal (Upstate, Lake Placid, USA). Secondary antibodies were HRP-conjugated goat anti-mouse (Dianova, Hamburg, Germany) and donkey anti-rabbit IgG (Promega, Mannheim, Germany).

RESULTS

Medium-throughput screening and hit confirmation For medium-throughput screening the 1-step version of the standard fluorogenic HDAC assay was used [16, 17] because the enzyme FB188 HDAH is resistant against trypsin digest. For the assay conditions used, a Z´-factor of 0.75 was determined as part of the overall screening program. A total of 3719 compounds containing mostly natural products were tested at a concentration of 0.04 µg/ml. Thirty-eight compounds (1 % of total tested samples) that displayed more than 75 % inhibition were selected as primary hits (see also supplementary material). From the selected primary hits 22 were confirmed to be FB188 HDAH inhibitors and do not show autofluorescence or trypsin inhibition. Unfortunately, 6 out of the 22 hits were no longer available from the supplier. The results of all 16 remaining primary hits were validated by repeated measurements in the screening assay and subsequently by dose- response curves using final inhibitor concentrations ranging from 1 nM to 100 µM. Based on the selection criterion of IC50 < 10 µM for the FB188 HDAH assay, a total of 10 compounds (0.26 % of total tested samples) were selected as confirmed hits for follow-up studies (Table 1). The most efficient HDAH inhibitors exhibited IC50 values of 610 nM (45E09) and 550 nM (46F08).

Selectivity of hit compounds To determine whether the confirmed hit compounds selectively inhibit only FB188 HDAH, two other HDAC preparations were tested: rat liver HDAC (Calbiochem, Bad Soden, Germany; this preparation mainly contains HDAC1, 2 and 3 [15]) and recombinant human HDAC8 [15]. Interestingly, seven compounds (21E08, 23F10, 26H05, 33D02, 33C08, 40H03, 45E09) showed a marked selectivity for FB188 HDAH (Table 1). Compound 22G03 exhibited a clear preference for HDAH but also inhibited HDAC8 with a 5-fold increased THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536 IC50 value. Two compounds (46F08; 50B09) proved to be rather unspecific pan-inhibitors. Both of these compounds showed a marked preference for human HDAC8.

Competition assay with selected compounds For several hit compounds sufficient material was available to perform competition binding assays as published previously [22]. Briefly, a fluorescent HDAC inhibitor (FAHA), which shows fluorescence resonance energy transfer (FRET) with tryptophans of the enzyme was used. Upon competition with other HDAC inhibitors the displacement of the fluorescent inhibitor is accompanied by a decrease in FRET. In our experiments, compounds 21E08, 22G03, 33D02 and 33C08 were able to displace fluorescent inhibitor FAHA from the enzyme complexStage (Fig.1). 2(a) POST-PRINT

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Structure classes of inhibitors The 10 hit compounds listed in table 1 fall into different structure classes. Surprisingly, only half of these inhibitors match the typical structure of many HDAC inhibitors, i.e. a cap and a Zn-binding moiety linked by a long spacer group [23]. Examples of this type are methylketones 45E09 and 23F10, trifluoromethyl ketone 46F08 and carboxylic acids 22G03 and 33C08 (Table 1). All other hit compounds belong to structural classes that have not been described as HDAC inhibitors so far. The HDAH-selective inhibitors 45E09 and 23F10 (Table 1) showed IC50 vlaues of 0.61 µM and 1.12 µM and comprise a pyran-4-one or a p-benzochinone ring as cap group, a C9-10 aliphatic spacer and a methylketone moiety. The latter of which in principle may interact with the active site zinc ion as this has been demonstrated for the trifluoromethyl ketone moiety [24]. Because an additional series of related compounds was available, we were able to identify structural elements that were important for activity of this inhibitor class (Table 2). Simultaneous changes in the substituents of the pyran-4-one ring, i.e. replacement of the ethyl groups in 3- and 5-position by methyl groups and the introduction of a hydroxyl group replacing the 6-methoxy group lead to a 7-fold increase in the IC50 value for An2 as compared to 23F10. The favourable effect of at least the ethyl group in 3-position as compared to a methyl group was also observed in the context of the structural scaffold shared by compounds An3, An4 and An5. Changing the pyran-4-one ring (23F10) into a p- benzochinone ring (45E09) obviously does not influence the inhibitory activity significantly. The opposite seems to be true for the length of the spacer. An inhibitor with a C6-spacer like An1 appeared to be inactive, whereas inhibitors like An2, 23F10 or 45E09 with a C9-10 aliphatic spacers show significant activity. Compound 46F08 (Table 1; [25] is a pan-inhibitor with IC50 values for HDAH, rat liver HDAC and human HDAC8 of 0.55 µM, 0.64 µM and 0.10 µM, respectively. It belongs to the group of trifluoromethyl ketone-based HDAC-inhibitors that have been described previously [10]. From a recent crystallographic study it is known that the oxygen atom of the trifluoromethyl ketone moiety binds to the zinc atom of the enzyme and also the fluoric atoms participate in various interactions with the enzyme [24]. In our screening, it was the most active inhibitor of FB188 HDAH with an IC50 of 0.55 µM for FB188 HDAH. For the rat liver HDAC preparation, the IC50 was similar to that of HDAH. In contrast, the IC50 for human HDAC8 showed a 5-fold decrease, i.e. a value of 0.10 µM. Thus, the potency of 46F08 is at least similar if not slightly improved as compared to previously published trifluoromethyl ketone-based HDAC-inhibitors [10], which already were the result of subsequent cycles of optimization. In addition, resembling the more extended type of HDAC inhibitors, compounds 22G03 and 33C08 show a high selectivity for HDAH with IC50 values of 9.24 µM and 3.66 µM,

respectively. As shown in table 1, compounds 22G03 and 33C08 (isolated from yeast THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536 Candida bombicola ATCC 22214; [26] are marked by the presence of carboxylic acid moiety at the end of a long spacer, suggesting that this moiety may contact the active site zinc ion. 22G03 contains an epoxide group in the middle of an aliphatic chain reminiscent of depudecin [27, 28], whereas 33C08 belongs to the class of sophorose lipids. The view that both compounds directly bind to the active site pocket of the enzyme is supported by the finding that 22G03 as well as 33C08 are active in the FAHA competition assay (Figure 1). However, the structural similarity between 33C08 and 40H03 (isolated from yeast Candida bombicola ATCC 22214; [29] argues against the importance of a direct binding to the active site zinc ion (see Table 1). Compound 40H03 shows an IC50 value of 6.18 µM for HDAH and contains a lipid chain without a polar end group, such as carboxylic acid functionality. In agreement with the view that a possible direct contact to the zinc ion in 33C08 does not significantlyStage contribute to binding,2(a) the IC50 ofPOST-PRINT 40H03 (6.18 µM) shows only an increase by a factor of 2 as compared to that of 33C08 (3.66 µM). Further support of this view comes from

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the fact that a cyclic “lactonic” derivative of 33C08 termed 33D02 (isolated from yeast Candida bombicola ATCC 22214; [26] was also among the most active hits. In this molecule, no free carboxylic acid end is present. In addition, it shows a second acetylation of the sophorose moiety. Similar to the open chain derivatives it proved to be a selective HDAH inhibitor with an IC50 value of 2.71 µM. Reexamination of the compound library indicated that it contained several compounds similar to 33C08 and 40H03, which appeared to be inactive under screening conditions (data not shown). Inactive variants of 40H03 included (i) derivatives with altered lipid chain length (± C2), and (ii) derivatives combining a second sophorose acetylation with either a longer lipid chain(± C2) or hydroxylation at position 12 of the lipid chain. Three types of inactive derivatives of 33C08 were identified, (i) a derivative with glucose replacing sophorose, (ii) a derivative with trehalose replacing sophorose and at the same time missing the carboxylic acid moiety, and (iii) derivatives containing a saturated lipid chain in which the carboxyl acid moiety was replaced by butyl amide or higher amide moieties. In summary, it appears that the nature of the sugar moiety as well as the length and the nature but not necessarily the cyclization of the lipid chain have a significant influence on inhibitory activity. Compound 26H05 (Table 1) is a chromone also termed pilopleurin originally isolated from the roots of Pilopleura kozo-poljanskii Schischk [30]. In our experiments compound 26H05 proved to be rather selective for FB188 HDAH (IC50 = 1.61 µM), not inhibiting eukaryotic HDACs. Unfortunately, no closely related compounds were available for structure-activity- relationship studies. Actinomycin X (compound 21E08; originally isolated by H. Zahner from Streptomyces michiganensis ATCC 14970; Table 1) also was among the most potent hit compounds with an IC50 value of 1.08 µM for HDAH. Basically, no inhibitory activity was observed for rat liver HDAC and human HDAC8. Actinomycin X is a close relative of actinomycin D, an approved antitumor agent (Lyovac-Cosmegen®, Cosmegen®) [31, 32]. In contrast to actinomycin X, actinomycin D carries an additional amino group in the phenoxazone system and a L-proline replacing one sarcosine to give to identical pentapeptidic cycles. Our results with actinomycin X prompted us to also test actinomycin D for a possible HDAC inhibitory activity. As a result, it appeared that actinomycin D inhibited FB188 HDAH with an IC50 of 81 µM and only weakly blocked rat liver HDAC (IC50 0 Hit compound 50B09 (Table 1) belongs to the class of pyrazolopyrimidinones. It has been identified previously as a potent inhibitor of coxsackievirus B3 replication [33]. In our study, it was among the two inhibitors of FB188 HDAH, which also blocked eukaryotic HDAC preparations. For HDAH, rat liver HDAC and human HDAC8 IC50 values of 2.77 µM, 4.00 µM and 0.31 µM were measured.

Effects of hit compounds on tumor cells THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536 Because 46F08 and 50B09 showed significant activity toward eukaryotic HDACs, we next focused our attention on the inhibitory activity of both compounds in cell culture models. Treatment of SHEP neuroblastoma cells with 46F08 (0.5 - 4µM) indeed generated an increase in acetylation of histone 4, indicating HDAC-inhibition. Similar results were obtained for T24 cells (see supplementary material). Acetylation, however, was weaker compared to that induced by TSA (Figure 2A). In contrast, treatment with compound 50B09 did not show an increase in histone 4 acetylation at concentrations of 25 – 100 µM. Here, it rather seems that 50B09 suppressed the increased level of histone H4 acetylation caused by DMSO [34, 35], which was used as solvent. HDAC inhibitors are known to have anti-cancer effects. Therefore, the influence of hit compounds 46F08 and 50B09 on the metabolic activity of SHEP neuroblastoma cells was investigatedStage using the WST 2(a)-1 assay. This POST-PRINT assay quantifies the enzymatic activity of

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mitochondrial dehydrogenases in the sample and correlates with the number of viable cells [20]. Treatment of cells with compound 46F08 resulted in a concentration-dependent, pronounced reduction of cell metabolic activity even at low concentrations of 0.1 – 4 µM. Similar results were obtained for T24 cells (see supplementary material). Compared to the well established HDAC inhibitor valproic acid (VPA) already used in clinical trials, 46F08 was approximately 104 fold more active (figure 2). Because tumor cell selective activity of HDAC inhibitors has been described [36], the effect of 46F08 on the metabolic activity of normal, non-transformed human skin fibroblasts was investigated. 46F08 did not obviously influence fibroblast metabolic activity even at the highest concentration applied suggesting tumor cell selective activity of the compound (figure 2). In contrast, 50B09 elicited no effect on the metabolic activity of SHEP cells at concentrations up to 10 µM (figure 2B).

DISCUSSION

We have successfully confirmed the feasibility of our screening assay to efficiently screen a large compound library. Using a robotic workstation (CyBiScreen, CyBio, Jena, Germany) the assay exhibited a very high Z′-factor of 0.75 indicating an excellent assay. The screening identified several compounds capable of inhibiting HDAH reporter enzyme activity. Here, 10 out of 3719 compounds showed IC50 values < 10 µM. The identification of inhibitors 46F08 and 50B09, both of which are highly potent inhibitors of human HDAC8, proved that FB188 HDAH indeed can be used as a model system to also find inhibitors of human HDAC. Altogether, hit compounds fall into different structural classes. Only half of the inhibitors identified followed the simple HDAC inhibitor architecture, realized e.g. in SAHA, in which a cap and a Zn-binding moiety are linked by an extended spacer. For the group of ketone-based inhibitors with pyran-4-one or p-benzochinone rings as cap moieties, a preliminary structure- activity study showed that structure elements like alkyl substituents in the ring system, linker length and the potential zinc-binding moiety are important for inhibitory activity. However, based on the relatively small number of derivatives examined so far it is not justified to draw more specific conclusions. One of our hit compounds, 46F08 belongs to the class of trifluoromethyl ketones. It has been shown that electrophilic ketones such as trifluoromethyl ketones are potent inhibitors of zinc- dependent enzymes [10, 37, 38]. In contrast to simple methylketones, trifluoromethyl ketones do not only show an increase in electrophilicity at the carbonyl carbon with favourable effects on hydrogen bonding capabilities of the carbonyl oxygen. The recently solved crystallographic structure of a complex of FB188 HDAH with a trifluoromethyl ketone inhibitor [24] also revealed that the fluoric atoms actively participate in direct protein binding. THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536 Compound 46F08 not only proved to be a potent inhibitor of FB188 HDAH but also blocked rat liver HDAC and human HDAC8 with submicromolar IC50 values. Furthermore, it significantly reduced the metabolic acitivity of SHEP neuroblastoma cells, which correlates with a decrease in the number of viable cells. Interestingly, normal human fibroblasts were not affected by the compound suggesting tumor cell selective activity of 46F08 as has been described for other HDAC inhibitors [36]. In T24 bladder carcinoma cells, a similar effect was observed. Thus, 46F08 is an interesting candidate for further development. However, previously developed trifluoromethyl ketones had exhibited an unfavourably short half-life in animals [10]. It remains to be shown, if compound 46F08 also is readily metabolized. In this regard, it will be interesting to test derivatives of 46F08 with substituents at the CH2- moiety next to the carbonyl group. CompoundsStage 40H03, 33C08 and2(a) 33D02 all belongPOST-PRINT to the class of sophorose lipids, a group of microbial glycolipids produced by yeasts [39]. Comparison with structurally similar but inactive derivatives of the compound library suggested that these molecules do not directly 8 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Biochemical Journal Immediate Publication. Published on 25 Mar 2008 as manuscript BJ20080536

bind to the active site zinc ion. Interestingly, earlier work by Isoda et al. [40, 41] and Scholz et al. [42, 43] had already demonstrated that certain sophorolipids induce cell differentiation of human leukemia cells by an “unknown mechanism”. We may now hypothesize that the anti-cancer activity of sophorolipids and maybe also of other glycolipids [40, 44, 45] is due to their inhibition of certain histone deacetylases, particularly those with similarities to FB188 HDAH. The same may be true for hit compound 26H05. In our experiments, it did show selectivity for HDAH and not for eukaryotic HDACs. However, several chromones have already been described as anticancer agents [46] with proposed mechanisms as diverse as inhibition of breast cancer resistance protein (ABCG2) [47] or inhibition of phosphatidylinositol 3‘-kinase [48]. It cannot be excluded, though, that chromones may also work as inhibitors of certain histone deacetylases, particularly those with similarities to FB188 HDAH. Neither actinomycin X nor actinomycin D have been described as HDAC inhibitors so far. On the contrary, actinomycin D has been shown to intercalate into DNA by virtue of its planar phenoxazone ring system [49, 50]. As a consequence, it is believed that actinomycin D blocks DNA-dependent RNA polymerase [51] and poisons topoisomerase II, which results in double-strand DNA breaks [52]. In principle, the action of actinomycin D should not be restricted to a certain phase in the cell cycle. However, there are experimental evidences that it is by far most active in the S- and G2-phases, indicating that it also may act in a different way [53]. In the light of our data, it can be speculated that compounds such as actinomycin X and D may also work as inhibitors of certain histone deacetylases. With compound 50B09 another new class of HDAC inhibitors has been discovered in our screening program. Previously 50B09 and related pyrazolopyrimidinones had been identified as potent inhibitors of coxsackie virus B3 replication [33]. In this previous study, cytotoxicity was also determined. Whereas compound 50B09 did not show a significant cytotoxicity against HeLa cells or a pronounced growth inhibition in L-929 and K-562 cells, derivatives of 50B09 did show inhibitory activity on these cell lines [33]. In our study, we also measured the effect of 50B09 on eukaryotic cells. Despite a good inhibitory activity against rat liver and human HDAC8, we neither detected a pronounced effect on the metabolic activity of SHEP neuroblastoma cells nor an increase in histone H4 acetylation. Structure-activity-relationship studies are on the way to identify derivatives with improved anti-tumor cell activities. In summary, several inhibitors have been discovered that block enzymes belonging to the HDAC family of proteins. Most of the identified inhibitor structures have not been described as HDAC inhibitors so far and therefore may be a good starting point for further development toward future generations of HDAC inhibitors with possible applications in the fields of malignant and other diseases. THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536 ACKNOWLEDGEMENTS This research was in part supported by grant BioFuture 0311852 from the Bundesministerium für Forschung und Technologie, Germany to A.S. and by a grant of the Nationales Genomforschungsnetzwerk (NGFN2) to O.W. A.S. would like to thank R. Jansen (Braunschweig), H.-P. Fiedler (Tübingen), G.-V. Roeschenthaler and D.V. Sevenard (Bremen) for providing hit compounds and for valuable discussions.

Stage 2(a) POST-PRINT

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Table 1: Most active hit compounds in the screening for HDAH inhibitors Compound structure Compound IC50 [µM] Nr. HDAH RL HDAC HDAC8 Sar Sar 50§ L-Pro L-(N-Me)Val Sar L-(N-Me)Val 21E08 1.08 NA* (4 )

D-Val O D-Val O L-Thr L-Thr O O

O O

CH3 CH3 Actinomycin X O 22G03 9.24 NA (46)50§ OH

O O 23F10 1.12 (10) 10§ (10) 10§

O O O 26H05 1.61 (6) 10§ (10) 10§ O OH O

O O CH3 33D02 2.71 (12) 50§ (19) 50§ O O

CH3 HO O HO O O O

CH3 O O O O HO

OH 50§ 50§ HO O 33C08 3.66 (10) (12) O HO OH O OH HO O O CH3 HO

OH OH 40H03 6.18 (22) 50§ NA O HO 10 12 O 1 HO CH3 CH3

CH3 O O O O HO HO

OH THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536 O 45E09 0.61 (13) 50§ (4) 50§

O O F N F 46F08 0.55 0.64 0.10 F N H O NO2 H2N 50B09 2.77 4.00 0.31 Cl N NH N

Cl O

NO2 *NA =Stage no activity at 50 µM inhibitor concentration;2(a) 50§ percent POST-PRINT inhibition at 50 µM; 10§ percent inhibition at 10 µM

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Table 2. Derivatives of compound 45E09 Compound structure Compound IC50 [µM] Nr. (HDAH) O 45E09 0.61

O O O 23F10 1.12

O O O O An1

O O

O O An2 7,76

HO O O O An3

O O O O O An4

O O O O O An5 11,98

O O O O THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536

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FIGURE CAPTIONS

Fig. 1. Fluorescence resonance energy transfer-based competition assay for selected hit compounds. Fluorescent HDAC inhibitor (FAHA), which shows fluorescence resonance energy transfer (FRET) with tryptophans of the enzyme was used. Upon competition with other HDAC inhibitors the displacement of the fluorescent inhibitor is accompanied by a decrease in FRET. Binding degrees and binding constants were calculated as described [22].

Fig. 2. Cellular effects produced by hit compounds 46F08 and 50B09. (A) Influence on cell metabolic activity. Upper panel: SHEP neuroblastoma cells and normal primary human fibroblasts (PHF) were treated with compound 46F08 for 48 h with the indicated concentrations. Cellular metabolic activity was determined using WST-1 assay. Vehicle: cells were treated with solvent only (MeOH, 0.1 ‰ v/v), corresponding to the MeOH concentration applied in 10µM 46F08 treated cells. Values for untreated cells were set to 100 %. Lower panel: SHEP neuroblastoma cells were treated with compound 50B09 for 48 h with the indicated concentrations. Vehicle: cells were treated with solvent only (DMSO, 0.1 % v/v), corresponding to the DMSO concentration applied in 10 µM 50B09 treated cells. (B) Influence on histone H4 acetylation. SHEP cells were treated for 4 h with indicated compounds and harvested for Western analysis using an anti-histone H4-acetyl antibody. MeOH (0.04 ‰ v/v) was used as a solvent control for 4 µM 46F08 treated cells. DMSO (0.25, 0.5, 1% v/v) were used as solvent controls for 25, 50, 100 µM 50B09 treated cells, respectively. Note that DMSO, in contrast to MeOH, harbors HDAC-inhibiting activity. β- actin served as loading control. THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536

Figure 1 Stage 2(a) POST-PRINT

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Figure 2 THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080536

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REFERENCES

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