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A novel mechanism of irinotecan targeting MDM2 and Bcl-xL
Biochemical and Biophysical Research Communications 514 (2019) 518e523
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A novel mechanism of irinotecan targeting MDM2 and Bcl-xL Boah Lee a, Jeong A. Min b, c, Abdullateef Nashed a, Sang-Ok Lee b, Jae Cheal Yoo a, Seung-Wook Chi b, c, **, Gwan-Su Yi a, * a
Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, South Korea Disease Target Structure Research Center, KRIBB, Daejeon, 34141, South Korea c Department of Proteome Structural Biology, KRIBB School of Bioscience, Korea University of Science and Technology, Daejeon, 34113, South Korea b
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a b s t r a c t
Article history: Received 27 March 2019 Accepted 2 April 2019 Available online 2 May 2019
Irinotecan is a strong anticancer drug whose mechanism of action has been reported only for the inhibition of DNA topoisomerase I (Topo I) through its active metabolite SN-38. In this study, we present a new mechanism of Irinotecan which inhibits the activities of MDM2, an E3 ligase of tumour suppressor p53, and Bcl-xL, an anti-apoptotic protein, through direct binding. In our structure modelling study, Irinotecan could fit to the binding sites of MDM2 and Bcl-xL for their known drugs, Nutlin-3 and ABT-737, with a better binding affinity than to Topo I. The direct binding of Irinotecan to both proteins was confirmed through a NMR study. We further showed that Irinotecan increased the amount of p53 only in the presence of MDM2 and inhibited the physical interaction of Bcl-xL with Bim, a core pro-apoptotic protein. In addition, we demonstrated that Irinotecan induced the down regulation of proliferation and strong G2/M arrest in HCT116 colon cancer cells shortly after treatment. Collectively, we suggest a new mechanism of action for Irinotecan as a dual target inhibitor of MDM2 and Bcl-xL facilitating the anticancer activities mediated by p53 and Bcl-xL interaction partners. © 2019 Elsevier Inc. All rights reserved.
Keywords: Irinotecan Dual-targets MDM2 Bcl-xL Structure modelling NMR
1. Introduction p53, a key tumour suppressor protein, plays a key role in the cellular stress response pathway. The proapoptotic activity of p53 is controlled by a number of proteineprotein interactions that constitute a network of negative and positive regulators [1]. MDM2 is located in the central part of this network by regulating p53 with direct interaction [2]. MDM2 is an E3 ubiquitin ligase [3] targeting p53 causing its proteasomal degradation [4]. Bcl-xL, a well-known antiapoptotic protein, is another direct interaction partner of p53; consequently, both MDM2 and Bcl-xL became the targets of anticancer drugs such as Nutlin-3 [5] and ABT-737 [6], respectively.
Abbreviations: Topo I, topoisomerase I; BH, Bcl-2 homology; MDM2, Mouse double minute 2 homolog; Bcl-xL, B-cell lymphoma extra-large; DS, Discovery Studio 3.1; IPTG, isopropyl-b-D-thiogalactopyranoside; KBSI, Korea Basic Science Institute; PS, penicillin-streptomycin; FBS, fetal bovine serum; RT, room temperature; BrdU, 5-bromo-20 -deoxyuridine; IP, immunoprecipitation; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; UGT 1A1, uridine diphosphate glucuronosyltransferase 1A1. * Corresponding author. ** Corresponding author. Disease Target Structure Research Center, KRIBB, Daejeon, 34141, South Korea. E-mail addresses: [email protected] (S.-W. Chi), [email protected] (G.-S. Yi). https://doi.org/10.1016/j.bbrc.2019.04.009 0006-291X/© 2019 Elsevier Inc. All rights reserved.
The p53 binding sites of both MDM2 and Bcl-xL have been characterized as Bcl-2 homology (BH) motifs [7], which is also the targeting site of their drugs, according to NMR studies [8,9]. With this mechanism, Nutlin-3 binds to the BH3 motif of MDM2, interfering with its interaction with p53, rescuing the tumour suppressor function of p53. As p53 does to Bcl-xL, ABT-737 binds to the BH3 motif of Bcl-xL, preventing any further inhibiting interactions of Bcl-xL with essential proapoptotic proteins such as Bax and Bim [10,11]. Despite the similar structure of the binding site, there have been few studies on compounds that bind commonly to both targets [12]. We searched for such compounds using in silico structure modelling, and Irinotecan was one of the best candidates that can bind to both targets. Irinotecan, an alkaloid derived from the extracts of the tree Camptotheca acuminata, is commonly used for the treatment of colorectal and lung cancer [13]. It is well known that this compound is converted by intracellular carboxylesterase activity to its active metabolite termed SN-38. This metabolite SN-38 binds to DNA Topo I, stabilizes the DNA Topo IeDNA complex and prevents DNA ligation. It has been reported that double-stranded DNA breakage induces ATM kinase activation and autophosphorylation of DNA Topo I resulting in the DNA damage check point response and apoptosis
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and cell cycle arrest [14]. Until now, the reported mechanism of Irinotecan, a strong anticancer drug, has only focused on the inhibition of DNA topoisomerase I (Topo I) through its active metabolite SN-38. In this study, we found that Irinotecan, is a dual specific inhibitor of MDM2 and Bcl-xL through structure modelling and confirmed the direct binding of Irinotecan to both proteins through a NMR study. Moreover, Irinotecan regulates apoptotic cell death, proliferation inhibition and G2/M cell cycle arrest in response to these interactions. With these results, we propose a new perspective on the anticancer mechanism of Irinotecan through its binding to MDM2 and Bcl-xL. 2. Materials and methods 2.1. Structure modelling Nutlin-3 and ABT-737 were used to compare the 2D structural similarities with Irinotecan using Pipeline Pilot [Pipeline Pilot; Accelrys, Inc.: San Diego, CA, 2000]. We converted the 2D structures to molecular descriptors: ECFP4, standard circular fingerprints based on the Morgan algorithm [15]. The similarity between the compound pairs was calculated by the Tanimoto similarity. Nutlin-3 and ABT-737 were also used to build pharmacophore features from 3D conformations of the molecules generated with Discovery Studio 3.1 (DS) from the Accelrys Co. It identified configurations or 3-dimensional spatial arrangements of the chemical features of Nutlin-3 and ABT-737. The protocol considered the hydrogen bond acceptor/donor, hydrophobic feature, negative/ positive ionizable feature and aromatic ring for feature types to generate a selective pharmacophore model from a single ligand. A set of candidate pharmacophore models were enumerated from the features. The protocol picked the pharmacophore with the highest selectivity as predicted by a Genetic Function Approximation model [16]. Docking simulations using the Libdock algorithm [17] in DS were performed with compounds against three proteins, MDM2, Bcl-xL, and Topo I, respectively. The X-ray crystal structure complex of MDM2 with Nutlin-3 was obtained from the protein data bank (PDB ID: 4HG7) [18], and the X-ray crystal structure complex of BclxL with ABT-737 was obtained from the PDB (PDB ID: 2YXJ) [19]. Moreover, the X-ray crystal structure complex of DNA and human Topo I was obtained from the PDB (PDB ID: 1A35) [20]. The proposed binding site was centered on the ligand, and a site sphere was created with the following coordinates: 23.835, 7.53, 14.053 with 9.5 Å diameter for MDM2; 39.1692, 4.14924, 10.7891 with 9 Å diameter for Bcl-xL and 39.1692, 4.14924, 10.7891 with 13.6 Å diameter for Topo I. The protocol included 100 hotspots with a docking tolerance 0.25. The FAST conformation method was also used with CHARMm. All other parameters followed the default settings. 2.2. Protein purification For the NMR experiments, 15N-labeled MDM2 and Bcl-xL were expressed in minimal media containing 15N-NH4Cl. The Escherichia coli BL21 CodonPlus (RP) cells were grown to an optical density at 600 nm (OD600) of ~0.9 at 37 C, and expression of the N-terminal domain of the MDM2 (residues 3e109) construct was induced at 20 C with 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG) for 16 h. The protein was precipitated with ammonium sulphate and further purified using ion exchange columns (HiTrap® SP and QSepharose, GE healthcare) and a gel-filtration column (HiLoad® 16/ 600 Superdex 75 pg, GE healthcare). The 15N-labeled truncated BclxL construct was expressed and purified for the NMR experiments
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as previously reported [21,22]. 2.3. NMR spectroscopy All the NMR spectra were collected on a Bruker Avance II 800 spectrometer at the Korea Basic Science Institute (KBSI). The 2D 1 H-15 N HSQC spectra of 0.1 mM MDM2 and Bcl-xL each obtained at 10 and 25 C, respectively, were with and without the addition of 0.4 mM Irinotecan. The NMR samples comprised 90% the H2O/10% D2O and were prepared in 20 mM MES (pH 6.5), 50 mM NaCl, and 1 mM DTT for MDM2 and 20 mM sodium phosphate (pH 6.5), 50 mM NaCl and 1 mM DTT for Bcl-xL. All the NMR data were processed and analysed using nmrPipe/nmrDraw and the SPARKY software. For the assignment of the 2D 1H-15 N HSQC spectra of MDM2 and Bcl-xL, we used the BMRB entry 18876 and 18250, respectively. 2.4. Cell culture and compounds The HCT116 cells were cultured in McCoy's 5a medium supplemented with 2 mM Glutamine, 1% penicillin-streptomycin, and 10% Fetal Bovine Serum at 37 C with 5% CO2. SW480 cells were cultured in L-15 medium in the same environment without CO2. Irinotecan was purchased from Biopurify Phytochemicals Ltd. ABT-737 was purchased from APExBIO and Nutlin-3 from Sigma Aldrich. 2.5. Western blot analysis and immunoprecipitation Cells were lysed with RIPA buffer containing a proteaseinhibitor cocktail. Whole-cell lysates were incubated on ice for 30 min and then cleared at 20,000 x g for 20 min at 4 C. For the immunoprecipitation (IP) analysis, the resulting supernatants were incubated with anti-Bcl-xL antibody for 3 h, at 4 C with an additional incubation for 2 h after the addition of protein A/G plus agarose (Santa Cruz Biotechnology). The immunocomplexes captured in the agarose gel were then washed three times with RIPA buffer and eluted with SDS gel-loading buffer. For the Western blot analysis, the supernatants were separated by SDS-PAGE using 10% gels and blotted onto PVDF membranes. The blots were then probed with anti-p53, anti-MDM2 (all from Thermo Fisher Scientific), anti-Bim, anti-Bcl-xL, and anti-GAPDH (all from Cell signaling) antibodies. Blots were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies, followed by additional washing and then detection with enhanced chemiluminescence (ECL; Amersham). 2.6. Viability assay Cell viability was performed using Cell Titer-Glo cell viability assay kit (Promega). HCT-116 cells were seeded into a 96 well plate at a density of 104 cells/well. The cells were incubated for 24 h before treatment with the compound. Treated plates were incubated for 48 h at 37 C with 5% CO2. Before the assay, cells were incubated at room temperature (RT) for 30 min and 100 ml of assay reagent were added to each well. The luminescence was measured using the VICTOR X Multilabel Reader (PerkinElmer). 2.7. Apoptosis assay Apoptosis assay was performed using thee Caspase-Glo 3/7 assay kit (Promega). HCT-116 cells were seeded into a 96 well plate at a density of 104 cells/well. The cells were incubated for 24 h before treatment with the compound. Cells were then treated with a concentration equivalent to the IC50 for each compound calculated from the viability dose response curve. Before the assay, cells
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were incubated at RT for 30 min, and 100 ml of assay reagent were added to each well. The luminescence was measured using the VICTOR X Multilabel Reader (PerkinElmer). 2.8. Cell proliferation assay The cellular proliferation was determined using cell proliferation ELISA (Roche) via 5-bromo-20 -deoxyuridine (BrdU) incorporation according to the manufacturer's protocol. Cellular incorporation of BrdU was visualized with a substrate solution and, the chemiluminescence was measured using a luminometer. Each experiment was performed in triplicate. 2.9. Cell cycle analysis Cells were seeded in a 25 cm2 flask (106 cells/flask) incubated for 24 h and were treated with compounds for 8 h. Cells were trypsinized, harvested, and fixed in 1 ml of 80% cold ethanol in test tubes and incubated at 4 C for 15 min. After incubation, the cells were centrifuged at 1500 rpm for 5 min, and the cell pellets were resuspended in 500 ml of propidium iodine (10 mg/ml) containing 300 mg/ml RNase (Sigma-Aldrich). Then, the cells were incubated on ice for 30 min and filtered with nylon mesh. Cell cycle distribution was calculated with the BD FACSDiva Clinical software. 3. Results 3.1. Identification of direct binding of Irinotecan to MDM2 and BclxL To find the molecules targeting MDM2 and Bcl-xL, we searched for compounds that are similar to Nutlin-3 and ABT-737. Among the best candidates, we selected Irinotecan for further investigation because it showed anti-cancer functions that matches with the effect of suppressing MDM2 and Bcl-xL in previous studies related to Topo I. To investigate the similarity of the compound with ABT-737 and
Nutlin-3, we first performed a structural similarity analysis based on the extended connectivity fingerprints. Tanimoto scores were calculated to measure the similarities of the compound pairs. In the similarity analysis, the Tanimoto scores were 0.18 and 0.11 for ABT737 and Nutlin-3, respectively. Because the similarities of the 2D structures were low, we conducted pharmacophore modelling to find the similarity considering the 3D arrangement of the structure. We generated the pharmacophore features of Nutlin-3 and ABT-737, including the hydrogen bond acceptor/donor features, positive/negative ionizable features, aromatic ring features, and hydrophobic features. Irinotecan was well suited to the derived model with fit values of 0.55 and 0.56, respectively. (Fig. 1A- B). In addition, docking simulations of Irinotecan further supported that it binds more stably to MDM2 and Bcl-xL than to Topo I based on the energy values: 100.37, 170.59 and 99.53 respectively. Especially, resides such as V53, L54, L57, F91, and V93 in MDM2 and L130, R139, A142, and Y195 in Bcl-xL in the binding region were proven to be also present in the NMR result (Fig. 1CeE). To examine the direct binding of Irinotecan to MDM2 and Bcl-xL, we performed 2D 1H-15 N HSQC experiments using NMR spectroscopy. During the binding titration with Irinotecan, we observed NMR chemical shift perturbations of 15 N -labeled MDM2 and BclxL (Fig. 2A and D, Fig. S1). The overlaid 2D 1H-15 N HSQC spectra showed that some cross-peaks of MDM2 and Bcl-xL disappeared, and others moved significantly, indicating that the binding involved a fast to intermediate exchange between the free and Irinotecan-bound forms on the NMR chemical shift time scale. These NMR chemical shift perturbations demonstrate the direct binding of Irinotecan to both MDM2 and Bcl-xL. The residues showing significant chemical shift perturbations upon Irinotecan binding were plotted against the amino acid sequences (Fig. 2B and E) and projected onto the structures of MDM2 (PDB ID: 4HG7) (Fig. 2C) and Bcl-xL (PDB ID: 2YXJ) (Fig. 2F). For MDM2, the NMR chemical shift perturbations induced by binding to Irinotecan were localized to a p53 peptide-binding hydrophobic binding pocket, which are in good agreement with that previously observed during
Fig. 1. (AeB) Pharmacophore models. Fitted pharmacophore model of Irinotecan to the models of Nutlin-3 and ABT-737, respectively. The orange colour represents the aromatic ring, cyan for hydrophobic, green for hydrogen bond acceptor and red for positive ionizable variables. Fit values are indicated in the parentheses. (CeE) Predicted binding modes: energies are indicated in the parentheses. The docking poses of Irinotecan in the MDM2, Bcl-xL and Topo I. Among the docking results, the residues mapped in the NMR results are shown in red.
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Fig. 2. Direct binding of Irinotecan to MDM2 and Bcl-xL proteins. The overlaid 2D 1H-15 N HSQC spectra for 15N-labeled MDM2 (A) and Bcl-xL (D) proteins without (blue) and with the addition of Irinotecan (red) (at the molar ratio of 1:4). NMR chemical shift perturbations on MDM2 (B) and Bcl-xL (E) induced by binding to Irinotecan. Weighted DCS ¼ [(D1H)2 þ 0.2(D15 N)2]0.5, in which the D1H and D15 N are the chemical shift changes on the 1H and 15 N dimensions, and plotted against the residue number of the MDM2 and Bcl-xL. Resonances of MDM2 and Bcl-xL disappeared upon binding of Irinotecan are shown as grey bars. Binding site mapping of Irinotecan on the structures of MDM2 (C) (PDB code: 4HG7) and Bcl-xL (F) (PDB code: 2YXJ). The residues that disappeared upon chemical binding are shown in red, and the residues showing the chemical shift changes of DCS >0.015 ppm in MDM2 and DCS >0.03 ppm in Bcl-xL are coloured in yellow.
the binding of Nutlin-3 (Fig. S2A). For Bcl-xL, the Irinotecaninduced NMR chemical shift perturbation occurred on the BH3 peptide-binding hydrophobic groove surrounded by the BH1, BH2 and BH3 domains, which overlapped with the ABT-737-binding site (Fig. S2B). These results proved a direct interaction between Irinotecan and specific hydrophobic interfaces of MDM2 and Bcl-xL proteins. 3.2. Irinotecan blocks the binding of MDM2 and Bcl-xL to their signaling partners To confirm the function of Irinotecan on MDM2 and Bcl-xL, we tested the effects of MDM2/p53 and Bcl-xL/Bim binding. First, to identify the Irinotecan specific effect in MDM2-p53
binding, p53 expression was measured by treatment with Irinotecan in HCT116 cells (Fig. 3A). Irinotecan increased the intact protein level of p53 and MDM2 in a dose dependent manner just like Nutlin-3 (Fig. 3A). These results indicate that Irinotecan is an inhibitor of the MDM2-p53 interaction just like Nutlin-3. To further confirm Irinotecan as an inhibitor of MDM2-p53 binding, we tested the effect of Irinotecan in SW480 cells, transcriptionally inactive p53 mutated cells (Fig. 3B). Interestingly, constant expression of p53 but no detectable MDM2 expression was found in the SW480 cells with the same concentration of Irinotecan that was used in the HCT116 cells (Fig. 3B). These results show that Irinotecan induces wild-type p53 accumulation, which is dependent on MDM2 expression, and that Irinotecan specifically regulates the MDM2-p53 binding.
Fig. 3. (A) Expression levels of p53 and MDM2 proteins in HCT116 cells. The indicated concentrations of the compound were incubated at the IC10, IC25 and IC50, respectively. Expression level of GAPDH was used as the loading control. (B) Expression levels of p53 and MDM2 proteins were confirmed by the indicated concentrations in SW480, p53 mutant cells. (C) Co-IP data show the interaction between Bcl-xL and Bim in HCT116 cells with the indicated concentrations of the compound.
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To investigate the Irinotecan-mediated inhibition of the Bcl-xL related apoptotic signal, the physical interaction of Bcl-xL and Bim in HCT116 cells was examined (Fig. 3C). Irinotecan treatment induced the disruption of the Bcl-xL and Bim interactions in a dosedependent manner just like ABT-737. (Fig. 3C). This result shows that Irinotecan is involved in direct targeting of Bcl-xL in HCT116 cells. 3.3. Irinotecan induces apoptosis, proliferation regulation, and cell cycle arrest To investigate the efficacy of Irinotecan in inducing Bcl-xL and/ or MDM2 related responses, viability, apoptosis, proliferation, and cell cycle arrest assays were performed. The viability of HCT116 cells were measured in a dosedependent manner. In addition, two control compounds were used, ABT-737 and Nutlin-3, which are Bcl-xL and MDM2 specific inhibitors, respectively. The dose response curves of each compound was plotted, and the IC50 values were calculated from the graph (Fig. 4A). The IC50 values were 10, 18 and 12 mM for Irinotecan, Nutlin-3 and ABT-737, respectively. This result shows that Irinotecan has a significant potency in reducing cell viability compared to Nutlin-3 and ABT-737. To investigate the contribution of apoptosis induction in the observed viability reduction, caspase 3/7 activity assays were performed in cells using the IC50 dosing compounds (Fig. 4B). ABT-737 showed the highest efficacy in the induction of apoptosis, and the ABT-737 response can be induced mainly by direct inhibition of BclxL. On the other hand, the weak apoptotic effect of Nutlin-3 may represent a secondary effect of Nutlin-3 through apoptosis related p53 signaling. In this condition, the apoptotic effect of Irinotecan was almost two times higher than that of Nutlin-3 but was half that of ABT-737 (Fig. 4B). BrdU is commonly used for the detection of proliferating cells. To investigate the contribution of cell growth inhibition in the observed viability reduction, proliferation assays were performed on the cells using the compounds at the IC50 values with BrdU
staining (Fig. 4C). BrdU incorporation was measured in Irinotecan or Nutlin-3 treated HCT-116 cells. The percentage of BrdU positive cells in Irinotecan or Nutlin-3 treated cells was nearly 10-fold lower than that of the control cells (Fig. 4C). Irinotecan showed an antiproliferative effect similar to Nutlin-3, a known inhibitor MDM2p53 binding, in the HCT116 cells (Fig. 4C). To determine whether the inhibitory effect of Irinotecan is mediated by cell cycle arrest, the effect of Irinotecan on cell cycle progression was analysed by flow cytometer. As shown in Fig. 4D, most of the HCT116 cells were in the G1 phase. The G2/M phase cell population was significantly increased in cells treated with Nutlin3. In addition, the Irinotecan treatment significantly increased the late phase of the cell cycle population in the HCT116 cells (Fig. 4D). Irinotecan showed a strong G2/M cell cycle arrest in this cell, like Nutlin-3, a known inhibitor and MDM2-p53 binding agent. 4. Discussions In this work, a NMR study was performed to obtain definite evidence of binding of Irinotecan to the two targets, and pharmacophore models were constructed using previously known compounds that bind to the two targets to understand the structural reasons. In addition, a docking simulation analysis showed that Irinotecan binds more stably to MDM2 and Bcl-xL than to Topo I. Furthermore, we demonstrated a novel dual target-based anticancer activity of Irinotecan with a short treatment time: (1) Irinotecan binds directly to antiapoptotic Bcl-xL, to release Bim (proapoptotic proteins) from complexes and induce apoptosis. (2) Irinotecan binds to MDM2, releasing p53 from the p53/MDM2 complex inducing proliferation inhibition and cell cycle arrest through the transcriptional activity of p53. Irinotecan itself can be further converted to several active/ inactive metabolites by the action of intracellular enzymes such as carboxylesterase, uridine diphosphate glucuronosyltransferase 1A1 (UGT 1A1) and CYP3A4. The mechanism of action of SN-38, the most well-known active metabolite of Irinotecan, is best described as an inhibitor of Topo I, forming a trimetric complex of DNA-
Fig. 4. Four different phenotype assessments of HCT-116 cells treated with Nutlin-3, ABT-737, or Irinotecan. (A) ATP assay to determine the cell viability after treatment with the compounds (B) The caspase 3/7 activity assay to determine cell apoptosis at the IC50 of the compounds (C) BrdU assay to determine cell proliferation at the IC50 of the compounds. (D) Cell cycle distribution was analysed by PI staining with cell cycle analysis at the IC50 of the compounds.
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Irinotecan-Topo I. When this cleavable complex collapses facing replication forks during S-phase, permanent double-strand DNA nicks form, and an irreversible arrest occurs. Activation of the DNA damage checkpoint response, which leads to cell cycle arrest, usually is relative to the other immediate cell death mechanisms such as necrosis and apoptosis. Therefore, SN-38 exerts a cytotoxicity activity greater than its parental compound. In these aspects, SN-38 has been believed to be a more potent Topo I inhibitor. It also has been reported that the accumulation of SN-38 causes many severe side-effects. To overcome these phenomena, we propose to reconsider Irinotecan's anti-cancer activity by suggesting new targets that control the cellular death mechanism. With our finding that Irinotecan itself can modulate the process of cell death, development of a non-esterified compound is recommended to overcome Irinotecan resistance such as in colorectal cancer. Moreover, treating patients with a carboxylesterase mutation, which lowers the activity, with a non-esterified compound can be a better indication for cancer as a precision medicine.
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Acknowledgement This study was supported by a grant from the Bio-Synergy Research Project (NRF-2012M3A9C4048759) of the Ministry of Science, ICT and Future Planning through the National Research Foundation. This work was also supported by NRF grants funded by the Korean government (MSIT) (NRF-2017R1E1A1A01074403) and by the KRIBB Research Initiative Program. Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.009.
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Transparency document
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Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.009.
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A novel mechanism of irinotecan targeting MDM2 and Bcl-xL Boah Lee a, Jeong A. Min b, c, Abdullateef Nashed a, Sang-Ok Lee b, Jae Cheal Yoo a, Seung-Wook Chi b, c, **, Gwan-Su Yi a, * a
Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, South Korea Disease Target Structure Research Center, KRIBB, Daejeon, 34141, South Korea c Department of Proteome Structural Biology, KRIBB School of Bioscience, Korea University of Science and Technology, Daejeon, 34113, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2019 Accepted 2 April 2019 Available online 2 May 2019
Irinotecan is a strong anticancer drug whose mechanism of action has been reported only for the inhibition of DNA topoisomerase I (Topo I) through its active metabolite SN-38. In this study, we present a new mechanism of Irinotecan which inhibits the activities of MDM2, an E3 ligase of tumour suppressor p53, and Bcl-xL, an anti-apoptotic protein, through direct binding. In our structure modelling study, Irinotecan could fit to the binding sites of MDM2 and Bcl-xL for their known drugs, Nutlin-3 and ABT-737, with a better binding affinity than to Topo I. The direct binding of Irinotecan to both proteins was confirmed through a NMR study. We further showed that Irinotecan increased the amount of p53 only in the presence of MDM2 and inhibited the physical interaction of Bcl-xL with Bim, a core pro-apoptotic protein. In addition, we demonstrated that Irinotecan induced the down regulation of proliferation and strong G2/M arrest in HCT116 colon cancer cells shortly after treatment. Collectively, we suggest a new mechanism of action for Irinotecan as a dual target inhibitor of MDM2 and Bcl-xL facilitating the anticancer activities mediated by p53 and Bcl-xL interaction partners. © 2019 Elsevier Inc. All rights reserved.
Keywords: Irinotecan Dual-targets MDM2 Bcl-xL Structure modelling NMR
1. Introduction p53, a key tumour suppressor protein, plays a key role in the cellular stress response pathway. The proapoptotic activity of p53 is controlled by a number of proteineprotein interactions that constitute a network of negative and positive regulators [1]. MDM2 is located in the central part of this network by regulating p53 with direct interaction [2]. MDM2 is an E3 ubiquitin ligase [3] targeting p53 causing its proteasomal degradation [4]. Bcl-xL, a well-known antiapoptotic protein, is another direct interaction partner of p53; consequently, both MDM2 and Bcl-xL became the targets of anticancer drugs such as Nutlin-3 [5] and ABT-737 [6], respectively.
Abbreviations: Topo I, topoisomerase I; BH, Bcl-2 homology; MDM2, Mouse double minute 2 homolog; Bcl-xL, B-cell lymphoma extra-large; DS, Discovery Studio 3.1; IPTG, isopropyl-b-D-thiogalactopyranoside; KBSI, Korea Basic Science Institute; PS, penicillin-streptomycin; FBS, fetal bovine serum; RT, room temperature; BrdU, 5-bromo-20 -deoxyuridine; IP, immunoprecipitation; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; UGT 1A1, uridine diphosphate glucuronosyltransferase 1A1. * Corresponding author. ** Corresponding author. Disease Target Structure Research Center, KRIBB, Daejeon, 34141, South Korea. E-mail addresses: [email protected] (S.-W. Chi), [email protected] (G.-S. Yi). https://doi.org/10.1016/j.bbrc.2019.04.009 0006-291X/© 2019 Elsevier Inc. All rights reserved.
The p53 binding sites of both MDM2 and Bcl-xL have been characterized as Bcl-2 homology (BH) motifs [7], which is also the targeting site of their drugs, according to NMR studies [8,9]. With this mechanism, Nutlin-3 binds to the BH3 motif of MDM2, interfering with its interaction with p53, rescuing the tumour suppressor function of p53. As p53 does to Bcl-xL, ABT-737 binds to the BH3 motif of Bcl-xL, preventing any further inhibiting interactions of Bcl-xL with essential proapoptotic proteins such as Bax and Bim [10,11]. Despite the similar structure of the binding site, there have been few studies on compounds that bind commonly to both targets [12]. We searched for such compounds using in silico structure modelling, and Irinotecan was one of the best candidates that can bind to both targets. Irinotecan, an alkaloid derived from the extracts of the tree Camptotheca acuminata, is commonly used for the treatment of colorectal and lung cancer [13]. It is well known that this compound is converted by intracellular carboxylesterase activity to its active metabolite termed SN-38. This metabolite SN-38 binds to DNA Topo I, stabilizes the DNA Topo IeDNA complex and prevents DNA ligation. It has been reported that double-stranded DNA breakage induces ATM kinase activation and autophosphorylation of DNA Topo I resulting in the DNA damage check point response and apoptosis
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and cell cycle arrest [14]. Until now, the reported mechanism of Irinotecan, a strong anticancer drug, has only focused on the inhibition of DNA topoisomerase I (Topo I) through its active metabolite SN-38. In this study, we found that Irinotecan, is a dual specific inhibitor of MDM2 and Bcl-xL through structure modelling and confirmed the direct binding of Irinotecan to both proteins through a NMR study. Moreover, Irinotecan regulates apoptotic cell death, proliferation inhibition and G2/M cell cycle arrest in response to these interactions. With these results, we propose a new perspective on the anticancer mechanism of Irinotecan through its binding to MDM2 and Bcl-xL. 2. Materials and methods 2.1. Structure modelling Nutlin-3 and ABT-737 were used to compare the 2D structural similarities with Irinotecan using Pipeline Pilot [Pipeline Pilot; Accelrys, Inc.: San Diego, CA, 2000]. We converted the 2D structures to molecular descriptors: ECFP4, standard circular fingerprints based on the Morgan algorithm [15]. The similarity between the compound pairs was calculated by the Tanimoto similarity. Nutlin-3 and ABT-737 were also used to build pharmacophore features from 3D conformations of the molecules generated with Discovery Studio 3.1 (DS) from the Accelrys Co. It identified configurations or 3-dimensional spatial arrangements of the chemical features of Nutlin-3 and ABT-737. The protocol considered the hydrogen bond acceptor/donor, hydrophobic feature, negative/ positive ionizable feature and aromatic ring for feature types to generate a selective pharmacophore model from a single ligand. A set of candidate pharmacophore models were enumerated from the features. The protocol picked the pharmacophore with the highest selectivity as predicted by a Genetic Function Approximation model [16]. Docking simulations using the Libdock algorithm [17] in DS were performed with compounds against three proteins, MDM2, Bcl-xL, and Topo I, respectively. The X-ray crystal structure complex of MDM2 with Nutlin-3 was obtained from the protein data bank (PDB ID: 4HG7) [18], and the X-ray crystal structure complex of BclxL with ABT-737 was obtained from the PDB (PDB ID: 2YXJ) [19]. Moreover, the X-ray crystal structure complex of DNA and human Topo I was obtained from the PDB (PDB ID: 1A35) [20]. The proposed binding site was centered on the ligand, and a site sphere was created with the following coordinates: 23.835, 7.53, 14.053 with 9.5 Å diameter for MDM2; 39.1692, 4.14924, 10.7891 with 9 Å diameter for Bcl-xL and 39.1692, 4.14924, 10.7891 with 13.6 Å diameter for Topo I. The protocol included 100 hotspots with a docking tolerance 0.25. The FAST conformation method was also used with CHARMm. All other parameters followed the default settings. 2.2. Protein purification For the NMR experiments, 15N-labeled MDM2 and Bcl-xL were expressed in minimal media containing 15N-NH4Cl. The Escherichia coli BL21 CodonPlus (RP) cells were grown to an optical density at 600 nm (OD600) of ~0.9 at 37 C, and expression of the N-terminal domain of the MDM2 (residues 3e109) construct was induced at 20 C with 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG) for 16 h. The protein was precipitated with ammonium sulphate and further purified using ion exchange columns (HiTrap® SP and QSepharose, GE healthcare) and a gel-filtration column (HiLoad® 16/ 600 Superdex 75 pg, GE healthcare). The 15N-labeled truncated BclxL construct was expressed and purified for the NMR experiments
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as previously reported [21,22]. 2.3. NMR spectroscopy All the NMR spectra were collected on a Bruker Avance II 800 spectrometer at the Korea Basic Science Institute (KBSI). The 2D 1 H-15 N HSQC spectra of 0.1 mM MDM2 and Bcl-xL each obtained at 10 and 25 C, respectively, were with and without the addition of 0.4 mM Irinotecan. The NMR samples comprised 90% the H2O/10% D2O and were prepared in 20 mM MES (pH 6.5), 50 mM NaCl, and 1 mM DTT for MDM2 and 20 mM sodium phosphate (pH 6.5), 50 mM NaCl and 1 mM DTT for Bcl-xL. All the NMR data were processed and analysed using nmrPipe/nmrDraw and the SPARKY software. For the assignment of the 2D 1H-15 N HSQC spectra of MDM2 and Bcl-xL, we used the BMRB entry 18876 and 18250, respectively. 2.4. Cell culture and compounds The HCT116 cells were cultured in McCoy's 5a medium supplemented with 2 mM Glutamine, 1% penicillin-streptomycin, and 10% Fetal Bovine Serum at 37 C with 5% CO2. SW480 cells were cultured in L-15 medium in the same environment without CO2. Irinotecan was purchased from Biopurify Phytochemicals Ltd. ABT-737 was purchased from APExBIO and Nutlin-3 from Sigma Aldrich. 2.5. Western blot analysis and immunoprecipitation Cells were lysed with RIPA buffer containing a proteaseinhibitor cocktail. Whole-cell lysates were incubated on ice for 30 min and then cleared at 20,000 x g for 20 min at 4 C. For the immunoprecipitation (IP) analysis, the resulting supernatants were incubated with anti-Bcl-xL antibody for 3 h, at 4 C with an additional incubation for 2 h after the addition of protein A/G plus agarose (Santa Cruz Biotechnology). The immunocomplexes captured in the agarose gel were then washed three times with RIPA buffer and eluted with SDS gel-loading buffer. For the Western blot analysis, the supernatants were separated by SDS-PAGE using 10% gels and blotted onto PVDF membranes. The blots were then probed with anti-p53, anti-MDM2 (all from Thermo Fisher Scientific), anti-Bim, anti-Bcl-xL, and anti-GAPDH (all from Cell signaling) antibodies. Blots were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies, followed by additional washing and then detection with enhanced chemiluminescence (ECL; Amersham). 2.6. Viability assay Cell viability was performed using Cell Titer-Glo cell viability assay kit (Promega). HCT-116 cells were seeded into a 96 well plate at a density of 104 cells/well. The cells were incubated for 24 h before treatment with the compound. Treated plates were incubated for 48 h at 37 C with 5% CO2. Before the assay, cells were incubated at room temperature (RT) for 30 min and 100 ml of assay reagent were added to each well. The luminescence was measured using the VICTOR X Multilabel Reader (PerkinElmer). 2.7. Apoptosis assay Apoptosis assay was performed using thee Caspase-Glo 3/7 assay kit (Promega). HCT-116 cells were seeded into a 96 well plate at a density of 104 cells/well. The cells were incubated for 24 h before treatment with the compound. Cells were then treated with a concentration equivalent to the IC50 for each compound calculated from the viability dose response curve. Before the assay, cells
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were incubated at RT for 30 min, and 100 ml of assay reagent were added to each well. The luminescence was measured using the VICTOR X Multilabel Reader (PerkinElmer). 2.8. Cell proliferation assay The cellular proliferation was determined using cell proliferation ELISA (Roche) via 5-bromo-20 -deoxyuridine (BrdU) incorporation according to the manufacturer's protocol. Cellular incorporation of BrdU was visualized with a substrate solution and, the chemiluminescence was measured using a luminometer. Each experiment was performed in triplicate. 2.9. Cell cycle analysis Cells were seeded in a 25 cm2 flask (106 cells/flask) incubated for 24 h and were treated with compounds for 8 h. Cells were trypsinized, harvested, and fixed in 1 ml of 80% cold ethanol in test tubes and incubated at 4 C for 15 min. After incubation, the cells were centrifuged at 1500 rpm for 5 min, and the cell pellets were resuspended in 500 ml of propidium iodine (10 mg/ml) containing 300 mg/ml RNase (Sigma-Aldrich). Then, the cells were incubated on ice for 30 min and filtered with nylon mesh. Cell cycle distribution was calculated with the BD FACSDiva Clinical software. 3. Results 3.1. Identification of direct binding of Irinotecan to MDM2 and BclxL To find the molecules targeting MDM2 and Bcl-xL, we searched for compounds that are similar to Nutlin-3 and ABT-737. Among the best candidates, we selected Irinotecan for further investigation because it showed anti-cancer functions that matches with the effect of suppressing MDM2 and Bcl-xL in previous studies related to Topo I. To investigate the similarity of the compound with ABT-737 and
Nutlin-3, we first performed a structural similarity analysis based on the extended connectivity fingerprints. Tanimoto scores were calculated to measure the similarities of the compound pairs. In the similarity analysis, the Tanimoto scores were 0.18 and 0.11 for ABT737 and Nutlin-3, respectively. Because the similarities of the 2D structures were low, we conducted pharmacophore modelling to find the similarity considering the 3D arrangement of the structure. We generated the pharmacophore features of Nutlin-3 and ABT-737, including the hydrogen bond acceptor/donor features, positive/negative ionizable features, aromatic ring features, and hydrophobic features. Irinotecan was well suited to the derived model with fit values of 0.55 and 0.56, respectively. (Fig. 1A- B). In addition, docking simulations of Irinotecan further supported that it binds more stably to MDM2 and Bcl-xL than to Topo I based on the energy values: 100.37, 170.59 and 99.53 respectively. Especially, resides such as V53, L54, L57, F91, and V93 in MDM2 and L130, R139, A142, and Y195 in Bcl-xL in the binding region were proven to be also present in the NMR result (Fig. 1CeE). To examine the direct binding of Irinotecan to MDM2 and Bcl-xL, we performed 2D 1H-15 N HSQC experiments using NMR spectroscopy. During the binding titration with Irinotecan, we observed NMR chemical shift perturbations of 15 N -labeled MDM2 and BclxL (Fig. 2A and D, Fig. S1). The overlaid 2D 1H-15 N HSQC spectra showed that some cross-peaks of MDM2 and Bcl-xL disappeared, and others moved significantly, indicating that the binding involved a fast to intermediate exchange between the free and Irinotecan-bound forms on the NMR chemical shift time scale. These NMR chemical shift perturbations demonstrate the direct binding of Irinotecan to both MDM2 and Bcl-xL. The residues showing significant chemical shift perturbations upon Irinotecan binding were plotted against the amino acid sequences (Fig. 2B and E) and projected onto the structures of MDM2 (PDB ID: 4HG7) (Fig. 2C) and Bcl-xL (PDB ID: 2YXJ) (Fig. 2F). For MDM2, the NMR chemical shift perturbations induced by binding to Irinotecan were localized to a p53 peptide-binding hydrophobic binding pocket, which are in good agreement with that previously observed during
Fig. 1. (AeB) Pharmacophore models. Fitted pharmacophore model of Irinotecan to the models of Nutlin-3 and ABT-737, respectively. The orange colour represents the aromatic ring, cyan for hydrophobic, green for hydrogen bond acceptor and red for positive ionizable variables. Fit values are indicated in the parentheses. (CeE) Predicted binding modes: energies are indicated in the parentheses. The docking poses of Irinotecan in the MDM2, Bcl-xL and Topo I. Among the docking results, the residues mapped in the NMR results are shown in red.
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Fig. 2. Direct binding of Irinotecan to MDM2 and Bcl-xL proteins. The overlaid 2D 1H-15 N HSQC spectra for 15N-labeled MDM2 (A) and Bcl-xL (D) proteins without (blue) and with the addition of Irinotecan (red) (at the molar ratio of 1:4). NMR chemical shift perturbations on MDM2 (B) and Bcl-xL (E) induced by binding to Irinotecan. Weighted DCS ¼ [(D1H)2 þ 0.2(D15 N)2]0.5, in which the D1H and D15 N are the chemical shift changes on the 1H and 15 N dimensions, and plotted against the residue number of the MDM2 and Bcl-xL. Resonances of MDM2 and Bcl-xL disappeared upon binding of Irinotecan are shown as grey bars. Binding site mapping of Irinotecan on the structures of MDM2 (C) (PDB code: 4HG7) and Bcl-xL (F) (PDB code: 2YXJ). The residues that disappeared upon chemical binding are shown in red, and the residues showing the chemical shift changes of DCS >0.015 ppm in MDM2 and DCS >0.03 ppm in Bcl-xL are coloured in yellow.
the binding of Nutlin-3 (Fig. S2A). For Bcl-xL, the Irinotecaninduced NMR chemical shift perturbation occurred on the BH3 peptide-binding hydrophobic groove surrounded by the BH1, BH2 and BH3 domains, which overlapped with the ABT-737-binding site (Fig. S2B). These results proved a direct interaction between Irinotecan and specific hydrophobic interfaces of MDM2 and Bcl-xL proteins. 3.2. Irinotecan blocks the binding of MDM2 and Bcl-xL to their signaling partners To confirm the function of Irinotecan on MDM2 and Bcl-xL, we tested the effects of MDM2/p53 and Bcl-xL/Bim binding. First, to identify the Irinotecan specific effect in MDM2-p53
binding, p53 expression was measured by treatment with Irinotecan in HCT116 cells (Fig. 3A). Irinotecan increased the intact protein level of p53 and MDM2 in a dose dependent manner just like Nutlin-3 (Fig. 3A). These results indicate that Irinotecan is an inhibitor of the MDM2-p53 interaction just like Nutlin-3. To further confirm Irinotecan as an inhibitor of MDM2-p53 binding, we tested the effect of Irinotecan in SW480 cells, transcriptionally inactive p53 mutated cells (Fig. 3B). Interestingly, constant expression of p53 but no detectable MDM2 expression was found in the SW480 cells with the same concentration of Irinotecan that was used in the HCT116 cells (Fig. 3B). These results show that Irinotecan induces wild-type p53 accumulation, which is dependent on MDM2 expression, and that Irinotecan specifically regulates the MDM2-p53 binding.
Fig. 3. (A) Expression levels of p53 and MDM2 proteins in HCT116 cells. The indicated concentrations of the compound were incubated at the IC10, IC25 and IC50, respectively. Expression level of GAPDH was used as the loading control. (B) Expression levels of p53 and MDM2 proteins were confirmed by the indicated concentrations in SW480, p53 mutant cells. (C) Co-IP data show the interaction between Bcl-xL and Bim in HCT116 cells with the indicated concentrations of the compound.
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To investigate the Irinotecan-mediated inhibition of the Bcl-xL related apoptotic signal, the physical interaction of Bcl-xL and Bim in HCT116 cells was examined (Fig. 3C). Irinotecan treatment induced the disruption of the Bcl-xL and Bim interactions in a dosedependent manner just like ABT-737. (Fig. 3C). This result shows that Irinotecan is involved in direct targeting of Bcl-xL in HCT116 cells. 3.3. Irinotecan induces apoptosis, proliferation regulation, and cell cycle arrest To investigate the efficacy of Irinotecan in inducing Bcl-xL and/ or MDM2 related responses, viability, apoptosis, proliferation, and cell cycle arrest assays were performed. The viability of HCT116 cells were measured in a dosedependent manner. In addition, two control compounds were used, ABT-737 and Nutlin-3, which are Bcl-xL and MDM2 specific inhibitors, respectively. The dose response curves of each compound was plotted, and the IC50 values were calculated from the graph (Fig. 4A). The IC50 values were 10, 18 and 12 mM for Irinotecan, Nutlin-3 and ABT-737, respectively. This result shows that Irinotecan has a significant potency in reducing cell viability compared to Nutlin-3 and ABT-737. To investigate the contribution of apoptosis induction in the observed viability reduction, caspase 3/7 activity assays were performed in cells using the IC50 dosing compounds (Fig. 4B). ABT-737 showed the highest efficacy in the induction of apoptosis, and the ABT-737 response can be induced mainly by direct inhibition of BclxL. On the other hand, the weak apoptotic effect of Nutlin-3 may represent a secondary effect of Nutlin-3 through apoptosis related p53 signaling. In this condition, the apoptotic effect of Irinotecan was almost two times higher than that of Nutlin-3 but was half that of ABT-737 (Fig. 4B). BrdU is commonly used for the detection of proliferating cells. To investigate the contribution of cell growth inhibition in the observed viability reduction, proliferation assays were performed on the cells using the compounds at the IC50 values with BrdU
staining (Fig. 4C). BrdU incorporation was measured in Irinotecan or Nutlin-3 treated HCT-116 cells. The percentage of BrdU positive cells in Irinotecan or Nutlin-3 treated cells was nearly 10-fold lower than that of the control cells (Fig. 4C). Irinotecan showed an antiproliferative effect similar to Nutlin-3, a known inhibitor MDM2p53 binding, in the HCT116 cells (Fig. 4C). To determine whether the inhibitory effect of Irinotecan is mediated by cell cycle arrest, the effect of Irinotecan on cell cycle progression was analysed by flow cytometer. As shown in Fig. 4D, most of the HCT116 cells were in the G1 phase. The G2/M phase cell population was significantly increased in cells treated with Nutlin3. In addition, the Irinotecan treatment significantly increased the late phase of the cell cycle population in the HCT116 cells (Fig. 4D). Irinotecan showed a strong G2/M cell cycle arrest in this cell, like Nutlin-3, a known inhibitor and MDM2-p53 binding agent. 4. Discussions In this work, a NMR study was performed to obtain definite evidence of binding of Irinotecan to the two targets, and pharmacophore models were constructed using previously known compounds that bind to the two targets to understand the structural reasons. In addition, a docking simulation analysis showed that Irinotecan binds more stably to MDM2 and Bcl-xL than to Topo I. Furthermore, we demonstrated a novel dual target-based anticancer activity of Irinotecan with a short treatment time: (1) Irinotecan binds directly to antiapoptotic Bcl-xL, to release Bim (proapoptotic proteins) from complexes and induce apoptosis. (2) Irinotecan binds to MDM2, releasing p53 from the p53/MDM2 complex inducing proliferation inhibition and cell cycle arrest through the transcriptional activity of p53. Irinotecan itself can be further converted to several active/ inactive metabolites by the action of intracellular enzymes such as carboxylesterase, uridine diphosphate glucuronosyltransferase 1A1 (UGT 1A1) and CYP3A4. The mechanism of action of SN-38, the most well-known active metabolite of Irinotecan, is best described as an inhibitor of Topo I, forming a trimetric complex of DNA-
Fig. 4. Four different phenotype assessments of HCT-116 cells treated with Nutlin-3, ABT-737, or Irinotecan. (A) ATP assay to determine the cell viability after treatment with the compounds (B) The caspase 3/7 activity assay to determine cell apoptosis at the IC50 of the compounds (C) BrdU assay to determine cell proliferation at the IC50 of the compounds. (D) Cell cycle distribution was analysed by PI staining with cell cycle analysis at the IC50 of the compounds.
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Irinotecan-Topo I. When this cleavable complex collapses facing replication forks during S-phase, permanent double-strand DNA nicks form, and an irreversible arrest occurs. Activation of the DNA damage checkpoint response, which leads to cell cycle arrest, usually is relative to the other immediate cell death mechanisms such as necrosis and apoptosis. Therefore, SN-38 exerts a cytotoxicity activity greater than its parental compound. In these aspects, SN-38 has been believed to be a more potent Topo I inhibitor. It also has been reported that the accumulation of SN-38 causes many severe side-effects. To overcome these phenomena, we propose to reconsider Irinotecan's anti-cancer activity by suggesting new targets that control the cellular death mechanism. With our finding that Irinotecan itself can modulate the process of cell death, development of a non-esterified compound is recommended to overcome Irinotecan resistance such as in colorectal cancer. Moreover, treating patients with a carboxylesterase mutation, which lowers the activity, with a non-esterified compound can be a better indication for cancer as a precision medicine.
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Acknowledgement This study was supported by a grant from the Bio-Synergy Research Project (NRF-2012M3A9C4048759) of the Ministry of Science, ICT and Future Planning through the National Research Foundation. This work was also supported by NRF grants funded by the Korean government (MSIT) (NRF-2017R1E1A1A01074403) and by the KRIBB Research Initiative Program. Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.009.
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Transparency document
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Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.009.
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