Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone

Article

Mitochondrial Chaperonin HSP60 Is the ApoptosisRelated Target for Myrtucommulone Graphical Abstract

Authors € ller, Katja Wiechmann, Hans Mu Stefanie Ko¨nig, Natalie Wielsch,  Svatos , Johann Jauch, Ales Oliver Werz

Correspondence [email protected]

In Brief Wiechman et al. exploited an unbiased, discriminative protein fishing approach to identify heat-shock protein 60 (HSP60) as a macromolecular mitochondrial target of the natural product mytrucommulone. Their experimental evidence proposes myrtucommulone as a valuable tool to study HSP60 biology.

Highlights d

Myrtucommulone and its inactive derivative were probed for protein fishing

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Heat-shock protein 60 (HSP60) was fished as binding partner of myrtucommulone

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Myrtucommulone inhibits the chaperonin activity of HSP60 in a cell-free assay

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Inhibition of HSP60 by myrtucommulone correlates to LONP and LRP130 aggregation

Wiechmann et al., 2017, Cell Chemical Biology 24, 1–10 May 18, 2017 ª 2017 Elsevier Ltd. http://dx.doi.org/10.1016/j.chembiol.2017.04.008

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

Cell Chemical Biology

Article Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone  Svatos ,3 Johann Jauch,2 and Oliver Werz1,4,* € ller,2 Stefanie Ko¨nig,1 Natalie Wielsch,3 Ales Katja Wiechmann,1 Hans Mu 1Pharmaceutical/Medicinal

Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, 07743 Jena, Germany € cken, Germany Chemistry II, Saarland University, Campus C 4.2, 66123 Saarbru 3Research Group Mass Spectrometry and Proteomics, Max Planck Institute for Chemical Ecology, Hans-Kno ¨ ll-Straße 8, 07745 Jena, Germany 4Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2017.04.008 2Organic

SUMMARY

The acylphloroglucinol myrtucommulone A (MC) causes mitochondrial dysfunctions by direct interference leading to apoptosis in cancer cells, but the molecular targets involved are unknown. Here, we reveal the chaperonin heat-shock protein 60 (HSP60) as a molecular target of MC that seemingly modulates HSP60-mediated mitochondrial functions. Exploiting an unbiased, discriminative protein fishing approach using MC as bait and mitochondrial lysates from leukemic HL-60 cells as target source identified HSP60 as an MC-binding protein. MC prevented HSP60-mediated reactivation of denatured malate dehydrogenase in a protein refolding assay. Interference of MC with HSP60 was accompanied by aggregation of two proteins in isolated mitochondria under heat shock that were identified as Lon protease-like protein (LONP) and leucine-rich PPR motif-containing protein (LRP130). Together, our results reveal HSP60 as a direct target of MC, proposing MC as a valuable tool for studying HSP60 biology and for evaluating its value as a target in related diseases, such as cancer.

INTRODUCTION The heat-shock protein 60 (HSP60) functions as a mitochondrial molecular chaperone, serving as a danger signal of stressed or damaged cells. HSP60 is involved in the correct folding of nascent polypeptides to native proteins imported into mitochondria and stabilizes proteins, thereby preventing protein aggregation which is especially required under cellular stress conditions (Martin et al., 1992). The corresponding cochaperonin for HSP60 is HSP10, and both proteins assemble to heptameric rings that are stacked to form a folding chamber (Nielsen and Cowan, 1998; Nisemblat et al., 2014). HSP60 is upregulated in various human cancers, exhibits anti-apoptotic activities, and supports tumor formation, progression, invasion, and metastases, associated with therapeutic resistance and poor survival (Ghosh et al., 2008; Arya et al., 2007; Cap-

pello et al., 2014; Lianos et al., 2015). During carcinogenesis, HSP60 accumulates outside mitochondria, for example, in the cytosol, plasma membrane, and in secretory vesicles, protecting tumor cells from external environmental stressors, thereby promoting cell proliferation (Cappello et al., 2014). Of interest, HSP60 participates in mitochondrial membrane permeabilization, in particular by its interaction with the mitochondrial permeability transition pore regulator cyclophilin D (Ghosh et al., 2010). Myrtucommulone A (MC, Figure 2A) is a non-prenylated acylphloroglucinol contained in myrtle (Myrtus communis, myrtaceae) displaying multiple bioactivities such as antibacterial (Rotstein et al., 1974; Appendino et al., 2002), antioxidant (Rosa et al., 2003), anti-inflammatory (Feisst et al., 2005; Rossi et al., 2009), and anti-tumoral properties (Grandjenette et al., 2015; Izgi et al., 2015; Tretiakova et al., 2008). The microsomal prostaglandin E2 (PGE2) synthase 1 (mPGES-1) and 5-lipoxygenase (5-LO) were identified as targets of MC, rationalizing the suppression of PGE2 and leukotriene biosynthesis as anti-inflammatory features (Feisst et al., 2005; Koeberle et al., 2009). The selective cytotoxic effects of MC against cancer cells were related to induction of the intrinsic pathway of apoptosis, involving caspase-9, along with cytochrome c release and loss of the mitochondrial membrane potential (DJm) (Tretiakova et al., 2008). Interestingly, non-transformed human foreskin fibroblasts and peripheral blood mononuclear cells were hardly susceptible for MC (Tretiakova et al., 2008). MC directly acts on isolated mitochondria derived from human leukemic cells, thereby affecting mitochondrial functions at submicromolar concentrations including reduction of mitochondrial viability, loss of DJm, and inhibition of mitochondrial ATP synthesis (Wiechmann et al., 2015a). However, the underlying modes of action and the molecular targets within mitochondria that mediate the effects of MC are unknown. Here we report about the identification of HSP60 as a direct mitochondrial protein target of MC using an unbiased, discriminative target fishing approach. We find that MC modulates HSP60 functions that are related to mitochondrial chaperonin activity. Along these lines, we discovered the mitochondrial proteins Lon protease-like protein (LONP) and leucine-rich protein 130 (LRP130) that are both seemingly regulated by HSP60, as they are remarkably influenced by MC. Cell Chemical Biology 24, 1–10, May 18, 2017 ª 2017 Elsevier Ltd. 1

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

Figure 1. Myrtucommulone Induces Cytochrome c Release in Isolated Mitochondria Mitochondria were incubated with compounds or vehicle (0.3% DMSO) for 1 hr at 37 C. Pellet (A) and supernatant (B) fractions were analyzed for cytochrome c (Cyt c) by western blot and quantified by densitometric analysis. Means ± SEM; n = 4; **p < 0.01, ***p < 0.001 versus vehicle control, ANOVA plus Bonferroni. Statistics applied to logarithmized data.

RESULTS Induction of Cytochrome c Release from Isolated Mitochondria MC causes cytochrome c release from leukemic cells by an unknown mode of action (Tretiakova et al., 2008). To investigate whether MC acts directly with mitochondria to induce cytochrome c release, we incubated isolated mitochondria from human leukemic HL-60 cells with MC and analyzed the release of cytochrome c by western blot. MC (10 mM) induced a significant loss of cytochrome c from the mitochondria with concomitant enrichment of the protein in the supernatant (Figure 1), indicating that MC acts directly on mitochondria, leading to cytochrome c release without the need of the entire cell or extramitochondrial factors. Design and Synthesis of Immobilized MC and Derivatives for Target Fishing To reveal mitochondrial target protein(s) of MC that upon interaction initiate(s) cytochrome c release, we sought to conduct protein fishing experiments using MC as bait (Figure 2A) and isolated mitochondria of HL-60 cells as target source. MC was immobilized to EAH Sepharose 4B (1) with a suitable linker (Figure 2B). As linker we chose the sebacoyl group connected to the phloroglucinol of the MC for (a) retention of the acyl group in MC and (b) provision of a carboxylic acid for amide coupling with EAH Sepharose 4B (1). Sebacoyl-MC (2) could be easily synthesized according to our previously developed strategy (Muller et al., 2010). Thus, acylating phloroglucinol with sebacic acid chloride monoethyl ester (3) (ethyl-10-chloro-10-oxodecanoate), readily obtained from sebacinic acid (4) according to Reininger and Hartl (1976), leads to monoethyl sebacoyl phloroglucinol (5) (ethyl 10-oxo-10-(2,4-6-trihydroxyphenyl)-decanoate). Hydrolysis of the ester function gives the free acid 6, which was coupled to freshly synthesized isobutylidene syncarpic acid (7) 2 Cell Chemical Biology 24, 1–10, May 18, 2017

from syncarpic acid (8) in a double Michael addition to produce sebacoyl-MC (2). Coupling with EAH Sepharose 4B (1) was achieved with EDCI in dioxane/water 1:1 (v/v) leading to immobilized myrtucommulone (9) (MC-immob.). The chemical structures of the MC probes are shown in Figure 2A and the entire synthesis is shown in Figure 2C. Structure elucidation of 2 was done with high-resolution mass spectrometry (HRMS), and additionally by conversion of 2 into the bis-pyrane derivative 10 (Figure S1) and nuclear magnetic resonance (NMR) spectroscopy thereof, since 2 does not give suitable NMR spectra due to a complex mixture of rotamers and keto-enol tautomers. 10 was also immobilized to EAH Sepharose 4B (1) to give immobilized MC-penta-immob. (11) (Figures S1 and 2A). For reference purposes, the non-immobilized n-butyl amides 12 (MC-linked) and 13 (MC-penta-linked) of 2 and 10, respectively, were synthesized (Figure S1). To ensure that MC-linked 12 remained bioactive, we analyzed the ability to induce cell death of HL-60 cells and cytochrome c release from isolated mitochondria. MC as well as the MC-linked 12 reduced cell viability (MTT assay) in a concentration-dependent manner equally well whereas MC-penta was markedly less active and, as expected, MC-penta-linked 13 was also inactive (Figure 3A). The apoptosis inducer staurosporine (3 mM) served as positive control, causing complete loss of cell viability (1.5% ± 0.8%, data not shown). Similarly, MC-linked 12 caused mitochondrial cytochrome c release but MC-penta-linked 13 failed in this respect. These results (1) ensure that the covalent modification of MC by insertion of the linker (MC-linked, 12) does not abolish the bioactivities, and (2) show that MC-pentalinked 13 is suitable as inactive negative control for protein fishing. Identification of HSP60 as Direct Binding Partner of MC Immobilized MC (MC-immob., 9) and the negative control consisting of immobilized bioinactive, pentacyclic MC derivative (MC-penta-immob., 11) as well as the untreated EAH Sepharose 4B (1) were incubated with mitochondrial lysates of HL-60 cells. After intensive washing, bound proteins were eluted from the beads, followed by separation using SDS-PAGE. Coomassie staining of the gel and densitometric analysis revealed a selective and strong enrichment of a 60 kDa protein that was pulled down using MC-immob. (9), compared with samples using MC-penta-immob. (11) or EAH Sepharose 4B (1) (Figure 4A). The amounts of essentially all other protein bands were not strikingly different, indicating that the 60 kDa protein was rather selectively enriched while supporting equal loading of pulleddown proteins with the various beads. The protein band at 60 kDa was cut out from the gel and subjected to nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/ MS). Analysis of detected peptide fragments using the NCBI (human) database suggested the human mitochondrial HSP60 as a potential binding partner of MC-immob. (9) (Figure S1). Analysis of pulled-down proteins by western blot confirmed the identity of HSP60, and densitometric analysis revealed strong enrichment of HSP60 in samples obtained with MC-immob. (9) compared with samples using MC-penta-immob. (11) or EAH Sepharose 4B (1) (Figure 4B). To exclude that MC-immob. binds to another mitochondrial protein that then hooks up HSP60, we aimed to confirm the direct binding of MC-immob. to isolated HSP60.

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

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Figure 2. Myrtucommulone Derivatives and the Synthesis Thereof (A) MC and derivatives as well as their corresponding pentacyclic structures used for cellular and biochemical studies. Immobilized derivatives were coupled to EAH Sepharose 4B and used for target fishing. Gray sphere represents Sepharose beads. (legend continued on next page)

Cell Chemical Biology 24, 1–10, May 18, 2017 3

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

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Figure 3. Impact of MC and Derivatives on Cell Viability of HL-60 Cells and Cytochrome c Release from Isolated Mitochondria

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Thus, human recombinant HSP60 was incubated with the beads in the presence of unspecific blocking proteins (non-fat dry milk powder). Western blot analysis of respective pull-downs showed that HSP60 specifically binds to MC-immob. (9) carrying active MC, but not to the negative controls MC-penta-immob. (11) and EAH Sepharose 4B (1) (Figure 4C). MC Inhibits HSP60 Chaperone Activity Next, we investigated whether the binding of MC to HSP60 has any consequences for the function of the protein affecting its bioactivity. The characteristic function of HSP60 is its chaperone activity, an ATP-driven protein-folding process in cooperation with the co-chaperonin HSP10 that refolds and reactivates misfolded proteins. A typical target of this chaperone complex is mitochondrial malate dehydrogenase (MDH) (Dubaquie et al., 1997). Preincubation of HSP60/HSP10 with MC prior to initiation of MDH refolding caused a concentration-dependent inhibition of HSP60 chaperone activity, thus restoring MDH catalysis, with significant effects at 3 mM. Of interest, in agreement with the structure-activity relationships (SARs) for reduction of the viability of HL-60 cells, the bioactive MC-linked (12) also strongly inhibited the HSP60-mediated MDH refolding activity, whereas the negative control MC-penta-linked (13) was hardly active (Figure 5A). The HSP60 inhibitor epolactaene tertiary butyl ester (ETB) (Nagumo et al., 2004, 2005) significantly reduced the chaperone activity at 23.2 mM as expected, with apparent lower efficiency versus MC (Figure 5A). To exclude direct inhibitory effects of MC on MDH function, we determined the effect of MC on MDH activity itself (in the absence of HSP60/HSP10). MDH activity was hardly affected, and only at the highest concentration of 30 mM did MC cause significant inhibition, by about 50% (Figure 5B).

(A) HL-60 cells were treated with test compounds or vehicle (0.3% DMSO) for 24 hr at 37 C, 5% CO2. MTT was added and incubation was continued until blue staining of the vehicle-treated control. Afterward, cell lysis absorbance was measured at 570 nm. Staurosporine served as positive control leading to complete cell death (1.5% ± 0.8% cell viability, data not shown). Means ± SEM; n = 3. + p < 0.05, +++p < 0.001, ###p < 0.001. (B) Mitochondria were incubated with test compounds or vehicle (0.3% DMSO) for 1 hr at 37 C, and supernatant (upper panel) and pellet (lower panel) fractions were analyzed by western blot (representative blots out of two to three experiments are shown) and quantified by densitometric analysis. means ± SEM; n = 2–3. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle control, ANOVA plus Bonferroni. Statistics applied to logarithmized data.

MC Induces Selective Mitochondrial Protein Aggregation under Heat Shock We then investigated whether inhibition of HSP60 chaperone activity by MC translates also to functional consequences, resulting in misfolding and aggregation of proteins in isolated mitochondria under heat-shock conditions. Thus, after addition of test compounds, mitochondria were heat-shock treated (42 C), subsequently lysed, and protein aggregates were separated from soluble native proteins. Since the pelleted protein aggregates could not be resuspended after high-speed centrifugation (125,000 3 g), the soluble proteins in the supernatant were subsequently analyzed by SDS-PAGE and Coomassie staining. Compared with vehicle, MC selectively prevented the appearance of two soluble proteins with an estimated size of approximately 130 and 105 kDa (Figure 6A), indicating that these proteins aggregated. Notably, when the experiment was conducted at 37 C the appearance of these two proteins was only slightly affected by MC, suggesting that MC caused aggregation predominantly under heat-shock conditions. Moreover, MC-linked 12 induced disappearance of the 130 and 105 kDa proteins but not the negative control MC-penta-linked 13 (Figure 6A). To identify the 130 and 105 kDa proteins, we cut the bands from the gel and after digestion analyzed them using nanoLCMS/MS. The 130 kDa protein band was revealed as LRP130 with a high score of 1,042 as potential candidate, while the 105 kDa band was identified as LONP (Figure 6B). Western blot analysis confirmed the identity of LRP130 and LONP as the respective proteins that were induced to aggregate in the presence of MC (Figure 6C). As expected, the aggregation of LRP130 and LONP under heat-shock conditions was induced by MC-linked 12 but not by MC-penta-linked 13. More detailed

(B and C) Synthesis of immobilized MC (9). Reagents and conditions: (a) EtOH, H2SO4 cat., cyclohexane, reflux, 96 hr, 58%; (b) SOCl2, CHCl3, reflux, 14 hr, quant.; (c) AlCl3, CH2Cl2, CH3NO2, reflux, 10 min, 58%; (d) NaOH, H2O, isoPrOH, reflux, 3 hr, 50%; (e) CH2Cl2, isobutyl aldehyde, piperidine, room temperature, 5 min, transfer to step (f) without further purification; (f) HCl, NH4Cl, room temperature, 15 min, transfer to step (g) without further purification; (g) NaH, THF, room temperature, 2 hr, 80%; (h) EAH Sepharose 4B, EDCI, dioxane/H2O (1:1, v/v), room temperature, 96 hr, 90%–100%. See also Figure S1.

4 Cell Chemical Biology 24, 1–10, May 18, 2017

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

Figure 4. Target Identification Using Immobilized MC Derivatives (A) Mitochondrial lysates derived from HL-60 cells were incubated with the fishing constructs (see Figure 2A) overnight at 4 C. After washing, bound proteins were eluted by SDS lysis (5 min, 95 C) and analyzed by SDS-PAGE followed by Coomassie staining. Protein bands of interest were cut out, digested, and analyzed by nanoLC-MS/MS. The Coomassie-stained gel shown is representative of at least three independent experiments; proteins (bands framed by dashed rectangles) were quantified by densitometric analysis. Means ± SEM; n = 3; ***p < 0.001 versus MC-penta-immob., ANOVA plus Bonferroni. Statistics applied to logarithmized data. (B) Fishing samples (see A) were analyzed by western blot for HSP60 and quantified by densitometric analysis; result shown is representative of three independent experiments. Means ± SEM; n = 3; ***p < 0.001 versus MC-penta-immob., ANOVA plus Bonferroni. Statistics applied to logarithmized data. (C) Beads were blocked with milk powder and then incubated with human recombinant HSP60 overnight at 4 C. Beads were washed and proteins eluted by SDS lysis (5 min, 95 C). Protein samples were analyzed by western blot; milk powder served as loading control. Densitometric analysis; result shown is representative of three independent experiments. Means ± SEM; n = 3; **p < 0.01 versus MC-penta-immob., ANOVA plus Bonferroni. See also Table S1.

investigation of the potency of MC to induce aggregation of LRP130 and LONP showed that significant effects were already evident at 0.1 and 0.03 mM MC, respectively, and 0.3 mM MC caused almost complete aggregation (Figure 6D). In contrast, at ambient temperature (37 C) MC was much less active, and 40% reduction of LRP130 and LONP aggregation required more than 30- to 100-fold higher concentrations (i.e., 10 mM). Together, these data imply that MC directly binds to HSP60 and blocks its chaperone activity, and is associated with the aggregation of sensitive proteins in mitochondria under heat stress. Although protein fishing experiments using MC-immob. (9) did not immediately reveal obvious enrichment of protein bands at 105 and 130 kDa by Coomassie staining (Figure 4A, lane b), it still appeared possible that LONP and LRP130 co-precipitate with HSP60 and are enriched as well. Therefore, we analyzed LONP and LRP130 in the protein precipitates by more sensitive western blot detection. Interestingly, both LONP and LRP130 were significantly enriched in samples obtained after pull-down with MC-immob. (9) but not so with MC-penta-immob. (11) or untreated beads (1) (Figure 6E). Such co-precipitation of LONP and LRP130 might confirm their interaction with (MC-bound) HSP60, but could also indicate that LONP and LRP130 bind directly to MC, independent of HSP60.

DISCUSSION Here, we succeeded in identification of HSP60 as a selective, direct MC-binding protein that inhibits HSP60 chaperonin activity in a cell-free assay and apparently also in a more complex biological environment, the mitochondrion. Moreover, by exploiting MC as a tool compound we propose a new possible biological function of HSP60: the protection of LONP and LRP130 against heat-shock-induced aggregation. Because current research intensively investigates HSP60 as a drug target (Nakamura and Minegishi, 2013), small molecules targeting HSP60 are in urgent demand as tools and potential therapeutics (Cappello et al., 2014). Accordingly, the unbiased discovery of HSP60 as a target of MC is of utmost interest, especially because MC has been reported to modulate pathologies that are connected to the dysfunction of HSP60, such as autoimmune diseases and inflammation, cardiovascular disease, diabetes, and cancer (Cappello et al., 2014). MC confers activation of the intrinsic apoptotic pathway involving caspase-9, loss of DJm, and cytochrome c release in cancer cells (Izgi et al., 2015; Tretiakova et al., 2008), and directly inhibited crucial functions of isolated mitochondria from HL-60 cells such as their viability, DJm, and mitochondrial ATP synthesis (Wiechmann et al., 2015a). However, the Cell Chemical Biology 24, 1–10, May 18, 2017 5

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

Figure 5. Myrtucommulone Inhibits HSP60 Chaperone Activity HSP60/HSP10 were preincubated with compounds or vehicle (1% DMSO) for 15 hr at 4 C. MDH was denatured with 10 mM HCl for 2 hr at 25 C. MDH refolding was initiated by 2 mM ATP for 30 min at 27 C. The reaction was terminated by addition of glucose/hexokinase, NADH and oxaloacetate were added, and kinetics was monitored at 360 nm at 30 C. (A) MC inhibits HSP60 chaperone activity. (B) Effect of MC on native MDH. Means ± SEM; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle control, ANOVA plus Bonferroni.

underlying mode of action and the respective target protein(s) of MC remain unknown. Here, we find that MC caused release of cytochrome c from isolated mitochondria, implying the interaction with a mitochondrial target, thereby inducing the intrinsic pathway of apoptosis. Nevertheless, MC may have additional extramitochondrial targets, such as 5-LO (Feisst et al., 2005), which are not considered in our approach using mitochondria as target source. Revealing the molecular target that mediates a phenotypic effect of a bioactive small molecule allows understanding of the precise mode of action but also enables the development of novel target-specific tool compounds or drugs. Besides genetic approaches applying phenotypic profiling studies to identify the target of a given compound, protein target fishing strategies using gels or gel-free multidimensional protein identification technology are valuable for this purpose (Schirmer et al., 2003; Burdine and Kodadek, 2004; Schmitt et al., 2015). The successful application of the unbiased, discriminative protein fishing approach in this study required the availability of (1) immobilized MC that retains bioactivity after linking and (2) a suitable inactive counterpart as negative control to discriminate unspecific protein binding. Previous SARs showed that replacing the butyryl group of MC by extended acyl chains retains pro-apoptotic properties (Wiechmann et al., 2015b), suggesting the acyl chain as appropriate group for immobilization of MC. Indeed, such linked MC (12) was equally bioactive versus MC in HL-60 cell death induction and mitochondrial cytochrome c release, supporting its usefulness as active probe. Ring closure of MC to MC-penta caused loss of apoptosis induction, qualifying MCpenta as negative control. In fact, the corresponding sebacoyln-butyl amide (MC-penta-linked 13) failed in HL-60 cell death induction and cytochrome c release. Together, by applying these active and inactive probes immobilized to EAH Sepharose 4B in a discriminative target fishing approach, HSP60 was revealed as selective MC-binding protein. Use of isolated HSP60 in the pulldown assay verified the direct interaction between HSP60 and MC, independent of any mitochondrial linker proteins. HSP60 prevents protein aggregation by facilitating the correct folding and stabilization of proteins (Martin et al., 1992). In fact, MC and MC-linked 12, but not the inactive MC-penta-linked 13, blocked HSP60-mediated reactivation of isolated denatured MDH, a typical target protein of HSP60 (Dubaquie et al., 1997), in a cell-free assay. Moreover, MC as tool compound revealed important insights into HSP60 biology, as the data imply for 6 Cell Chemical Biology 24, 1–10, May 18, 2017

the first time the protection of LRP130 by HSP60 under heat stress and support the recently proposed interrelation between HSP60 and LONP (Kao et al., 2015). It is noteworthy that LONP and LRP130 were selectively enriched in the pull-downs using MC-immob. (9) as well, in addition to HSP60. This may confirm the interaction of HSP60 with these two proteins causing their co-precipitation within the MC-immob-HSP60 complex. On the other hand, LONP and LRP130 could also directly bind to MCimmob. (9) without HSP60. In this respect it is notable that HSP60, LONP, and LRP130 share the common ability to bind nucleic acids (Kaufman et al., 2003; Liu et al., 2004; Mili and Pinol-Roma, 2003), and these DNA/RNA binding sites might be contact sites also for MC. LRP130 is a mitochondrial protein that binds to mRNA (Mili and Pinol-Roma, 2003), is involved in expression of mitochondrial cytochrome c oxidase (Xu et al., 2004), and participates in mitochondrial transcription, leading to mitochondrial remodeling and impairment of oxidative metabolism (Liu et al., 2011). Of interest, LRP130 is involved in apoptosis resistance in hepatic cancer cells (Michaud et al., 2011), providing a possible link to the pro-apoptotic effects of MC that, potentially via inhibition of HSP60, may lead to inactivation of LRP130. LONP is a mitochondrial matrix protein possessing chaperone activity, mtDNA-binding capacity (Liu et al., 2004), and ATPdependent protein degrading functions (Gur et al., 2012; Venkatesh et al., 2012). Proteins modified by oxidation can be hydrolyzed by LONP, preventing cytotoxic protein aggregation (Bota and Davies, 2002) and maintaining mitochondrial protein homeostasis (Gur et al., 2012). LONP activity is crucial for mitochondria, in particular under stress conditions when accumulation of misfolded and oxidized proteins is impending. Similar to HSP60, LONP is upregulated by cellular stresses (Ngo and Davies, 2009), and high levels of LONP were found in hypoxic microenvironments (Hori et al., 2002) typically found in solid tumors (Dhani et al., 2015), as well as in several cancers such as lung adenocarcinoma and breast cancer (Cheng et al., 2013). MC-induced aggregation of LONP might support accumulation of misfolded proteins and lead to mitochondrial unfolded protein response (Arnould et al., 2015). Interestingly, LONP deficiency increased the activity of AMP-activated protein kinase (AMPK) (Gibellini et al., 2015), and we recently found AMPK activation in HL-60 cells treated with MC, supporting the hypothesis that MC reduces the level of soluble, functional LONP fostering AMPK activation. Also, depletion of LONP via short hairpin RNA in colon

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

Figure 6. Myrtucommulone Induces Mitochondrial Protein Aggregation Mitochondria were incubated with compounds or vehicle (0.3% DMSO) for 20 min at the indicated temperatures. Samples were centrifuged and pelleted mitochondria were lysed. After centrifugation at 125,000 3 g, the supernatants were analyzed by SDS-PAGE and Coomassie staining. (A) Coomassie-stained polyacrylamide gel. Results are representative of three independent experiments. (B) Proteins showing different amounts compared with the control samples (lane a and b, bands framed by dashed rectangles) were cut out and identified by LCMS/MS. (C) Effect of MC and derivatives on mitochondrial protein aggregation. Samples were analyzed by western blot for HSP60, LRP130, or LONP and quantified by densitometric analysis. (D) MC triggers mitochondrial protein aggregation in a concentration-dependent manner. Samples were analyzed as described above. Means ± SEM; n = 3. (E) Mitochondrial lysates derived from HL-60 cells were incubated with the fishing constructs (see Figure 2A) overnight at 4 C. After washing, bound proteins were eluted by SDS lysis (5 min, 95 C) and analyzed by western blot for LONP and LRP130. Loading control: unspecific protein band at approximately 50 kDa. Densitometric analysis; means ± SEM; n = 3. #, *p < 0.05, ##,**p < 0.01, ###,***p < 0.001 versus vehicle control, Student’s t test. Statistics applied to logarithmized data. See also Table S2.

Cell Chemical Biology 24, 1–10, May 18, 2017 7

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

cancer cells caused alterations in the architecture, proteome, and metabolism of mitochondria as well as decreased ATP synthesis (Gibellini et al., 2014). In fact, we observed decreased ATP synthesis by MC in isolated mitochondria (Wiechmann et al., 2015a), and, as observed for MC, inhibition of LONP by the anti-cancer agent 2-cyano-3,12-dioxooleana-1,9,-dien-28oic acid induces morphological changes in mitochondria and triggers the mitochondrial apoptosis pathway (Bernstein et al., 2012). Together, based on the congruent effects of MC and interference with LONP, it is tempting to speculate that inhibition of HSP60 by MC abrogates LONP functions, thereby contributing to apoptosis in cancer cells. Again, as discussed for LRP130, MC may bind directly to LONP and thus cause aggregation of the protease complex, independent of HSP60. Future studies addressing direct binding of MC to LONP and LRP130 in detail and specific interference with HSP60 (e.g., knockdown) will eventually confirm the requirement of HSP60 for MC-induced mitochondrial LONP and LRP130 aggregation under heat shock. Based on the multiple roles in common diseases, HSP60 has been suggested as a valuable drug target. Two classes of HSP60 inhibitors are known: (1) compounds that covalently bind to certain cysteine residues such as oxidizable sites in HSP60, and (2) compounds that block ATP binding and hydrolysis, thereby affecting ATP-dependent conformational changes that are required for protein folding (Cappello et al., 2014). Cysteine residues in HSP60 are targeted, for example, by avrainvillamide, which alkylates cysteines (Wulff et al., 2007), by the electrophilic a,b-unsaturated aldehyde 4-hydroxynonenal (Vila et al., 2008), and by epolactaene or its derivative ETB that covalently binds to Cys442 (Nagumo et al., 2005). On the other hand, mizoribine and the pyrazolopyrimidine EC3016 block protein-folding activity of HSP60 by interference with ATP binding and/or hydrolysis (Chapman et al., 2008; Itoh et al., 1999). The exact binding site of MC at HSP60 and the molecular mode of interaction remains to be determined in future studies. The chemical structure of MC reveals no alkylating or sulfating moieties, rather excluding modification of cysteine residues. Our previous data, however, revealed suppression of ATP synthesis by MC in isolated mitochondria at submicromolar concentrations (Wiechmann et al., 2015a), implying possible interference with adenine nucleotidebinding sites. The dual actions of MC, that is, direct binding to HSP60 and affecting mitochondrial ATP levels, might synergize and result in the efficient modulation of LONP and LRP130 at submicromolar MC concentrations. Together, our studies highlight the target fishing approach with MC as bait that allowed identification of HSP60 as an apoptosisrelated target from mitochondria of cancer cells. MC directly binds to HSP60 and inhibits its chaperone activity, and induces aggregation of LRP130 and LONP in mitochondria under heat stress. By using MC as a tool compound, further investigations revealing the roles of HSP60 are feasible, to enable better understanding of the potential of HSP60 as a drug target for anti-cancer therapy.

of apoptosis by direct interference with mitochondrial functions. However, the molecular mode of action and the macromolecular target(s) of MC are unknown. By exploiting the knowledge of previously established SARs of MC and the strategy for its total synthesis, we designed suitable immobilized MC probes for conducting an unbiased protein fishing approach using the proteome of isolated mitochondria from cancer cells. The mitochondrial chaperonin HSP60 was identified and validated as a functional, direct molecular target of MC. Our data suggest that interference with HSP60 function may underlie the induction of the mitochondrial pathway of cancer cell apoptosis by MC. HSP60 is critically involved in protein transport and correct folding of imported proteins, and promotes the refolding and correct assembly of unfolded polypeptides generated under stress conditions. By employing MC as chemical tool to study HSP60 biology in mitochondrial protein aggregation, we revealed two HSP60-regulated proteins that are seemingly protected against heat-stressinduced aggregation: LONP and LRP130. Based on the important roles of HSP60 and LONP in mitochondria to promote survival and proliferation of cancer cells, interference of MC with these proteins may explain the induction of the intrinsic pathway by directly affecting mitochondria. Current research intensively investigates the potential of HSP60 as a drug target with relevance for intervention in a variety of diseases including cancer. MC might serve as a suitable tool compound for better understanding the biological roles of HSP60 and for assessment of its potential as a drug target for (anti-cancer) pharmacotherapy. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell Lines METHODS DETAILS B Protein Determination B Cytochrome c Release from Mitochondria B Cell Viability Assay B Preparation of Mitochondrial Lysates B Target Fishing B SDS-PAGE and Gel Staining B In-gel Digestion B Protein Identification by nanoLC-MS/MS B Western Blot B Determination of HSP60 Chaperone Activity B Mitochondrial Protein Aggregation Assay B Chemistry Materials B Synthesis of Compounds and Characterization QUANTIFICATION AND STATISTICAL ANALYSIS

SIGNIFICANCE Myrtucommulone (MC) is a well-known naturally occurring acylphloroglucinol with remarkable cytotoxic activity and selectivity for cancer cells. MC induces the intrinsic pathway 8 Cell Chemical Biology 24, 1–10, May 18, 2017

SUPPLEMENTAL INFORMATION Supplemental Information includes one figure and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.chembiol.2017.04.008.

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

AUTHOR CONTRIBUTIONS K.W. and S.K. designed and conducted experiments, evaluated the data, and wrote the paper. H.M. conducted the synthesis of MC and derivatives. N.W. and A.S. performed LC-MS/MS and corresponding data processing, and wrote parts of the paper. J.J. designed and supervised the synthesis of MC and derivatives and wrote parts of the paper. O.W. designed and supervised all studies and wrote the paper. ACKNOWLEDGMENTS H.M. and J.J. are grateful to the Saarland University. S.K. received a doctoral fellowship by the Excellence Graduate School of the Jena School for Microbial €ller and Thomas Hoffmann Communication (JSMC). We thank Prof. Dr. Rolf Mu (Pharmaceutical Biotechnology, Saarland University), and Prof. Dr. Dietrich Volmer and Tobias Dier (Analytical Chemistry, Saarland University) for preparation of the ESIMS spectra. Received: November 1, 2016 Revised: February 18, 2017 Accepted: April 6, 2017 Published: April 27, 2017

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10 Cell Chemical Biology 24, 1–10, May 18, 2017

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

cytochrome c

Epitomics

1896-1; RRID: AB_1267098

heat shock protein 60

Epitomics

S1470

LONP

Abcam

ab103809; RRID: AB_10858161

LRP130

Abcam

ab97505; RRID: AB_10688419

IRDye 800CW Goat anti-Mouse IgG (H+L)

LI-COR

[P/N 925-32210]

IRDye 680LT Goat anti-Rabbit IgG (H+L)

LI-COR

[P/N 925-68020]

Antibodies

Chemicals, Peptides, and Recombinant Proteins epolactaene tertiary butyl ester

Wako

051-07671

staurosporine

VWR

569396

DMSO

VWR

1029500500

human recombinant HSP60

Enzo

ADI-NSP-540-E

human recombinant HSP10

Enzo

ADI-SPP-110-F

RPMI-1640

GE Healthcare Life Sciences

R8758-6

fetal calf serum

Sigma

F7524

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT reagent)

Thermo Fisher

15214654

bovine serum albumin

AppliChem

A1391.0500

malate dehydrogenase

Sigma

M1567-5KU

ATP disodium salt

Sigma

10519987001

NADH disodium salt

Sigma

N8129

oxaloacetate

Sigma

O4126

Coomassie Brilliant Blue G-250

Fisher Scientific

20279

EAH Sepharose 4B

GE Healthcare Life Sciences

17-0569-01

penicillin/streptomycin

GE Healthcare Life Sciences

A2213

Biorad

5000111

ATCC

CCL-240

MassLynx

Waters

version 4.1

ProteinLynx Global Server Browser

Waters

version 2.5

MASCOT version 2.3

Mascot Server

http://www.matrixscience.com/server.html

GraphPad InStat 3

GraphPad Software Inc

https://www.graphpad.com/scientific-software/ instat/

Odyssey 3.0 software

LI-COR

https://www.licor.com/bio/products/software/ image_studio/index.html

Critical Commercial Assays DC protein assay kit Experimental Models: Cell Lines HL-60 Software and Algorithms

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Oliver Werz ([email protected])

Cell Chemical Biology 24, 1–10.e1–e6, May 18, 2017 e1

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EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Lines The human acute promyelocytic leukemia cell line HL-60 (from a female Caucasian, obtained at ATCC) was cultured in RPMI-1640 (GE Healthcare Life Sciences, South Logan, UT) supplemented with heat-inactivated fetal calf serum (FCS; 10%, v/v), penicillin (100 U/mL), and streptomycin (100 mg/mL). Cells were grown in a humidified atmosphere at 37  C and 5% CO2. Cells were counted using the Vi-CELL XR (Beckman Coulter GmbH, Krefeld, Germany). METHODS DETAILS Protein Determination Protein contents of mitochondrial samples were determined based on the Lowry assay using the DC (detergent compatible) protein assay kit (Bio-Rad, Munich, Germany). Samples were treated according to the manufacturer’s protocol prior to photometric analysis (Multiskan microplate spectrophotometer, Thermo Scientific, Ulm, Germany). Bovine serum albumin (BSA; AppliChem, Darmstadt, Germany) served as external standard. Cytochrome c Release from Mitochondria Mitochondria were isolated from HL-60 cells; buffers were prepared according to Park and Kim, 2005. Cells were centrifuged (314 3 g, 10 min, 4  C) and washed in Dulbecco’s phosphate buffered saline (PBS) (450 3 g, 10 min, 4  C). Cells were resuspended in buffer A (250 mM mannitol, 70 mM sucrose, 0.5 mM EGTA, 5 mM HEPES, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.2) and homogenized in a dounce homogenizer (4 3 10 strokes, on ice). The suspension was diluted approx. 1:5 with buffer A and centrifuged (1,000 3 g, 10 min, 4  C). The resulting supernatant was centrifuged again (10,000 3 g, 10 min, 4  C). The pellet containing the crude mitochondrial fraction was resuspended in buffer B (250 mM sucrose, 10 mM HEPES, 2 mM KH2PO4, 5 mM sodium succinate, 25 mM EGTA, 0.1 mM PMSF, pH 7.5) and protein content was determined. Mitochondria were immediately used for determination of cytochrome c release. Thus, mitochondrial protein concentration was adjusted to 500 mg/mL and 100 mL samples were aliquoted in 1.5 mL Eppendorf tubes. Mitochondria were incubated with compounds and vehicle control (0.3% DMSO) at 37  C for 1 h. After incubation mitochondria were pelleted (10,000 3 g, 10 min, 4  C) and supernatant and pellet fractions were separated. Pellets were resuspended in equal volumes of Dulbecco’s PBS and the pellet as well as supernatant fractions were analyzed by Western blot (see below). Cell Viability Assay HL-60 cells were seeded (3 3 105/mL, 100 mL/well) into a 96-well plate in RPMI medium supplemented with heat-inactivated FCS (10%, v/v), penicillin (100 U/mL), and streptomycin (100 mg/mL). Compounds were added (0.3% DMSO, final concentration) and samples were incubated for 24 h at 37  C, 5% CO2. 20 mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (5 mg/mL in Dulbecco’s PBS) were added and the incubation was continued until blue staining of the DMSO-treated sample. MTT formazan formation was stopped by addition of 100 mL of lysis buffer (SDS, 10%, w/v in 20 mM HCl) and samples were shaken overnight. Absorbance was measured at 570 nm in a Multiskan microplate spectrophotometer. Experiments were performed in triplicates. Preparation of Mitochondrial Lysates HL-60 cells were pelleted (500 3 g, 10 min, 4  C) and resuspended in Dulbecco’s PBS containing 5 mM EDTA. Cells were washed (750 3 g, 10 min, 4  C), resuspended in mitochondria isolation buffer (MIB; 210 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EDTA, pH 7.4) and washed again (750 3 g, 10 min, 4  C). The cell pellet was resuspended in MIB containing 0.1 mM PMSF and cells were disrupted mechanically by passing the cell suspension 6 times through a 26 gauge needle. Afterwards, the suspension was centrifuged (2,000 3 g, 10 min, 4  C), the supernatant containing mitochondria was collected in a tube, and the cell pellet was again resuspended in MIB containing 0.1 mM PMSF. This homogenization procedure was repeated 3 times. The obtained supernatants were pooled and centrifuged (16,500 3 g, 10 min, 4  C). Mitochondrial pellet was resuspended in MIB containing 0.1 mM PMSF and centrifuged (2,000 3 g, 10 min, 4  C) to remove remaining cell debris. The resulting supernatant was transferred to a fresh tube and mitochondria were pelleted (16,500 3 g, 10 min, 4  C). Mitochondrial mass was determined and 1 mL of lysis buffer (50 mM HEPES, 200 mM NaCl, 1 mM EDTA, pH 7.4, 1% Triton X-100, 5% protease inhibitor cocktail) was added per mg mitochondria. Mitochondrial lysate was homogenized by sonication (3 3 3 sec) and centrifuged (15,000 3 g, 60 min, 4  C). The resulting supernatant was immediately used for target fishing experiments. Target Fishing EAH Sepharose 4B beads carrying MC or the negative control MC-penta as well as untreated EAH Sepharose 4B beads (50 mL each) were washed (7,000 3 g, 5 min, 4  C) three times in binding buffer (50 mM HEPES, 200 mM NaCl, 1 mM EDTA, pH 7.4). For target fishing, beads were resuspended in 735 mL of binding buffer and 15 mL of mitochondrial lysate were added. Samples were incubated by overhead rotation at 4  C overnight.

e2 Cell Chemical Biology 24, 1–10.e1–e6, May 18, 2017

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

For target fishing with isolated human recombinant HSP60, beads were added to 750 mL of binding buffer containing 0.01% Triton X-100 and 250 mg milk powder per sample, and blocked for 1 h at 4  C to saturate unspecific binding sites. After blocking, 250 ng of human recombinant HSP60 were added and samples were incubated by overhead rotation at 4  C overnight. Beads incubated with mitochondrial lysates or isolated HSP60 were pelleted and washed three times in 500 ml binding buffer (7,000 3 g, 5 min, 4  C). 50 mL of elution buffer (10 mM Tris, 1 mM EDTA, 5% SDS (m/V), pH 8.0) containing 5% 2-mercaptoethanol were added. Samples were boiled at 95  C for 5 min and centrifuged (7,000 3 g, 5 min, 4  C). Proteins in the resulting supernatant were applied to SDS-PAGE and analyzed by Coomassie staining or by Western blot (see below). SDS-PAGE and Gel Staining Samples were incubated with Laemmli buffer, boiled at 95  C for 5 min and applied to SDS-PAGE. Samples obtained from the mitochondrial protein aggregation assay were separated on an 8% polyacrylamide gel. For samples obtained from fishing experiments, 10% polyacrylamide gels were used, and cytochrome c was separated on 16% polyacrylamide gels. For Coomassie staining, gels were rinsed two times with water prior to washing twice in water while shaking. Water was discarded and colloidal Coomassie solution (0.2 g/L Coomassie Brilliant Blue G-250, 50 g/L Al2(SO4)3-14-18 hydrate, 2% phosphoric acid, 10% ethanol) was added. Gels were incubated up to 5 h while shaking at RT. Then, background was destained by replacement of colloidal Comassie by water. Stained gels were imaged and quantified using the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE). In-gel Digestion Protein bands of interest were cut from the gel and tryptic digestion was carried out as described (Shevchenko et al., 2006). Proteins were in-gel reduced by 10 mM dithiothreitol and alkylated by 55 mM iodoacetamide. Washed and dehydrated gel pieces were rehydrated for 60 min in 0.5 mM solution of bovine trypsin in 25 mM ammonium bicarbonate buffer at 4  C and then digested overnight at 37  C. The tryptic peptides were extracted from gel pieces with extraction buffer (50% ACN / 5% formic acid) and the extracts were dried down in a vacuum centrifuge. For LC-MS/MS analysis samples were reconstructed in 10 mL aqueous 1% formic acid. Protein Identification by nanoLC-MS/MS The samples were analyzed on a nanoAcquity nanoUPLC system (Waters) online coupled to a Q-ToF Synapt HDMS mass spectrometer (Waters). Peptides were initially transferred with 0.1% aqueous formic acid for desalting onto a Symmetry C18 trap-column (20 3 0.18 mm, 5 mm particle size) at a flow rate of 15 mL/min (0.1% aqueous FA), and subsequently eluted onto a nanoAcquity C18 analytical column (200 mm 3 75 mm ID, BEH 130 material, 1.7 mm particle size) at a flow rate of 350 nL/min with the following gradient: 1–30% B over 13 min, 30–50% B over 5 min, 50–95% B over 5 min, isocratic at 95% B for 4 min, and a return to 1% B over 1 min (phases A and B composed of 0.1% FA and 100% acetonitrile in 0.1% FA, respectively). The analytical column was re-equilibrated for 9 min prior to the next injection. The eluted peptides were transferred to the nano electrospray source of a Synapt HDMS tandem mass spectrometer (Waters) equipped with metal-coated nanoelectrospray tip (Picotip, 50 3 0.36 mm, 10 mm internal diameter, New Objective). The source temperature was set to 80  C, cone gas flow 20 L/h, and the nanoelectrospray voltage was 3.2 kV. For all measurements, the mass spectrometer was operated in V-mode with a resolving power of at least 10,000. The acquisition cycle consisted of a survey scan covering the range of m/z 400-1500 Da followed by MS/MS fragmentation of the three most intense precursor ions collected over a 1 sec interval in the range of 50-1700 m/z. 650 fmol/mL human Glu-Fibrinopeptide B in 0.1% formic acid/acetonitrile (1:1 v/v) was infused at a flow rate of 0.5 mL/min through the reference NanoLockSpray source every 30 seconds to compensate for mass shifts in MS and MS/ MS fragmentation mode. Data were collected using MassLynx version 4.1 software (Waters) and processed using ProteinLynx Global Server Browser version 2.5 (Waters) under baseline subtraction, smoothing and deisotoping of acquired spectra. Pkl-files of MS/MS spectra were generated and searched against a species restricted NCBI (human) database (updated January, 28, 2011 installed on a local server) using MASCOT version 2.3. Mass tolerances for precursor and fragment ions were 15 ppm and 0.03 Da, respectively. Other search parameters were: instrument profile, ESI-Trap; fixed modification, carbamidomethyl (cysteine); variable modification, oxidation (methionone); up to 1 missed cleavage were allowed. Hits were considered as confident if at least three peptides were matched with ion scores above 30, or proteins were identified by one or two peptides with score of 55 or better. Western Blot Proteins separated by SDS-PAGE were transferred onto nitrocellulose membranes. After blocking for 1 h at room temperature (RT) with 5% BSA in Tris-buffered saline (TBS) containing 0.1% Tween 20, membranes were incubated with primary antibodies (antiHSP60, 1:1000; anti-LRP130, 1:1000; anti-LONP, 1:1000; anti-cytochrome c, 1:1000) overnight at 4  C. Membranes were washed and incubated with the corresponding secondary antibodies. Visualization and densitometric analysis were performed using the Odyssey Infrared Imaging System. Determination of HSP60 Chaperone Activity MDH refolding assay was performed as described (Hayer-Hartl, 2000; Nagumo et al., 2004, 2005). A mixture of purified HSP60 and its co-chaperonin HSP10 was preincubated with the compounds or vehicle (1.0% DMSO) in siliconized tubes for 15 h at 4  C. MDH, Cell Chemical Biology 24, 1–10.e1–e6, May 18, 2017 e3

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

denatured under acidic conditions for 2 h, was added and samples were incubated for 5 min at 27  C. Then, chaperone activity was initiated by addition of ATP, and the refolding of MDH was performed for 30 min at 27  C. The reaction was quenched by addition of glucose and hexokinase to consume the remaining ATP. Samples were transferred into a 96-well plate, NADH was added and catalytic activity of the refolded MDH was simultaneously initiated by oxaloacetate, and absorbance was immediately monitored at 360 nm at 30  C in a Multiskan microplate spectrophotometer. Initial linear decrease of absorbance served as measure for the enzymatic activity. In order to investigate direct effects of MC on the catalytic activity of MDH, the assay was performed under the same experimental conditions as described above, but using native MDH in the absence of HSP60 and HSP10. Because of the fact that no chaperones were present the preincubation of the buffer with the compounds was decreased to 1 h. Mitochondrial Protein Aggregation Assay Mitochondria were isolated from HL-60 cells as described by Rotem et al. (2005). The mitochondria were resuspended and protein aggregation was determined as described by Bender et al. (2011). Mitochondria (0.666 mg protein/mL) were aliquoted and preincubated with compounds or vehicle (0.3% DMSO) for 20 min at the indicated temperature. Samples were centrifuged (20,000 3 g, 10 min, 4  C) and mitochondrial pellets were lysed in 100 mL of lysis buffer A containing 0.5 mM PMSF and 1% protease inhibitor cocktail while vortexing thoroughly. Protein aggregates were pelleted by centrifugation (125,000 3 g, 30 min, 4  C). Supernatants were applied to SDS-SPAGE and analyzed by Coomassie staining or by Western blot. Chemistry Materials All solvents and reagents were from Merck, Darmstadt, Germany, in the highest purity available. EAH Sepharose 4B was from GE Healthcare Life Sciences. All reactions were run under an atmosphere of N2 or Ar in solvents, which were dried by the usual laboratory methods. Solvents for flash chromatography were distilled before use. NMR spectra (1D: 1H, 13C, DEPT 135; 2D: H,H-COSY, HSQC, HMBC, NOESY) were recorded on a Bruker AVANCE II 400 (1H NMR at 400 MHz, 13C NMR at 100 MHz). Chemical shifts are given in ppm and coupling constants in Hz. For processing the NMR spectra, MestReC 4.9.9.6 was used. ESIMS data were measured with a Shimadzu LC-MS 2020 from Shimadzu, Duisburg, Germany, and HRESIMS data were measured on an LTQ Orbitrap XL from Thermo Scientific, Dreieich, Germany. TLC was carried out on silica glass plates (Si60 F254 from Merck) and RP18 silica glass plates (RP18-F254 from Merck). For flash chromatography, silica gel with 40-63 m particle size from Merck was used. Synthesis of Compounds and Characterization Ethyl-10-chloro-10-oxodecanoate (3) Sebacinic acid 4 (6.7 g, 30 mmol) was suspended in a mixture of ethanol (100 mL) and water (120 mL). After addition of conc. sulfuric acid (1 mL), the reaction mixture was extracted with cyclohexane (400 mL) in a Kutscher-Steudel apparatus for four days. After cooling to room temperature, the organic phase was extracted with sat. aq. NaHCO3 solution. The aqueous solution was carefully acidified with 6N HCl and extracted with diethyl ether. The extract was dried with MgSO4, filtered and evaporated in vacuo. A colorless oil of sebacinic monoethyl ester was obtained (4.1 g, 17.4 mmol, 58%). The oil was dissolved in chloroform (200 mL), thionyl chloride (5 mL, 68 mmol) was added and the mixture was refluxed over night. After cooling to room temperature, the solvent and excess thionly chloride was removed in vacuo, which gave a yellowish oil of 3 in quantitative yield. Spectroscopic data are in accordance to Reininger and Hartl, 1976. 1H-NMR (CDCl3, 400 MHz): = 4.12 (quart, J = 7.1 Hz, 2H), 2.34 and 2.28 (two tr., J = 7.5 Hz, 4H), 1.62 (m, 4H), 1.36-1.27 (m, 8H), 1.25 (tr. J = 7.1 Hz, 3H). Ethyl 10-oxo-10-(2,4-6-trihydroxyphenyl)-decanoate (5) Phloroglucinol (1.26 g, 10 mmol) and AlCl3 (2.67 g, 20 mmol) were suspended in CH2Cl2 (40 mL). Nitromethane (1.28 g, 20 mmol) was added dropwise with stirring and the mixture was heated to 40-45  C (reflux, the solids dissolved and HCl evolution was observed). Next, ethyl-10-chloro-10-oxodecanoate (3) (2.74 g, 11 mmol) was added dropwise and the mixture was refluxed for additional 10 min. The cooled reaction was quenched with 2N HCl/ice (ca. 100 mL) and stirred at room temperature for ca. 2 h. The organic solvents were evaporated in vacuo and the aqueous residue was extracted with diethyl ether. The organic extracts were dried with MgSO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography with petroleum ether/acetone (2:1, v/v). Yield: 1.96 g (58%) as a yellowish solid (mp. 85-87  C). 1H-NMR (CDCl3, 400 MHz): = 10.69 (s, 2H), 5.91 (s, 2H), 4.18 (quart, J = 7.1 Hz, 2H), 3.07-2.95 (m, 2H), 2.39-2.25 (m, 2H), 1.73-1.57 (m, 2H), 1.41-1.30 (m, 8H), 1.25 (tr, J = 7.1 Hz, 3H). 13C-NMR (CDCl3, 100 MHz): = 207.3, 175.9, 163.5, 104.7, 95.8, 61.1, 43.9, 34.5, 29.4, 29.1, 29.0, 28.9, 25.1, 25.0, 24.9, 14.2. 3-Oxo-3-(2,4,6-trihydroxyphenyl)-decanoic Acid (6) Ethyl 10-oxo-10-(2,4-6-trihydroxyphenyl)-decanoate (5) (340 mg, 1 mmol) was dissolved in iso-propanol (10 mL) and 50% aq. NaOH (5 mL) was added. This mixture was refluxed for 3 h. After cooling to room temperature it was acidified with 2N HCl and extracted with diethyl ether. The etheral extracts were dried with MgSO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography with petroleum ether/acetone (1:1, v/v) + 1% acetic acid. Yield: 155 mg (50%) of a yellowish solid with mp 170-173  C. 1 H-NMR (Acetone-D6, 400 MHz): = 11.19 (s, 3H), 5.92 (s, 2H), 3.11-3.00 (m, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.73-1.61 (m, 2H), 1.64-1.53 (m, 2H), 1.42-1.28 (m, 8H). 13C-NMR (Aceton-D6, 100 MHz): = 206.6, 174.8, 165.4, 165.1, 105.2, 95.8, 44.4, 34.2, 25.7, 25.6.

e4 Cell Chemical Biology 24, 1–10.e1–e6, May 18, 2017

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

Sebacoyl-myrtucommulone (2) a) Isobutylidene syncarpic acid (7): syncarpic acid (Jain and Seshadri, 1955; Riedl and Risse, 1954; Murin et al., 1959; Benbakkar et al., 1989) (8) (1.09 g, 6 mmol) was suspended in CH2Cl2 (18 mL) prior to addition of isobutyraldehyde (0.65 g, 9 mmol). To this mixture, piperidine (1.02 g, 12 mmol) was added dropwise with stirring. Stirring at room temperature was continued for 5 min meanwhile the solids dissolved. 1N HCl, which had been saturated with NH4Cl, was added in excess (20 mL) and the mixture was stirred vigorously for 15 min. The phases were separated and the CH2Cl2-phase was filtered through a short plug of silica gel. The aqueous phase was extracted once with CH2Cl2 (ca. 20 mL) and the separated CH2Cl2-phase was filtered through the same plug of silica gel as above. The combined organic filtrates were evaporated in vacuo and used immediately in the next step. b) Sebacoyl-myrtucommulone (2): Sodium hydride (0.13 g of a 60% suspension in mineral oil, 3.0 mmol) was washed free of mineral oil and suspended in THF (20 mL) and sebacoyl phloroglucinol (6) (0.31 g, 1 mmol) was added. After stirring for 5 min at room temperature, the isobutylidene syncarpic acid from step a) was dissolved in THF (5 mL) and added to the deprotonated sebacoyl phloroglucinol. The reaction mixture was stirred at room temperature until all sebacoyl phloroglucinol had been consumed (TLC with petroleum ether/acetone 1:1 (v/v) + 1% HOAc) and was evaporated in vacuo. The residue was purified by flash chromatography (petroleum ether/acetone 2:1 v/v). Yield: 0.63 g (80%) of a yellow solid with a melting range 140-170  C. 1H- and 13C-NMR are too complicated, due to rotamers and keto-enol tautomers present in 2. HRMS (ESI+): calcd for [C44H63O12]+ 783.4314, found 783.4313. Immobilized Sebacoyl-myrtucommulone (9) EAH Sepharose 4B (10 mL of a suspension in 20% ethanol in water) was washed with distilled water (100 mL, pH adjusted to 4.5 with 0.1N HCl) and then with 0.5 M NaCl solution (800 mL) in a sintered glass filter G3. Sebacoyl-myrtucommulone (2) (156.4 mg, 0.2 mmol) was dissolved in 1,4-dioxane (10 mL) and distilled water (10 mL, pH adjusted to 4.5 with 0.1N HCl) was added (the dissolved sebacoyl-myrtucommulone partly precipitates). To this mixture, the washed EAH Sepharose 4B from above was added carefully prior to addition of EDCI (310.5 mg, 2 mmol), dissolved in a minimum amount of 1,4-dioxane/water (1:1 v/v). The reaction mixture was shaken carefully for 96 h and then filtered and washed successively with 1,4-dioxane/water (1:1 v/v), water and a solution of 20% ethanol in 0.5 M aq. NaCl solution. The immobilized sebacoyl-myrtucommulone (9) is suspended in ca. 20 mL of 20% ethanol in 0.5 M aq. NaCl solution and stored in the refrigerator. The combined filtrates from the washing steps were extracted with diethyl ether, the combined extracts were dried with MgSO4, filtered and evaporated to dryness. The recovered sebacoyl-myrtucommulone (2) (ca. 75-80 mg) corresponds to ca. 0.1 mmol immobilized sebacoyl-myrtucommulone (9), which is in accordance to 7-10 mol of NH2-groups per mL of EAH Sepharose 4B (1) (Instructions No.71-7097-00 AE from GE Life-Sciences (http://www.gelifesciences.com). Bis-pyrane Derivative (10) Sebacoyl-myrtucommulone (2) (200 mg, 0.25 mmol) was suspended in benzene (20 mL) and pTsOH (340 mg, 1.8 mmol) was added. The mixture was refluxed for 1 h. After cooling to room temperature, the solvent was evaporated and the residue was purified by flash chromatography with petroleum ether/acetone 5:1 (v/v). Yield: 170 mg (89%) of a white solid with mp 152-155  C. 1H-NMR (CDCl3, 400 MHz): = 4.50 (d, J = 3.8 Hz, 2H), 2.93 (t, J = 7.3 Hz, 2H), 2.34 (t, J = 7.4Hz, 2H), 1.95 (ddt, J = 10.9, 6.8, 3.9 Hz, 2H), 1.681.26 (m, 12H), 1.54 (s, 3H), 1.44 (s, 3H), 1.41 (s, 3H), 1.36 (s, 3H), 0.86 (d, J = 6.8 Hz, 6H), 0.79 (d, J = 6.9 Hz, 6H). 13C-NMR (CDCl3, 100 MHz): = 211.9, 201.6, 198.6, 177.7, 169.1, 153.1, 147.8, 111.4, 111.2, 108.7, 56.1, 47.7, 45.9, 35.3, 33.8, 32.7, 29.5, 29.4, 29.3, 29.2, 25.1, 25.0, 24.9, 24.8, 24.4, 19.3, 19.0. Immobilized bis-pyrane Derivative (11) EAH Sepharose 4B (10 mL of a suspension in 20% ethanol in water) was washed with distilled water (100 mL, pH adjusted to 4.5 with 0.1N HCl) and then with 0.5 M NaCl solution (800 mL) in a sintered glass filter G3. Bis-pyrane derivative (10) (148.9 mg, 0.2 mmol) were dissolved in 1,4-dioxane (10 mL) and distilled water (10 mL, pH adjusted to 4.5 with 0.1N HCl) was added (the dissolved sebacoylmyrtucommulone partly precipitates). To this mixture, the washed EAH Sepharose 4B from above was added carefully prior to addition of EDCI (310.5 mg, 2 mmol), dissolved in a minimum amount of 1,4-dioxane/water (1:1 v/v). The reaction mixture was shaken carefully for 96 h and then filtered and washed successively with 1,4-dioxane/water (1:1 v/v), water and a solution of 20% ethanol in 0.5 M aq. NaCl solution. The immobilized bis-pyrane derivative (11) is suspended in ca. 20 mL of 20% ethanol in 0.5 M aq. NaCl solution and stored in the refrigerator. The combined filtrates from the washing steps were extracted with diethyl ether, the combined extracts were dried with MgSO4, filtered and evaporated to dryness. The recovered bis-pyrane derivative (10) (ca. 70-75 mg) corresponds to ca. 0.1 mmol immobilized bis-pyrane derivative, which is in accordance to 7-10 mol of NH2-groups per mL of EAH Sepharose 4B (1) (Instructions No.71-7097-00 AE from GE Life-Sciences (http://www.gelifesciences.com). (Sebacoyl-n-butylamide)-myrtucommulone (12) Sebacoyl-myrtucommulone (2) (235 mg, 0.3 mmol) was dissolved in THF (10 mL) and n-butylamine (75 mL, 0.45 mmol) was added. To this mixture was added HOBT (61 mg, 0.45 mmol) and DCC (93 mg, 0.45 mmol) and the mixture was stirred for three days at room temperature. The solvent was removed in vacuo and the residue was purified by flash chromatography with petroleum ether/acetone 2:1 (v/v) as eluent. Yield: quantitative. 1H-NMR and 13C-NMR 1H- and 13C-NMR are too complicated, due to rotamers and keto-enol tautomers present in 12. MS (ESI+): 837.14 [M+H]+. Bis-pyrane-n-butylamide Derivative (13) (Sebacoyl-n-butylamide)-myrtucommulone (12) (126 mg, 0.15 mmol) was suspended in benzene (20 mL) and pTsOH (200 mg, 1.0 mmol) was added. The mixture was refluxed for 1 h. After cooling to room temperature, the solvent was evaporated and the residue was purified by flash chromatography with petroleum ether/acetone 4:1 (v/v). Yield: 115 mg (95%) of a white solid with mp 127130  C. 1H-NMR (CDCl3, 400 MHz): = 4.50 (d, J = 3.9 Hz, 2H), 2.93 (t, J = 7.3 Hz, 2H), 3.24 (t, J = 7.1 Hz, 2H), 2.14 (t, J = 7.6 Hz, 2H), 1.96 (ddd, J = 12.3, 10.5, 5.6 Hz, 2H), 1.64-1.23 (m, 16H), 1.56 (s, 3H), 1.45 (s, 3H), 1.41 (s, 3H), 1.36 (s, 3H), 0.92 (t, J = 7.3 Hz, 3H) 0.87 Cell Chemical Biology 24, 1–10.e1–e6, May 18, 2017 e5

Please cite this article in press as: Wiechmann et al., Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone, Cell Chemical Biology (2017), http://dx.doi.org/10.1016/j.chembiol.2017.04.008

(d, J = 6.8 Hz, 6H), 0.79 (d, J = 6.9 Hz, 6H). 13C-NMR (CDCl3, 100 MHz): = 211.8, 202.1, 198.3, 177.4, 168.9, 158.3, 147.6, 111.3, 111.0, 108.8, 56.0, 47.5, 45.9, 39.3, 36.8, 35.1, 32.6, 31.7, 29.4, 29.3, 29.2, 29.1, 25.1, 25.0, 24.8, 24.7, 24.3, 20.1, 19.3, 18.9, 13.7. QUANTIFICATION AND STATISTICAL ANALYSIS Results are expressed as mean S.E.M. of n observations, where n represents the number of experiments performed (see Figure Legends) on different days. Statistical analyses were performed using GraphPad InStat 3 (GraphPad Software, La Jolla, CA) conducting repeated measures one-way analysis of variance (ANOVA) followed by Bonferroni for selected pairs post-hoc test. Where appropriate, 2-tailed Student’s t test was applied. Statistical significance was defined as p < 0.05.

e6 Cell Chemical Biology 24, 1–10.e1–e6, May 18, 2017