key: cord-0819066-7vndh3hp
authors: Fu, Lifeng; Ye, Fei; Feng, Yong; Yu, Feng; Wang, Qisheng; Wu, Yan; Zhao, Cheng; Sun, Huan; Huang, Baoying; Niu, Peihua; Song, Hao; Shi, Yi; Li, Xuebing; Tan, Wenjie; Qi, Jianxun; Gao, George Fu
title: Both Boceprevir and GC376 efficaciously inhibit SARS-CoV-2 by targeting its main protease
date: 2020-09-04
journal: Nat Commun
DOI: 10.1038/s41467-020-18233-x
sha: 3e129cc7772601ba52d8a4b94b9d8606bf194a35
doc_id: 819066
cord_uid: 7vndh3hp

COVID-19 was declared a pandemic on March 11 by WHO, due to its great threat to global public health. The coronavirus main protease (M(pro), also called 3CLpro) is essential for processing and maturation of the viral polyprotein, therefore recognized as an attractive drug target. Here we show that a clinically approved anti-HCV drug, Boceprevir, and a pre-clinical inhibitor against feline infectious peritonitis (corona) virus (FIPV), GC376, both efficaciously inhibit SARS-CoV-2 in Vero cells by targeting M(pro). Moreover, combined application of GC376 with Remdesivir, a nucleotide analogue that inhibits viral RNA dependent RNA polymerase (RdRp), results in sterilizing additive effect. Further structural analysis reveals binding of both inhibitors to the catalytically active side of SARS-CoV-2 protease M(pro) as main mechanism of inhibition. Our findings may provide critical information for the optimization and design of more potent inhibitors against the emerging SARS-CoV-2 virus.

I n December 2019, a novel coronavirus was discovered due to emerging viral pneumonia cases [1] [2] [3] [4] [5] . The virus has rapidly spread to more than 200 countries in the world 6, 7 . The World Health Organization (WHO) named the infectious disease as COVID-19, and declared a global pandemic on 11 March 2020 8 . As of 28 March, the disease has caused more than 500,000 human infections with over 20,000 deaths globally 9 .

Human coronavirus 2019 (HCoV-19) 10 is the seventh coronavirus capable of infecting humans, which was later named SARS-CoV-2 by the International Committee on Taxonomy of Viruses (ICTV) 11 though with different name being proposed. The other six coronaviruses are the low-pathogenicity members including HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, and highly pathogenic SARS-CoV and MERS-CoV 12 . The clinical manifestations of COVID-19 include fever, fatigue, dry cough, headache, and diarrhea 13 . The median incubation period of the disease is 4 days, and the longest is no more than 41 days 13 . The patient at incubation period is contagious, and the median duration of viral shedding was 20 days in survivors, but the SARS-CoV-2 was detectable until death in non-survivors 14 . The longest observed duration of viral shedding in survivors was 37 days 14 . Some mildly ill patients do not have fever and obvious respiratory symptoms 14 . Most of the patients have a good prognosis, and the symptoms of children are relatively mild 14 . The patients with older age and comorbidities such as hypertension, diabetes, and coronary heart disease will have high risk of death 14 .

Sequence comparison has showed that the SARS-CoV-2 has the closest relationship (96.2%) with the bat SARS-like coronavirus 4, 12 , but the origin of this virus was yet to be identified. Recent studies have shown that the SARS-CoV-2 uses the same Angiotensin-converting enzyme 2 (ACE2) receptor as SARS-CoV 4 , and the structural basis of receptor recognition was quickly elucidated to provide important basis for the molecular understanding of virus entry process and the development of potential antiviral inhibitors [15] [16] [17] [18] . Some old drugs such as Remdesivir, Favipiravir, and Chloroquine/Hydroxychloroquine, and also traditional Chinese medicines showed potential for the treatment of COVID-19 [19] [20] [21] [22] . However, to date, no clinically approved specific drugs or vaccines are available to treat the disease. Therefore, it is urgent to develop specific drugs against the virus.

The coronavirus main protease (M pro , 3CLpro) is essential for viral polyproteins processing and maturation, therefore, it is recognized as an attractive drug target. Actually, viral proteases are also promising targets for many different viruses including hepatitis C virus (HCV)and human immunodeficiency virus (HIV) 23 . As there is neither specific antiviral agents nor available vaccines, repurposing of clinically approved drugs to combat the COVID-19 is urgently needed. Here we show both Boceprevir and GC376 can inhibit M pro activity and SARS-CoV-2 in Vero cells. Moreover, combination of GC376 with Remdesivir treatment can enhance antiviral activity. Additionally, the highresolution crystal structures of SARS-CoV-2 M pro complex with these two inhibitors are solved to figure out their mechanism of inhibition. Taken together, these data provide critical information for the optimization and design of more potent inhibitors against the emerging pathogen SARS-CoV-2.

High throughput drug screening. In the beginning, we obtained soluble and pure M pro protein of the SARS-CoV-2 with an extra glycine residue at the N-terminus (GM pro ) after TEV cleavage by expression in E. coli cells. At the same time, we found that the M pro of SARS-CoV-2 has high homology with other CoV M pro ( Supplementary Fig. 1 ). So, we selected a cluster of 18 chemical drugs that were designed to target the different viral proteases and proteasome (Table 1) . Then, we screened these chemical drugs by in vitro fluorescence resonance energy transfer (FRET) enzymatic assays at a single concentration (100 μM; Fig. 1a , b). We identified two inhibitors, Boceprevir and GC376, can inhibit the enzymatic activity well (Fig. 1b) . In contrast, other drugs such as Aluvia ® (HIV protease inhibitors, lopinavir, and ritonavir) did not show detectable inhibitory activity, which is consistent with the reports of recent clinical trials that Aluvia ® has no obvious effect on the treatment of COVID-19 24 .

Enzyme activity study and its structural basis. Before further evaluating protein level inhibitory activity of Boceprevir and GC376, we found that extra residues at the N-terminus would impair the enzyme activity in previous report 25 . Therefore, we expressed the native M pro without any extra amino acids at the N-terminus (a gift from Dr Haitao Yang in ShaghaiTech University) according to the literature 26 . Then, we compared GM pro activity with the native M pro . Substrate hydrolysis rate of the native M pro (frist 1000 s initial speed v 0 = 1618 ± 84 RFU s −1 ) is almost three times faster than that of GM pro (first 1000 s initial speed v 0 = 449 ± 19 RFU s −1 ), which must be caused by extra Gly at the N terminus ( Supplementary Fig. 2a) . Again, our data readdress the importance of the free N-terminus after previous report of 2 or 5 extra amino acids, showing 20-fold or 150-fold slower catalytic efficiency 25 . In order to figure out how the glycine at the N-terminus affects protease activity, we crystallized the GM pro protein and solved the crystal structure at a resolution of 2.0 Å. The overall structure of GM pro is highly similar to those of SARS-CoV-2 native M pro and SARS-CoV native M pro (Supplementary Fig. 3a ). The first residue Ser is disordered in GM pro structure, indicating this residue cannot stabilize Glu166 of the S1 subsite in the neighboring protomer, which may be the main reason for the decrease of enzyme activity ( Supplementary  Fig. 3a) . However, the extra glycine did not induce the main chain of residues 141-142 moves towards the S1 subsite as seen in previous report with five extra amino acids 25 ( Supplementary  Fig. 3b ).

Enzyme inhibitory activity of Boceprevir and GC376. Boceprevir is a serine protease inhibitor that was approved by FDA to treat HCV infection in 2011. It was reported that the ketoamide group of Boceprevir can reversibly bind covalently to the Ser139 of HCV NS3/4A protease, whose hydroxyl group function as nucleophilic groups in enzymatic reaction 27 . Boceprevir was shown to be a time-dependent inhibitor with a fast-initial binding followed by a slow formation of the covalent adduct against NS3/ 4A protease 28 . GC376 is a cysteine protease covalent inhibitor against picornaviruses, noroviruses, and coronaviruses, and has shown promise in treating cats with fatal feline infectious peritonitis (FIP) caused by FIPV 29, 30 . For time-dependent inhibitors, we tried to evaluate the equilibrium-binding constant K i and the inactivation rate constant k inac for covalent bond formation of Boceprevir and GC376 without preincubation. Progress curve of peptide hydrolysis by M pro showed the inactivation processes of both two inhibitors are very slow ( Supplementary Fig. 2b, c) . So we used the IC 50 values of these two inhibitors, 8.0 μM and 0.15 μM, to represent the inhibitory activity (Fig. 1d, f) , which has also been used in other covalent inhibitor studys 31, 32 . Meanwhile, we have performed the inhibition assay to test if these two compounds inhibit bovine chymotrypsin at the concentrations of 20 and 50 μM ( Supplementary Fig. 2d) . The results showed that GC376 does not have any inhibition activity against the bovine chymotrypsin, while Boceprevir shows very trivial inhibition activity, indicating the M pro inhibitory activity of these two compounds are specific. Fig. 4 ). In order to further validate the antiviral effect of these inhibitors, we performed the traditional plaque assay stained with crystal violet. The plaque produced by the SARS-CoV-2 does not look like circles, which caused that it is difficult to quantify the antiviral efficacy for GC376 and Boceprevir. The possible reason is that the virus has been incubated with cells for a long time and multiple plaques are connected together. However, in presence of 40 μM Boceprevir or 1.6 μM GC376, the size and number of plaques are obviously smaller and less ( Supplementary Fig. 5a , b). In addition, combination of 1 μM GC376 and 1 μM Remdesivir can completely inhibit viral replication in virus plaque assay ( Supplementary Fig. 5d) , showing additive effect of the joint application of RdRp inhibitors and protease inhibitors targeting different viral proteins.

Inhibition mechanism of Boceprevir and GC376 against M pro . In order to elucidate the inhibitory mechanisms of these two compounds, we determined the crystal structures of M pro -Boceprevir and M pro -GC376 complexes at resolutions of 1.60 Å and 2.00 Å, respectively (Supplementary Fig. 6 and Supplementary Table 1 ). The unambiguous electron density maps show that the two inhibitors bind in the active site of M pro in different conformations ( Supplementary Fig. 7 ). In the M pro -Boceprevir complex structure, the Sγ atom of the nucleophilic Cys145 in M pro forms a C-S covalent bond with the keto carbon of Boceprevir, which is a typical Michael addition (Fig. 3a , b and Supplementary Fig. 8a ). Residues His41, Gly143, and His164 form four hydrogen-bonding interactions with the amide backbone of Boceprevir in one side, and residue Glu166 form three hydrogenbonding interactions with Boceprevir in the other side. The small size of cyclobutylalanice (c-Bua) residue can be tolerated by S1 subsite. However, the P1 c-Bua residue has no interaction with S1 subsite (Fig. 3b) . The hydrophilic Glu166 pushes the P1 hydrophobic group away and exposes the c-Bua residue to solvent. This can explain why Boceprevir displays a moderate activity against M pro . The rigid P2 dimethylcyclopropylproline (DMCP) residue can fit S2 subsite well (Fig. 3a) and has hydrophobic interaction with Met149 and Asp187 ( Supplementary  Fig. 8a ). The hydrophobic P3 and P4 tert-butyl (tBu) residue can interact with Met165 in S3 subsite and Gln189, Gln192, Thr190 in S4 subsite ( Supplementary Fig. 8a ). Compared with HCV NS3/ 4A-Boceprevir complex 27 , the binding pocket are quite different due to low structural similarity, and the compound has large conformational changes to fit the HCV NS3/4A binding pocket (Fig. 3c) . In all, 80% P1 c-Bua residue of Boceprevir is buried in S1 subsite which contributes the largest factor to binding. By contrast, in the SARS-CoV-2 M pro -Boceprevir complex structure, the P2 DMCP residue and P4 tBu cap are 80% buried other than P1 c-Bua residue. About 40% of P2, P3, and P4 residues are buried in S2, S3, and S4 subsite of HCV NS3/4A protease, which are highly exposed to solvent (Fig. 3c) 27 .

In the M pro -GC376 complex structure, the bisulfite group of GC376 was removed and GC376 created a covalent bond with Cys145 as aldehyde form in the complex structure (Fig. 3d , e and Supplementary Fig. 8b ). The glutamine surrogate ring of GC376 fits into the S1 pocket and has hydrogen bonding interactions with the carboxyl group of Glu166. The Leu of GC376 fits into S2 hydrophobic pocket, which is consists of residues Arg40, His41, Met49, Tyr54, and Asp187. Compared with transmissible gastroenteritis virus (TGEV) M pro -GC376 complex, both the binding pocket and the configuration of GC376 are highly similar, suggesting a conserved interaction mode for GC376 against different coronaviruses (Fig. 3f) . The crystal structures of GM pro -Boceprevir and GM pro -GC376 complexes at resolutions of 1.80 Å and 1.40 Å was also determined to evaluate the influences of the extra glycine at N terminal. Compared to M pro -Boceprevir, only P4 residue of Boceprevir has slight changes in GM pro -Boceprevir complex (Supplementary Fig. 3c) . The binding mode of GC376 with GM pro or M pro has no obvious changes ( Supplementary Fig. 3d ).

We have performed the SAR analysis of other 3CLpro inhibitors reported recently. Compared with the inhibitors N3 26 , 13a (IC 50 = 0.67 μM) 31 , 11a (IC 50 = 0.053 μM), and 11b (IC 50 = 0.04 μM) 32 , the P1 c-Bua residue is very important for inhibition activity. P2 moieties bulkier than cyclohexane of 13b and cyclopropane of 11a may be tolerant, which has been proved in the M pro -Boceprevir complex structure. Introduction of hydrogen bond acceptor F at P2 residue in 11b which has hydrogen bond interaction with Q189 can increase inhibition activity. The indole group of 11a/11b and the benzene ring group of GC376 showed that P3 is more suitable for aromatic conjugated group.

During our initial screen, there are four other HCV protease inhibitors which showed much lower activity against the main protease of SARS-CoV-2. Telaprevir, also a covalent inhibitor of serine protease 33 , loses inhibition activity against M pro of SARS-CoV-2 which may be caused by steric hindrance of the large P2 quinoxaline moiety. The active pocket of HCV serine protease is larger and more solvent exposed than that of SARS-CoV-2 M pro according to the complex structures of HCV serine protease cocrystallization with the inhibitors simeprevir, asunaprevir, and grazoprevir [34] [35] [36] . Therefore, the macrocyclic compound simeprevir, asunaprevir, and grazoprevir can not be accommodated in the relatively narrower pocket of SARS-CoV-2 M pro .

As we presented in the text, GC376 has more potent inhibition efficiency than Boceprevir, which makes GC376 might be more advantageous than Boceprevir in the clinical practice. However, GC376 has shown side effects such as retarded development of adult teeth in the animal tests 37 . The delayed eruption of some adult teeth was observed only in cats treated for 3 months or longer, not in cats treated for 2 weeks 38 . Therefore, GC376 has the potential to be a short-term treatment of COVID-19 for 1-2 weeks in combination with Remdesivir, because of COVID-19 is an acute disease.

In a word, the structural basis of interaction between the compounds and the main protease provides a good starting point 

Protein expression and purification. To express the SARS-CoV-2 M pro protein with an extra glycine residue at the N-terminus, the cDNA encoding residues 3264-3569 of ORF1ab (GenBank: MN908947.3) was synthesized and codonoptimized for expression in E. coli. The coding sequence was then cloned into the NcoI and NotI sites of the pET-52b vector (Genscript) with a N-terminal tag followed by TEV cleavage sequences. The recombinant protein was expressed in E. coli strain BL21 (DE3) as soluble proteins after inducing with 0.2 mM isopropyl-βd-thiogalactopyranoside (IPTG) at an OD 600 of 0.6-0.8 and expressing at 16°C for 18 h. The cells were lysed by sonication in the lysis buffer (20 mM Tris, 50 mM NaCl, 20 mM imidazole, pH 8.0). After 19,802 × g centrifugation 30 min, the supernatants were then purified by affinity chromatography using the HisTrap HP 5 ml columns (GE Healthcare). The target protein was eluted with the elution buffer (20 mM Tris, 50 mM NaCl, 300 mM imidazole, pH 8.0). The purified proteins per mg were incubation with 2 μL reconstruction TEV protease (Solarbio, P2060) at 30°C for 2 h after centrifugation. The hydrolyzed proteins were then purified by affinity chromatography using the HisTrap HP 5 ml columns (GE Healthcare) again and further purified by size-exclusion chromatography using a Hiload 16/600 Superdex 75 PG column (GE Healthcare) equilibrated with the binding buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and pH 7.8). Expression and purification of the native SARS-CoV-2 M pro protein were performed according to literature 26 . The recombinant protein was expressed in E. coli strain BL21 (DE3) as soluble proteins after inducing with 0.5 mM IPTG at an OD600 of 0.6-0.8 and expressing at 16°C overnight. The cells were lysed by sonication in the lysis buffer (20 mM Tris, 50 mM NaCl, pH 8.0). After 19,802 × g centrifugation 30 min, the supernatants were then purified by affinity chromatography using the HisTrap HP 5 ml columns (GE Healthcare). The target protein was eluted with the elution buffer (20 mM Tris, 50 mM NaCl, 300 mM imidazole, and pH 8.0). The purified proteins per mg were incubation with 20 μL recombinant human rhinovirus protease (Genscript, Z03092) at 30°C for 2 h after centrifugation. The hydrolyzed proteins were then purified by affinity chromatography using the HisTrap HP 5 ml columns (GE Healthcare) again and further purified by sizeexclusion chromatography using a Hiload 16/600 Superdex 75 PG column (GE Healthcare) equilibrated with the binding buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.8). Table 1 were diluted in 25 mM Tris buffer (pH = 8.0) to a final concentration of 100 μM for screen. DMSO was used as a solvent control. In all, 10 μL compound solution was add to black 96well plate (Greiner). In total, 30 μL of 2 μM GM pro was added to the plate and incubated with the compounds at 37°C incubator for 30 min. In total, 330 μL of 25 mM Tris buffer was also added as blank control. Crystallization. In all, 8 mg/ml and 12 mg/ml GM pro or M pro (in 10 mM Tris, 1 mM EDTA, 1 mM DTT, pH 7.8) was incubated with 2 mM inhibitor at 4°C for 18 h. All the crystals were obtained by using the sitting drop vapor diffusion method with 1 μL protein mixing with 1 μL reservoir solution and then equilibrating against 100 μL reservoir solution at 18°C. The initial crystallization screenings were carried out using the commercially available kits. The native GM pro was crystallized in 7% PEG6000, 100 mM MES (pH 6.1) 40 . The M pro -Boceprevir and GM pro -Boceprevir were crystallized in 20% PEG5000, 0.1 M BIS-TRIS (pH 6.5). While the M pro -GC376 and GM pro -GC376 complexes were crystallized in 14% PEG4000, 0.1 M MES monohydrate (pH 6.0) and 10% PEG6000, 0.1 M MES (pH 6.1), and 3% DMSO respectively.

Data collection and structure determination. Diffraction data were collected at Shanghai Synchrotron Radiation Facility (SSRF) BL17U (wavelength, 0.97918 Å). For data collection, the crystals were cryo-protected by briefly soaking in reservoir solution supplemented with 20% (v/v) glycerol before flash-cooling in liquid nitrogen. The dataset was processed with HKL2000 software 41 . The native M pro structure was determined by the molecular replacement method using Phaser 42 with the previously reported SARS-CoV M pro structure (PDB code, 3F9F), while the complexes were further determined with the solved M pro structure. The atomic models were completed with Coot 43 and refined with phenix.refine in Phenix 44 , and the stereochemical qualities of the final models were assessed with MolProbity 45 . Data collection, processing, and refinement statistics are summarized in Supplementary Table 1 . All structural figures were generated using Pymol software (http://www.pymol.org).

Cells and viruses. African green monkey kidney Vero cells were maintained in DMEM (Gibco, C11995500BT) supplemented with 10% fetal bovine sera (FBS) (Gibco, A31608-02), 200 mg/ml streptomycin, and 200 IU/ml penicillin (Gibco, 15140122) at 37°C. SARS-CoV-2 was isolated from Wuhan seafood market by China CDC. All the infection experiments were performed in a biosafety level-3 (BLS-3) laboratory.

Cell viability assay. The Vero cells were seeded in 96-well plate and cultured overnight. In total, 100 μL different concentration of compound solution in DMEM was added to the Vero cells after PBS (Gibco, C10010500BT) wash and incubated for 48 h at 37°C, 5% CO 2 . Meanwhile, 100 μL DMEM was added to the Vero cells as negative control. In total, 10 μL CCK8 assays (TargetMol, C0005) was added to each well directly. OD 450 was measured by using a microplate reader (TECAN Infinite 200 Pro, Switzerland) after 3 h incubation at 37°C. Experiments were performed in triplicate. OD 450 value in present of different concentration of compound is divided by the OD 450 value of the negative control to calculate the percentage cytotoxicity with Microsoft Excel 2016. The cytotoxicity curve was plotted by GraphPad Prism 8.0.

In vitro antiviral assays. In all, 20 mM GC376 and Boceprevir in DMSO were diluted to 200 μM to 0.00256 μM by DMEM contained 1% FBS. In total, 10 mM remdesivir in DMSO were diluted to 100 μM to 0.032 μM. DMSO medium (1%) was used as negative control. Vero cells cultured overnight in 96-well plate, were infected by 0.01 MOI virus for 2 h. The medium was removed, and fresh inhibitorcontaining medium was added to the cells then. Experiments were performed in quadruplicate. After 48 h, the cell in triplicate was lysed in lysis buffer. Viral RNA was extracted from 100 μL supernatant of infected cells using the automated nucleic acid extraction system (TIANLONG, China), following the manufacturer's recommendations. SARS-CoV-2 detection was performed using the One Step PrimeScript RT-PCR kit (TaKaRa, Japan) on the LightCycler® 480 Real-Time PCR system (Roche, Rotkreuz, Switzerland). ORF 1ab was amplified from cDNA and cloned into MS2-nCoV-ORF1ab and used as the plasmid standard after its identity was confirmed by sequencing. A standard curve was generated by determination of copy numbers from serially dilutions (103-109 copies) of plasmid. The following primers used for quantitative PCR were ORF1ab-F: 5′-AGAAGATTGGTTAGAT GATGATAGT-3′, ORF1ab-R: 5′-TTCCATCTCTAATTGAGGTTGAACC-3′, and probe 5′-FAM-TCCTCACTGCCGTCTTGTTGACCA-BHQ1-3′. In all, 72 h later, the cytopathic effect changes of the rest replicate were observed by microscope.

The Ct value was changed to virus copy number (10 y ) by formula y = (Ct-35.461)/−3.4546 with Microsoft Excel 2016. The virus copy number in percent of different concentration inhibitors is divided by that of negative control to get percentage inhibition or percentage relative RNA copies with Microsoft Excel 2016. The inhibition curve was plotted by fitting log(inhibitor) vs. response (three parameters) mode with GraphPad Prism 8.0. The EC 50 was also calculated in the table of results by GraphPad Prism 8.0. The column graph of relative RNA copies in percent of inhibitors and unpaired t test between two columns were performed by GraphPad Prism 8.0.

Plaque-reduction assays. In all, 2 × 10 5 Vero cells were seeded in a 24-well plate and cultured overnight in 24-well plate. The cells were infected by 200 μL SARS-CoV-2 (100 PFU) for 2 h. The medium was then removed, and fresh medium (DMEM contained 2% FBS and 1.2% Avicell) containing appropriate concentrations of inhibitors was added to the cells. The cells were incubated for 72 h at 37°C with 5% CO 2 . The overlay was discarded. The cells were fixed for 30 min with 4% polyoxymethylene (Solarbio, P1110) and stained with crystal violet working solution for 15 min. 

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We thank the staff of BL17U beamlines at Shanghai Synchrotron Radiation Facility. We also thank Mr. Yi Han at the Institute of Biophysics Core-Facility for the support during in-house data collection. We also thank Hanxing Zhang 

The authors declare no competing interests.

Supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-18233-x.Correspondence and requests for materials should be addressed to X.L., W.T., J.Q. or G.F.G.Peer review information Nature Communications thanks Haitao Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Reprints and permission information is available at http://www.nature.com/reprintsPublisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/.