UHPLC-MS-based HDAC Assay Applied to Bio-guided Microfractionation of Fungal Extracts:UHPLC-MS-based HDAC assay for...

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UHPLC-MS-based HDAC Assay Applied to Bio- guided Microfractionation of Fungal Extracts:

UHPLC-MS-based HDAC assay for...

Article in Phytochemical Analysis · December 2016

DOI: 10.1002/pca.2652

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UHPLC-MS-based HDAC Assay Applied to Bio- guided Microfractionation of Fungal Extracts

Vincent Zwick, ^a Pierre-Marie Allard, ^a Lucie Ory, ^a Claudia A. Simões-Pires, ^a * Laurence Marcourt, ^a Katia Gindro, ^b Jean-Luc Wolfender ^a

and Muriel Cuendet ^a

ABSTRACT:

Introduction

–

Histone deacetylases (HDAC) are considered as promising targets for cancer treatment. Today, four HDAC inhibitors, vorinostat, romidepsin, belinostat, and panobinostat, have been approved by the Food and Drug Administration (FDA) for cancer treatment, while others are in clinical trials. Among them, several are naturally occurring fungal metabolites.

Objective

–

To develop and optimise an enzyme assay for bio-guided identification of HDAC inhibitors in fungal strains.

Methods

–

Fluorescence and MS-based HDAC enzymatic assays were compared during the bio-guided fractionation of

Penicillium griseofulvum. The MS-based approach was then optimised to evaluate HDAC selectivity using the human recombinant class I

isoform HDAC1 and the class II isoform HDAC6.

Results

–

Fluorescence-based assays have several drawbacks when used for bio-guided fractionation because of the native fluorescence and the trypsin inhibitory ability of compounds present in many extracts. The MS-based method led to the isolation of gliocladride C, which is selective for HDAC1 and salirepol, which showed an HDAC6 selectivity. Their activity and presence in

P.

griseofulvum

is described here for the first time.

Conclusion

–

The UHPLC-ESI-MS/MS-based method using specific HDAC isoforms is suitable to isolate selective HDAC inhibitors by bio-guided fractionation of fungal strains. Also, it decreases potential interferences with natural products compared to the fluorescence-based assay.

Supporting information can be found in the online version of this article.

Keywords: histone deacetylase; bio-guided fractionation; isoform selectivity; UHPLC-ESI-MS/MS; fungi

Introduction

Histone acetylation and deacetylation constitute a dynamic and reversible process catalysed by two classes of enzymes, histone acetyltransferase (HATs) and histone deacetylases (HDACs), respectively. HDACs deacetylate the lysine residues in the N- terminal tails of the core histones. In humans, 18 HDAC isoforms have been identified and classified into four classes (I

–

IV). Class I isoforms (HDAC1

–

3, HDAC8) are found essentially in the nucleus where histones constitute their main substrate proteins (de Ruijter et al., 2003; Haberland et al., 2009). Class II is divided into two sub- classes, IIa (HDAC4, HDAC5, HDAC7, and HDAC9) and IIb (HDAC6 and HDAC10) (Gregoretti et al., 2004; Yang and Grégoire, 2005).

Contrary to class I, class II isoforms are found to shuttle between nucleus and cytoplasm upon certain cellular signals (de Ruijter et al., 2003; Haberland et al., 2009; Yang and Grégoire, 2005).

HDAC11 is the only member of class IV. Class III (SIRT1

–

7), also known as sirtuins, are NAD

⁺

-dependent. The deregulation of the HDAC/HAT balance is involved in the development of several diseases including cancer, cardiovascular, and neurodegenerative disorders (Colarossi et al., 2014; Donmez and Outeiro, 2013;

Govindarajan et al., 2013; Kang et al., 2014; Kilgore et al., 2010;

Simões-Pires et al., 2013; Stubbs et al., 2015). Today, four HDAC in- hibitors, vorinostat, romidepsin, belinostat, and panobinostat, have been approved by the Food and Drug Administration (FDA) for cancer treatment (Ganai, 2016; Mottamal et al., 2015). Among them, none showed a significant class or isoform selectivity (Furumai et al., 2002; Khan et al., 2008). However, the search for

selective HDAC inhibitors is of interest. Such compounds could be tools for the discovery of unknown functions of a specific HDACs isoform. They could also reduce the toxicity attributed to the pan-HDAC inhibitors by selectively targeting the isoform involved in the disease pathogenesis without acting on uninvolved isoforms (Balasubramanian et al., 2009).

Natural products have been important for the early discovery of selective and/or potent HDAC inhibitors. Romidepsin, a natural product isolated from Chromobacterium violaceum, is used to treat cutaneous and peripheral T-cell lymphoma (Ueda et al., 1994).

Another example is trapoxin B, a selective HDAC1 inhibitor produced by Helicoma ambiens, also studied in the context of can- cer therapy (Butler et al., 2014; Itazaki et al., 1990). The discovery of both romidepsin and trapoxin B was not driven by bio-guided fractionation based on HDAC activity. Instead, the HDAC inhibitory activity of these natural products, like several others, was discov- ered in a second step after being identified as cytotoxic agents

* Correspondence to: Claudia A. Simões-Pires, School of Pharmaceutical Sciences, University of Geneva, CMU–Rue Michel Servet 1, CH-1211 Geneva 4, Switzerland.

Email: claudia.avello@unige.ch

a School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU–Rue Michel Servet 1, 1211, Geneva 11, Switzerland

bMycology and Biotechnology group, Institute for Plant Production Sciences IPS, Agroscope, Route de Duillier 50, P.O. Box 1260, Nyon, Switzerland Received: 17 June 2016, Revised: 6 September 2016, Accepted: 18 September 2016 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/pca.2652

(Itazaki et al., 1990; Ueda et al., 1994). To accelerate the search for HDAC inhibitors of natural origin, a more systematic implementa- tion of bio-guided fractionation would be of great value. This necessitates assays that are effective, reasonably priced and adaptable to high-throughput screening (HTS). In the present pro- ject, an MS-based assay was optimised for bio-guided identifica- tion of HDAC inhibitors in fungi. Given the matrix complexity of natural extracts and fractions, the previously published cell-based multi-substrate MS-HDAC inhibition assay (Zwick et al., 2016) was modified to use a single isoform enzymatic source and a single substrate. In addition, steps for measuring matrix effect of fractions and the influence of individual blanks were included in the new approach. Natural HDAC inhibitors were identified, and the UHPLC

–

MS/MS approach was compared to the traditional fluorescence-based assay.

Experimental

Chemicals and materials

Potato Dextrose Broth (PDB) was purchased from Lifesystem (Newton, MA, USA). MAL (BOC-Ac-Lys-AMC), trypsin from bovine pancreas (7500 BAEE units/mg), trichostatin A (TSA), protease inhibitor cocktail (P8340), HDAC1 and HDAC6 recombinant human isoforms were purchased from Sigma-Aldrich (St Louis, MO, USA). dMAL (BOC-Lys-AMC) was purchased from Bachem (Bubendorf, Switzerland). HeLa nuclear cell extracts were purchased from Enzo Life Science (Lausen, Switzerland). Acetonitrile (MeCN), methanol (MeOH), ethyl acetate (EtOAc) and formic acid (FA) (analytical grade) were purchased from Merck (Darmstadt, Germany).

Distilled water was further purified using a Milli-Q system (Millipore, Milford, MA, USA). Deuterated dimethyl sulphoxide (DMSO-d6) was obtained from Cambridge Isotope Laboratories, Inc (Andower, MA, USA) and Wilmad nuclear magnetic resonance (NMR) tubes were obtained from VWR (Radnor, PA, USA).

Fungal culture

A total of 40 fungal species belonging to the Mycoscope dynamic mycotheca of Agroscope in Changins (Nyon, Switzerland, www.

mycoscope.bcis.ch) were cultivated in triplicates (3 × 20 mL of PDB in 100 mL shots). The scale-up culture of the selected speciesPenicillium griseofulvumDierckx was realised in 30 shots containing 200 mL each of PDB media. They were allowed to grow in static mode for 15 days with artificial day/night cycles (12 h each) at 23 °C.P. griseofulvumwas isolated in 1964 in Lausanne (Vaud, Switzerland) from a wheat crop and authenticated by molecular sequencing of the ITS1 and ITS2 regions. The strain is stored under accession number 506.

Extraction and isolation

The mycelial mat of each fungal culture was filtered on Büchner and the culture media was extracted by liquid/liquid partition with equal volume of EtOAc. The extraction process was repeated three times and the pooled EtOAc fractions were evaporated under reduced pressure. The same methodology was applied to the selected scale-up culture of P.

griseofulvum. In this case, reduced pressure evaporation of the EtOAc fraction afforded 3.6 g of crude extract (brown liquid gum), which was fractionated by flash chromatography on a Puriflash 4100 preparative chromatographic system (Interchim, Montlucon, France) equipped with a quaternary pump, a PDA detector, and a fraction collector. This extract was loaded by dry load using celite and injected on a 120 g C18 column.

A linear gradient of H2O + FA 0.1% and MeCN + FA 0.1% from 98:2 to 2:98 was applied at 14 mL/min for 200 min and fractions were collected by volume in 20 mL tubes. Fractions were combined according to their UV profile into 31 fractions (F1–F31), which were dried using rotary

evaporator. An aliquot (200μL) of each fraction was sampled and plated into 96 well plates for UHPLC–MS/UV/ELSD (evaporative light scattering detector) analysis. F2 purification was performed on an Armen Spot System (Armen Instrument, Saint-Ave, France) with a Kinetex Axia Core-Shell C18 column (5μm, 250 mm × 21.2 mm; Phenomenex, Torrance, CA, USA) with elution in isocratic mode (H2O + 0.1% FA:MeOH + 0.1% FA, 97:3) at 10 mL/min to afford (1) (2.1 mg) and (2) (1.6 mg). F26 was purified on a Shimadzu semi-preparative system using an X-Bridge C18 column (5μm, 250 x 10 mm; Waters, Milford, MA, USA) to afford (3) (0.6 mg).

Purity control of the fractions was carried on a HPLC-PDA-ELSD Agilent 1100 series system (Santa Clara, CA, USA) consisting of an autosampler, high-pressure mixing pump, and photodiode array (PDA) detector connected to an ELSD (Sedex 85, Sedere Omnilab, Alfortville, France).

Identification of the isolated substances

Optical rotations were measured on a JASCO P-1030 (Easton, MD, USA) po- larimeter (at 20 °C in MeOH,cin g/100 mL). The isolated compounds were identified by means of high resolution mass spectrometry (HRMS), and one- dimensional (1D) and two-dimensional (2D)-NMR spectroscopy (1H, HSQC, HMBC, NOESY and ROESY experiments). HRMS measurements were per- formed on a Thermo Dionex Ultimate 3000 UHPLC system interfaced to a Q-Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany), using a heated electrospray ionisation (HESI-II) source. The optimised HESI-II parameters were as follows: source voltage, 4.0 kV (positive); sheath gas flow rate (N2), 50 units; auxiliary gas flow rate, 12 units; spare gas flow rate, 2.5 units; capillary temperature, 266.25 °C (positive), S-Lens RF Level, 50%.¹H- and¹³C-NMR spectra were recorded on a Varian Unity Inova 500 MHz NMR instrument (Varian, Palo Alto, CA, USA). Chemical shifts are reported in parts per million (δ) using the residual DMSO-d6signal (δH2.50;δC39.52) as internal standards for¹H- and¹³C-NMR, respectively, and coupling constants (J) are reported in Hertz.

HDAC fluorescence-based assay

Crude extracts, fractions or pure compounds were tested for HDAC inhibi- tion in nuclear cell extracts from HeLa cells. Reactions were carried out in 96-well ½ volume microplates in duplicate. Test samples were diluted in DMSO and added at a final concentration of 100μg/mL (5% DMSO in each well) for screening purposes. MAL was added to a final concentration of 10.5μM. The reaction was initiated by the addition of HeLa nuclear extract diluted in assay buffer (50 mM Tris at pH 8.0 adjusted with HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) followed by incubation at 37 °C for 4 h.

The reaction was stopped with 1μM TSA. Trypsin from bovine pancreas was added 45 min prior to plate reading. HDAC inhibition was calculated by comparing the amount of deacetylated substrate between control (100% HDAC activity) and test sample. The relative amounts of deacetylated substrate were obtained by fluorescence reading with excita- tion at 360 nm and emission at 460 nm. TSA was used as positive control.

Considering the risk of auto-fluorescence interferences, an individual blank was done for each test sample.

HDAC UHPLC

–

MS/MS-based assay

Crude extracts, fractions or pure compounds were tested in duplicate for HDAC inhibition in nuclear cell extracts from HeLa cells, as well as with the specific isoforms HDAC1 and HDAC6, adapted from the previous method that uses living cells as an enzymatic source (Zwicket al., 2016). Test samples, single substrate (MAL) and enzymes were added similarly as in the HDAC fluorescence-based assay, except for the specific isoforms that were added to a final concentration of 50 U/well. The enzyme concentration was adjusted to provide a signal to noise ratio≥20 for the chromatographic peaks of both acetylated and deacetylated MAL. Samples were then incu- bated at 37 °C for 4 h. The reaction was stopped by the addition of cold MeCN. The plate was placed for 10 min at 80 °C and then centrifuged at 11500 rpm for 10 min. Supernatants were transferred into UHPLC- compatible 96-well plates to be analysed on a UHPLC-ESI-MS/MS system

Zwick V. et al.

Phytochem. Anal.2016 Copyright © 2016 John Wiley & Sons, Ltd.

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consisting of an Acquity UPLC System (Waters) connected to a Quattro Mi- cro triple quadrupole mass spectrometer equipped with an ESI source oper- ating in positive-ion mode (Waters). Samples were injected (5μL) into a C18 Kinetex column (2.6μm, 100 mm × 3 mm i.d.; Phenomenex) and eluted (0.8 mL/min, 40 °C) with MeCN and H2O both containing 0.1% FA. A gradi- ent of 5 to 45% MeCN in 2 min followed by 1 min with 98% MeCN was used.

This was followed by a washing step with 98% MeCN for 2 min. After the washing step, the column was equilibrated with 5% MeCN during 4 min be- fore the next injection. The conditions used for ESI-MS/MS detection were:

cone voltage, 30 V; capillary temperature, 350 °C; source voltage, 3.0 kV; ar- gon pressure in the collision cell (Q2), 1.4 bar; nitrogen was used as the sheath gas (800 L/h); MAL was detected by MS/MS at mass transition 446→346 (MAL) and 404→304 (dMAL) at retention times of 2.52 min and 1.92 min, respectively. Peak areas were determined using automatic peak area detection of Masslynx 4.1 (Micromass, Manchester, UK). For each substrate, the ratio between areas (deacetylated peptide/acetylated peptide) was determined to obtain the amount of deacetylated substrate. HDAC activity was calculated by dividing the amount of deacetylated substrate between control (100% HDAC activity) and test samples. IC50values were calculated using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). TSA was used as positive control.

Trypsin inhibitory assay

Fractions were tested for trypsin inhibition with trypsin from bovine pan- creas. Reactions were carried out in 96-well ½ volume microplates in dupli- cate. Trypsin was dissolved in assay buffer (50 mM Tris at pH 8.0 adjusted with HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) with TSA (1μM) and added to each microplate well, except for blanks. Fractions were diluted in DMSO and added at a final concentration of 100μg/mL (5% DMSO) in each well. The reaction was initiated by the addition of dMAL substrate.

The plate was then incubated at 25 °C for 45 min. Trypsin inhibition was calculated by comparing the amount of deacetylated substrate between control (100% HDAC activity) and test sample. The relative amounts of deacetylated substrate were obtained by fluorescence reading with excita- tion at 360 nm and emission at 460 nm. A protease inhibitor cocktail (P8340) was used as positive control. Considering the risk of optical interfer- ences, an individual blank was done for each test sample.

Matrix effect assay

Various amounts of MAL and dMAL were dissolved in assay buffer (50 mM Tris at pH 8.0 adjusted with HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2).

Fractions and crude extract were then diluted in DMSO and added at a final concentration of 100μg/mL (5% DMSO) in each well in duplicate. Cold MeCN was added after 4 h incubation at 37 °C. Samples were then treated and analysed by UHPLC-ESI-MS/MS as described in the HDAC UHPLC– MS/MS-based assay.

Results and discussion

Selection and fractionation of

P. griseofulvum

A preliminary HDAC screening assay was performed on the EtOAc extracts of 40 terrestrial fungi from our in-house collection using both fluorescence- and MS-based detection methods. P.

griseofulvum was the only extract showing an inhibitory activity higher than 70% (77.3%) at 100

μ

g/mL with the fluorescence assay.

The activity was further confirmed by the UHPLC

–

MS/MS HDAC assay with a slightly lower inhibition (63.3%). P. griseofulvum is a worldwide occurring fungus. It is well known to produce several bioactive metabolites including patulin, a mycotoxin usually found in apples and apple-derived products (Puel et al., 2010), and griseofulvin, an antibiotic used against various dermatophytes (El-Nakeeb et al., 1965; Shim et al., 2006). The active EtOAc extract of P. griseofulvum was selected to evaluate bio-guided

fractionation approaches aiming at the identification of natural HDAC inhibitors. Flash liquid chromatography fractionation of this extract led to 31 fractions (F1

–

F31).

Fluorescence-based assay for bio-guided fractionation P. griseofulvum fractions were tested at 100

μ

g/mL in the fluorescence assay using HeLa nuclear extract as HDAC source.

Two large zones, corresponding to F1

–

F13 and to F18

–

F21, displayed more than 50% HDAC inhibitory activity. Within these zones, the 10 fractions showing more than 70% inhibition (F1, F2, F4

–

F11) were considered for further fractionation. A small peptide possessing a N-acetylated lysine residue coupled to the fluorophore 7-amino-4-methylcoumarin (AMC) was used (Fig. 1) to measure HDAC inhibition. The HDAC reaction allows deacetylation of the substrate that undergoes a second reaction through a peptidase able to release the fluorophore from the pep- tide sequence (Fig. 1) (Heltweg et al., 2005). This approach is rele- vant for high-throughput screening thanks to its reproducibility and its rapidity to measure HDAC activity (Wegener et al., 2003).

However, fluorescence-based assays suffer from potential interfer- ences between the samples and the reaction mixture. Some natural extracts and fractions are known to have fluorescence background or fluorescence quenching, making them difficult to be evaluated in fluorescence-based assays (Krasteva et al., 2011).

Thus, we introduced an optimised step for evaluating the auto- fluorescence of each fraction by the measurement of individual blanks. This analysis revealed that F6

–

F16 had high background fluorescence signals (Supporting Information Fig. S1), even prior to any enzymatic reaction. Therefore, the results obtained by fluo- rescence reading may be misleading. Moreover, results could also be affected by peptidase inhibitory activity of constituents of the fractions. Numerous natural products are already known to inhibit trypsin (Gupta et al. 2000; Klomklao et al., 2015; Kodani et al., 1998;

Liu et al., 2014; Shee and Sharma, 2007). To verify if the tested frac- tions were interfering with the assay by direct trypsin inhibition, fractions were subjected to an in vitro trypsin inhibition assay. Ac- cording to this assay, 11 fractions (F16

–

F26) displayed more than 50% trypsin inhibition at 100

μ

g/mL (Fig. S2). The most active frac- tion (F20) was tested in a dose

–

response for trypsin inhibition and displayed an IC

₅₀

of 46.7 ± 3.2

μ

g/mL. To see if the trypsin inhibi- tory activity could have an impact on HDAC inhibition, a protease inhibitor cocktail was tested in both trypsin and HDAC inhibition assays in a dose

–

response. The IC

50

for trypsin inhibition (IC

₅₀

= 0.51 ± 0.059% v/v) was quite similar to that of the HDAC in- hibition (0.57 ± 0.049% v/v). These results suggested that the HDAC inhibition measured was mainly due to the protease inhibi- tion. Therefore, the fluorescence-based assay could not clearly re- flect the real HDAC activity. Thus, an HDAC inhibition assay using MS as a detection method was evaluated for the bio-guided frac- tionation of P. griseofulvum.

MS-based assay for bio-guided fractionation

Many of the disadvantages linked to fluorescence detection can be

overcome by the use of specific MS monitoring of the enzyme sub-

strate, an approach that tends to be commonly used for enzymatic

screening in pharmaceutical research (Greis, 2007). This very sensi-

tive and selective way of detection is able to distinguish the acet-

ylated substrate from its deacetylated product upon HDAC

reaction (Fig. 1C). Considering this, each fraction of P. griseofulvum

was investigated with the UHPLC

–

MS/MS-based HDAC inhibition

assay using the HeLa nuclear extract and was compared with the fluorescence method.

Fractions tested on HeLa nuclear extract were expected to pro- vide a similar inhibitory profile between assays. However, F18

–

F27 showed different results when fluorescence- and MS-based methods were compared. These fractions showed some inhibition in the fluorescence approach while their HDAC activity with the MS strategy was higher than the non-treated controls suggesting an activation of HDAC. This difference could be the result of either an analytical interference with the assay or an HDAC activation by compounds contained in the fraction. Several hypotheses to explain this are presented later.

It has been previously reported that matrix effects caused by the presence of co-eluting compounds may alter the signal response, affecting directly the peak area ratios (Taylor, 2005), which in our assay would interfere with the results of HDAC activity. To see if matrix effects had an influence over the activity results, we intro- duced an analysis step for method optimisation in which several amounts of deacetylated and acetylated substrate standards were

subjected to either an inhibitory fraction (F2), a non-active fraction (F11) or activator fractions (F23, F25) in absence of HDAC source (HeLa nuclear extract and recombinant human HDAC isoform). Un- der these conditions and for each fraction, the original peak area ratio (deacetylated peptide/acetylated peptide) was not affected by the various samples. Thus, matrix effects were not observed in the HDAC assay using the UHPLC

–

MS/MS approach. It could also be possible that the HDAC activation observed in the MS-based method could come from an indirect increase of the HDAC activity.

The HeLa nuclear extract and extraction buffers used with recombinant isoforms are particularly complex. The interaction of complex mixtures, such as extracts and fractions, with those com- ponents can result in an apparent higher HDAC activity compared to the non-treated control. For example, protein extracts obtained from cells are well known to contain protease inhibitors. A total of nine fractions (F18

–

F26) were recognised as protease inhibitors (Fig. 2) in the trypsin inhibitory assay. Those fractions were within the HDAC activation zone (F18

–

F27, F29, F31) (Fig. 3) in the MS- based HDAC assay. Thus, over 4 h incubation, this protease

Figure 1. (A) Step 1:deacetylation of the histone deacetylase substrate MAL by histone deacetylases. (B) Step 2: Cleavage of the deacetylated substrate by trypsin leads to the release of the fluorophore (AMC, 360/460 nm). (C) MAL and its deacetylated product (dMAL) are both detected by UHPLC–MS/MS. The peak areas of test samples (A–D) were retrieved.

Figure 2. HDAC activity ofP. griseofulvumfractions with the fluorescence-based assay using HeLa nuclear extract as HDAC sources. F6–F16 and F16–F26 show a self-fluorescence (high blank fluorescence signal) and/or a trypsin inhibitory activity, respectively.

Zwick V. et al.

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inhibitory activity could protect HDACs from being degraded by the naturally occurring proteases within the nuclear extract, resulting in an apparent higher HDAC activity. To check this hypothesis, a protease inhibitor cocktail was used, and no HDAC activation was observed, proving that the results obtained are not an artefact provided by protease inhibitors. The last explana- tion of the result differences could be a direct activation of one or more of the HDAC isoforms contained in the protein extracts.

Some compounds have already been identified as direct activa- tors. For example, N-acetylthiourea derivatives directly activated HDAC8 (Singh et al., 2011) and some natural compounds such as acetyl-CoA, butyryl-CoA, HMG-CoA, malonyl-CoA or NADPH are known for their allosteric activation of HDAC1 and HDAC2 (Vogelauer et al., 2012). However, direct enzymatic activation is not well understood and not confirmed for all HDAC isoforms.

For this reason and because there is to date no real therapeutic ap- plication for HDAC inducers, the research was focused on inhibi- tive fractions in order to identify HDAC inhibitors.

Four fractions inhibited HDAC (F1

–

F3 and F6) in the MS-based assay using the HeLa nuclear extract (Fig. 3). Given that a nuclear extract is composed of a complex matrix, and that it only con- tains nuclear HDAC isoforms, class representative isoforms were chosen for the follow-up of the activity profile. Indeed, the search for class-selective compounds is a current interest in drug discovery. The inhibition of class I HDAC1 isoform has been linked to the efficacy of anti-cancer drugs in drug-resistant tu- mours (Cacan, 2016; Krumm et al., 2016). Accordingly, class II HDAC6 inhibition has been currently investigated in the field of cancer, cardiovascular and neurodegenerative diseases (Demos- Davies et al., 2014; Imai et al., 2016; Simões-Pires et al., 2013).

Therefore, the activity of the fractions was evaluated against the human recombinant isoforms HDAC1 and HDAC6. With HDAC1, F1

–

F4, F6, F26

–

F28 inhibited HDAC activity. In the case of HDAC6, F1

–

F13 and F27

–

F31 had inhibitory activities (Fig. 4). These differences allowed the identification of fractions selectively inhibiting one of the isoforms tested.

Figure 3. HDAC activity ofP. griseofulvumfractions with the MS-based assay using HeLa nuclear extract. The inhibitory activity of fractions was considered significant if the % inhibition was≥60% at 100μg/mL. Fractions were considered as HDAC activators when the % inhibition was≤ 100% at the same concentration.

Figure 4. HDAC activity ofP. griseofulvumfractions with the MS-based assay using either HDAC1 or HDAC6 human recombinant isoforms. For both isoforms, the inhibitory activity of fractions was considered significant if the % inhibition was≥70% at 100μg/mL. Fractions were considered as HDAC activators when the % inhibition was≤ 100% at the same concentration.

To isolate the compounds responsible for the observed HDAC inhibition, F2 (inhibiting HeLa nuclear extract, HDAC1 and HDAC6) and F26 (preferably inhibiting HDAC1) were selected for chemical investigation.

Compounds isolation from

P. griseofulvum

From F2, two compounds were isolated. The structure of the compounds was established by comparison of HRMS and NMR data with those of the literature (Alfaro et al., 2003; Bennett et al., 1991) and allowed the identification of patulin (1) and salirepol (2) (Fig. 5). Patulin had previously been reported in P. griseofulvum (Welke et al., 2011), while the occurrence of salirepol in this particular species is reported for the first time.

From F26, compound 3 at m/z 385.2123 [M + H]

⁺

(calcd for C

22

H

29

N

2

O

4

, 385.2122;

Δ

ppm = 0.2) was detected. A query in the Dictionary of Natural Products database limited to fungal metabo- lites putatively identified this protonated MS feature to the piperazine-2,5-dione derivative gliocladride A or B, previously iso- lated from a Gliocladium strain (Yao et al., 2009). After isolation of the compound, NMR data (Table 1) indicated that 3 shared the same geranyloxy side chain than glioclaride A or B as indicated by the three methyl groups at

δH

1.57 (3H, s, H-21), 1.63 (3H, s, H-20) and 1.70 (3H, s, H-22), the three methylene groups at

δH

2.03 (2H, m, H-16), 2.08 (2H, m, H-17) and 4.57 (2H, d, J = 6.5 Hz, H-13), and the two vinyl groups at

δH

5.07 (1H, t, J = 6.5 Hz, H-18) and 5.42 (1H, t, J = 6.5 Hz, H-14). The 1,4-disubstituted aromatic group was also observed at

δH

6.95 (2H, d, J = 8.5 Hz, H-10,10

′

) and 7.44 (2H, d, J = 8.5 Hz, H-11,11

′

). Nevertheless the vinyl singlet as well as signals from the 3-methyl-2,5-diketonpiperazine were observed at chemical shifts slightly different from those of gliocladride A or B: 6.64 (1H, s, H-8), 9.74 (1H, brs, OH-N1), 8.34 (1H, s, NH-4), 4.11 (1H, qd, J = 6.9, 1.4 Hz, H-2) and 1.33 (3H, d, J = 6.9 Hz, H-7). The HMBC correlations from these protons to the two carbonyls at

δC

160.5 (C-5) and 167.5 (C-2) and to the quater- nary carbon at

δC

125.1 (C-6) were similar to those observed for gliocladride A but the ROESY correlation from NH-4 to H-3 and CH

3–

7 indicated that, when compared to the known compound, 3 was hydroxylated in N-1 instead of N-4 (Fig. 5). The Z configura- tion of the double bond was established by comparison with the E isomer present with 3 in very small amount (0.1/1). For the Z iso- mer, a ROESY correlation was observed between the vinyl singlet H-8 at

δH

6.31 and OH-N1 at

δH

10.20. In order to establish the absolute configuration of 3, optical rotation and electronic circular dichroism spectra were measured ([

α

]

_D

= 2.2 (c 0.1, MeOH)) and indicated that 3 was probably a racemic mixture. Compound 3 was identified as a new natural product named gliocladride C.

Compound 1 did not inhibit any HDAC isoform when tested as a pure compound. Compound 2 showed a 14-fold selectivity for

HDAC6 (IC

50

= 3.4

μ

M) versus HDAC1 (IC

50

= 76.4

μ

M) (Table 2).

Remarkably, the active profile of 2 was coherent with results obtained for F2, which inhibited HDAC activity with HeLa nuclear extract, HDAC1 and HDAC6. From F26, the major compound (3) was isolated (Fig. 5) and characterised as a selective HDAC1 inhibitor (HDAC1 IC

₅₀

= 83.1

μ

M) (Table 2). These results were consistent with the HDAC activity measured for F26. Among the isolated compounds, only the non-active compound 1 (patulin) is known for its toxicity, being considered as a ubiquitous mycotoxin responsible for food contamination (Wright, 2015).

Compound 3 is described here for the first time; however gliocladride derivatives have been reported as cytotoxic towards cancer cell lines (Yao et al., 2009; Yao et al., 2007), an activity that is known to be related with HDAC class I inhibition.

To our knowledge, 2 and 3 have never been reported in this fun- gus and their HDAC inhibitory activity is described here for the first

Figure 5. Structures of the isolated compounds (1–3).

Table 1.

¹

H- (500 MHz) and

¹³

C- (125 MHz) NMR data of compound 3

Position

δH

Multiplicity, J (Hz)

δC

1 9.74 brs

2 167.5

3 4.11 qd, J = 6.9,1.4 Hz 50.1

4 8.34 s

5 160.5

6 125.1

7 1.33 d, J = 6.9 Hz 19.1

8 6.64 s 114.2

9 125.6

10, 10

’

7.44 d, J = 8.5 Hz 130.6

11, 11

’

6.95 d, J = 8.5 Hz 114.7

12 158.1

13 4.57 d, J = 6.5 Hz 64.3

14 5.42 t, J = 6.5 Hz 119.4

15 140.3

16 2.03 m 38.8

17 2.08 m 25.6

18 5.07 t, J = 6.5 Hz 123.6

19 130.9

20 1.63 s 25.3

21 1.57 s 17.4

22 1.71 s 16.2

23 9.74 s

Table 2. HDAC inhibition (IC

₅₀

in

μ

M) of isolated compounds in HeLa nuclear extract and towards various isoforms

Compounds IC

₅₀

(

μ

M)

HeLa nuclear extract HDAC1 HDAC6

1

>

300

>

300

>

300

2 64.9 ± 4.3 76.4 ± 3.3 3.4 ± 0.8

3

>

300 83.1 ± 3.9

>

300

Trichostatin A

^a

6.9 ± 0.9 13.6 ± 1.5 14.4 ± 2.3 Results are the means ± standard deviation of three independent experiments.

a

Positive control (IC

50

in nM).

Zwick V. et al.

Phytochem. Anal.2016 Copyright © 2016 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/pca

time. The inhibition of 2 was detected by using all the approaches during the fractionation. However, 3 could only be detected with the MS-based assay using specific HDAC isoforms, which led to the identification of fractions not detected with the HeLa nuclear extract. Considering these results, the MS-based assay using spe- cific HDAC isoforms appeared to be a more adequate approach for bio-guided fractionation by minimising potential interferences.

Acknowledgments

The authors would like to thank Miss Nicole Lecoultre for her very valuable technical assistance.

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Supporting information

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’

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