Novel Atg4B inhibitors potentiate cisplatin therapy in lung cancer cells through blockade of autophagy

Computational Toxicology 12 (2019) 100095

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Novel Atg4B inhibitors potentiate cisplatin therapy in lung cancer cells through blockade of autophagy

T

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Satoshi Endoa, , Mai Uchiboria, Miho Suyamaa, Mei Fujitaa, Yuki Araia, Dawei Hub, Shuang Xiab, Biao Mac, Aurangazeb Kabirc, Yuji O. Kamatarid, Kazuo Kuwatac, Naoki Toyookab, Toshiyuki Matsunagaa, Akira Ikaria a

Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan Graduate School of Innovative Life Science, University of Toyama, Toyama 930-8555, Japan c United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan d Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Atg4B Autophagy inhibitor Cisplatin Overcoming chemoresistance

Autophagy induction in tumors is implicated in tumor malignancy and acquisition of anticancer-drug resistance, and hence, autophagy inhibitors are expected as novel anticancer agents. Atg4B, a cysteine proteinase, is a key enzyme in autophagy process, and considered as one of desirable targets for development of selective autophagy inhibitors. In this study, to search novel inhibitors of Atg4B, we employed the virtual screening approach and differential scanning fluorimetry analysis, and found two compounds 1 (2-methyl-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-6-carboxylic acid) and 17 (methyl 4-({2-[(4,6-diaminopyrimidin-2-yl)sulfanyl]propanoyl} amino)benzoate), showing IC50 values of 10 and 12 µM, respectively, in in vitro LC3 cleavage assay of Atg4B. Molecular docking analysis suggested that 1 and 17 bind to the catalytic domain of Atg4B. The treatments with 1 and 17 significantly suppressed autophagy induction to lung cancer A549 cells by tamoxifen and cisplatin, in which their cotreatments promoted tamoxifen- and cisplatin-induced apoptotic cell death. Moreover, the autophagy suppression by 1 and 17 remarkably restored the sensitivity to cisplatin in cisplatin-resistant A549 cells. Thus, 1 and 17 represent promising leads for future development of potent and selective Atg4B inhibitors that augment existing chemotherapy efficacy.

1. Introduction Autophagy is a tightly orchestrated process that sequesters proteins, and damaged or aged organelles in double-membrane vesicles called autophagosomes, which ultimately fuse to lysosomes, leading to the degradation of the sequestered components [1]. The recycling capacity of autophagy regulates cellular homeostasis in both physiologic and pathophysiologic contexts. Dysregulated autophagy has been implicated in several diseases, including cancer [2]. It has been suggested that autophagy induction in tumors is involved in tumor malignancy and acquired anticancer-drug resistance by suppressing accumulation of abnormal proteins, resultant oxidative stress, and enhancement of DNA destabilization [3,4]. Pancreatic cancer primary tumors and cell

lines also show elevated autophagy, and inhibition of autophagy leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models [5]. Therefore, autophagy inhibitors are expected as candidates of novel anticancer agents. In fact, clinical trials of autophagy inhibitors such as chloroquine and hydroxychloroquine against metastatic pancreatic cancer and non-small cell lung cancer are underway in the United States [6]. Phosphoinositide 3kinase (PI3K) inhibitors such as wortmannin (Wo) and 3-methyladenine (3MA) are also used as autophagy inhibitors [7,8], but it is expected that clinical application of the PI3K inhibitors will be difficult, because the PI3K signal is deeply involved in survival of both normal and cancer cells. In development of anticancer drugs targeting autophagy in cancer cells, it is considered that it is very important to selectively inhibit

Abbreviations: 3MA, 3-methyladenine; A549/CDDP, CDDP-resistant A549 cells; BSA, bovine serum albumin; CDDP, cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle medium; DMSO, dimethyl sulfoxide; DPBS, Dulbecco’s phosphate buffered saline; DS, docking simulation; DSF, differential scanning fluorimetry; GST, glutathione-S-transferase; MD, molecular dynamics; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; QC, quantum chemistry; RT, room temperature; TBS-T, Tris-buffered saline with Tween-20; Tm, thermal denaturation midpoint; Wo, wortmannin ⁎ Corresponding author. E-mail address: [email protected] (S. Endo). https://doi.org/10.1016/j.comtox.2019.100095 Received 22 November 2018; Received in revised form 4 June 2019; Accepted 26 June 2019 Available online 27 June 2019 2468-1113/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Molecular machinery of autophagosome formation. Cysteine protease Atg4B converts nascent LC3 (pro-LC3) into LC3-I, which exposes its C-terminal glycine residue essential for subsequent binding to Atg7 and Atg3. LC3-I is then converted to LC3-II, which was formed by an amide bond with an amino group of the hydrophilic head of phosphatidylethanolamine (PE) through binding reactions, in which Atg7, Atg3 and Atg12/Atg5/Atg16L1 complex are used as E1 ubiquitinactivating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase, respectively. LC3-II functions as a part of the membrane component of autophagosomes and is recycled through its delipidation by Atg4B.

(DeLano Scientific, San Carlos, CA) [22,23] and integrates three main GUIs: 1) Docking simulation (DS), a GUI for DSs and large-scale screening via AutoDock Vina [24]; 2) Molecular dynamics (MD), a GUI for obtaining the stable structure of protein–ligand complexes and the binding free energy in MD simulations via the Amber package [25]; and 3) Quantum chemistry (QC), a PAICS GUI for optimizing the MC molecular structure by QC calculations [26]. The structure of Atg4B was picked up from the crystal structure of human Atg4B-LC3(1–124) complex (PDB ID: 2Z0E). The ligand database was obtained from Asinex subset (~360,000 compounds) of LigandBox (Asinex Ltd, Moscow, Russia) [27]. The docking area covered the binding area of Atg4B with LC3. In the parameter setting of AutoDock Vina, the exhaustiveness value and maximum number of generated binding modes were set to 8 and 9, respectively, the maximum difference between energies of the best and the worse binding modes was as large as 4 kcal/mol, and other optional settings were set to their default values. The inhibitor-docked models were generated using PyMOL. The compounds 1–18 were obtained from Asinex Ltd.

autophagy [6]. In autophagy, the biogenesis and maturation of autophagosomes are controlled by class III PI3K complex, Unc51-like kinase complex and two ubiquitin-like conjugating systems including autophagy-related gene (Atg) proteins, Atg12/Atg5/Atg16L1 and Atg4B/microtubule-associated protein 1 light chain 3 (LC3) [9] (Fig. 1). It has been reported that Atg5 gene deficiency and loss-of-function mutation (Cys74Ala) of Atg4B gene hamper autophagosome closure and consequently suppress autophagy [10,11]. Therefore, inhibition of autophagosome formation is thought to be a promising strategy to suppress enhanced autophagy in cancers. Among Atg proteins, Atg4, the sole protease, functions as an essential factor in the LC3 conjugation system (Fig. 1), and thereby has been regarded as a potential therapeutic target for cancers [12]. The crystal structures of Atg4B and Atg4B-LC3 complex were resolved [12,13], and some small compounds were identified as Atg4B inhibitors by in silico screening based on the structure of Atg4B. Such inhibitors are NSC185058 (N-pyridin-2-ylpyridine 2-carbothioamide) [14] and LV-320 (a styrylquinoline derivative) [15], of which NSC185058 shows antitumor effects on osteosarcoma in vivo. However, the inhibitory potencies of NSC185058 and LV-320 are weak with IC50 values of 51 and 24.5 µM, respectively, for the Atg4B activity. More potent Atg4B inhibitors with IC50 values of 0.1–4.4 µM were found by highthroughput screening and subsequent chemical modification. They are Z-L-Phe-chloromethyl ketone [16], fluoromethylketone derivatives [17,18], LC3 peptide mimics [19] and benzotropolone derivatives [20], but have following issues for their use in vivo: irreversible binding to Atg4B [16–19], lack of selectivity [18], induction of autophagy [18] or cytotoxicity [16]. Therefore, it would be desirable to identify reversible, potent and selective Atg4B inhibitors, which are not cytotoxic. In order to identify potential leads for new Atg4B inhibitors, we adapted a virtual screening-based approach, and discovered two small molecules, 1 and 17, as potent inhibitors of Atg4B. In addition to biochemical and cellular assays of their inhibitory potencies, the two compounds were evaluated in terms of their combination effects on efficacy of cisplatin (CDDP) to lung cancer A549 cells and their effects on CDDP resistance of the cells.

2.2. Construction of Atg4B and LC3-GST expression plasmids The cDNA for human Atg4B (NCBI accession no. NM_013325.4) was isolated from a total RNA sample of human brain (Takara Bio Inc., Otsu, Japan) by reverse transcription-PCR. The RNA and DNA techniques were performed as described by Sambrook et al. [28]. PCR was performed using KOD FX neo DNA polymerase (Toyobo, Osaka, Japan) and primers, 5′-CCCCCATATGATGGACGCAGCTACTCTGA-3′ (forward) and 5′-CCCCGTCGACTCAAAGGGACAGGATTTCAA-3′ (reverse), which contain underlined NdeI and SalI sites, respectively. The PCR products were purified, and ligated into the pET28a vectors (Novagen, Madison, WI) that had been digested with NdeI and SalI. The insert of the cloned cDNA was sequenced by using a Beckman CEQ8000XL DNA sequencer, to confirm that the full-length sequence of Atg4B fused to the N-terminal 6-His tag is encoded. For the in vitro cleavage assay, human LC3 cDNA was constructed with GST tags fused to the C-terminal end except for a stop codon. At first, the cDNA for GST was amplified from pGEX-2T vector (Pharmacia LKB Biotechnology, Piscataway, NJ) using KOD FX neo DNA polymerase and primers, 5′-CCCCGAATTCGATCTGGTTCCGCGTGGATCCA TGTCCCCTATACTAGG-3′ (forward) and 5′-GGGGGTCGACTTATTTTG GAGGATGGTCG-3′ (reverse), which contained underlined EcoRI and SalI sites, respectively and thrombin recognition site indicated in italics. pColdI-GST vector was constructed by ligation the GST cDNA fused

2. Materials and methods 2.1. Virtual screening and molecular docking In silico large-scale screening of ligands was performed using an original program “NAGARA” [21]. NAGARA is a plugin for PyMOL 2

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of tamoxifen or CDDP. DMSO was used as a vehicle of the agents. Drug sensitivity of the cells was estimated by monitoring the cell viability, which was evaluated by tetrazolium dye-based cytotoxicity assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium [35].

with the thrombin recognition site into the pCold I vectors (Takara Bio Inc.) that had been digested with EcoRI and SalI. Next, the cDNA for LC3 (NCBI accession no. NM_032514.3) was isolated from the total RNA sample of human heart (Takara Bio Inc.) by RT-PCR. PCR was performed using KOD FX neo DNA polymerase and primers, 5′-CCCCCAT ATGATGCCCTCAGACCGGCC-3′ (forward) and 5′-CCCCGAATTCGAAG CCGAAGGTTTCCTGG-3′ (reverse), which contained underlined NdeI and EcoRI sites, respectively. The PCR products were purified, and ligated into the pCold I-GST vectors that had been digested with NdeI and EcoRI. The insert of the cloned cDNA was sequenced to confirm that the full sequence of LC3 fused to the C-terminal GST tag is encoded.

2.7. Western blot analysis Cells were washed twice with Dulbecco's phosphate buffered saline (DPBS), suspended in Urea buffer (10 mM Tris-HCl pH 8.0, containing 50 mM NaH2PO4 and 8 M urea), and homogenized by sonication. The cell extract was prepared by centrifugation (12,000×g for 15 min) of the homogenate and protein concentration was determined with the bicinchoninic acid protein assay reagent (Nacalai Tesque, Kyoto, Japan). Proteins in the extract (20 μg) were separated by SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane by electroblotting. The membrane was blocked with 50 mM Tris-buffered saline with 0.05% Tween-20 (TBS-T) containing 5% skim milk, and incubated overnight at 4 °C with the TBS-T supplemented with primary antibodies against LC3 (#PM036, MBL, Nagoya, Japan), Bax (#CSBPA000976, Wuhan Huamei Biotech Co., Hubei, China), Bcl-2 (#D0383, MBL) or β-actin (#4970, Cell Signaling Technology, Beverly, MA) as the loading control. After washing with the TBS-T, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody against rabbit immunoglobulin. The immunopositive bands were visualized using an enhanced chemiluminescence reagent (GE Healthcare, Buckinghamshire, UK). The densities of the bands were quantified using the ImageJ software.

2.3. Production of recombinant proteins Recombinant Atg4B and LC3-GST were expressed in Escherichia coli BL21 (DE3) pLysS cells (Life Technologies, Gaithersburg, MD) transformed with the expression plasmids harboring the cDNAs as described previously [29]. The proteins were purified to homogeneity from the cell extracts using Ni-Sepharose 6FF resin (GE Healthcare, Waukesha, WI) according to the manufacturer’s manual, and the purity was analyzed by SDS-PAGE according to standard procedures. Protein concentration was determined by Bradford’s method using bovine serum albumin as the standard [30]. 2.4. DSF DSF was carried out according to our previous study [31] using Applied Biosystems® StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA). The SYBR green melting curve protocol was performed in each experiment to measure the Tm using the PCR system. The Atg4B proteins (4 μM) were mixed with phosphate buffered saline, pH 7.2, including 4 × SYPRO Orange (diluted from 5000 × stock in dimethyl sulfoxide (DMSO), Sigma-Aldrich, St. Louis, MO) and additives in a 96-well plate. DSF was carried out from 25 to 95 °C in increments of 1 °C per minute. The Tm was determined using the software, StepOnePlus or Igor Pro V.6 (HULINKS Inc., Tokyo, Japan). DSF measurements were repeated at least three times.

2.8. Immunofluorescence analysis For immunofluorescence of cleaved caspase-3 proteins, A549 cells on 1.3 cm diameter glass coverslips were seeded into a 24-well multiplate at a density of 7.5 × 104 cells/well, and then treated for 24 h with Atg4B inhibitors in the presence or absence of tamoxifen. The cells were fixed with 4% phosphate-buffered paraformaldehyde solution for 10 min at room temperature (RT), and permeabilized with DPBS containing 0.1% Triton X-100 and 100 mM glycine for 10 min. After blocking with DPBS containing 0.1% Tween 20 and 1% bovine serum albumin (BSA) for 30 min, the cells were incubated with anti-cleaved caspase-3 antibodies (#9661, Cell Signaling Technology) for 1 h at RT. They were then incubated with DyLight 488-conjugated secondary antibodies against rabbit immunoglobulin (Thermo Fisher Scientific) for 1 h at RT. The coverslips were mounted in 4′,6-diamidino-2-phenylindole (DAPI) fluoromount-G medium (Southern Biotech, Birmingham, AL), and the immunolabelled and DAPI-stained cells were visualized on LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany). The green fluorescence intensity of cleaved caspase-3 was calculated using ImageJ software.

2.5. In vitro LC3-GST cleavage assay The reaction mixture (a total volume of 20 μL) consisted of recombinant Atg4B (1 pmole), LC3-GST (62 pmoles), 10 mM Tris-HCl, pH 7.4 and/or inhibitor, and was incubated at 37 °C for 4 h. Reactions were stopped by adding the 3 × Laemmli sample buffer (150 mM Tris-Cl pH 6.8, 6% SDS, 6% 2-mercaptoethanol, 0.3% bromophenol blue, and 30% glycerol) and the proteins were separated by SDS-PAGE, followed by Coomassie brilliant blue staining [32]. The amounts of the substrate (LC3-GST) and cleaved products (LC3 and GST) were quantified using ImageJ software from National Institutes of Health (available in the public domain at http://rsbweb.nih.gov/ij/).

2.9. Statistical analysis 2.6. Cell culture Data are expressed as the means ± S.D. of at least three independent experiments. Statistical evaluation of the data was performed by using the unpaired Student t-test and ANOVA followed by Fisher’s test. A p value < 0.05 was considered statistically significant.

Lung cancer A549 cells were obtained from American Type Culture Collection (Manassas, VA), and grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a humidified incubator containing 5% CO2. The A549/CDDP cells were established as reported previously [33,34]. For establishment of the CDDP-resistant cells, the A549 cells were continuously treated in the growth medium supplemented with CDDP, whose concentration was increased in a stepwise manner (0.05–5 μM). To evaluate sensitivity of A549 cells and A549/CDDP cells to tamoxifen or CDDP, the cells were seeded into a 96-well multiplate at a density of 2 × 104 cells/well, and then treated for 24 h with PI3K inhibitors (3MA and Wo) or Atg4B inhibitors in the presence or absence

3. Results 3.1. Inhibitory potency of the screened compounds for Atg4B The search in an Asinex ligand database (comprising approximately 360,000 structures) using a ligand-screening program NAGARA [21] generated 100 compounds that were ranked according to their energy scores. Out of the top scoring compounds, commercially available 18 compounds (Table 1) were first examined their binding potencies 3

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Table 1 Candidate compounds identified as Atg4B inhibitors by in silico screening. No.

Structure

Interaction energy (kcal/mol)

a

No.

Structure

Interaction energy (kcal/mol)

1

–32.1

10

−32.88

2

−35.22

11

−32.15

3

−33.61

12

−44.93

4

−34.3

13

−33.81

5

−33.55

14

−32.03

6

−32.02

15

−32.57

7

−35.43

16

−37.64

8

−34.41

17

−33.66

9

−32.54

18

−33.21

a

a

The interaction energy between the compound and Atg4B obtained in the in silico analysis.

against Atg4B by differential scanning fluorimetry (DSF), in which the thermal denaturation midpoint (Tm) values of Atg4B were measured in the absence or presence of the compounds. Among them, compounds 1, 2, 5, 9 and 17 significantly increased the Tm value of Atg4B, and the increases in Tm values by compounds 1 and 17 were most remarkable (Fig. 2). To elucidate the structural reasons for the high increases in Tm value, the binding modes of compounds 1 and 17 were next examined by constructing their docked models in the active conformation of Atg4B of its LC3 complex structure (PDB: 2Z0E [13]). Docking simulations of 1 and 17 in the Atg4B revealed that the two compounds similarly occupied in an active site of Atg4B, which is overlaid by the C-terminal loop of LC3 in the Atg4BLC3 complex (Fig. 3). In the 1-docked model, 1 was surrounded by the side-chains of Trp72, Met75, Trp142, Tyr143, Ser262, Ala263 and catalytic Cys74 [13] of Atg4B, of which the main-chain of Cys74 and Met75 formed hydrogen bonds with the carboxyl group and carbonyl on the aromatic ring, respectively, of 1, in addition to hydrophobic interaction between Trp142 and the aromatic ring (Fig. 3C). Compound 17, a larger molecule than 1, was surrounded by the side-chains of

Tyr54, Cys74, Trp142, Tyr143, Asn261 and Asp314, of which Tyr54, Asn261 and Asp314 were different from the 1-binding residues and their side-chains formed two hydrogen bonds (for Tyr54 and Asn261) and a salt-bridge between Asp314 and the amino group of the pyrimidine ring of 17 (Fig. 3D). Inhibitory potency of compounds 1 or 17 on Atg4B catalytic activity was estimated by in vitro cleavage assay of LC3-GST, which was a LC3 protein fused with GST to the C-terminus. Since Atg4B cleaves the Cterminal amino acid of LC3 [12], it formed LC3 and GST from the LC3GST substrate (Fig. 4A). Both 1 and 17 potently inhibited the Atg4B activity and showed IC50 values of 10 ± 1.3 and 12 ± 0.6 µM, respectively (Fig. 4B). The reversibility of the inhibition of Atg4B by 1 and 17 was examined with dilution of the enzyme-inhibitor mixture. When a mixture of Atg4B (10 pmoles) and 20 µM 1 was diluted 10-fold with 10 mM Tris-HCl buffer, pH 7.4, its LC3-GST cleavage activity was almost the same as that of the control enzyme prepared Atg4B and without the inhibitor (Supplementary Fig. 1). Similar results were obtained with 20 µM 17 as the inhibitor. The results indicate that 1 and 17 behave as reversible inhibitors. 4

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Fig. 2. Tm values of Atg4B in the absence or presence of candidates for Atg4B inhibitors. Tm was analyzed by DSF. The concentrations of candidates were 100 μM. ** p < 0.01, * p < 0.05 versus Tm without candidate, which is also shown with a dashed line.

Fig. 3. Docked models of compounds 1 and 17 in Atg4B-LC3 complex. (A) Overall-structure of complex of Atg4B (magenta)-LC3 (right blue) (PDB:2Z0E). Dashed circle: catalytic domain of Atg4B. (B) Chemical structures of 1 and 17. (C, D) Close-up view of docking of 1 (C) and 17 (D) in the catalytic domain. The residues (magenta) of Atg4B within 4.0 Å from the compound (1 or 17, green) are depicted with possible hydrogen bond and salt-bridge interactions, which are shown in dotted lines with distances (Å). 5

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Fig. 4. Inhibition of Atg4B-mediated LC3GST cleavage by compounds 1 and 17. (A) Representative SDS-PAGE in cleavage assay of LC3-GST by Atg4B in the absence or presence of 20 µM 1 and 17. The proteins in the reaction mixtures were stained by Coomassie Brilliant Blue dye. (B) Dose response curves of 1 (▲) and 17 (○). The densities of the bands of LC3-GST, GST and LC3 were measured. The conversion rate was estimated as (GST + LC3)/(LC3GST + GST + LC3), and expressed relative to that in the control without inhibitor.

3.2. Effects of compounds 1 and 17 on tamoxifen-induced autophagy and apoptosis

50% (Fig. 6A). The treatment with tamoxifen increased Bax expression (Fig. 6B) and cleaved-caspase-3 positive cells (Fig. 6C). Next, effects of the Atg4B inhibitors 1 and 17 on the tamoxifen-induced apoptotic cytotoxicity to A549 cells were evaluated. The cell viability was not affected by 1 and 17 at concentrations below 20 μM (Supplementary Fig. 2). The cotreatment of the cells with known autophagy inhibitors (3MA and Wo) or the Atg4B inhibitors (1 and 17) significantly enhanced tamoxifen-induced cytotoxicity (Fig. 6A). The tamoxifen-induced increase in Bax level was further accelerated by the cotreatment with 1 and 17. In contrast, Bcl-2 level was significantly decreased by the cotreatment with 1 and 17, although it was not altered by the treatment with tamoxifen alone (Fig. 6B). Furthermore, the number of cleaved-caspase-3-positive cells by the cotreatment of 1 or 17 with tamoxifen was larger than that by the treatment with tamoxifen alone (Fig. 6C). No significant change in the cleaved-caspase-3-positive cell number was observed by the treatment with 1 or 17 alone (data not shown).

Tamoxifen, an estrogen receptor antagonist used as an anticancer drug, is known to induce autophagy and, in general, has been used as a positive control of autophagy inducers [36]. Once autophagy is induced, LC3-I is directly conjugated to the lipid phosphatidylethanolamine and inserted into autophagic membranes to produce LC3-II (Fig. 1), a protein marker of autophagy [37]. However, as Atg4B takes part in both processing and delipidation of LC3, the inhibitors have been shown to increase levels of LC3-II in cells [38]. Then, monitoring protein levels of autophagy-specific substrate p62 was used for evaluation of autophagy inhibitory efficacy of the Atg4B inhibitors. The treatment with tamoxifen of lung cancer A549 cells decreased p62 expression levels in a dose-dependent manner (Fig. 5A). The decrease in p62 by 5 μM tamoxifen was significantly suppressed by the cotreatment of the cells with 20 μM 1 or 17 (Fig. 5B). Tamoxifen also induces apoptosis in many breast cancer cells [39]. Therefore, we first examined whether tamoxifen induces apoptosis in A549 cells in terms of its cytotoxicity and effect on expression of proapoptotic Bax, anti-apoptotic Bcl-2 and cleaved-caspase-3, a key executor in apoptosis (Fig. 6). Tamoxifen was cytotoxic to A549 cells, as the treatment with 10 μM tamoxifen decreased their viability to about

3.3. Effects of compounds 1 and 17 on CDDP sensitivity Recent studies indicated that acute CDDP treatment activates an autophagic response that serves as a survival factor to counteract CDDP-induced cell death [4,40]. To investigate whether 1 and 17 Fig. 5. Effects of compounds 1 and 17 on tamoxifen (Tam)-induced autophagy in A549 cells. (A) Autophagy induction by Tam. A549 cells were treated with the indicated concentrations of Tam for 24 h. p62 in the cell extracts was detected by Western blotting (upper panel). Lower bar graph shows the p62 level that is expressed as a percentage of the band density relative to that in the control cells (shown as 0 μM). **p < 0.01 versus DMSO. (B) Effect of 1 and 17 on Tam-induced p62 decrease. The cells were pretreated with 20 μM 1 or 17 for 2 h prior to the 24-h treatments with 5 μM Tam, and the p62 level was determined as described above. **p < 0.01 versus DMSO. ## p < 0.01 versus Tam alone.

6

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Fig. 6. Enhancement of tamoxifen (Tam)induced apoptosis by compounds 1 and 17. (A) Effects of PI3K inhibitors (3MA and Wo), 1 and 17 on Tam-mediated toxicity to A549 cells. The cells were treated with 10 μM Tam for 24 h after 2 h-pretreatment with 5 mM 3MA, 1 μM Wo, 1 or 17 (10 or 20 μM). The viability is expressed as a percentage of the value in the control cells. ** p < 0.01 versus Tam alone. (B) Effects of 1 and 17 on expression of Bax and Bcl-2. The cells were treated with 5 μM Tam for 24 h after 2 hpretreatment with 20 μM 1 or 17. Bax and Bcl-2 in the cell extracts were detected by Western blotting (upper panel). Lower graph shows the protein levels that are expressed as a percentage of the band density relative to those in the control cells. *p < 0.05 versus DMSO (shaded bar). # p < 0.05 versus Tam alone (open bar). (C) Expression of cleaved-caspase-3. The cells were treated as described in Fig. 5B, and subjected to immunostaining of cleavedcaspase-3 (green) and DNA-staining with DAPI (blue). Merged images were also shown. Scale bar indicates 20 μm. The fluorescence intensity of cleaved-caspase 3 (green) was calculated using ImageJ. *p < 0.05 versus DMSO (shaded bar). # p < 0.05 versus Tam alone (open bar).

inhibition of CDDP-induced autophagy and promotion of CDDP cytotoxicity.

contribute to enhancing the anticancer potency of CDDP for lung cancer treatment, we examined their effects on CDDP-induced autophagy and cytotoxicity in A549 cells. The treatment with 10 μM CDDP enhanced autophagy in A549 cells, in which the p62 level was lower than that in the non-treated control cells, and the decreased p62 expression was significantly recovered by the pretreatment with 1, 17 or Wo (Fig. 7A). As CDDP was reported to induce apoptotic cell death in A549 cells [41], the CDDP treatment decreased the viability of the cells to about 70% (Fig. 7B). The CDDP sensitivity of the cells was enhanced by the cotreatment with 1, 17 or known autophagy inhibitors, 3MA and Wo, similarly to the effect of the combined treatment of the Atg4B inhibitors with tamoxifen (Fig. 6A). These results suggest that 1 and 17 augment the anticancer efficacy of CDDP against A549 cells through both

3.4. Overcoming CDDP resistance of A549 cells by compounds 1 and 17 In lung cancer cells, autophagy has been suggested to play a role in acquired CDDP resistance [42,43] and to be used as a novel therapeutic target to overcome CDDP-resistant lung cancer [44,45]. The efficacy of the Agt4B inhibitors 1 and 17 in preventive treatment of CDDP resistance was examined using the resistant A549 (A549/CDDP) cells, which were less sensitive to CDDP than the parental A549 cells (Fig. 8A). The cotreatment of 1 or 17 with 50 μM CDDP resulted in decreases in the cell viability of A549/CDDP cells in a dose-dependent 7

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Fig. 7. Enhanced sensitivity to CDDP by compounds 1 and 17. (A) Effects of 1 and 17 on CDDP-induced p62 decrease. A549 cells were pretreated with 20 μM 1, 17 or wortmannin (Wo) for 2 h prior to the 24-h treatment with 10 μM CDDP. p62 in the cell extracts were detected by Western blotting (upper panel). Lower graph shows the protein levels that are expressed as percentages of those in the control cells. (B) Effects of PI3K inhibitors, 1 and 17 on CDDP-mediated toxicity. The cells were treated with 10 μM CDDP for 24 h after 2 h-pretreatment with 5 mM 3MA, 1 μM Wo, 1 or 17 (10 and 20 μM). The viability is expressed as a percentage of the value in the control cells. ** p < 0.01 versus DMSO (shaded bar). ## p < 0.01 versus CDDP alone (open bar).

promotes cancer progression and survival through modulation of the tumor microenvironment by promoting angiogenesis, nutrient supply and protection of tumors from oxidative stress and inflammatory responses [2,46]. Based on these findings, dozens of clinical trials using autophagy inhibitors, chloroquine or hydroxychloroquine alone, or their combination with anticancer drugs (e.g., erlotinib, bortezomib, temsirolimus) have been conducted mainly in the United States, and demonstrated the effectiveness of autophagy inhibitors as novel anticancer drugs [6]. On the other hand, Pfizer and Novartis groups have recently reported that autophagy is not related to the proliferation of KRAS mutant tumor and the antitumor effect of chloroquine [47,48].

manner (Fig. 8B). When autophagy induction was compared between the resistant and parental cells, A549/CDDP cells showed higher LC3-II level than A549 cells (Fig. 8C), which suggests autophagy was greatly induced in the A549/CDDP cells compared to the parental cells. Moreover, the treatment with 50 μM CDDP increased LC3-II level. The cotreatment of A549/CDDP cells with CDDP and 1 or 17 markedly increased the p62 level (Fig. 8D). 4. Discussion To date, numerous studies have demonstrated that autophagy

Fig. 8. Overcoming CDDP resistance of A549 cells by treatments with compounds 1 and 17. (A) CDDP sensitivity. The cells were treated with the indicated concentrations of CDDP for 24 h. Data are expressed as percentages of the viability value in the control cells treated with the vehicle DMSO alone (shown as 0 μM). ** p < 0.01 versus the parental cells. (B) Effects of 1 and 17 on CDDP sensitivity of A549/CDDP cells. The A549/CDDP cells and parental A549 cells were pretreated for 2 h without or with the indicated concentrations of 1 or 17, and then treated for 24 h with 50 µM CDDP. The viability values are expressed as percentages to that of the parental cells treated without inhibitor. **p < 0.01, *p < 0.05 versus A549/CDDP cells treated with CDDP alone. (C) CDDP-induced LC3-II production in A549/CDDP cells. The cells were treated with 50 μM CDDP. LC3-II in the cell extracts were detected by Western blotting. (D) Effects of 1 and 17 on CDDP-induced p62 decrease in A549/CDDP cells. The cells were pretreated with 20 μM 1 or 17 for 2 h prior to the 24-h treatments with 50 μM CDDP. p62 in the cell extracts were detected by Western blotting (upper panel). Lower graph shows the protein levels that is expressed as a percentage of the band density relative to that in the control cells. ** p < 0.01 versus A549/CDDP cells treated with DMSO. ## p < 0.01 versus CDDP alone.

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cancer chemoprevention and chemotherapy, it would be valuable to investigate whether the combination treatment of the present Atg4B inhibitors, 1 and 17, exerts overcoming effects on resistance to other anticancer drugs in other cancerous cells in future. In conclusion, we discovered two novel Atg4B inhibitors, 1 and 17, by in silico screening. Their cell-based analyses revealed that 1 and 17 improve therapeutic efficiency of CDDP by enhancing apoptosis of CDDP and tamoxifen and overcoming CDDP resistance, which have not been studied with previously reported Atg4B inhibitors [14–20]. The newly identified compounds represent promising leads for the development of more potent and selective agents targeting Atg4B by structure-based drug design.

To demonstrate whether autophagy is a novel and reliable target for cancer therapies, development of selective autophagy inhibitors, unlike non-selective inhibitors such as chloroquine or hydroxychloroquine, is desired. Maturation of autophagosomes is one of the key and characteristic events in autophagy, and thereby the development of compounds that inhibit LC3 processing by Atg4B, an essential process in the event, has been drawing attention. In this study, we used a combination of in silico screening and both cell-free and cell-based activity assays to identify two candidate small molecule inhibitors of Atg4B. The two inhibitors 1 and 17 with IC50 of around 10 μM (Fig. 4) are more potent than NSC185058 [14] and LV320 [15] previously found by the same screening method. The high inhibitory potency may be attributed to several interactions (hydrogenbond, hydrophobic bond and/or salt bridge) between the inhibitor molecules and residues in the active site of Agt4B (Fig. 3). In addition, these non-covalent interactions suggest that, unlike irreversible inhibitors [16–19], 1 and 17 are reversible inhibitors, which were also evident from the recovery of the activity by the Atg4B-inhibitor mixtures. The two inhibitors 1 and 17 did not show autophagy induction and cytotoxicity to A549 cells (Fig. 6A), in contrast to the actions of FMK-9a [18] and Z-L-Phe-chloromethyl ketone [16]. The reduction of LC3-II level by 1 and 17 in the tamoxifen-treated A549 cells (Fig. 5B) indicates that tamoxifen-induced autophagy is effectively suppressed by inhibition of Atg4B by 1 and 17. Since mTOR, a serine/threonine protein kinase, regulates autophagy [44,45], we examined phosphorylation of p70S6 kinase, a substrate of mTOR in A549 cells, but the treatment with 1 and 17 (20 μM) did not affect the phosphorylation level of p70S6 kinase (Supplementary Fig. 3). In addition, the enhancement of apoptosis by cotreatment of 1 and 17 with tamoxifen in A549 cells was accompanied by activation of caspase 3 (Fig. 6) suggests that the two compounds do not inhibit the cysteine protease caspase 3. Thus, 1 and 17 may specifically inhibit Atg4B, although their selectivity using other cysteine proteases must be studied in future. The present cell-based analysis of 1 and 17 also revealed that the inhibition of autophagy by 1 and 17 enhances cytotoxicity of tamoxifen and CDDP to A549 cells through upregulation of apoptosis, in contrast to augmentation of both autophagy and apoptosis by tamoxifen or CDDP alone (Figs. 6 and 7). Since 1 or 17 alone were not cytotoxic to the cells, their inhibition of the autophagy upregulated by the anticancer drugs may trigger the promotion of apoptosis. A number of studies have recently been demonstrated that CDDP-induced apoptosis in cancer cells including A549 cells is promoted through inhibition of autophagy by knockdown of Atg5 and/or Beclin-1 (Atg6) [41,49] and by autophagy inhibitors such as 3MA [49], luteolin [50] and chloroquine [51,52]. However, the relationship between autophagy and apoptosis in cell death on cancer cells remains poorly understood, because of complex autophagy network and its integration with other cellular networks [1,53]. Our data using 1 and 17 confirm the enhancement of the CDDP-induced apoptotic cell death by inhibition of autophagy, and suggest that the Atg4B inhibitors can be used as combined drugs for improvement of therapeutic efficiency of CDDP and tamoxifen. The cell-based assay using A549/CDDP cells furthermore indicated that 1 and 17 resensitize the resistant cells to CDDP (Fig. 8). This finding is consonant with previous studies demonstrating that CDDPresistance of its resistant lung cancer cells is sensitized by addition of autophagy inhibitors, 3MA [42], chloroquine [45] and andrographolide [44]. The overcoming effect of 1 and 17 on CDDP resistance, together with their stimulatory effect on CDDP sensitivity, may lead to improvement of the therapeutic efficiency of CDDP. As recently reviewed by Li et al. [54], autophagy promotes resistance of many types of cancer cells to not only CDDP but also other chemotherapeutic drugs, and inhibition of autophagy (by siRNAs, 3MA and chloroquine) facilitates the efficiency of chemotherapy in multidrug resistance cancer, although the exact mechanisms of the interaction between autophagy and chemoresistance reversal remain obscure. Therefore, from the viewpoint of

Author contributions S. E. participated in research design and mainly contributed to writing the paper. M. U., M. S., Y. A., D. H. and S. X. performed the biological analyses. R. H., A. K. and Y. K. performed physicochemical analysis. B. M. and K. K. contributed to the molecular docking. N. T., T. M. and A. I. contributed to the experiment planning and manuscript preparation. All authors read and approved the final manuscript. Acknowledgements We are indebted to Dr. Ryo Honda for valuable discussion of the manuscript. This work was partly funded by Grant-in-Aid for Scientific Research (C) from JSPS KAKENHI, Japan (no. 26460149 and 17K11151 for S.E.), and was also supported by a special grant from Gifu Pharmaceutical University and grants from the Koshiyama Science and Technology Foundation and the OGAWA Science and Technology Foundation (for S.E.). S. X. Wishes to thank Otsuka Toshimi Scholarship Foundation for its generous support. Declaration of Competing Interest The authors declare no competing financial interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.comtox.2019.100095. References [1] C. Behrends, M.E. Sowa, S.P. Gygi, J.W. Harper, Network organization of the human autophagy system, Nature 466 (2010) 68–76, https://doi.org/10.1038/ nature09204. [2] G. Kroemer, Autophagy: a druggable process that is deregulated in aging and human disease, J. Clin. Investig. 125 (2015) 1–4, https://doi.org/10.1172/ JCI78652. [3] M.T. Rosenfeldt, K.M. Ryan, The role of autophagy in tumour development and cancer therapy, Expert Rev. Mol. Med. 11 (2009) e36, , https://doi.org/10.1017/ S1462399409001306. [4] T.R. O'Donovan, G.C. O'Sullivan, S.L. McKenna, Induction of autophagy by drugresistant esophageal cancer cells promotes their survival and recovery following treatment with chemotherapeutics, Autophagy 7 (2011) 509–524. [5] S. Yang, X. Wang, G. Contino, M. Liesa, E. Sahin, H. Ying, A. Bause, Y. Li, J.M. Stommel, G. Dell'antonio, J. Mautner, G. Tonon, M. Haigis, O.S. Shirihai, C. Doglioni, N. Bardeesy, A.C. Kimmelman, Pancreatic cancers require autophagy for tumor growth, Genes Dev. 25 (2011) 717–729, https://doi.org/10.1101/gad. 2016111. [6] J.M.M. Levy, C.G. Towers, A. Thorburn, Targeting autophagy in cancer, Nat. Rev. Cancer 17 (2017) 528–542, https://doi.org/10.1038/nrc.2017.53. [7] A. Petiot, E. Ogier-Denis, E.F. Blommaart, A.J. Meijer, P. Codogno, Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells, J. Biol. Chem. 275 (2000) 992–998. [8] G. Powis, R. Bonjouklian, M.M. Berggren, A. Gallegos, R. Abraham, C. Ashendel, L. Zalkow, W.F. Matter, J. Dodge, G. Grindey, et al., Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase, Cancer Res. 54 (1994) 2419–2423. [9] N. Mizushima, T. Yoshimori, Y. Ohsumi, The role of Atg proteins in autophagosome formation, Annu. Rev. Cell Dev. Biol. 27 (2011) 107–132, https://doi.org/10.1146/

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