Target Identification of Yaku'amide B and Its Two Distinct Activities

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Target Identification of Yaku’amide B and Its Two Distinct Activities against Mitochondrial FoF1-ATP Synthase Kai Kitamura, Hiroaki Itoh, Kaori Sakurai, Shingo Dan, and Masayuki Inoue J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b07339 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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Journal of the American Chemical Society

Target Identification of Yaku’amide B and Its Two Distinct Activities against Mitochondrial FoF1-ATP Synthase Kai Kitamura1, Hiroaki Itoh1, Kaori Sakurai2, Shingo Dan3, and Masayuki Inoue*1 1

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan. 3 Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan. 2

ABSTRACT: Yaku’amide B (1b) is a structurally unique tetradecapeptide bearing four ,-dialkylated ,-unsaturated amino acid residues. Growth-inhibitory profile of 1b against a panel of 39 human cancer cell lines is distinct from those of clinically used anticancer drugs, suggesting a novel mechanism of action. We achieved total syntheses of chemical probes based on 1b and elucidated the cellular target and mode of action of 1b. Fluorescent (3, 4) and biotinylated (5, 6) derivatives of 1b were prepared for cell imaging studies and pull-down assays, respectively. In addition, the unnatural enantiomer of 1b (ent-1b) and its fluorescent probe (ent-3) were synthesized for control experiments. Subcellular localization analysis using 3 and 4 showed that 1b selectively accumulates in the mitochondria of MCF-7 human breast cancer cells. Pull-down assays with 6 revealed FoF1-ATP synthase as the major target protein of 1b. Consistent with these findings, biochemical activity assays showed that 1b inhibits ATP production catalyzed by mitochondrial FoF1-ATP synthase. Remarkably, 1b was also found capable of enhancing the ATP hydrolytic activity of FoF1-ATP synthase. On the other hand, ent-1b inhibits ATP synthesis more weakly than 1b, and does not affect ATP hydrolysis, suggesting the stereospecific requirement for the characteristic multimodal functions of 1b. These findings corroborate that 1b causes growth arrest in MCF-7 cells by inhibiting ATP production and enhancing ATP hydrolysis, thereby depleting the cellular ATP pool. This study provides, for the first time, a structural basis for the design and development of anticancer agents exploiting the novel mode of action of 1b.

INTRODUCTION Peptides have profoundly influenced the modern pharmaceutical industry and contributed significantly to the advancement of biological and chemical science.1,2,3 Peptidic secondary metabolites in peptides contain not only the 20 proteinogenic amino acids, but also hundreds of different non-proteinogenic building blocks.4,5 The presence of non-proteinogenic amino acids confers diverse bioactivities as well as increased resistance to enzymatic degradation. Accordingly, these compounds have attracted increased attention as potentially promising drug leads, such as antibiotic, immunosuppressive, and anticancer agents. Exploiting the utility of bioactive peptides in the drug discovery processes first requires the identification of their cellular targets. 6 , 7 , 8 Such biological studies, however, are challenging when availability of the peptides from natural sources is scarce, or their synthetic accessibility is impeded due to their non-proteinogenic components. Yaku’amides A (1a) and B (1b) are structurally unique dehydropeptides (Figure 1) isolated as minute components (1a: 1.3 mg; 1b: 0.3 mg) from a rare deep-sea sponge, Ceratopsion sp.9 These compounds exert potent growth inhibitory activity against a P388 mouse leukemia cell line (IC50 = 0.88 nM for 1a and 0.51 nM for 1b). A JFCR39 cancer cell panel assay of 1a indicated the strong activity of 1a against a broad range of human cancer cell lines. Despite higher activity in the initial screening assay, 1b was not evaluated against the panel due to its more limited availability. Both of the 13-residue peptides are characterized by four -dialkylated ,-unsaturated

amino acids [(Z)-Ile-2/9, (E)-Ile-4 and Val-13], seven other non-proteinogenic -amino acids (L-OHIle-1, D-Val-5/11, Dallo-Ile-6, D-OHVal-7, L-OHVal-8, D-Ala-10), an N-terminal acyl group (NTA), and a C-terminal amine group (CTA). They differ by one amino acid with Gly-3 for 1a and L-Ala-3 for 1b. The four -dialkylated ,-unsaturated amino acids represent a particularly unusual structural feature among the peptide natural products,10,11 and significantly heighten synthetic challenges for their efficient assembly.12,13 The characteristic structures and biological activities of 1a and 1b prompted us to launch synthetic studies of these natural products. Total synthesis of 1a and 1b was accomplished by our group, and led to a revision of their proposed structures to those shown in Figure 1.14,15 In this synthesis, four dipeptides containing the ,-unsaturated moieties were constructed via Cu-mediated stereoselective enamide formation. 16 , 17 , 18 , 19 Subsequently, Boc-protected amino acids and enamides were condensed via stepwise elongation to produce 1a and 1b. The thus-established synthetic strategy enabled us to conduct structure-activity relationship (SAR) studies on biologically more potent 1b rather than 1a.20 Namely, 14 analogues of 1b that differ in their chiralities of -amino acids from 1b were prepared and biologically evaluated. Importantly, only the analogue sharing the same C-stereochemistries of the peptide sequence of 1b displayed potent growth inhibitory effect against P388 cells. These results suggested that 1b exerts its biological activity through stereospecific interactions with chiral biomolecules and not through non-

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specific interactions with achiral receptors such as plasma membrane. Z H N L O

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yaku'amide A (1a) NTA OHIle-1

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Ile-4 Val-5 allo-Ile-6 OHVal-7 OHVal-8

Ile-9 Ala-10 Val-11 Val-12

Val-13 CTA

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Figure 1. Structures of yaku'amides A (1a) and B (1b), propargyl yaku'amide B (2), fluorescent probes 3 and 4, biotinylated probes 5 and 6, enantiomeric yaku’amide B (ent-1b), and the enantiomeric fluorescent probe (ent-2). CTA = C-terminal amine group, NTA = N-terminal acyl group.

The detailed biological behavior of 1a/b had not been explored largely due to the scarcity of the naturally occurring samples and the lack of an appropriate functionality for structural derivatization. Because of the novel activities and structures, we anticipated that identifying the cellular targets of 1a/b should have implications for the development of new biological

tools and anticancer drug leads. To decipher the mechanism of actions of 1b, we herein prepared fluorescent (3, 4) and biotinylated (5, 6) derivatives of 1b as well as the unnatural enantiomer of 1b (ent-1b) and its fluorescent derivative (ent-3) through total syntheses (Figure 1). The development of a set of the fully synthetic chemical probes enabled us, for the first time,

2


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to reveal the cellular localization, the biological targets, and the function of 1b.

RESULTS AND DISCUSSION Cancer panel assay and cytostatic effect of yaku’amide B. The JFCR39 cancer panel screening of 1b was not conducted in previous studies.21,22 The total synthesis allowed us to apply 1b to the JFCR39 assay as an initial step to gain preliminary insight into yet unknown mode of action of 1b. In this assay, 1b potently inhibited the growth of a broad range of cancer cell lines (Figure S1). The 50% growth inhibition concentration (GI50) of 1b against 36 of 39 cell lines was below 100 nM (mean GI50 = 26 nM). In particular, MCF-7 and HBC-5 (breast), SF-295 (brain), NCI-H522 and DMS114 (lung), MKN-A and MKN-B (stomach), and PC-3 (prostate) cell lines exhibited higher susceptibility to 1b (GI50 values 10 nM). The COMPARE analysis for 1b revealed that the highest-ranked clinically-used anticancer drugs were paclitaxel (correlation coefficient value R = 0.632), docetaxel (R = 0.587), and vincristine sulfate (R = 0.551), all of which are tubulin binders. The modest correlations of the fingerprint of 1b with those of the known drugs (R < 0.70) made it difficult to predict the mode of action of 1b based on this analysis.23 In contrast, the correlation coefficient between 1a and 1b was 0.77, which reflected similar selectivity observed for 1a and 1b against the 39 cancer cell lines. This finding corroborated that structurally closely-related 1a and 1b share a similar biological mechanism. Furthermore, the data corresponding to median lethal dose (LC50) against all 39 cell lines could not be determined even at micromolar concentrations. These results strongly indicated that 1b arrested cell growth, but did not induce cell death, in the tested range of concentrations.

calcein. On the other hand, PI can only permeate the disrupted membrane of dead cells, where it intercalates double-stranded DNA and emits red fluorescence. Fluorescence microscopic and flow-cytometric analyses of the double-stained cells showed that PI-stained cells were absent after treatment with 1b (100 nM) for 48 hours. Hence, cell death was not induced by 1b at the tested concentration. In addition, our microscopic analysis indicated that 1b is unlikely to be a simple membrane disruptive agent, which perturbs the membrane integrity by non-selective actions.24,25 In parallel, we confirmed that 1b does not cause apoptotic cell death. Because mitochondrial membrane potential is diminished during apoptosis, we monitored the fluorescence from a mitochondrial membrane potential-dependent dye, tetramethylrhodamine methyl ester (TMRM) in MCF-7 cells (Figure 3).26,27 In a positive control experiment, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which is a known disrupting agent of the proton gradient across the inner mitochondrial membrane (IMM), indeed abolished the fluorescence from TMRM (Figure 3b).28,29 In sharp contrast, adding 1b (100 nM) did not change the fluorescence staining of MCF-7 cells for 3 or 24 hours (Figures 3c and 3d, respectively), indicating that 1b had no apparent effect on the mitochondrial membrane potential, and validating that 1b does not cause apoptosis.

Figure 3. Confocal fluorescence images of tetramethylrhodamine methyl ester (TMRM) obtained after treatment of MCF-7 cells with (a) vehicle control (DMSO) for 24 h, (b) a protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 10 M) for 24 h, (c) 1b (100 nM) for 3 h, and (d) 1b (100 nM) for 24 h, respectively. Scale bar represents 10 m.

Figure 2. Calcein/propidium iodide (PI) double staining. (a) Fluorescence images of MCF-7 cells stained with calcein/PI after treatment with 1b (100 nM) for 48 h. Scale bar represents 50 m. (b) Flow cytometric analysis of MCF-7 cells treated with (left) 1b (100 nM) for 48 h, (center) vehicle (DMSO) for 48 h, (right) 0.1% digitonin for 10 min as a control for dead cells. a.u. = arbitrary unit.

To further characterize if 1b is a cytostatic agent, fluorescent microscopic and flow-cytometric analyses were conducted using calcein-acetoxymethyl ester (calcein-AM) and propidium iodide (PI) staining (Figures 2a and 2b). The MCF-7 human breast cancer cell line was selected because it is one of the most 1b-sensitive cell lines in the JFCR39 panel. In this assay, membrane permeable calcein-AM is internalized into live cells, where it is hydrolyzed by intracellular esterase and is kept encapsulated as membrane-impermeable green-fluorescent

As shown in Table 1, cell cycle analysis by flow cytometry using HCT-116 cells found that 1b induces cell cycle arrest at the G1 phase in a dose-dependent fashion. Based on the panel, flow cytometry, TMRM, and cell cycle assays together, we concluded that 1b exerts a potent growth-inhibitory effect rather than a cytotoxic effect on the cancer cell lines through a specific mode of action.

Table 1. Cell cycle analysisa concentration of 1b (M) 0 (untreated control) 0.12 0.6 3

phase of the cell cycle (%) G1

S

G2/M

49 56 82 82

44 35 4 1

7 9 14 17

3



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a

The analysis was performed using HCT116 human colon cancer cells.

SCHEME 1. Total synthesis of yaku’amide B (1b) and 1b-based chemical probes (3–6, ent-1b, and ent-3)a

4


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a Boc = tert-butoxycarbonyl, COMU = (1-cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate, DMEDA = N,N-dimethylethylenediamine, DMF = N,N’-dimethylformamide, TBTA = Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, TFA = trifluoroacetic acid.

Design and synthesis of yaku’amide-based chemical probes. Next, we planned the synthesis and application of a set of 1bbased chemical probes to elucidate the molecular target and mechanism of 1b. A previous SAR study suggested that the stereostructure of the peptide sequence is essential for the potent biological activity of 1b.20 Thus, the NTA and CTA moieties were selected as sites of functionalization. Since a prerequisite of the chemical probes is to retain the biological activity of the parent natural product, their terminal structures would need to be modified without changing the original physicochemical properties of 1b.30 Probes 3 and 4 with a fluorescent group at the N- and Ctermini, respectively, were designed to investigate cellular uptake and distribution by live cell imaging (Figure 1). The hydrophobic NTA moiety of 1b was replaced with hydrophobic 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) group31,32 and an alkyl linker of 3 to prevent attenuation of its activity. On the other hand, 4 was designed to possess the BODIPY fluorophore with a hydrophilic polyethylene glycol (PEG) linker. Since the CTA moiety is presumably of ionic character with the protonated dimethylamine group under physiological conditions, the PEG linker was employed to endow hydrophilicity. To synthesize 4, derivative 2 possessing an alkyne at its C-terminus was to be prepared and derivatized by Cu-mediated azide-alkyne cyclization33 to link the yaku’amide core to the PEG-attached BODIPY fluorophore. Biotinylated probes 5 and 6 were also designed for affinity pull-down assays to isolate the unknown target proteins of 1b using the specific interaction between biotin and avidin (Figure 1). To maintain the hydrophilic nature of biotin and the hydrophobic nature of NTA, we decided to connect the two structures by a linker comprising both hydrophilic PEG and hydrophobic alkyl chains. Two different lengths of linkers were adopted for 5 and 6. This was because the length of the linker would affect the physicochemical properties of the biotin probe and steric repulsion between the target protein and avidin, but the appropriate linker length could not be estimated a priori. In addition, enantiomers of 1b (ent-1b) and fluorescent probe 3 (ent-3) were designed to distinguish 1b-specific and nonspecific functions in the assays. An enantiomer is in general a suitable negative control in biological assays, because it has the same molecular behavior as the natural product except for chiral recognition by the target molecules, such as proteins.34 We first prepared 21a and 21b as precursors for the synthesis of chemical probes 3–6 (Scheme 1a). While the synthesis of 21a was previously described in the synthesis of 1b,14,15 its alkyne counterpart 21b needed to be constructed by replacing methyl amine 12a with propargyl amine 12b.35 Both syntheses started from alkenyl iodide 7. Hydrolysis of 7, condensation with 8a, and subsequent Cu-mediated coupling with amide 10 afforded dipeptide unit 12a.36 Alternatively, for the synthesis of 12b, introduction of CTA unit 8b was performed after Cumediated coupling to avoid undesired coordination of the alkyne moiety with the Cu species. The peptide was elongated from the thus-obtained 12a and 12b by repeated steps of Bocremoval and amide coupling using eight building blocks 13–19 to produce intermediates 20a and 20b in 7.0% and 1.7% yields in 19 steps, respectively. The Boc groups of 20a and 20b were

then removed by TFA to deliver free N-terminal amines 21a and 21b, respectively. N-terminal substituted probes 3, 5, and 6 were derivatized from 21a under conditions similar to those used for the transformation of 21a and 22 into 1b. Specifically, BODIPYsubstituted alkanoic acid 23 was condensed at the N-terminal amine of 21a in the presence of (1-cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate (COMU)37 and 2,4,6-collidine, giving rise to 3 in 65% yield in two steps. For the synthesis of 5 and 6, amine 21a was treated with the biotin tags 24 and 25 using COMU and 2,4,6-collidine to produce 5 and 6 in 50% and 45% yields in two steps, respectively. Prior to preparation of the C-terminal substituted probe 4, propargylic 2 was synthesized from 21b by condensing carboxylic acid 22 by the action of COMU and 2,4,6-collidine. BODIPY-substituted alkyl azide 26 was then conjugated with the C-terminal alkyne of 2 via Cu-mediated azide-alkyne cyclization. Standard CuSO4 and sodium ascorbate conditions, however, resulted in partial elimination of the N-propargyl moiety probably due to the Lewis acidity of Cu(II). To maximize the desired triazole formation, tris[(1-benzyl-1H-1,2,3-triazol4-yl)methyl]amine (TBTA)38 was added as a ligand for stabilization of the catalytically active Cu(I). As a result, C-terminal BODIPY-functionalized 4 was obtained in 28% yield in three steps. Enantiomeric yaku’amide B (ent-1b) and its fluorescent derivative ent-3 were constructed via the same route by employing enantiomeric building blocks (Scheme 1b). First, ent-8a, ent10, ent-15, ent-16, ent-18, ent-19, and ent-22 were synthetically prepared. Then, ent-12a was built from 7 through coupling reactions with ent-8a and ent-10. Next, ent-20a was elaborated from ent-12a by eight cycles of Boc-removal and amidation using ent-13–ent-19 in 1.8% overall yield from 7 (19 steps). TFAmediated detachment of the Boc group of ent-20a in turn furnished free amine ent-21a. Finally, installation of the enantiomeric NTA (ent-22) and BODIPY (23) structures at the N-terminus of ent-21a gave rise to ent-1b and ent-3 in 52% and 62% in two steps, respectively. Overall, the successful syntheses of the six chemical probes (3–6, ent-1b and ent-3) demonstrated the robustness of the established strategy for the total synthesis of 1b. Growth inhibitory activity against cancer cells. To evaluate the suitability of the functionalization for subsequent biological studies, growth inhibitory activities of all the synthetic peptides against MCF-7 cells were assessed (sulforhodamine B (SRB) assay, Table 2).39 The GI50 number of the parent 1b was 10.7 nM, confirming its potent growth inhibitory activity. In contrast, ent-1b exhibited a 3-fold increase in the GI50 value (29.2 nM) compared with 1b. The 3-fold difference was attributable to the existence of the cellular chiral target of 1b. Incorporating BODIPY at the N-terminus conferred a 20fold higher GI50 value (3, 202 nM), but the potent growth inhibitory activity was retained despite this structural alteration. The GI50 value of ent-3 was 1260 nM – 6-fold larger than that of 3. Comparing the results of 1b, 3, and their enantiomeric counterparts, reversal of the chirality had a consistent negative effect on the growth inhibition activity. On the other hand, the GI50 of 2 was 10.2 nM, suggesting that changing the methyl group

5



to a propargyl group at the C-terminus did not affect the activity. Similarly, introducing the BODIPY group at the C-terminus had a negligible impact on the GI50 value (4, 12.6 nM), and biotin-labeled 6 maintained a submicromolar GI50 value (GI50 = 149 nM). Alternatively, 5 showed a complete loss of activity (GI50 = >9000 nM), indicating that a shorter PEG linker is detrimental to the growth inhibition of 1b when labeled with biotin. Like 1b, none of the derivatives exhibited cytotoxicity, or provided LC50 values at the tested concentrations (Figure S2). This observation was in accordance with the results of flowcytometric analysis and fluorescence imaging of TMRM (Figures 2 and 3). Table 2. Growth inhibitory activity of yaku’amide B (1b) and 1b-based chemical probes compounds yaku’amide B (1b) ent-1b 2 3 ent-3 4 5 6

GI50 (nM)a MCF-7 cells

MCF-10A cells

10.7 ± 3.1b 29.2 ± 2.9c 10.2 ± 2.9c 202 ± 8.0c 1260 ± 46c 12.6 ± 4.4c >9000c 149 ± 54c

37.1 ± 7.8c 109 ± 6.1c – – – – – –

aConcentrations

that cause 50% growth inhibition (GI50) were determined by sulforhodamine B (SRB) assays performed on MCF-7 and MCF-10A cells. bThe value is displayed as mean ± SD of six independent experiments. cThe values are displayed as mean ± SD of three independent experiments.

Moreover, we evaluated the cancer cell selectivity of 1b and ent-1b using an MCF-10A immortalized human breast epithelial cell line as a model of normal cells (Tables 2 and S2). Both 1b and ent-1b exhibited growth inhibitory activity toward MCF-10A cells rather than cytotoxicity (Figure S3). The GI50 values of 1b and ent-1b against MCF-10A cells were determined to be 37.1 nM and 109 nM, respectively, and the higher value of ent-1b compared with that of 1b again reflected the importance of the chirality of the molecules. Significantly, the 3-fold differences in the potency of 1b against MCF-7 and MCF-10A cells clarified that 1b was more selective against cancer cells, which is a potentially promising feature as a seed compound of anticancer agents. Mitochondrial localization of yaku’amide B. To investigate the cellular sites of action of 1b, fluorescence imaging analysis in MCF-7 cells was carried out by confocal microscopy using fluorescent probes 3, ent-3, and 4 in the presence of specific organelle markers. Preliminary studies indicated that the fluorescence intensity of 3 reached a plateau after 8 hours (Figure S4). Thus, the probes were incubated in MCF7 cells for 3 or 8 hours. Colocalization was quantitatively assessed using Pearson’s correlation coefficient (R)40 calculated in Coste’s quantitative analysis.41 We identified that 1b has a dimethyl amine moiety at its Cterminus, which is a lysosome-targeting functionality found in a lysosome-specific marker, LysoTracker Red DND-99 (Figure 4e). Basic compounds localize to acidic lysosomes with the pH

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differences as a driving force. Therefore, we initially evaluated the co-localization of 3 at 100 nM with LysoTracker Red DND99. Contrary to our expectation, 3 did not accumulate in lysosomes (Figure 4a, R = 0.069). Instead, the fluorescence signal of 3 (shown in green) exhibited excellent overlap with that of MitoTracker Red CMXRos (Figure 4e) (shown in red), demonstrating that 3 selectively accumulates in mitochondria (Figure 4b, R = 0.87). Moreover, more potent fluorescent probe 4 localized in mitochondria at a lower concentration (20 nM) than 3 with a high R value (Figure 4c, R = 0.88). These findings raised the possibility that 1b inhibits cell growth by targeting mitochondria. Comparative analysis of the subcellular localizations of 3 and ent-3 showed a lower correlation coefficient for ent-3 (Figure 4d, R = 0.49), indicating that it localizes to mitochondria less selectively, and implying that chiral recognition by a mitochondrial target of 1b underlies the superior growth inhibitory potency and localization selectivity of 3 compared with ent-3. Since peptides are in general not readily cell-permeable, we wondered if 4 might be internalized into cells by active transport pathways such as endocytosis. We thus studied the mode of cell internalization for the fluorescent probe 4 under conditions that are known to block active transport. Interestingly, cellular uptake of 4 was not inhibited by ATP depletion conditions or by other endocytosis inhibitors (Figure S5), 42 showing that 4 is internalized into cells via a spontaneous diffusion pathway. This unusual behavior for a large peptide should be functionally coded by the unique peptide sequence of yaku’amide B, but further SAR studies are required to determine the responsible structural factors. Identification of subunits  and  of FoF1-ATP synthase by affinity pull-down from MCF7 cell lysate. To explore the protein targets of 1b, we utilized biotinylated probes 5 and 6 immobilized on NeutrAvidin-coated agarose resins for the affinity pull-down analysis. In doing so, a biotin derivative conjugated to the longer PEG linker with no yaku’amide structure was prepared as a control probe (27, Figure 5a). The probe-immobilized resins were incubated in the MCF-7 whole cell lysate, and then washed to remove nonspecific proteins. The specifically bound proteins were subsequently eluted and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis. As a result, doublet bands around 50 kDa (indicated by arrow heads, Figure 5b, lane 1, and Figure 5c, lane 1) were detected by silver staining for probe 6 and not for 27 (Figure 5b, lane 3, and Figure 5c, lane 8). Consistent with the SRB assay data, inactive probe 5 with the shorter PEG linker did not provide these protein bands (Figure 5b, lane 2). It is conceivable that the linker length critically affected the target binding efficiency, which resulted in the loss of growth inhibitory activity of 5. Next, the specificity of yaku’amide B-binding activity of the proteins detected as the doublet bands was successfully demonstrated by competition experiments using increasing concentrations of the parent 1b (Figure 5c, lanes 2–4). Interestingly, ent-1b was also able to interfere with the enrichment of the 50 kDa doublet proteins in a dose-dependent manner, although to a lesser degree than 1b (Figure 5c, lanes 5–7). The weaker inhibitory activity of ent1b is in accordance with its less potent activity in the SRB assay. Furthermore, we confirmed the reversible nature of binding between 1b and the the 50 kDa doublet proteins by their elution from 6-immobilized resins with excess amounts of 1b (Figure S6).

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Figure 4. Subcellular localization analysis of fluorescence probes with organelle markers in MCF-7 cells. (a) Probe 3 (100 nM) with the lysosome marker LysoTracker Red DND-99. (b) Probe 3 (100 nM) with MitoTracker Red CMXRos. (c) C-terminus modified 4 (20 nM) with mitochondria marker MitoTracker Red CMXRos. (d) Enantiomer of 3 (ent-3, 100 nM) with MitoTracker Red CMXRos. Bright field, fluorescence probes, organelle markers, and merged images are shown. Probes were incubated for 3 h for 4 (c) and 8 h for 3 and ent-3 (a, b, and d). Subcellular regions colored in yellow in the merged images indicate co-localization of BODIPY probes and organelle indicators. The nuclei were stained with Hoechst 33342. Scale bar represents 10 m. R values represent Pearson’s correlation coefficients calculated from the green and red fluorescence in each merged image for (a)–(d). (e) Structures of organelle markers.

To identify the target proteins of 1b, the pair of 50 kDa protein bands were cut out and separately subjected to in-gel digestion followed by LC-MS/MS sequencing analysis. The findings revealed that the top band represented subunit  (ATP5A1) and the bottom band corresponded to subunit (ATP5B) of FoF1ATP synthase (Figure S7 and Table S3). For further validation of these results, the proteins pulled down by 6 were analyzed by western blotting using anti-ATP5A1 and anti-ATP5B antibodies. In the anti-ATP5A1 blot, a single band corresponding to the top band of the 50 kDa doublet bands was specifically detected only when using 6, and not when using control probe 27 (Figure 5d left, lanes 1 and 2). Similarly, in the anti-ATP5B blot, a single band was observed for the bottom band with 6 and not with 27 (Figure 5d right, lanes 1 and 2). FoF1-ATP synthase is an IMM-associated protein. 43 Accordingly, we also performed an affinity pull-down analysis with 6 using mitochondrial lysate prepared by subcellular fractionation, and detected the same 50 kDa doublet bands from this fraction (Figure S8). These outcomes agree well with our findings in the intracellular localization analysis that probe 3, a fluorescent analogue of 1b, targets mitochondria.

Figure 5. Affinity pull-down assays using resin-immobilized biotinylated probes. (a) Structure of control probe 27. (b) Affinity pull-down from whole-cell lysate of MCF-7 cells using probes 5 and 6 visualized by silver staining. (c) Competitive pull-down assay using 1b and ent-1b (25, 2.5, and 0.25 M) visualized by silver staining. The bands corresponding to subunits  (ATP5A1) and (ATP5B) ofFoF1-ATP synthase are indicated by arrowheads. (d)

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Target validation by western blotting analysis using anti-ATP5A1 and anti-ATP5B antibodies.

Inhibition of ATP production in mitochondria and enhancement of ATP hydrolysis of purified FoF1-ATP synthase by yaku’amide B. FoF1-ATP synthase, also known as respiratory chain Complex V, is responsible for ATP production during oxidative phosphorylation in mitochondria, which are essential energy-producing organelles (Figure 6a).44,45 This membrane protein is an extremely large (600 kDa) complex comprising an Fo part in the membrane and an F1 head in the matrix. While the Fo domain mediates the translocation of protons across the IMM, the F1 domain constituted by the subunits  and  catalyzes both ATP synthesis and hydrolysis. Hence, FoF1-ATP synthase can produce ATP in response to the electrochemical proton gradient across the IMM, and can hydrolyze ATP as a reverse reaction to generate a membrane potential. We first evaluated the ability of 1b and ent-1b to perturb ATP synthesis using a coupled enzymatic assay (Figures 6a and 6b).46,47 In this assay, MCF-7 cells are permeabilized with digitonin to give a solution of intact mitochondria, which is applied to an enzymatic ATP-detection system composed of glucose, hexokinase, glucose-6-phosphate dehydrogenase, and NADP. Because the production of NADPH is coupled with ATP consumption, the amount of mitochondria-derived ATP can be assessed by monitoring the absorption of NADPH at 340 nm. As a control inhibitor, we used oligomycin A, a widely used inhibitor of FoF1-ATP synthase (Figure 7).48,49 Titration of 1b, ent1b, and oligomycin A in this assay system provided EC50 values of 15.5 ± 6.5 nM (pink), 135 ± 11 nM (cyan), and 1.23 ± 0.39 nM (black), respectively (Figure 6c). These data showed that both 1b and ent-1b have strong inhibitory effects on ATP production, whereas 1b is 9-fold more potent than ent-1b. Importantly, the EC50 values of 1b and ent-1b correlated well with their corresponding GI50 values (10.7 nM for 1b; 29.2 nM for ent-1b), strongly suggesting that the cell growth inhibition activity caused by 1b and ent-1b is mainly attributable to the inhibition of ATP production by binding 1b and ent-1b to FoF1ATP synthase. Next, we investigated the effect of 1b on the individual protein complexes involved in oxidative phosphorylation by biochemical assays. Each of the Complexes I, IV, and V (FoF1ATP synthase) was purified from bovine heart mitochondria by immunoprecipitation, 50 , 51 and their activities were evaluated with spectrophotometric assays. The coupled function of Complexes II and III was assessed by monitoring the reduction of cytochrome c using intact mitochondria.49,52 As expected, 1b had no effect on Complexes I, II, III, and IV at the tested concentrations (10–100 nM, Figures S9–S11). The purified FoF1ATP synthase catalyzes ATP hydrolysis, which is coupled with pumping of protons (Figure 6d). In the ATP hydrolysis activity assay with FoF1-ATP synthase, pyruvate kinase uses ADP to convert phosphoenolpyruvate to pyruvate, which in turn serves as a substrate for lactate dehydrogenase to oxidize NADH to NAD+ (Figure 6e). An increase in NAD+ can be calculated by

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a decrease in the UV absorption of NADH. Remarkably, 1b was found to dose-dependently enhance ATP hydrolysis by FoF1-ATP synthase up to 300% compared to the background hydrolysis of ATP (Figure 6f, pink). As exemplified by oligomycin A (black), many inhibitors of FoF1-ATPsynthase are known to inhibit both ATP synthesis and hydrolysis.53 To our knowledge, 1b is the first organic compound that increases ATP hydrolysis activity of the mammalian FoF1-ATP synthase.54,55 It is important to note that ent-1b had no effect on ATP consumption despite displaying inhibitory activity on ATP production by FoF1-ATP synthase (Figure 6f, cyan). Therefore, these findings together revealed that 1b has two distinct modes of function: suppressing ATP synthesis and enhancing ATP hydrolysis. Exertion of these functions should both lead to depletion of the cellular ATP pool. Mitochondrial FoF1-ATP synthase generates most cellular energy in the form of ATP to sustain cellular activity. It has been reported that the cell cycle progression of tumor cells depends on the cellular ATP level and hence a severe drop in the ATP concentration would arrest cells. 56 , 57 Therefore, downregulation of ATP through FoF1-ATP synthase is likely the mechanism by which 1b causes cell growth inhibition. We speculate that selective regulation of the ATP production/hydrolysis by 1b might originate from perturbation of ATP or ADP binding/release at the F1 catalytic domain, although the binding sites of 1b remain to be determined. Enantiomers of bioactive natural products usually exhibit no biological activity or different activities when chiral targets are involved.58,59 Intriguingly, ent-1b interacted with the same molecular targets of naturally occurring 1b, leading to inhibition of ATP production. Based on the observation that ent-1b competed with 1b in the pull-down assay, 1b and ent-1b have a common binding site on FoF1-ATP synthase, suggesting that these compounds might have partially similar three-dimensional structures. The striking differences in the activity of 1b and ent-1b lie in their effects on the ATP hydrolysis activity of FoF1-ATP synthase; whereas 1b remarkably enhanced this activity, ent-1b had no effect. It is possible that activation of ATP hydrolysis by FoF1-ATP synthase may require more stringent stereospecificity compared with the inhibition of ATP synthesis. Many natural products (e.g., oligomycin A,48,49 apoptolidin,60,61 efrapeptin,62,63 and resveratrol64,65) act as inhibitors of the mammalian FoF1-ATP synthase.53 These compounds show cytotoxic or cytostatic activities depending on their structure and on the cell lines. Thus, the cellular responses to these inhibitors appear complex and are not well understood. Nonetheless, all these compounds are reported to inhibit both the ATP synthesis and hydrolysis activities of FoF1-ATP synthase. Among the FoF1-ATP synthase-targeting natural products, 1b is unique because it not only inhibits ATP synthesis, but it also enhances ATP hydrolysis activity of the mammalian FoF1-ATP synthase. Based on its unique functions toward ATP synthase and its potent antiproliferative activity against diverse cancer cell lines, 1b likely belongs to a novel class of anticancer agents.66

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Figure 6. Inhibition of ATP production and enhancement of ATP hydrolysis by 1b. (a) Schematic representation of ATP synthesis by FoF1ATP synthase. IMM = inner mitochondrial membrane. (b) Schematic representation of the enzymatic system for the ATP production assay. G6DP = glucose-6-phosphate dehydrogenase. (c) Dose-dependent inhibition of ATP synthesis of permeabilized MCF-7 cells by 1b and ent1b. The ATP production rate was normalized against the rates obtained by adding vehicle (DMSO) as 100% and 5 M oligomycin A as 0%. Data are displayed as mean ± SD. (d) Schematic representation of ATP hydrolysis by FoF1-ATP synthase. (e) Schematic representation of the enzymatic system for monitoring ATP hydrolysis using immunoprecipitated FoF1-ATP synthase. LDH = lactate dehydrogenase. (f) Enhancement of ATP hydrolysis induced by 1b. The ATP hydrolysis rate was normalized against the rate obtained by adding vehicle (DMSO) as 100%. Data are displayed as mean ± SD.

Figure 7. Structure of FoF1-ATP synthase inhibitor oligomycin A.

CONCLUSION In conclusion, we synthesized a series of chemical probes of a highly unsaturated tetradecapeptide yaku’amide B (1b), and uncovered the cellular target and biological mode of action of 1b for the first time. By applying our previously established route to 1b, fluorescent (3, 4), biotinylated (5, 6), and enantiomeric (ent-1b, ent-3) derivatives were assembled for cell imaging studies, pull-down assays, and control experiments, respectively. The fingerprint based on the GI50 values of 39 cancer cell lines indicated that 1b has a mode of action distinct from clinically used anticancer drugs. The antiproliferative assay also suggested that 1b has potent growth inhibitory activity rather than cytotoxicity in cancer cells. Subsequent cellular

localization analyses revealed that fluorescent probes 3 and 4 accumulated in the mitochondria of MCF-7 cells via a passive internalization pathway. The biotinylated probe 6 in turn achieved specific pull-down of two proteins, which were revealed as mitochondrial FoF1-ATP synthase by LC-MS/MS analysis. Furthermore, enzymatic assays clarified that 1b inhibited ATP production in mitochondria, supporting the notion that 1b targets FoF1-ATP synthase. Intriguingly, 1b markedly enhanced the ATP hydrolysis of purified FoF1-ATP synthase. These unprecedented multimodal functions of 1b against FoF1ATP synthase potentially cooperate together to deplete cellular ATP, thereby exerting a potent growth inhibitory effect. In contrast, ent-1b with 3-fold less growth inhibitory activity inhibited ATP production in the mitochondria, and had no effect on ATP hydrolysis. It is possible that 1b and ent-1b bind to FoF1-ATP synthase at a similar site, yet they differentially modulate its cellular functions. Mitochondria are energy-producing organelles with essential functions in cell biology, and promising therapeutic targets.67,68 Because of the unique mitochondrial FoF1-ATP synthase-regulating activity found in this study, 1b and its derivatives offer promising platform structures for the development of novel FoF1-ATP synthase modulators and anticancer agents. Moreover, the present work demonstrated the crucial role of a robust synthetic route for preparing the architecturally complex chemical probes of scarce, but highly valuable, natural products,

9



and for elucidating a new mode of action of bioactive compounds.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Supplementary Figures and Tables, experimental procedures, characterization data, NMR spectra, and HPLC charts of newly synthesized compounds.

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

ORCID

This research was financially supported by Grant-in-Aids for Scientific Research (S) (JSPS, 17H06110) to M.I. and K.S., and for Young Scientists (B) (JSPS, 17K15421) to H.I. The JFCR39 cancer cell panel assays were performed by Molecular Profiling Committee, and supported by a Grant-in-Aid for Scientific Research on Innovative Areas (MEXT, 16H06276). We gratefully acknowledge Regina M. Kanada-Sonobe (Eisai Co., Ltd. Tsukuba Res. Labs.) for conducting the cell cycle analysis and the activity assays of Complexes I–IV, and Dr. Tomomi Goto (The University of Tokyo) for conducting preliminary experiments for preparation of ent-1b.

Hiroaki Itoh: 0000-0002-1329-6109 Masayuki Inoue: 0000-0003-3274-551X

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AUTHOR INFORMATION Corresponding Author *[email protected].

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Target Identification of Yaku'amide B and Its Two Distinct Activities

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