On the Histone Lysine Methyltransferase Activity of Fungal

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On the histone lysine methyltransferase activity of fungal metabolite chaetocin Fanny L. Cherblanc, Kathryn Chapman, Jim Reid, Aaron Borg, Sandeep Sundriyal, Laura Alcazar Fuoli, Elaine Bignell, Marina Demetriades, Christopher J. Schofield, Peter DiMaggio, Robert Brown, and Matthew J Fuchter J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401063r • Publication Date (Web): 07 Oct 2013 Downloaded from http://pubs.acs.org on October 21, 2013

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Journal of Medicinal Chemistry

On the histone lysine methyltransferase activity of fungal metabolite chaetocin. Fanny L. Cherblanc1, Kathryn L. Chapman2, Jim Reid3, Aaron J. Borg4, Sandeep Sundriyal1, Laura Alcazar-Fuoli5†, Elaine Bignell5, Marina Demetriades6, Christopher J. Schofield6, Peter A. DiMaggio Jr4, Robert Brown7, Matthew J. Fuchter1*

1

Department of Chemistry, Imperial College London, South Kensington Campus, London SW7

2AZ, UK 2

Mechanism and Functional Screening Facility, Department of Surgery and Cancer, Imperial

College London, Hammersmith Hospital Campus, London, W12 ONN, UK 3

Domainex Ltd, 162 Cambridge Science Park, Milton Road, Cambridge CB4 0GH, UK

4

Department of Chemical Engineering, Imperial College London, South Kensington Campus,

London SW7 2AZ, UK 5

Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road,

London SW7 2AZ, UK 6

Department of Chemistry and the Oxford Centre for Integrative Systems Biology, Chemistry

Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, UK OX1 3TA.

ACS Paragon Plus Environment

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Ovarian Cancer Action Research Centre, Department of Surgery and Cancer, Imperial College

London, Hammersmith Hospital Campus, London W12 ONN, UK

Abstract. Histone lysine methyltransferases (HKMTs) are an important class of targets for epigenetic therapy. 1 (Chaetocin), an epidithiodiketopiperazine (ETP) natural product, has been reported to be a specific inhibitor of the SU(VAR)3-9 class of HKMTs. We have studied the inhibition of the HKMT G9a by 1 and functionally important analogues. Our results reveal that only the structurally unique ETP core is required for inhibition, and such inhibition is timedependent and irreversible (in the absence of DTT), ultimately resulting in protein denaturation. Mass spectrometric data provide a molecular basis for this effect, demonstrating covalent adduct formation between 1 and the protein. This provides a potential rationale for the selectivity observed in the inhibition of a variety of HKMTs by 1 in vitro and has implications for the activity of ETPs against these important epigenetic targets.

Introduction The epipolythiodioxopiperazines (ETPs) are a broad class of fungal toxins, which contain a characteristic diketopiperazine scaffold bridged by two or more sulfur atoms (Figure 1).1, 2 There has been considerable interest in the biosynthesis of this functionally important ETP core 3-5 and its role in ETP derived toxicity.2, 6, 7 Sporidesmin, an ETP produced by Pithomyces chartarum on infected grasses has, for example, been implicated in the development of facial eczema and liver disease in sheep.8 A variety of molecular mechanisms have been proposed for ETP biological


2

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activity. These include protein cross-linking through reaction of the disulfide functionality with cysteine residues, the generation of reactive oxygen species via redox cycling, or the ejection of structurally important zinc ions from the protein.1,

2, 9, 10

All these mechanisms rely on the

established chemical reactivity of the ETP disulfide bridge.

Figure 1. Examples of ETP containing natural products and structure of compound 1a – 4.

ETP 1 (Chaetocin, Figure 1) is an ETP metabolite first isolated from fermentation of Chaetomium minutum.11 1 was reported to be the first specific inhibitor of histone lysine methyltransferase (HKMT) SU(VAR)3-912 Histone lysine methyltransferases are emerging as important and druggable targets through their central role in epigenetic gene regulation13-15 Indeed, since dynamic epigenetic modifications, such as histone methylation, can potentially be reversed using small-molecule inhibitors of enzymes involved in maintenance of a disease-


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associated epigenetic state, suitable epigenetic inhibitors are gaining increasing attention in epigenetic therapy.16-18 Originally identified in a compound library screen, 1 was reported to inhibit SU(VAR)3-9 from Drosophila melanogaster with an IC50 of 0.6 µM.12 Furthermore, it was shown that 1 could inhibit the human ortholog of dSU(VAR)3-9 (SUV39H1) with a similar IC50 value (0.8 µM), as well as other members of the SU(VAR)3-9 class of HKMTs including mouse G9a (IC50 = 2.5 µM) or Neurospora crassa DIM5 (IC50 = 3 µM). 1 was shown to be far less active against HKMTs not within the SU(VAR)3-9 class, such as Drosophila E(z)-complex, PRSET7 or SET7/9. Such apparently differential IC50 values for different HKMTs led to the suggestion that 1 is specific for the SU(VAR)3-9 class of enzymes. HKMT selectivity aside, we became particularly interested in the inhibitory mechanism of action of 1. Based on the fact that DTT had no effect on the inhibitory activity of 1 against SU(VAR)3-9,12 it has been hypothesized that the disulfide bridge of this ETP molecule is not required for HKMT activity. In principle, should this hypothesis prove correct, removal of the disulfide functionality from 1 (which endows this molecule with broad non-selective cytotoxicity), while retaining the broad skeletal framework should result in the discovery of a novel HKMT inhibitor chemotype with low off-target toxicity. Natural or synthetic ETP analogues whereby the disulfide functionality has been modified or removed however, have almost exclusively been shown to be inactive compared to their parental ETP counterparts.2, 19 We recently reported preliminary data on the activity of 1 and key analogues against the human ortholog of SU(VAR)3-9.20 Our data was supportive of non-competitive and non-specific inhibition of this enzyme by 1. Unfortunately, further mechanistic studies were hampered by the limited access we had to significant quantities of SUV39H1, coupled with the intrinsic low catalytic activity of this enzyme under our optimized assay conditions. We therefore selected an


4

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alternative HKMT, G9a, to use as a representative model system for further study. G9a is closely related to SUV39H1,21 and has also previously been reported to be inhibited by 122 and other ETP natural products.23, 24 Furthermore, we had previously used this protein construct in robust screening assays to identify novel G9a inhibitors. Here we report our full data on the inhibitory activity of 1, and a series of mechanistically important analogues, against G9a. In agreement with our preliminary data on SUV39H1,20 the disulfide bridge of the ETP unit is central to G9a inhibition by 1, resulting in time dependent inhibition in vitro. Furthermore, we found that the mechanism of inhibition is dependent on the precise assay conditions employed. As such, we believe these results give further insight into the molecular mode of action of this class of natural products against these important biological targets.

Results ETP 1 was isolated from the culture of Chaetomium virescens var. thielavioideum and two analogues lacking the disulfide bridge (Compounds 2 and 3, Figure 1) were prepared via semisynthesis on the isolated material (see supplemental experimental procedures).19, 25 Compound 2 was designed to retain the bicyclic structure of the ETP cores while lacking the reactive disulfide bonds. Compound 3 is an analogue of the reduced dithiol 1a, where the reactive thiol moieties have been converted into unreactive thioethers. A structurally simple ETP model compound (4, Figure 1) was also prepared following literature procedures as a useful control to measure the activity of a structurally simple molecule bearing an ETP core.9 Compounds 1 – 4 were assayed against G9a in an AlphaLISA assay.26 The inhibition curves are presented in Figure 2. pIC50s


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were derived for compounds 1, 2 and 4 (Table 1), since no inhibition was observed for the methylthioether analogue 3. In the case of the monosulfide analogue 2, the curves are not sigmoidal (low Hill slope value) and even at a concentration of 25 µM, 36 % enzyme activity remains (Table 1). The IC50 derived from such a curve should be interpreted with caution.

Figure 2. Concentration-inhibition curves for 1 and analogues. Enzymatic activity was monitored over a concentration range of 0.02 µM up to 100 µM of compound (representative data set from one experiment, in duplicates), in absence of DTT, with 30 min preincubation time. See Table 1 for pIC50 values.

Table 1. pIC50 values and characteristics of inhibition of compound 1, 2 and 4.


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1

2

4

pIC50 ± standard error (n=5)

5.59 ± 0.18

4.97 ± 0.29

5.31 ± 0.10

IC50 (µM)

2.6

10.6

4.9

Hill Slope ± standard error

-1.20 ± 0.17

-0.60 ± 0.09

-0.85 ± 0.12

% Activity at 25 µM

8

36

19

n represents the number of independent experiments (run as duplicates). See Figure 2 for curves.

As we found for SUV39H1,20 our results confirm that the disulfide bridge of ETP compounds is essential for their HKMT inhibitory activity.1,

12, 22

The fact that structurally simple ETP

compound 4 is able to significantly inhibit G9a activity, supports the critical role of the ETP core for inhibition, and suggests much of the rest of the complex structure of 1 to be superfluous. It should be noted that epimonothiodioxopiperazines compounds such as analogue 2 are known to be relatively unstable due to their highly strained sulfur containing ring.27 We therefore suspect that the observed weak inhibition and low Hill slope value for this compound is linked to its chemical reactivity. It had been previously reported that the HKMT activity of 1 was not dependent on the presence of increasing concentrations of DTT.12 This result was used to infer an inhibitory mechanism of action. In light of this, we felt it was important to conduct our subsequent mechanistic assays with and without the chemical reductant DTT in the assay. The time-dependency of G9a inhibition by 1 was therefore assessed both in the presence and absence of DTT. Inhibition was


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measured after a variety of preincubation timepoints (0, 5, 15, 30 min), where the enzyme and inhibitor had been premixed (Figure 3 and supplemental Table S1). Inhibition by SAH, an endogenous competitive HKMT inhibitor,14 and the ETP model compound 4 was also measured at 0 min and 30 min preincubation, with or without DTT.

Figure 3. Influence of the preincubation of 1 on G9a activity. Collated data from representative experiments, activity normalized to DMSO control: a) and b) the effect on the potency of 1 with or without 30 minutes preincubation time before measuring G9a activity inclusive and exclusive of DTT (1 mM in the buffer); c) the effect of preincubation of SAH with G9a, also at 0 or 30 min preincubation, as a negative control for time dependency; d) the effect of preincubation of compound 4 with G9a, also at 0 or 30 min preincubation. See Supplemental Table S1.


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The potency of 1 significantly (P < 0.05; assessed by 1 way ANOVA) increased at longer preincubation with G9a, showing only very weak inhibition when the substrate was added to the enzyme prior to the inhibitor (i.e. at 0 min preincubation). The statistically significant difference in pIC50 for 1 between 0 and 30 min of preincubation, was apparent regardless of whether DTT was present or not (supplemental Table S1). The ETP model compound 4 showed analogous behavior with little or no inhibition detected without preincubation, with or without DTT. Since the activity of 1 was dependent on the chemically reactive disulfide ETP bridge and exhibited time dependence, we decided to check whether such inhibitory effects were reversible. A “dilution test”28 was used to assess the reversibility of G9a inhibition by 1. Briefly, G9a, at 100-fold over the standard assay concentration, was incubated for 30 minutes with the inhibitor, at a concentration of 10-fold the IC50. The mixture was then diluted 100-fold into the reaction buffer containing the substrates. At this final dilution, the concentration of the enzyme is at the standard assay concentration, and the concentration of the inhibitor is 10 times lower than the IC50. For reversible inhibition, the enzyme should recover its activity (Figure 4).


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Figure 4. Reversibility test of 1 with and without DTT. Enzymatic activity was normalised to DMSO controls. Experiments were run as duplicates. Data shown are mean values of n=3 independent experiments. 1 (a), SAH (b) and compound 4 (c) were incubated at a concentration of 10 times their respective IC50 with G9a for 30 min then diluted 100-fold with the substrates. Remaining inhibition after dilution is suggestive of irreversibility. Controls experiments were run with the compound at 0.1 times the respective IC50 without dilution to illustrate that no significant inhibition is expected at this concentration.


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In the presence of the reducing agent DTT, the inhibition is clearly reversible, with full G9a activity fully recovered, however in the absence of such a reducing agent, inhibition is maintained, which is suggestive of irreversible inhibition under these conditions. ETP model compound 4 displays comparable behavior with the reversibility of its inhibition dependent on the presence of DTT. Conversely, SAH inhibition is reversible under all conditions examined. This indicates clearly that the reversibility of G9a inhibition for ETP-containing compounds is different depending on the conditions employed in the assay. To further define the mechanism of action of 1, SAM competition experiments were conducted. pIC50s were measured at different concentrations of SAM (concentration range 10 – 160 µM) and plotted against [SAM]/Km (Figure 5). The experiments were run in presence of 1 mM DTT in the buffer to ensure the reversibility of the inhibition and a constant preincubation (enzyme and inhibitor) time of 30 min was applied prior to starting the reaction by addition of the substrates.

Figure 5. 1-SAM competition studies. pIC50s were determined as stated above (6 point curve) at various concentration of SAM, in presence of 1 mM DTT, with a 30 min preincubation time and plotted against [SAM]/Km. The error bars represents 95% confidence interval. pIC50s were derived from collated data of 3 replicates.


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pIC50s were not dependent on SAM concentration, which indicates that 1 is not competitive with SAM. The pIC50s obtained for the different [SAM] were not found to be significantly different. Comparably, G9a inhibition by the ETP model compound 4 was assessed at high (160 µM) and low (10 µM) SAM concentrations and no significant difference could be observed (data not shown), with pIC50 (±standard deviation) values of 5.292 (±0.2312) and 5.363 (±0.2836) respectively). Initial denaturing MS studies were carried out following incubation of 1 with G9a (data not shown). In agreement with the reversibility studies, irreversible adduct formation was observable in the absence of DTT. No observable adduct was apparent in the presence of DTT, also in agreement with our reversibility studies (see Figure 4). These results suggested the presence of a mixed disulfide linkage between 1 and G9a, which is reversibly cleaved by DTT. Further MS studies were therefore carried out to map the cysteine residues engaged in disulfide bonding with 1 (Figure 6 and supplemental Table S3). Following incubation of 1 with G9a, the protein was denatured and N-ethylmaleimide (NEM) was added to covalently label cysteines not engaged in disulfide bonding with 1. The samples were then reduced with DTT and subsequently treated with iodoacetamide to label the free cysteine residues released from disulfide linkages. The samples were digested and analysed by MS relative to untreated controls.


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Figure 6. G9a (913-1193) sequence highlighting cysteines involved in binding with 1. All 20 cysteine residues are highlighted in boldface, and those cysteines involved in the formation of disulfide bonds with 1 are scaled according to their fold increase in carbamidomethyl labeling upon treatment with 1 (the numbers above these residues represent the fold change observed relative to control). Singly and doubly underlined sequences indicate identified and quantitated peptides from the LC-MS/MS data, respectively. Shaded sequences highlight the SET domain, which is flanked on the left by the pre-SET domain and on the right by the post-SET domain.


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The arrows and twists represent β sheet and α helical secondary structural elements, respectively, as identified by RSCB PDB accession number 2O8J. See Supplemental Table S3.

Of the 280 amino acid residues contained within G9A (913-1193), 249 were identified from the LC-MS/MS data resulting in 89% sequence coverage (see underlined residues in Figure 6). G9A (913-1193) contains a total of 20 cysteines distributed on 12 peptides, and the fold change in labeling of 12 cysteines in response to treatment with 1, relative to untreated control, were quantitated on 9 of these peptides (see doubly underlined residues in Figure 6). The 3 remaining cysteine-containing peptides could not be accurately quantitated, as two peptides were too large and thus exhibited poor ionization efficiencies (i.e. a 23 amino acid peptide containing cysteines 937 and 946, and a 27 amino acid peptide containing cysteines 974, 976, 980, 985 and 987), and one peptide containing cysteine 1027 was only 3 amino acids in length and could not be fragmented for identification. Although these 3 peptides could not be accurately quantitated using chromatographic peak areas, manual inspection of the available MS/MS data did not reveal significant evidence of carbamidomethyl labeling (i.e. adduct formation with 1). Out of the 12 quantitated cysteines, 5 exhibited a greater than 2 fold change in carbamidomethyl labeling upon treatment with 1 (see Figure 6). These corresponded to Cys994 (4.8 fold increase), Cys1017, 1021 and 1023 (3.1 fold increase) and Cys1115 (2.1 fold increase).

Since ETPs such as 1, have been reported to generate reactive oxygen species (ROS),29, 30 a hydrogen peroxide generation assay was therefore run in order to assess the presence of ROS under our in vitro assay conditions.31 The hydrogen peroxide generation assay relies on the


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ability of Horse Radish Peroxidase (HRP) to catalyze the oxidation of phenol red by hydrogen peroxide. No generation of H2O2 was detected at 10 or 100 µM of 1 (see supplemental Figure S1), which suggests that the in vitro inhibitory activity of 1 towards HKMTs is not ROS dependent. A common mechanism of nonspecific inhibition is protein denaturation.32 ETP natural products have previously been reported to alter the folding of proteins by NMR spectroscopy and circular dichroism.33, 34 A denaturation test in presence of urea was therefore performed. It relies on the assumption that if a compound acts as denaturant, its potency should increase in the presence of another denaturant, in this case, urea.32 Urea was first titrated to determine the optimal concentration at which G9a still displays enzymatic activity (see supplemental Figure S2) and a concentration of 0.7 M was chosen for the denaturation assay. The presence 0.7 M of urea in the assay led to a statistically significant increase (**) in potency for 1, which was not observed for SAH (see supplemental Figure S3, and supplemental Table S2). This suggests that 1 inhibits G9a at least in part by nonspecific denaturation.

Discussion ETP 1 is part of a large family of natural product toxins, which all contain the reactive epipolythiodioxopiperazine (ETP) “warhead”. This ETP functional unit is central to the biological activity of this class of natural products. In accordance with this fact, we have found that the disulfide bond of the ETP unit(s) is critical for the HKMT inhibitory activity of 1, resulting in time dependent and non-specific inhibition, through chemical modification of G9a in vitro. Indeed, a structurally simple ETP molecule (i.e. 4) had significant inhibitory activity


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against G9a (and comparable mechanism of action), while the unreactive bisthioether derivative of 1 (i.e. 3) displayed no activity whatsoever. While contrary to the original report of the HKMT activity of this natural product,12 such a result is in agreement with our initial data on the activity of 1 against an alternative HKMT, SUV39H1.20 Furthermore, Sodeoka and co-workers have previously reported the first total synthesis of 1, both as the natural and unnatural enantiomeric forms, as well as the preparation of sulfur-free analogues.22 While their synthetic sample of 1 had comparable inhibitory activity against G9a to that disclosed herein (IC50 = 2.4 µM), the sulfurfree analogue was inactive, thus corroborating our results on the requirement of the disulfide bridges for G9a inhibition. In light of the dependence of inhibitory activity on the reactive ETP disulfide, we have attempted to investigate a plausible molecular mechanism(s) that underlies the inhibitory effect of this class of molecules. Since the generation of reactive oxygen species under the assay conditions was not observed, it is unlikely that 1 causes general assay interference or protein degradation in vitro through oxidative stress.2 Instead, our data supports a direct (inhibitory) interaction between 1 and G9a. Our mass spectrometric data demonstrates 1-G9a adduct formation through non-selective disulfide linkages. This reactivity is in accordance with the known chemical reactivity of ETPs.10, 35 The reversibility of inhibition observed in the presence of DTT is also consistent with disulfide bond formation. Importantly, as shown by the MS data, more than one cysteine residues are engaged in disulfide bond formation upon 1 treatment. It is therefore apparent that 1 does not exhibit specificity towards a single cysteine residue. It should be noted that all of the cysteines observed in disulfide bond formation with 1 are located in unstructured regions of the SET domain, as determined by available structural data for G9a.36 Adduct formation likely causes protein denaturation over time, as supported by the increased


16

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inhibitory activity of 1 in the presence of urea. Indeed, protein denaturation by ETPs has been observed previously by a number of techniques including circular dichroism and NMR studies33, 34

Previously, it was suggested that 1 binds to the co-factor (SAM) binding cleft.12 Our SAM competition studies refute such competitive binding. In addition, the fact that we observe significant and mechanistically analogous inhibitory activity against G9a for a structurally simple ETP molecule (i.e. 4), lacking the complex skeletal framework of 1, also argues against a well-defined binding pocket on the protein. In concordance with this, the study by Sodeoka and co-workers found both the natural and unnatural enantiomeric forms of 1 to be equally potent against G9a.22 A well-defined protein-binding site should result in significant different activities for enantiomeric inhibitors, which was clearly not observed. Furthermore, other structurally diverse ETP compounds (including gliotoxin, chetomin and 11,11’-dideoxyverticillin A) and synthetic ETPs have been reported to inhibit G9a.23, 24, 37 Therefore instead of a protein binding site conferring HKMT selectivity to 1, we believe any apparent in vitro selectivity against a range HKMTs (dSU(VAR)3-9, SUV39H1, G9a, DIM5)12 would likely be related to a given protein’s sensitivity to the ETP reactive disulfide ‘warhead’. Our MS data (Figure 6) revealed cysteines 994, 1017, 1021 and 1023 to exhibit the largest reactivity towards 1 and these residues are all located in the C-terminal region of the pre-SET domain of G9a. Such pre-SET cysteines are implicated in the binding of zinc atoms, which is thought to have a structural function in linking random coils of the protein and stabilising the SET domain.38 Interestingly, we observed minor adduct formation with Cys1115, which is part of the highly conserved NHS/CxxPN region in the pseudo-knot motif of the SET domain. This motif is involved in hydrogen bonding interactions with the adenine ring of S-adenosyl methionine (SAM) and it is plausible adduct


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formation with this cysteine may have some direct inhibitory role.38 We did not observe adduct formation at any of the other SET domain cysteines however. In light of this data, the sensitivity of a given HKMT to this natural product is likely related to the presence of a structurally important cysteine rich pre-SET domain and possibly also the postSET domain (although we observe no binding of 1 to the post-SET domain in G9a), through mixed disulfide formation with ETPs.39 For example, the three conserved cysteine residues in the post-SET domain within DIM5 have been shown to be essential for HKMTase activity.40 While G9a and SU(VAR)3-9 contain such pre-SET and post-SET domains,39 the HKMTs SET7/9 and PR-SET7 do not. It is perhaps telling therefore that the former two HKMTs are inhibited by 1, whereas the latter two are not. Indeed, gliotoxin and other ETP natural products have been reported to have a similar effect, inhibiting G9a and SU(VAR)3-9 but not SET7/9.23, 24 While the results contained herein focus on cell-free biochemical assays, it should be noted that 1 has been reported to effect histone methylation levels in cells.12, 41-43 Although one plausible explanation for this result is a direct inhibitory effect on HKMTs, an alternative possibility is that the observations are related to the high toxicity of the ETP class of compounds,2 coupled with their extensive off-target effects. Other than HKMTs, ETPs including 1 have been reported to target numerous structurally, functionally, and evolutionary unrelated enzymes,1, kinase6,

2

including thioredoxin reductase,44 p300:HIF-1α9 and inactivate muscle creatine

10, 23

to name but a few. Rather than a direct effect on HKMTs in cells, the complex

effect of compound treatment on histone methylation may instead be an indirect readout of ongoing cell death. Indeed, the release of histones from nucleosomes during apoptosis is well established,45 and a wide variety of changes in specific histone modifications accompany the apoptotic process.46 Regardless, it is clear that the large number of potential cellular targets for


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ETPs, their high cellular toxicity (at least at reportedly effective doses), and the fact they induce oxidative stress in cells, means it will be highly difficult to robustly characterize the origin of the effect such compounds have on the epigenetic landscape within a cellular environment.

Conclusion On the basis of our data, we conclude that 1 and other ETP molecules exhibit time dependent inhibition of HKMTs in vitro through protein-ETP adduct formation. In the absence of DTT, such effects are irreversible and ultimately result in denaturation of the protein. The presence of DTT appears to render adduct formation reversible, in accordance with disulfide bond formation being central, but this effect is not sufficient to rescue G9a from inhibition by 1. While these results highlight that 1 (and other ETPs) are not fit for purpose as selective molecule probes of HKMTs,47 they do raise interesting questions surrounding HKMT stability and structure. In particular, the clear reactivity of G9a (and SUV39H1) towards a thiol reactive (ETP) reagent, and the effect this has on its function, suggests that selectively targeting structurally and/or functionally important cysteine residues within HKMTs may be an alternative inhibition strategy to explore. Furthermore, the fact these proteins form adducts with thiol reactive reagents highlights an opportunity to employ activity-based protein profiling strategies48,

49

to further

delineate the function of these important epigenetic enzymes.

Experimental Procedures


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Materials – G9a Human recombinant, N-terminal GST tag used for all Alphascreen experiments was purchased from BPS Bioscience; Histone H3 (1-21) Biotinylated Peptides (H3K9Mex x=0, 2, 3) were purchased from AnaSpec; Anti-methyl-Histone H3 Lysine 9 (H3K9me2) AlphaLISA Acceptor Beads, Alpha Streptavidin Donor beads and AlphaLISA Epigenetics Buffer were purchased from PerkinElmer; S-(5'-Adenosyl)-L-methionine chloride (SAM) was purchased from Sigma and New England Biolabs (UK) Ltd, S-(5'-Adenosyl)-Lhomocysteine (SAH), H2O2 solution, NaCl solution, Tris buffer, Tween20 were purchased from Sigma. White low-volume 384-well plates (Greiner Bio-One, Gloucester, UK) were read on Pherastar Plus with AlphaScreen module.

Data Analysis – Data was normalized to high (no inhibitor) and low (no enzyme) controls and plotted using GraphPad Prism V5.0. pIC50 (-log(IC50)) values were derived from 10 point curve using a four parameter fit. Assays were run in duplicates in at least 3 independent experiments. The statistical significance was assessed by paired t test or 1 way ANOVA followed by Bonferroni's multiple comparison test. P value summary: ***: P < 0.001 (Extremely significant), **: 0.001 < P < 0.01 (Very significant), *: 0.01 < P < 0.05 (Significant), ns: P > 0.05 (Not statistically significant)

Compound handling – Compounds were stored as 10 mM DMSO solutions under N2 atmosphere or at – 20 ˚C. Purity (>95% pure) was assessed by HPLC-MS. Isolation, synthesis and characterisation of compound 1 – 4


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Isolation of 1

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– Chaetomium virescens var. thielavioideum (CBS 623.80) was cultured at

room temperature (~22˚C) in complete media (2.5% glucose, 0.5% yeast extract), according to Pontecorvo et al.,50 containing 1% vitamin solution, 1% glucose as carbon source and 5 mM ammonium tartrate as nitrogen source. Conidia stocks were prepared from agar surfaces of 14day-old cultures and preserved in sterile distilled water at 4˚C. A conidial suspension was then inoculated in ten 1l bottles slants with solid complete media and incubated for three weeks at room temperature. The cultures were then extracted with CH2Cl2 (2 × 500 ml per bottle) for 24 h and 48 h with orbital shaking. The resulting mixtures were then filtered, dried over MgSO4, and concentrated in vacuo. The extracts were then purified by column chromatography (CH2Cl2 : MeOH, 98 : 2). The resulting solid was further purified by trituration in hexanes with sonication and HPLC to afford 210 mg of 1. [α]D20 = + 542 (c = 0.82 in CHCl3); 1H NMR (400 MHz, CDCl3)  = 7.42 (d, J = 7.5 Hz, 2H), 7.25 (t, J = 7.5 Hz, 2H), 6.92 (t, J = 7.5 Hz, 2H), 6.74 (d, J = 7.5 Hz, 2H), 5.25 (s, 2H), 5.24 (s, 2H), 4.25 (dd, J = 12.5, 6.0 Hz, 2H), 4.17 (dd, J = 12.5, 9.4 Hz, 2H), 3.83 (d, J = 15.0 Hz, 2H), 3.28 (dd, J = 9.4, 6.0 Hz, 2H), 3.08 (s, 6H), 2.74 (d, J = 15.0 Hz, 2H);

13

C (100 MHz, CDCl3)  = 165.6, 162.8, 149.1, 130.4, 127.4, 125.1, 120.4, 110.7,

80.5, 75.7, 73.3, 60.6, 59.8, 39.2, 27.3; IR (neat) 3383, 3336, 1670, 1066, 749 cm-1; CD (MeOH): λmax (mdeg) = 237 (+434), 263 (0), 272 (42), 284 (0), 304 (+78), 350 (+1) nm; LCMS: Rt = 5.75 min; MS (ESI) m/z 697 (M+H)+; HRMS (ESI) m/z calcd (%) for C30H29N6O6S4: 697.1031, found: 697.1038 Synthesis of 2

19, 25

– PPh3 (28 mg, 0.11 mmol) was added to a solution of 1 (20 mg, 0.029

mmol) in CH2Cl2 (5 ml) and the resulting mixture was stirred for 2 h at room temperature. The solvent was removed in vacuo and the resulting pink solid was purified by column chromatography (CH2Cl2 : EtOAc, 60 : 40). 2 was obtained as a white solid (17 mg, 93%). m.p.


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210 - 212˚C (dec.); [α]D20 = + 484 (c = 0.0039 in CHCl3); 1H NMR (400 MHz, CDCl3)  = 7.47 (d, J = 7.8 Hz, 2H), 7.22 (td, J = 7.8, 0.7 Hz, 2H), 6.91 (td, J = 7.8, 0.7 Hz, 2H), 6.74 (d, J = 7.8 Hz, 2H), 5.46 (s, 2H), 4.89 (s, 2H), 4.13 (m, 4H), 3.63 (d, J = 14.9 Hz, 2H), 3.00 (s, 6H), 2.85 (m, 2H), 2.49 (d, J = 14.9 Hz, 2H);

13

C (100 MHz, CDCl3) 8 = 174.9, 173.5, 149.2, 130.2,

128.1, 124.9, 120.3, 110.6, 80.7, 79.4, 78.5, 63.6, 59.1, 31.6, 28.3 (dimeric structure); IR (neat) 3388, 2921, 1712, 1468, 1322, 753 cm-1; UV/Vis (CHCl3): λmax (A): 243.4 (0.49), 298 (0.21) nm, CD (MeOH): λmax (mdeg) = 217 (0), 223.5 (43.7), 230 (0), 248.5 (+184.9), 285 (+59.5), 304 (+81.6), 350 (+0.8) nm; LCMS: Rt = 4.77 min; MS (ESI) m/z 633 (M+H)+; HRMS (ESI) m/z calcd for C30H29N6O6S2: 633.1590 found: 633.1598. Synthesis of 3

19

– NaBH4 (16 mg, 0.42 mmol) was added to a solution of 1 (20 mg, 0.028

mmol) in CH2Cl2 / MeOH (6 ml / 2 ml) at 0 ˚C. After 30 min, MeI (1 ml, excess) was added dropwise and the solution was stirred for 16 h and then treated with 1N HCl (5 ml). The aqueous layer was extracted with CH2Cl2 (2 × 5 ml). The combined organic layer was dried (MgSO4), rotary evaporated and purified by column chromatography (CH2Cl2 : MeOH, 99 : 1) to afford the methylated compound as a white solid (10 mg, 47 %). 1H NMR (400 MHz, CDCl3) δ = 7.48 (d, J = 7.6 Hz, 2H), 7.20 (t, J = 7.6 Hz, 2H), 6.85 (t, J = 7.6 Hz, 2H), 6.63 (d, J = 7.6 Hz, 2H), 5.73 (s, 2H), 4.90 (br. s, 2H), 4.65 (br. s, 2H), 4.29 (d, J = 10.7 Hz, 2H), 3.78 (d, J = 10.7 Hz, 2H), 3.09 (s, 6H), 2.64 (d, J = 15.2 Hz, 2H), 2.44 (d, J = 15.2 Hz, 2H), 2.19 (s, 6H), 1.87 (s, 6H); 13C (100 MHz, CDCl3) δ = 166.5, 165.4, 150.9, 129.7, 128.7, 125.1, 119.1, 110.1, 80.5, 77.2, 69.2, 64.6, 60.6, 43.7, 28.6, 15.8, 12.8 (dimeric structure); [α]D20 = + 175 (c 0.002, CHCl3, 25 ˚C); UV λmax (CHCl3) (A): 238.6 (1.07), 303.3 (0.44) nm, CD (MeOH) ∆ε20 (nm): +180 (258), +53 (280), +126 (306), 0 (332); LCMS: Rt = 5.58 min, MS (ESI) m/z 779 (M+Na)+; HRMS (ESI) m/z calc. for C34H40N6O6S4Na 779.1790, found: 779.1777.


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Synthesis of (4) – The ETP model compound was prepared following literature procedure.9. 1H NMR (400 MHz, CDCl3) δ = 7.41-7.28 (m, 10H), 5.24 (s, 2H), 4.86 (d, J = 14.8 Hz, 2H), 4.50 (d, J = 14.8 Hz, 2H); 13C (100 MHz, CDCl3) δ = 163.8, 134.1, 129.2, 128.7, 128.5, 64.7, 47.7; MS (CI ammonia) m/z 357 (M+H)+, 374 (M+NH4)+; HRMS (CI ammonia) m/z calculated for C18H20N3O2S2 374.0997, found 374.0996.

AlphaLISA inhibition assay – 1 nM G9a was incubated with compounds (up to 100 µM, 1% DMSO), 100 nM biotinylated H3 peptide and 15 µM SAM in assay buffer (50 mM Tris-HCl, pH 9.0, 50 mM NaCl, 1 mM DTT, 0.01% Tween-20) with a total volume of 5 µl for 30 min at 25 ˚C in white low-volume 384-well plates (Greiner Bio-One, Gloucester, UK). The reaction was quenched by addition of 10 µg/ml Anti-methyl-Histone H3 Lysine 9 (H3K9me2) AlphaLISA® Acceptor Beads in epigenetic buffer and incubated for 1 hour. 10 µg/ml AlphaScreen® Streptavidin Donor beads in epigenetic buffer were added and incubated for 30 min. The plates were then read on Pherastar Plus (Alphascreen module).

ROS generation test – The ROS generation test was performed as decribed.31 In brief, compounds (100 and 10 µM) in Hanks’ balanced salt solution (HBSS) were incubated with 0.5 mM DTT for 15 mins in 384-well flat bottomed, clear polystyrene microtiter plates (Greiner, Bio-One, Gloucester, UK). 100 µM H2O2 in HBSS was used as control (100%). Phenol red/HRP detection buffer (100 µg/ml phenol red and 60 µg/ml HRP in HBSS) was added and incubated


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for 5 minutes. The reaction was stopped with addition of 1 M NaOH. Absorbance was then read at 610 nm on a BMG NOVOstar and values were normalized to 100 µM H2O2 value.

Time-dependency test – Enzyme and inhibitor in assay buffer were preincubated for 5, 15, or 30 minutes prior to addition of the substrate in assay buffer. For 0 minute preincubation data, the substrate was added just before adding the inhibitor. The assay was otherwise performed as stated above.

Reversibility - dilution test – G9a (100 nM) was incubated with compound (1 and SAH) at a concentration of 10 × IC50 (total volume 2 µl) for 30 min in assay buffer (50 mM Tris-HCl, pH 9.0, 50 mM NaCl, 0.01% Tween-20) with or without 1 mM DTT at room temperature in V-shape 96-well plates. The enzyme was also incubated with DMSO as a positive control. After preincubation, the samples were diluted 100-fold with peptide H3 and SAM in assay buffer (with and without DTT) to decrease all reaction components to their usual assay concentrations (as described previously) and the inhibitor concentration to 0.1 × IC50. After 30 min incubation, 5 µl of each reaction mixture was transferred to a white low-volume 384-well plate and acceptor and donor beads were added as described in the inhibition assay. The percentage activity was determined relative to DMSO control.28

SAM competition assay – IC50s were determined as stated above at various concentrations of SAM (concentration range 10 – 160 µM) and with G9a at a concentration of 0.5 nM. The assay


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was run in presence of 1 mM DTT to ensure the reversibility of the inhibition by 1. Enzyme and inhibitor were incubated for 30 min prior to addition of the substrates and the reaction were run for 30 min. Linearity was checked at the highest SAM concentration. In this assay, pIC50 (log(IC50)) values were derived from a 6 point curve.

Inhibition assay in presence urea – A titration with increasing amount urea was performed to determine a concentration at which the overall enzyme activity was not too affected, typically about 70–80 % remaining activity (concentration range: 0.29 – 5 M for urea, data not shown). The assays were then run as described above, with 0.7 M urea in assay buffer and 30 minutes preincubation of enzyme and compound prior to addition of the substrates.

Expression and purification of G9a for mass spectrometry experiments – Cloning and baculoviral expression. An N-terminally His-tagged DNA fragment, encoding a.a. 913-1193 of G9a, was subcloned into pFastBac for baculoviral expression using the Bac-to Bac system (Invitrogen). Recombinant G9a was expressed in Sf9 cells grown to mid-log phase in InsectXpress media supplemented with 5% FCS (Lonza). The cells were infected with an MOI of 2.5 and grown for further 72 h at 27 oC. Protein purification – Harvested cells were resuspended in lysis buffer (50 mM HEPES pH 7.4, 300 mM NaCl, 5 mM MgCl2, 10% Glycerol, 1 mM TCEP supplemented with EDTA-free protease inhibitors). The cells were lysed by sonication and the clarified lysate bound to Ni-NTA resin (Qiagen). The resin was washed with lysis buffer supplemented with 50 mM imidazole and eluted in 50 mM HEPES pH 7.4, 300 mM NaCl, 5 mM MgCl2, 10% Glycerol, 1 mM TCEP, 250 mM imidazole. The protein was further purified to


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homogeneity by gel filtration using a Superdex S75 26/60 column (GE Healthcare) in 50 mM HEPES pH 7.4, 300 mM NaCl, 5 mM MgCl2, 10% Glycerol, 1 mM TCEP. Fractions corresponding to dimeric G9a were pooled and concentrated.

MS analysis – Recombinant G9a was dialyzed into 100 mM NaCl, 1 mM tris(2carboxyethyl)phosphine (TCEP) (Sigma-Aldrich) and 50 mM Hepes pH 7.3 at 4 oC. 1 was added to half of the G9a sample in a molar ratio of 2:1 and allowed to react at 25 oC for 30 mins. The second half of the G9a sample was used as control experiment to which no 1 was added. To both the control and the treated samples, guanidine HCl (Promega) was added to a final concentration of 6 M to denature the G9a protein, and N-ethylmaleimide (NEM) (SigmaAldrich) was added to a final concentration of 14 mM and allowed to react for 30 mins at room temperature to covalently label cysteines not engaged in disulfide bonding. The samples were then reduced with a final concentration of 5 mM DTT for 1 hour at 51 oC, and iodoacetamide (Sigma-Aldrich) was added at a final concentration of 14 mM and allowed to react in the dark for 45 mins to label the reduced cysteines. Proteomics grade trypsin (Promega) was added at a 1:20 ratio to digest the G9a protein for 6 hours at 37 oC. After quenching digestion, ZipTips were used to reconstitute both samples into the correct loading solvent for nanoLC-MS. The peptides were chromatographically resolved using a linear gradient on an Ultimate 3000 RSLCnano System (Dionex), with an Acclaim PepMap100, C18 stationary phase, 3 µm particle size, 100 Å pore size, 75 µm internal diameter × 15 cm length column (Thermo Fisher). The LC conditions comprised of a flow rate of 0.3 µL/min and a linear gradient starting at 1% B (5% H2O, 95% MeCN, 0.1% Formic Acid and 99% A (0.1% FA, 100% H2O) and increased to 95%B


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over 70 mins. Real time tandem mass spectra were acquired on an LTQ Velos Pro linear ion trap (Thermo Scientific) with an 80 minute acquisition time over a 240-1800 m/z scan range and CID fragmentation collision energy of 35%. Tandem mass spectra were collected using top 5 datadependent acquisition, with a dynamic exclusion list (repeat count of 2, repeat duration of 10 secs, exclusion list size 100 and exclusion duration of 100 secs) to provide sufficient MS/MS peptide coverage. Initial peptide identification from the LC-MS/MS data was performed using a Sequest search in Proteome discoverer 1.3 (Thermo Fisher) against the Uniprot database with G9a appended. Reverse decoy false discovery rate values of 0.01/0.05 were used (strict/relaxed), allowing for 2 missed cleavages and a 2 Da precursor mass tolerance. Dynamic side chain modifications included in the search were carbamidomethyl (+57.021; from iodoacetamide treament), NEM (+125.048) and NEM in oxidized form (143.058) on cysteine residues, and oxidation (+15.995) on methionine residues. We manually assessed the accuracy of the tandem MS identifications reported by Sequest, and the 2 most abundant charge states observed for each cysteinecontaining peptide were then quantitated by peak integration of the precursor ion intensity in the extracted ion chromatogram. This was done for all possible combinations of modifications on the cysteine and methionine residues, and the raw abundances were normalized across all observed modified states of the same peptide sequence to determine the relative percentage of NEM (indicating an exposed cysteine) and carbamidomethyl (indicating a cysteine involved in disulfide bond formation) labeling for each cysteine residue. The fold change in carbamidomethyl-to-NEM labeling (i.e. disulfide bond formation) upon treatment with 1 was evaluated by taking ratio between the abundances of the treated and control samples, and the results summarised in Figure 6. See additional details in supplemental Table S3.


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ASSOCIATED CONTENT Supporting Information. Additional biochemical and MS data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Matthew J. Fuchter, Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, UK, Tel: (+44) 207-594-5815, Fax: (+44) 207-594-5805, E-mail: [email protected]

Present Addresses †Mycology Reference laboratory, National Centre for Microbiology, Instituto de Salud Carlos III. Crta. Majadahonda-Pozuelo, Km2. Majadahonda 28220. Madrid, Spain

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest


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ACKNOWLEDGMENT We would like to acknowledge Cancer Research UK (grant C21484A6944 and C536/A13086) for funding and the European Union for a Marie Curie International Incoming Fellowship (to SS, PIIF-GA-2011-299857). ABBREVIATIONS HKMT, histone lysine methyltransferase; H3, histone 3; H3K9, lysine 9 on histone 3; DTT, dithiothreitol; ETP, epipolythiodioxopiperazine; SAM, S-(5'-Adenosyl)-L-methionine chloride; SAH, S-(5'-Adenosyl)-L-homocysteine.


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On the Histone Lysine Methyltransferase Activity of Fungal

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