Mathieu Panel

Isaac Ruiz

Rozenn Brillet

Fouad Lafdil

Fatima Teixeira-Clerc

Trung Cong

Julien Nguyen

Muriel Caldéraro

Fred Gelin

Jean-François Allemand

Guichou

Nguyen

Julien Calderaro

Muriel Gelin

Fred Allemand

Jean-François Guichou

Bijan Ghaleh

Abdelhakim Ahmed-Belkacem

# Didier Morin

Jean-Michel Pawlotsky
email: jean-michel.pawlotsky@aphp.fr

Small-Molecule Inhibitors of Cyclophilins Block Opening of the Mitochondrial Permeability Transition Pore and Protect Mice From Hepatic Ischemia/Reperfusion Injury

Keywords: drug, mitochondrial swelling, PPIase activity, mouse model

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

4

-Fouad Lafdil: contribution to the experimental design of murine ischemia-reperfusion experiments; revision of the manuscript.

-Fatima Teixeira-Clerc: contribution to the experimental design of murine ischemiareperfusion experiments; revision of the manuscript.

-Cong Trung Nguyen: acquisition, analysis and interpretation of histological data.

-Julien Calderaro: acquisition, analysis and interpretation of histological data.

-Muriel Gelin: acquisition, analysis and interpretation of chemical data.

-Fred Allemand: acquisition, analysis and interpretation of chemical data.

-Jean-François Guichou: compound design and synthesis; generation of structural data; revision of the manuscript.

-Bijan Ghaleh: study supervision; analysis and interpretation of data; drafting of the manuscript.

-Abdelhakim Ahmed-Belkacem: study supervision; acquisition, analysis and interpretation of data; drafting of the manuscript.

-Didier Morin: study supervision; analysis and interpretation of data; drafting of the manuscript.

-Jean-Michel Pawlotsky: study supervision; analysis and interpretation of data; critical revision of the manuscript for important intellectual content.

5

Abstract

Background & Aims: Hepatic ischemia-reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia-reperfusion injury.

Methods:

We measured the activity of 9 small-molecule inhibitors of Cyps in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif -/-(CypD knock-out) mice, and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemiareperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion, and collected samples of blood and liver for histologic analysis.

Results:

The compounds inhibited peptidyl-prolyl isomerase activity (IC50 values, 0.2 to 16.2 µM) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (IC50 values, 1.4 to 132 µM) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif -/-mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia-reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle.

Conclusions: Recently created small-molecule inhibitors of CypD reduced calciuminduced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia-reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia-reperfusion injury after liver surgery or for other hepatic or non-hepatic disorders related to abnormal mPTP opening. KEY WORDS: drug, mitochondrial swelling, PPIase activity, mouse model.

Introduction

Hepatic ischemia-reperfusion injury is a major complication of liver surgery, including liver resection, liver transplantation, and trauma surgery [START_REF] Konishi | Hepatic ischemia/reperfusion: mechanisms of tissue injury, repair, and regeneration[END_REF] . Ischemia results from the interruption of the blood flow that perturbs the cellular metabolism as a result of oxygen shortage.

Reperfusion restores the blood flow, oxygen delivery and tissue pH, thereby exacerbating cellular damage initiated during hypoxia or anoxia [START_REF] Cannistra | Hepatic ischemia reperfusion injury: a systematic review of literature and the role of current drugs and biomarkers[END_REF][START_REF] Go | Mitochondrial dysfunction and autophagy in hepatic ischemia/reperfusion injury[END_REF] . Hepatic ischemia-reperfusion injury is an important cause of liver dysfunction or functional failure post-liver surgery, which affects perioperative morbidity, mortality and recovery [START_REF] Cannistra | Hepatic ischemia reperfusion injury: a systematic review of literature and the role of current drugs and biomarkers[END_REF][START_REF] Go | Mitochondrial dysfunction and autophagy in hepatic ischemia/reperfusion injury[END_REF] . There are two types of ischemia: the most frequent form, warm ischemia, is observed when vascular occlusion occurs during hepatic resection surgery or during exposure to low-flow incidences such as trauma, hemorrhagic shock, cardiac arrest or hepatic sinusoidal obstruction syndrome; cold ischemia is observed exclusively during orthotopic liver transplantation, when the graft is subjected to hypothermic preservation before warm reperfusion [START_REF] Go | Mitochondrial dysfunction and autophagy in hepatic ischemia/reperfusion injury[END_REF] . Their mechanisms and main target cells differ.

Hepatocytes are a major target of warm ischemia-reperfusion injury through anoxia, nutrition depletion and cytosolic acidosis [START_REF] Go | Mitochondrial dysfunction and autophagy in hepatic ischemia/reperfusion injury[END_REF] . In particular, sodium, chloride and calcium homeostasis are significantly altered. Mitochondrial dysfunction plays a major role in hepatic ischemia-reperfusion injury [START_REF] Go | Mitochondrial dysfunction and autophagy in hepatic ischemia/reperfusion injury[END_REF] . Indeed, ischemia-reperfusion triggers the mitochondrial permeability transition, characterized by an increase in the permeability of the inner mitochondrial membrane mediated by the opening of a channel, called the "mitochondrial permeability transition pore" (mPTP) [START_REF] Halestrap | What is the mitochondrial permeability transition pore?[END_REF] . Once mitochondrial permeability transition initiates, solutes with a molecular mass of up to 1.5 kDa diffuse across the mitochondrial inner membrane, inducing mitochondrial depolarization, uncoupling and swelling, which in turn induce ATP depletion and necrotic (and, to a lesser extent, apoptotic) cell death [START_REF] Go | Mitochondrial dysfunction and autophagy in hepatic ischemia/reperfusion injury[END_REF] . During hepatic ischemia-reperfusion, mPTP opening is triggered by calcium-mediated mitochondrial reactive oxygen species formation. The same phenomenon 8 has been reported to play an important role during ischemia-reperfusion affecting other organs (including heart and brain) [START_REF] Friberg | Mitochondrial permeability transition in acute neurodegeneration[END_REF][START_REF] Kim | Role of the mitochondrial permeability transition in apoptotic and necrotic death after ischemia/reperfusion injury to hepatocytes[END_REF][START_REF] Rauen | New insights into the cellular and molecular mechanisms of cold storage injury[END_REF][START_REF] Halestrap | A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection[END_REF] , neurodegenerative diseases [START_REF] Rao | Mitochondrial permeability transition pore is a potential drug target for neurodegeneration[END_REF] and drug-induced liver injury [START_REF] Jaeschke | Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity[END_REF] .

Therapies avoiding the consequences of hepatic ischemia-reperfusion injury remain limited. Given the key role of mPTP opening in ischemia-reperfusion injury, pharmacological inhibition of mPTP opening is a promising target for new therapies. However, the molecular structure and functioning of mPTP remain largely unknown. Several proteins have been suggested to be involved in the structure of mPTP, including ATP synthase, the adenine nucleotide translocase, a phosphate carrier, and cyclophilin D (CypD) [START_REF] Giorgio | Dimers of mitochondrial ATP synthase form the permeability transition pore[END_REF] . Cyclophilins are peptidyl prolyl cis/trans isomerases (PPIases) that catalyze the interconversion of the two energetically preferred conformers (cis and trans) of the planar peptide bond preceding an internal proline residue. Seventeen human cyclophilins have been identified, but the function of most of them is unknown [START_REF] Wang | The cyclophilins[END_REF][START_REF] Davis | Structural and biochemical characterization of the human cyclophilin family of peptidyl-prolyl isomerases[END_REF] . CypD is located within the mitochondrial matrix where it acts as a key component and regulator of the mPTP; mPTP formation appears to be catalyzed or stabilized by CypD through lowering of its calcium threshold [START_REF] Javadov | Mitochondrial permeability transition and cell death: the role of cyclophilin D[END_REF] . Thus, CypD represents an attractive target for mPTP opening inhibition and cellular protection in the context of hepatic ischemia-reperfusion injury.

Cyclosporine A (CsA) and its derivatives (together with sanglifehrin A) are known macromolecular CypD ligands. They have been shown to efficiently desensitize mPTP opening in various cellular and in vivo models [START_REF] Griffiths | Protection by cyclosporin A of ischemia/reperfusion-induced damage in isolated rat hearts[END_REF][START_REF] Matsuda | Mechanisms of action of cyclosporine[END_REF][START_REF] Azzolin | Antamanide, a derivative of Amanita phalloides, is a novel inhibitor of the mitochondrial permeability transition pore[END_REF][START_REF] Piot | Effect of cyclosporine on reperfusion injury in acute myocardial infarction[END_REF] . However, CsA is a potent immunosuppressor and its non-immunosuppressive derivatives suffer from many disadvantages, including their size, complex multistep synthesis, cell toxicity and lack of chemical plasticity. Thus, potent cyclophilin inhibitors unrelated to CsA or sanglifehrin A and lacking the disadvantages of nonimmunosuppressive macromolecular CsA derivatives are needed.

By means of a fragment-based drug discovery approach based on X-ray crystallography and nuclear magnetic resonance, we generated a new family of nonpeptidic small-molecule cyclophilin inhibitors (SMCypIs) unrelated to CsA or sanglifehrin A, with potent inhibitory 9 activity against CypA, CypB and CypD [START_REF] Ahmed-Belkacem | Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities[END_REF] . These compounds lack cellular toxicity and immunosuppressive activity and bear druggable pharmacological properties [START_REF] Ahmed-Belkacem | Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities[END_REF] . In this study, we assessed the ability of the new SMCypIs to inhibit liver mitochondria mPTP opening through CypD inhibition, studied their mechanisms of inhibition, and evaluated in vivo their protective properties in the context of experimental hepatic ischemia-reperfusion injury.

Materials and Methods

Drugs and cells

Unless specified, all reagents were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Calcein AM (C3100MP) and calcium Green 5N (C3737) were obtained from Invitrogen (Cergy-Pontoise, France). SMCypIs were synthesized as previously described [START_REF] Ahmed-Belkacem | Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities[END_REF] .

Primary human hepatocytes were obtained from Biopredict International (Saint-Grégoire, France).

Animals

Male C57BL/6J mice (8 to 10-week-old) were purchased from Janvier (Le Genest-St-Isle, France). Ppif -/-mice (i.e., CypD knock-out mice) were obtained from Jackson Laboratories (Bar Harbor, Maine). Animals were co-housed in an air-conditioned room with a 12-h lightdark cycle and received standard rodent chow and drinking water ad libitum. All animal procedures in this study were in strict accordance with the Directives of the European Parliament (2010/63/EU-848 EEC) and approved by the Animal Ethics Committee ANSES/ENVA/Université Paris-Est Créteil (approval numbers 09/12/14-02 and 11/10/16-01).

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Molecular modeling and docking of C31 into CypD

Molecular modeling and docking experiments were performed by means of the @TOME-2 server [START_REF] Pons | @TOME-2: a new pipeline for comparative modeling of proteinligand complexes[END_REF] . In each structural model, the boundaries of the active site were deduced from the vicinity of co-crystallized ligands (compounds C30, C27, and C24 selected as templates, with PDB accession numbers 4J5C, 4J5B and 4J5E, respectively) using @TOME-2 comparative option. They were used to guide docking in automatically computed models. The ligand files were generated with MarvinSketch 6.2.2 for SMILES and Frog2 server for mol2 [START_REF] Miteva | Frog2: efficient 3D conformation ensemble generator for small compounds[END_REF] .

The Figure was generated by means of Pymol.

CypD PPIase enzyme assay

Inhibition of CypD PPIase activity was measured at 20°C by means of the standard chymotrypsin coupled assay. The assay buffer (25 mM HEPES, 100 mM NaCl, pH 7.8) and purified CypD (1900 nM stock solution) were pre-cooled at 4°C in the presence of serial dilutions of the SMCypIs. Five µL of 50 mg/mL chymotrypsin in 1 mM HCl was added. The reaction was initiated by adding 20 µL of 3.2 mM peptide substrate (N-succinyl-Ala-Ala-Cis-Pro-Phe-p-nitroanilide). P-nitroanilide absorbance was measured at 390 nm for 1 min. CsA was used as a positive control of PPIase inhibition. The percent inhibition of CypD PPIase activity was calculated from the slopes and the IC50s were determined from percent inhibition curves using Sigmaplot software.

Isolation of liver mitochondria

Mouse livers were scissor-minced and homogenized on ice in a buffer (220 mM mannitol, 70 mM sucrose, 10 mM HEPES, 4 mM EGTA, pH 7.4 at 4°C) using a Potter-11 Elvehjem glass homogenizer in a final volume of 10 mL. The homogenate was centrifuged at 1000 g for 5 min at 4°C. The supernatant was centrifuged at 10,000 g for 10 min at 4°C. The mitochondrial pellet was resuspended in 600 µl of homogenization buffer without EGTA and protein concentration was determined.

Measurement of mitochondrial oxygen consumption

Oxygen consumption of isolated mitochondria was measured with a Clark-type electrode fitted to a water-jacketed reaction chamber (Hansatech, Cergy, France). Mitochondria (1 mg protein/mL) were incubated at 30°C in a respiration buffer containing 100 mM KCl, 50 mM sucrose, 10 mM HEPES and 5 mM KH2PO4 at pH 7.4. Respiration was initiated by the addition of glutamate/malate (5 mM each). After 1 min, ATP synthesis was induced by the addition of 1 mM ADP (state 3 respiration rate), and 1 µM carboxyatractyloside was added to measure the substrate-dependent respiration rate (state 4). The respiratory control ratio (state 3/state 4) was then calculated.

Mitochondrial swelling assays

Mitochondrial swelling was assessed by measuring the change in absorbance at 540 nm (A540) using a Jasco V-530 spectrophotometer equipped with magnetic stirring and thermostatic control. Mitochondria (0.5 mg/mL) energized with pyruvate/malate (5 mM each) were incubated 30 sec in the respiration buffer before the induction of swelling with 10 mM phosphate, 100 µM atractyloside or 40 µM tert-butyl hydroperoxide in the presence of 50 µM of CaCl2.

In de-energized conditions, mitochondria (0.5 mg/mL) were incubated 1 min at 30°C in a swelling buffer containing 100 mM KCl, 50 mM sucrose, 10 mM HEPES, 5 mM KH2PO4, 1 12 µM rotenone, and 1 µM antimycin at pH 7.4. Then, 50 µM or 100 µM CaCl2 was added 1 min before swelling induction with 1 µM of A23187 or 10 µM phenylarsine oxide, respectively.

In both conditions, SMCypIs or CsA were introduced at the beginning of the incubation period.

Measurement of the calcium retention capacity of isolated mitochondria

Mitochondria were loaded with increasing concentrations of calcium until mPTP opening and fast calcium release occurred 22 . Liver mitochondria (1 mg/mL) energized with 5 mM glutamate/malate were incubated in the respiration buffer supplemented with 1 µM Calcium Green-5N fluorescent probe. The calcium concentration in the extra-mitochondrial medium was monitored by means of a Jasco FP-6300 spectrofluorimeter (Jasco, Bouguenais, France) at excitation and emission wavelengths of 506 and 532 nm, respectively. The calcium signal was calibrated by addition of known calcium amounts to the medium.

Isolation of primary mouse hepatocytes

Murine hepatocytes from C57BL6 animals were isolated by portal vein perfusion of collagenase [START_REF] Seglen | Preparation of isolated rat liver cells[END_REF] . Freshly isolated hepatocytes were cultured in DMEM supplemented with 10% fetal calf serum, 10 U/mL penicillin, 10 μg/mL streptomycin, 10 μg/mL insulin, 5.5 μg/mL transferrin and 5 ng/mL sodium selenite. Four hours post-perfusion, the medium was removed and fresh medium, supplemented with 0.1 μM dexamethasone and 50 ng/mL epidermal growth factor, was added.

Measurement of mPTP opening in primary mouse and human hepatocytes

mPTP opening was assessed in primary mouse and human hepatocytes by inducing mitochondrial localization of calcein fluorescence [START_REF] Petronilli | Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence[END_REF] . Cells were loaded with 2 mM CoCl2 at 30°C in 1 mL of Tyrode's solution (NaCl 130 mM, KCl 5 mM, HEPES 10 mM, MgCl2 1 mM, CaCl2 1.8 mM, pH 7.4 at 37°C) for 30 min. After 10 min, the cells were supplemented with 1 µM calcein acetoxymethyl ester, washed free of calcein and CoCl2, placed in a thermostated chamber and incubated in Tyrode's solution. After 5 min, 2 µM of calcium ionophore A23187 was added to induce mPTP opening [START_REF] Petronilli | The mitochondrial permeability transition, release of cytochrome C and cell death. Correlation with the duration of pore openings in situ[END_REF] . When specified, either C31 or CsA was added at the beginning of the incubation period.

Cells were imaged with an Olympus IX-81 motorized inverted microscope equipped with a mercury lamp as the light source for epifluorescence illumination, combined with a 12bit cooled Hamamatsu ORCA-ER camera; 460-490 nm excitation and 510 nm emission filters were used for detection of calcein fluorescence. Images were acquired every min for 30 min after 30 ms of illumination per image using a digital epifluorescence imaging software (Cell M, Olympus, Rungis, France). Fluorescence from each cell in each region of interest (≈ 80 µm 2 ) was measured and background fluorescence was subtracted. Average fluorescence changes per field were calculated. Fluorescence was normalized to the maximal fluorescence value.

Ex vivo assessment of mPTP opening in mice treated with C31

The mice were anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg). Increasing doses of C31 (10, 20, 50 and 150 mg/kg) or vehicle were randomly administered as a 3-min infusion through the jugular vein. Two min after the end of the infusion, the livers were excised and mitochondria were isolated to measure their capacity to retain calcium, as a marker of the ability of C31 to inhibit mPTP opening in vivo.

14

In vivo assessment of the mito-and hepatoprotective effect of SMCypIs in the context of hepatic ischemia-reperfusion

The mice were anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg), intubated and mechanically ventilated. After section of the liver ligaments, hepatic normothermic ischemia of segments I to V was induced for 60 min by hilum clamping of the hepatic pedicle. Reperfusion occurred at removal of the clamp. One min before reperfusion, C31 or the vehicle was infused through the jugular vein and the infusion was maintained for the first 8 min of reperfusion.

Two sets of experiments were conducted. In the first set, livers were excised after 10 min of reperfusion and mitochondria were isolated for ex vivo experiments. In the second set, reperfusion was stopped after 60 min; 500 µL of blood was collected to measure alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, while livers were excised, formalin-fixed, paraffin-embedded and liver sections were stained with hematoxylin and eosin. "Hepatocyte clarification", a histological feature of severe hepatocellular damage characterized by hepatocyte ballooning degeneration, was measured on whole sections of mice livers. Histological slides were scanned at an x20 magnification by means of an Aperio Slide Scanner (Leica Biosystems, Shanghai, China). Slides in ".svs" format were analyzed by means of Qupath open source software [START_REF] Bankhead | QuPath: open source software for digital pathology image analysis[END_REF] . Areas with hepatocyte ballooning were then manually annotated independently by two pathologists specialized in liver diseases.

Statistical analysis

Results are expressed as mean±SEM of at least 5 independent experiments. Statistical analysis was performed using one-way ANOVA followed by the Scheffé post-hoc test.

Differences were considered significant for p <0.05.

Results

SMCypIs bind CypD and inhibit its PPIase activity

We recently created a new family of small-molecule, nonpeptidic cyclophilin inhibitors (SMCypI) [START_REF] Ahmed-Belkacem | Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities[END_REF] . As shown in Supplementary Table 1 and Supplementary Figure 1, 9 members of the new SMCypI family, including compound C22, compounds C23 to C27 and compounds C29 to C31 (which all resulted from structure-guided chemical optimization of C22), inhibited CypD PPIase activity in an enzyme assay, with IC50 values ranging from 0.2 to 16.2 µM. The most potent PPIase inhibitor was C31, with an IC50 of 0.2±0.08 µM. Molecular modeling and docking experiments revealed that C31 binds both the catalytic site and the gatekeeper pocket of CypD (Figure 1A).

SMCypIs inhibit calcium-induced swelling as a result of the inhibition of CypD PPIase activity in isolated mouse liver mitochondria

As shown in Figure 1B, 1 µM CsA, used as a positive control of CypD inhibition, fully inhibited calcium-induced swelling of energized isolated mouse liver mitochondria. The 9 SMCypIs also inhibited mitochondrial swelling in a concentration-dependent manner (Supplementary Table 1). The most effective compound was C31, with an IC50 of 1.4±0.2 µM (Figure 1B). SMCypI inhibition of calcium-induced mitochondrial swelling strongly correlated with SMCypI inhibition of CypD PPIase activity (r=0.8535; p <0.004; Figure 1C).

These results indicate that the new SMCypI family inhibits calcium-induced mitochondrial swelling as a result of CypD PPIase activity inhibition by binding to both the 16 PPIase catalytic site and the gatekeeper pocket of CypD. C31, the most potent SMCypI, was selected for subsequent experiments.

SMCypI inhibition of mitochondrial swelling is due to the inhibition of mPTP opening

To assess whether mitochondrial swelling inhibition by SMCypIs is related to the inhibition of mPTP opening, swelling was induced by different known mPTP opening inducers.

As shown in Figures 2A and2B, C31 inhibited mitochondrial swelling induced by both the prooxidant drug tert-butyl hydroperoxide and carboxyatractyloside, a ligand of adenine nucleotide translocase, in energized conditions.

Because mitochondrial parameters, such as the membrane potential or the ATP/ADP ratio, can modulate mPTP opening under energized conditions, thereby influencing the observed effect of the inhibitors, the inhibitory effect of C31 was analyzed under de-energized conditions, i.e., in the absence of substrate. Mitochondria were incubated in the presence of rotenone and antimycin to fully block the respiratory chain and supplemented with the calcium ionophore A23187 or the cross-linking thiol agent phenylarsine oxide in the presence of calcium. In these conditions (Figure 2C and 2D, respectively), mitochondrial swelling was slower than under energized conditions, but still inhibited by SMCypI C31.

These results indicate that SMCypI compound C31 inhibits mitochondrial swelling by inhibiting mPTP opening.

SMCypI compound C31 inhibits mPTP opening in both primary mouse and human hepatocytes

Primary mouse hepatocytes were loaded with calcein in the presence of CoCl2 and mPTP opening was induced by the addition of 1µM A23187 (Figure 3A). As shown in Figure 17 3B, CsA inhibited the drop in calcein fluorescence associated with mPTP opening by only 55.4±0.8% at 30 min at the most effective concentration of 2 µM. C31 inhibited the drop in calcein fluorescence in a concentration-dependent manner, a result indicating that C31 inhibits mPTP opening in living mouse liver cells (Figure 3B). The inhibitory effect of C31 was stronger than that of 2 µM CsA, with full inhibition of mPTP opening obtained with 100 µM of C31.

The same experiments were performed with primary human hepatocytes (Figure 3C).

CsA inhibited mPTP opening by approximately 50% at the most effective concentration of 0.2 µM. C31 inhibited mPTP opening in a concentration-dependent manner in primary human hepatocytes, with full inhibition achieved at the concentration of 100 µM (Figure 3C). This indicated that results obtained in mouse liver cells are representative of the human liver situation.

SMCypI compound C31 is more potent than CsA at increasing mitochondrial calcium retention capacity in isolated mouse mitochondria

The calcium retention capacity of isolated mouse mitochondria (i.e., the maximal calcium load achievable in the presence of a drug before mPTP opens) was measured, as previously described 22 . As shown in Figures 4A and4B, 6±1 additions of 20 µM calcium, corresponding to 109±15 nmol/mg of mitochondrial proteins, were required to induce mPTP opening in the absence of CypD inhibitors. One µM of CsA increased the mitochondrial calcium retention capacity by 3-fold (337±47 nmol/mg of mitochondrial proteins). C31 at concentrations up to 100 µM also increased the mitochondrial calcium retention capacity. The maximum effect of C31 was markedly greater than that of CsA (550±37 nmol/mg of mitochondrial proteins; Figures 4A and4B). C31 also more potently increased mitochondrial calcium retention capacity than CsA when the respiratory chain was fed with succinate in the presence of rotenone, indicating that this effect is independent from activated respiratory complex I or II (data not shown).

Maximum mitochondrial calcium retention capacities of the same order were achieved in the presence of 10 µM of C31 and 1 µM of CsA, respectively. At these concentrations, that correspond to approximately 10 times the respective compound IC50 values, CypD saturation is achieved. The combination of both C31 and CsA at the same concentrations did not result in any additive effect (Figure 4B), a result in keeping with them sharing CypD as a target.

Together, these results indicate that SMCypI compound C31 inhibits mPTP opening at least partly through CypD inhibition, and that this inhibition is more potent than that achieved by CsA.

The additional effectiveness of C31 as compared to CsA in inhibiting mPTP opening is unrelated to CypD inhibition

The mitochondrial calcium retention capacity in the presence of 100 µM C31 was not modified by the addition of 1 µM CsA (Figure 4B). This suggests that the greater capacity of C31 to inhibit mPTP opening as compared to CsA is unrelated to CypD inhibition. To verify this hypothesis, the effects of C31 and CsA were assessed on liver mitochondria isolated from Ppif -/-mice, which have been knocked-out for CypD, in comparison with wild-type animals.

As previously shown [START_REF] Baines | Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death[END_REF] , the mitochondrial calcium retention capacity of Ppif -/-mice was approximately 4-fold greater than that of wild-type animals (Figure 5A and5B). CsA 1 µM had no effect on Ppif -/-mice mitochondrial calcium retention capacity. In contrast, C31 increased the calcium retention capacity by approximately 1.5-fold at concentrations >20 µM in mitochondria isolated from Ppif -/-mice livers (Figure 5A and5B). The C31 concentrations at which this effect was observed were higher than those required to inhibit CypD PPIase activity and mitochondrial swelling and to enhance calcium retention capacity in wild-type mouse 19 mitochondria (see Figures 1 and3 for comparison). In addition, C31 inhibited swelling induced by very high calcium concentrations in mitochondria isolated from Ppif -/-mice (Figure 5C).

These results indicate that, in addition to blocking CypD, C31 interacts with another target at the mPTP level and that this interaction increases C31 blocking effectiveness on mPTP opening as compared to exclusive CypD inhibitors, such as CsA.

The additional effectiveness of C31 as compared to CsA in inhibiting mPTP opening is related to the chemical structure of its phenyl moiety

Comparison of the chemical structures of the SMCypIs (Supplementary Figure 1) suggested that the phenyl moiety of C31 is involved in this additional effect: indeed, SMCypI compound C34, that lacks the aromaticity of the cycle, lost the additional effect of C31 over CypD inhibition in isolated Ppif -/-mouse liver mitochondria shown in Figure 5D.

The additional effectiveness of C31 as compared to CsA in inhibiting mPTP opening is unrelated to a ubiquinone-like effect or an interaction with the mitochondrial respiratory chain.

Ubiquinones have been shown to inhibit mPTP opening to a greater extent than CsA [START_REF] Walter | Regulation of the mitochondrial permeability transition pore by ubiquinone analogs. A progress report[END_REF] .

The proximity between the urea and phenyl motifs of C31 would be compatible with a shared mPTP target with ubiquinones. As shown in Figure 5E, ubiquinone 0 (Ub0), the most efficient ubiquinone, strongly increased the calcium retention capacity of liver mitochondria isolated from wild-type mice, but had no effect in liver mitochondria isolated from Ppif -/-mice (Figure 5E). This result indicates that the additional, CypD-independent effect of C31 on mPTP opening is not ubiquinone-like.

20

Because it has been reported that inhibiting complex I with rotenone limits mPTP opening [START_REF] Li | Inhibition of complex I regulates the mitochondrial permeability transition through a phosphate-sensitive inhibitory site masked by cyclophilin D[END_REF] , we investigated whether C31 has an effect on respiratory chain functions. C31 did not alter substrate-dependent respiration rates or ADP-induced O2 consumption, suggesting no C31-induced alteration of mitochondrial respiration (Supplementary Figure 2). No drop in the electron transport chain activity (lower ∆Ψm and slower calcium absorption) was detected in our experiments. Thus, the greater potency of C31 as compared to CsA in inhibiting mPTP opening is not related to an interaction with mitochondrial complex I or the respiratory chain.

SMCypI compound C31 inhibits mPTP opening in vivo in mice

Anesthetized mice were infused with increasing doses of C31 for 3 min and sacrificed 2 min later. Liver mitochondria were isolated and their calcium retention capacity was measured. Mitochondria isolated from C31-treated mouse livers exhibited higher calcium retention capacities than those from vehicle-treated mice, and this effect was dose-dependent.

The effect of C31 was more potent than that of CsA (Figure 6A). This result, showing in vivo inhibition of mPTP opening by SMCypI compound C31, indicates that C31 reaches its mitochondrial target after infusion in living mice.

SMCypI compound C31 bears mito-and hepatoprotective properties during experimental liver ischemia-reperfusion in mice

Our final series of experiments aimed at demonstrating the protective effect of SMCypI compound C31 in an experimental murine model of hepatic ischemia-reperfusion injury. First, mice were subjected to 60 min of liver ischemia followed by 10 min of reperfusion. Ischemiareperfusion reduced the calcium retention capacity of isolated mouse mitochondria by 59% (96.1±4.5 to 39.0±2.7 nmol/mg of protein; p <0.001). This reduction was associated with an 21 alteration of oxidative phosphorylation, as demonstrated by a 31% decrease of the rate of ADPstimulated respiration (p=0.0016 vs Sham) and a 52% decrease of the respiratory control ratio (p=0.0026 vs Sham) (Figure 6B). Infusion of the most effective dose of C31 (150 mg/kg) 1 min before and during the first 8 min of reperfusion restored normal calcium retention capacity (84.9±8.9 nmol/mg of protein). The protective effect of C31 also translated into a restoration of oxidative phosphorylation parameters (Figure 6B).

Secondly, the hepatoprotective effect of C31 was assessed in mice subjected to 60 min of liver ischemia, followed by 60 min of reperfusion. Blood samples were collected for ALT and AST level measurement and livers were excised for liver histology assessment (Figure 7A). C31 treatment significantly reduced ischemia-reperfusion-induced ALT elevation at the end of reperfusion (mean ALT levels: 2,297±290 versus 4,805±1,088 in C31-treated and vehicletreated mice, respectively; p=0.0375) (Figure 7B). The effect of C31 on serum ALT levels was significantly greater than that of CsA, which was not significantly different from that of the vehicle (data not shown). C31 also substantially reduced AST elevation, but the difference did not reach significance (Supplementary Figure 3). 7C, 7D and7E, C31 protected mouse livers from hepatic damages induced by ischemia-reperfusion. Indeed, 60 min after reperfusion, hepatic clarification areas that measure hepatocyte ballooning degeneration, a feature of acute liver injury (Figure 7D), covered a significantly smaller surface in C31-treated than in vehicle-treated mice (30.4±4.3% versus 50.7±3.2% of liver section surface, respectively; p=0.0012). CsA also significantly reduced the surface of hepatic clarification in the model, to an extent similar to the effect of C31 (data not shown).

As shown in Figures

These findings indicate that SMCypI compound C31 exerts both mito-and hepatoprotective effects in mice exposed to ischemia-reperfusion.

Discussion

Hepatic ischemia-reperfusion injury is a major complication of liver surgery with limited therapeutic options. Ischemia-reperfusion injury results in great part from hepatic mitochondrial dysfunction related to abnormal mPTP opening. CypD is an important regulator of the mPTP complex that plays a key role in its opening in several pathological conditions, including hepatic ischemia-reperfusion [START_REF] Giorgio | Dimers of mitochondrial ATP synthase form the permeability transition pore[END_REF][START_REF] Javadov | Mitochondrial permeability transition and cell death: the role of cyclophilin D[END_REF] . Indeed, CypD translocation to the inner mitochondrial membrane triggers mPTP opening and promotes cell death. Both effects can be prevented by pharmacological CypD inhibitors or CypD knock-out [START_REF] Baines | Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death[END_REF][START_REF] Nakagawa | Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death[END_REF][START_REF] Hansson | The nonimmunosuppressive cyclosporin analogs NIM811 and UNIL025 display nanomolar potencies on permeability transition in brain-derived mitochondria[END_REF][32] . Thus, CypD represents an attractive target for inhibition of mPTP opening and hepatocellular protection in the context of hepatic ischemia-reperfusion injury.

We designed a new family of SMCypIs made of two linked fragments binding the cyclophilin catalytic and gatekeeper pockets, respectively [START_REF] Ahmed-Belkacem | Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities[END_REF] . Our aim was to develop CypA inhibitors with antiviral activity against the hepatitis C virus. We showed that our SMCypIs bind CypA and potently inhibit both CypA PPIase activity and the replication of hepatitis C virus and related viruses in cell culture [START_REF] Ahmed-Belkacem | Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities[END_REF][START_REF] Nevers | Characterization of the anti-hepatitis C virus activity of new nonpeptidic small-molecule cyclophilin inhibitors with the potential for broad anti-Flaviviridae activity[END_REF] . Because the different cyclophilins are structurally very close (essentially differing by their cellular localizations and functions), we assessed whether the new SMCypIs inhibit CypD activity. As shown here, their inhibitory activity was potent and concentration-dependent. SMCypI compound C31 was the most potent CypD inhibitor in our experiments. Its cytotoxic concentration 50% was >100 µM in Huh7 cells, suggesting a favorable therapeutic index [START_REF] Ahmed-Belkacem | Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities[END_REF] . We also showed that C31 binds both the catalytic and gatekeeper pockets of Cyp D.

The ability of our SMCypIs to inhibit mPTP opening and its mitochondrial consequences in a concentration-dependent manner correlated with their ability to block PPIase activity. Inhibition of mPTP opening was observed in different models and conditions, including energized and de-energized ones. This suggests that the SMCypIs act directly on CypD and mPTP opening, downstream of the site of action of the inducers used in the 23 experiments. Importantly, this effect was observed in both human and mouse primary hepatocytes, a result validating the human relevance of our findings.

Although the CypD affinity of the SMCypIs was lower than that of CsA, C31 achieved more potent mPTP opening inhibition than CsA at its maximum soluble concentration in the medium. Because non-immunosuppressive derivatives of CsA, e.g., PKF220-384 or NIM811, have been shown to be equipotent to CsA in inhibiting mPTP opening 32 , these compounds are also likely to have a weaker effect on mPTP opening than C31 (not tested). Importantly, our findings suggest that the additional effect of C31 on mPTP opening as compared to CsA is independent from CypD inhibition. Indeed, first, the concentrations of CsA and C31 that fully inhibited CypD PPIase activity retained the same amount of calcium in mitochondria; secondly, CsA did not alter the maximal calcium retention capacity induced by C31; thirdly, C31 had an effect on calcium accumulation in liver mitochondria isolated from Ppif -/-mice that do not express CypD.

The fact that CsA fully inhibited CypD PPIase activity in isolated mouse mitochondria (Supplementary Figure 3) suggests that the additional effect of C31 as compared to CsA is not related to its ability to inhibit PPIase activity. Because the structure and functioning of mPTP remain largely unknown, we could not identify the second target of C31 responsible for its greater potency as compared to CsA. We could however rule out an alteration of mitochondrial respiration [START_REF] Li | Inhibition of complex I regulates the mitochondrial permeability transition through a phosphate-sensitive inhibitory site masked by cyclophilin D[END_REF] or a ubiquinone-like mechanism [START_REF] Walter | Regulation of the mitochondrial permeability transition pore by ubiquinone analogs. A progress report[END_REF] . Many potential components or regulators of the mPTP, such as the recently identified SPG7 [START_REF] Shanmughapriya | SPG7 is an essential and conserved component of the mitochondrial permeability transition pore[END_REF] , could be involved, while many questions remain unanswered: is mPTP organized as a true physical channel? Is opening just a different state of the mitochondrial membrane with increased permeability? Which cellular components are actual constituents of the mPTP? Which ones only interact with it and/or regulate it? What are the mechanisms involved? Answering these questions was beyond the scope of our study, but we are confident that the new family of SMCypIs will be helpful in future mechanistic studies aimed at exploring these questions. Altogether, our result identify SMCypI C31 as a 24 particularly promising mPTP opening blocking agent, because its effect on mPTP opening related to CypD PPIase activity inhibition is enhanced by another complementary mechanism.

Key results in this study were obtained in vivo in murine models. We showed that C31 rapidly reaches its target after systemic administration. This is particularly important in the context of ischemia-reperfusion, because mPTP was reported to open within the first minutes of reperfusion after ischemia [START_REF] Halestrap | A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection[END_REF][START_REF] Glantzounis | The contemporary role of antioxidant therapy in attenuating liver ischemia-reperfusion injury: a review[END_REF] . For this reason, proof-of-concept efficacy experiments were performed in vivo in a murine model of ischemia-reperfusion. The administration of C31 at reperfusion restored the mitochondrial function. In addition, ALT elevations and hepatic damage were significantly reduced in C31-treated mice, proving the hepatoprotective properties of the new SMCypI, as a result of mPTP opening inhibition.

In this study, liver damage was assessed by measuring "hepatocyte clarification", i.e. hepatocyte ballooning degeneration, a feature of severe liver cell injury involving ATP depletion and a rise in intracellular calcium concentrations leading to a loss of cell volume control by plasma membranes and a disruption of the hepatocyte intermediate filament network.

Hepatocyte ballooning degeneration has been observed in the early post-transplant period , and described as an important morphological characteristic of ischemia-reperfusion injury measuring the degree of liver damage [START_REF] Datta | Molecular mechanisms of liver ischemia reperfusion injury: insights from transgenic knockout models[END_REF][START_REF] Saeed | Does necroptosis have a crucial role in hepatic ischemia-reperfusion injury?[END_REF][START_REF] Arab | Histological and biochemical alterations in earlystage lobar ischemia-reperfusion in rat liver[END_REF] . As shown in Figure 7D in our model, hepatocyte ballooning degeneration was characterized by hepatocellular swelling, cytoplasm vacuolization and blebbing of the cell membrane. It was significantly improved by C31 administration in our murine model of ischemia-reperfusion injury. Because the half-life of C31 is short, requiring continuous infusion, it was not possible to determine how long the protective effects might last. Chemical, galenic and pharmacological optimization of the compounds is in progress and studies with more stable derivatives will be conducted to address this important question as part of future preclinical development.

In summary, we showed that our new family of small-molecule, non-peptidic cyclophilin inhibitors binds CypD and inhibits its PPIase activity, and that this effect is, at least in part, responsible for their concentration-dependent inhibitory effect on calcium-induced swelling due to mPTP opening in both human and mouse hepatocytes. We also showed that the most potent SMCypI, compound C31, exerts an additional effect on mPTP opening, independent of its inhibitory effect on CypD, making it a promising pharmacological agent for hepatocellular protection in the context of diseases involving mitochondrial dysfunction related to abnormal mPTP opening. Finally, our study is the first to provide the in vivo proof-of-concept of mitochondrial and hepatocellular protection by SMCypI compound C31 in an experimental model of ischemia-reperfusion injury.

The new family of SMCypIs appears as a promising candidate for new drug development in the indication of hepatic protection in the context of warm ischemia-reperfusion after liver surgery. Whether similar protection can be obtained in the context of cold ischemiareperfusion related to orthotopic liver transplantation remains to be assessed. Other applications in liver and non-hepatic diseases related to mPTP opening involving CypD deserve to be explored. They include chronic alcohol consumption, which enhances sensitivity to calciummediated mPTP opening and increases CypD expression 43 , drug-induced liver injury, myocardial ischemia-reperfusion, brain injury and neurodegenerative disorders.

Figure 1 .

 1 Figure 1. (A) Molecular modeling and docking of SMCypI compound C31 bound to the

Figure 2 .

 2 Figure 2. Inhibitory effect of SMCypI C31 on mitochondrial swelling triggered by mPTP opening inducers in isolated mouse mitochondria incubated in the presence of Ca 2+ . The x axis

Figure 3 .

 3 Figure 3. Inhibition of mPTP opening by CsA and SMCypI compound C31 in primary mouse

Figure 4 .

 4 Figure 4. Calcium retention capacity of isolated mouse liver mitochondria in the presence of

Figure 5 .

 5 Figure 5. Investigation of the mechanisms underlying the more potent effect of SMCypI

Figure 6 .

 6 Figure 6. In vivo effect of C31 on mPTP opening and mitochondrial alterations related to liver

Figure 7 .

 7 Figure 7. In vivo hepatoprotective effect of C31 in the context of liver ischemia-reperfusion.

M it o c h o n d r ia l s w e llin g in h ib it io n IC 5

 5

Fig. 1 0

 1 Fig. 1

Acknowledgement: The authors are grateful to Drs Joseph Benga and Stéphane Moutereau from the Department of Biochemistry at Henri Mondor hospital for ALT and AST level measurements and to Prof. René Tolba and Dr Zoltan Czigany from the Aachen University Hospital, Germany, for helpful discussions regarding the in vivo ischemia-reperfusion injury model.

Grant support: Mathieu Panel was supported by a grant from the French Ministry for Higher Education and Research (Grant #2014-140). Isaac Ruiz was supported by grants from the National Agency for Research on AIDS and Viral Hepatitis (ANRS) and the Mexican National Council of Science and Technology (CONACYT).

Conflict of interest disclosures:

Inserm Transfert is the owner of patent EP 09306294.1 covering the family of cyclophilin inhibitors described, for which Abdelhakim Ahmed-

Authors contributions:

-Mathieu Panel: acquisition, analysis and interpretation of data; drafting of the manuscript; statistical analysis.

-Isaac Ruiz: contribution to the experimental design of murine ischemia-reperfusion experiments; acquisition, analysis and interpretation of data.

-Rozenn Brillet: acquisition, analysis and interpretation of data.


Kai Zhang

Romain Bernard

Yves Borensztein

Hervé Cruguel

Geoffroy Prévot

Growth of germanium-silver surface alloys followed by in situ scanning tunneling microscopy: Absence of germanene formation

come   L'archive ouverte pluridisciplinaire

Introduction

Germanene, the two-dimensional allotrope of germanium, has been widely studied in the last decade, since initial works based on density functional theory (DFT) [1,2] have predicted that Ge could exist in a free-standing (FS) metastable configuration, where Ge atoms are hexagonally organized. Such germanene would possess an electronic structure showing Dirac cones at the Fermi level, and a high electrical conductivity. Experimentally, due to the lack of a graphitic form of germanium, which would permit its synthesis by exfoliation, germanene needs to be synthesized by deposition on a substrate. This can be realized by physical vapor deposition of Ge atoms on a crystal surface. The formation of germanene has been first reported on Au(111) [3,4] and Pt(111) [5]. The growth of germanene has also been attested on Al(111) [6][7][8][9], Ag(111) [10,11], h-AlN [12], and on lamellar substrates such as MoS2 [13] and highly oriented pyrolytic graphite (HOPG) [14]. Germanene films were also claimed to be obtained by segregation of germanium through a thick Ag/Ge(111) deposit caused by high temperature annealing [10,15,16], and on the surface of Ge2Pt droplets formed by high temperature annealing of a thin Pt/Ge(110) deposit [17].

However, some of these results have been recently questioned. On HOPG, it has been shown that the corrugation observed after Ge deposition was due to a charge density wave, and not to germanene [18]. On Au(111), a recent study concluded to the absence of germanene formation in a large temperature range (297 K-500 K) [19]. For this metallic substrate, the observed structures would in fact correspond to a surface Au-Ge alloy [19]. The hypothetical formation of a surface alloy has also been recently discussed for Ge/Al(111) [20,21]. Similar questions may arise for germanene on Pt(111) due to the existence of a stable Ge2Pt bulk phase.

The case of germanene growth on Ag(111) is also controversial: some authors describe the result of depositing Ge on Ag(111) as an alloy, while others claim there is formation of a germanene single layer. In the following, the germanium coverage Ge  is given with respect to the atomic density of a Ag(111) plane; the coverage needed for a germanene monolayer (ML) is then 06 . 1 Ge   ML, assuming a germanene lattice constant of 0.397 nm [2]. The first study has been performed by depositing Ge  =1/3 ML at room temperature [22]. After deposition, low energy electron diffraction (LEED) indicated the presence of a     30 R 3 3 p reconstruction related to the Ag(111) surface (hereafter named as  

3 3 
), but no contrast associated with this structure could be evidenced in the scanning tunneling microscopy (STM) images, which showed a   1 1 structure, similar to the one of bare Ag(111) [22]. X-ray photoelectron spectroscopy (XPS) revealed the metallic character of the Ge atoms in the   3 3  superstructure, while from DFT computation, the authors concluded that the formation of a surface Ag2Ge alloy was favored with respect to adsorbed Ge adatoms. The model of Ag2Ge surface alloy was confirmed by angle-resolved photoelectron spectroscopy (ARPES), even if all observed features could not be reproduced by the simulation, in particular an unexpected surface band split at the -M points along the --K-M line of the  

3 3 
surface Brillouin zone (SBZ) [23]. Annealing the  

3 3  structure at 473K resulted in a   3 6 3  LEED pattern,
corresponding to satellite spots around the initial spots of the  

3 3 
reconstruction, and to a striped pattern in the STM images due to a long range modulation [24]. Contrary to the previous observation [22], the local   3 3  structure was clearly seen in the STM images, with protrusions that could correspond to Ge atoms in the model of Ag2Ge surface alloy, with a small distortion. It was thus concluded that the complex surface band structure, with several split bands, most likely originates from the structural distortions of the alloy layer [24]. More recently, other STM investigations during growth at 300 K showed clear evidence of Ge substitution in the very initial stage of deposition, followed by the formation of a triangular pattern where amorphous-like and   3 3 

phases alternate [25]. For deposition at 600 K, the formation of the   3 6 3  striped pattern was confirmed and also interpreted as the substitutional Ag2Ge surface alloy [25].

In contradiction with these reports, some studies have concluded to the formation of germanene after Ge deposition on Ag(111). A STM and LEED study questioned the alloy model for the

  3 3 
reconstruction [26]. Other authors observed the  

3 6 3 
striped phase for deposition at 423 K, but for a coverage of 0.74 ML, which was interpreted as germanene with a large tensile strain of 12% to 23 % as a function of the direction [11]. In the same study, for a larger deposit of about 1.08 ML, this striped phase was replaced by a "quasi free-standing germanene phase", also identified as an honeycomb germanene layer, but with a slight compressive strain and with slight disorder [11]. This transition was confirmed in another study [27] where the structure that replaced the   3 6 3  striped phase was called "disordered honeycomb". However, it was interpreted as the replacement of all Ag atoms of the Ag2Ge alloy by Ge atoms.

Other studies proposed alternative structures for deposits above 1/3 ML. At 300K, the early works indicated that the   show that they suffer from a lack of real-time observation of the surface, performed at the growth temperature and during the growth. This investigation should indeed permit to distinguish the processes in play and to determine the nature of the different Ge/Ag(111) structures, in relation with the amount of Ge. Such studies have previously been shown to be powerful for following the growth of Si layers on Ag(110) and on Ag(111) [29][30][31]. In the present study, we have followed in real-time by STM the surface evolution upon Ge deposition around 400 K on Ag(111). From the evolution of the Ag(111) step edges, we determined that the  

3 3 
reconstruction is a Ag2Ge surface alloy, and that the disordered hexagonal structure also corresponds to a surface alloy, with a larger proportion of Ge atoms.

Experimental

Sample preparation and STM experiments have been performed in an ultra-high vacuum (UHV) system with a base pressure less than 1×10 -10 mbar equipped with an Omicron variable temperature STM. The Ag(111) single crystal was prepared by several cycles of Ar ion sputtering (  P 7×10 -5 mbar, 600 eV) and annealing (  T 870 K). Germanium was evaporated from a graphite crucible using an Omicron Nanotechnology e-beam evaporator installed in front of the STM with a very low flux  F 0.1 ML/h. All coverages have been calibrated using the completion of the striped phase as a reference, set to a coverage of 1/3 ML.

STM imaging was performed in real-time during Ge evaporation, with a substrate held at a fixed temperature in the [380 K -430 K] range. The pressure was always below 1×10 -10 mbar during the experiments. Depending on the tip shape, small or large shadowing effects can occur during deposition. Indeed, as the STM tip remains very close to the surface (a few Å), the area located behind the tip with respect to the direction of the incoming flux is not covered by Ge atoms. However, as the tip moves over the surface while scanning, the shadow also moves, which gives rise to local inhomogeneities on the surface. In order to check that the growth is not influenced by the tip, we have performed experiments for which the tip was removed during evaporation, in order to avoid shadowing effects. For all cases, comparison between images of the same area performed at different times have been made by carefully correcting the STM images from the drift using a home-made procedure [30].

Results

Evolution of the Ge/Ag(111) structures

Fig. 1 shows the evolution of the same area during continuous Ge evaporation on a Ag(111) surface held at 380K. It corresponds to a detailed view (480 x 480 nm 2 ) of the larger area ( 680x 680 nm 2 ) continuously scanned during Ge evaporation (See Movie in the Supplemental Material [START_REF]STM movie and image analysis[END_REF] for the complete film). Due to shadowing effects, the Ge flux is higher on the top left side of the image than on the bottom right side. Before evaporation, the surface displays flat terraces separated by single atomic steps. The colors correspond to terraces at different levels, the lower ones being at the up-right part of the images. As imaging is performed during evaporation, Ge atoms also adsorb on the STM tip, which can lead to noisy parts on the STM images. The dark triangle at the center of the image is a defect that serves as a marker to follow the surface evolution. Note that step edges are also pinned at atomic defects of the surface.

Whereas before Ge deposition, atomic steps display only small fluctuations, during deposition, the step edges are mobile and move towards the right part of the image, i.e. toward the descending direction, forming outgrowths on the inferior terraces, as shown by the white arrows in Fig. 1.b. Such behavior has already been observed during Si deposition on Ag(110) [29] and on Ag(111) [30]. For Ag(110), it has been explained by the formation of a Ag missing-row reconstruction during which released silver atoms form outgrowths from the step edges. For Ag(111), it has been explained by the exchange between Si and Ag atoms in the surface plane, leading to the release of Ag adatoms that further condense at the step edges or grow new terraces. For both cases, the outgrowths and new terraces are initially free of silicon, and then covered with Si, as Si deposition continues.

In the present case, the situation is different. In Fig. 1b-1d, where outgrowths have developed at step edges, especially in the top of the images, the presence of Ge on the surface is also clearly visible. This is in particular the case in Fig. 1c, where dark twig-like dendrites appear (see Fig. S2a [32] for a detailed view), or in the bottom right part of Fig. 1d, where triangles have formed (see Fig. S6a [32] for a detailed view). As can be seen in Fig. S1 andS3, the Ge coverage is the same in the regions of the outgrowths and in the initial terraces, which indicates that the growing outgrowths are instantaneously (at the timescale of the observation) covered with Ge. This evolution has been observed for all substrate temperatures studied (see Fig. S4 for growth at 416 K). Supplemental Material [START_REF]STM movie and image analysis[END_REF] for the complete film.

Fig. 2 displays detailed views of the surface evolution for different evaporation times. At the very beginning of the evaporation, some dark spots are visible on the STM images (Fig. 2a).

They have been shown to correspond to Ge atoms in substitutional position [25] that occupy randomly the surface. Similar observations have been performed in the case of Si/Ag(111), after deposition of a very low Si coverage at room temperature [START_REF] Satta | [END_REF]. Tunneling conditions: (a, g)

 S V 1.5 V- I 30 pA; (b)  S V 0.4 V- I 20 pA; (c)  S V -0.3 V  I 0.5 nA; (d)  S V 0.4 V- I 0.2 nA; (e)  S V 1.0 V- I 50 pA; (f)  S V 0.3 V- I 0.2 nA; (h)  S V 0.4 V- I 0.1 nA.
As deposition time increases, this dilute Ge phase becomes denser, Ge atoms form denser areas separated by nearly pure Ag regions, with a characteristic length of 2-3 nm (Fig. 2b).

A first order transition occurs for a coverage of ~0.11 ML, where twig-like dendrites form on the surface, as shown in Fig. 1c. A detailed view of the surface shows that they are roughly oriented along the <112> directions of Ag(111) (see Fig. S2a [START_REF]STM movie and image analysis[END_REF]). In the STM images, they appear as darker areas (i.e., at a lower apparent height). They display a  

3 3  structure,
while the lighter areas in their vicinity have a   1 1 structure. This is clearly visible in Fig. 2c where a detailed view of the frontier between a   1 1 region at the bottom left part of the image and a   For 3 / 1 Ge   ML, another sudden transition occurs where triangle domains are replaced with the striped phase (Fig. 2e, 2f and S7a [START_REF]STM movie and image analysis[END_REF]). The detailed view of Fig. 2e, where the coverage is slightly higher at the top of the image than at the bottom, shows that the striped phase initially follows the orientation of the initial twig-like dendrites, but very rapidly transforms into larger domains with a single orientation (the stripes are oriented along <112>). This demonstrates that this reorganization into large areas does not imply a large amount of material transport.

Finally, local patches of a disordered-hexagonal (DH) reconstruction form. They start to grow at step edges, as already reported [11], but they also form inside large terraces. Depending on the STM tip termination, the DH phase appear as a disordered hexagonal array of protrusions, with a lattice constant of 0.97±0.02 nm (Fig. 2g andS7d-f [START_REF]STM movie and image analysis[END_REF]), or as a more organized hexagonal array, with a smaller lattice constant of 0.43±0.02 nm (Fig. 2h andS7g-i). This is significantly higher than the value found previously (0.391 nm) [11], and also higher than the lattice constant of free-standing germanene (0.397 nm) [2]. From the analysis of the evolution of the fraction of each phase as a function of coverage, we have estimated that this DH reconstruction is completed for 0.6±0.1 ML. This is far from the coverage 06 . 1 Ge   ML needed for a germanene monolayer. This disordered hexagonal layer must thus contain a significant amount of Ag atoms.

For higher coverage, a second layer starts to form above the disordered hexagonal layer, as already reported. [27] In Fig. 3 is plotted the surface fraction of each structure as a function of Ge coverage. The values have been obtained from the analysis of the successive images obtained during Ge deposition at 380 K (and corresponding to the movie [START_REF]STM movie and image analysis[END_REF]). The succession of the different phases is clearly visible. As there is a gradient of coverage in each of the successive recorded STM images, the transitions between the different structures are smoothed. The extrapolation of the linear growth of the DH phase is also in a good agreement with a completion of this phase Red dots: diluted phase, blue crosses: twig-like dendrites, green triangles: triangles, black squares: striped phase SP, pink hexagons: disordered hexagonal (DH) phase. Lines are guides for the eyes. Note that although consisting in the mixing of two phases, the triangle phase is counted as a whole.

Ag content in the structures

In order to determine the fraction of Ag atoms in the observed structures, we have precisely measured the advance of Ag step edges, which corresponds to the quantity of Ag atoms that are expelled by Ge atoms during the deposition. The Ag concentration in the observed structures is thus the complement of the concentration of expelled Ag atoms ( [START_REF]STM movie and image analysis[END_REF]). The dependence of Ag concentration with Ge coverage, measured on STM images obtained during Ge deposition at 380 K (see Fig. 1 and movie [START_REF]STM movie and image analysis[END_REF]) is drawn in Fig. 4. relationship, where 1 Ge atom replaces around 0.9±0.1 Ag atom. Note that this latter quantity could be slightly underestimated due to the formation of some Ag clusters on the surface, which can be seen by STM during deposition. As it is not possible to determine whether they contain Ag or Ge, we have excluded them from the measurements. Taking into account the uncertainty related to the measurement, this is in correct agreement with a Ag2Ge surface alloy for the

  3 3 
structure obtained for 1/3 ML. Concerning the DH structure, the extrapolation of the data presented in Fig. 4 indicates that the completion should be obtained for a Ge coverage nearly equal to twice the coverage for the   3 3  structure, i.e., 0.6±0.1 ML, largely smaller than the expected coverage of 1.06 ML for germanene. This DH structure is thus an Ag-Ge alloy and as the decrease of Ag coverage in the structures is linear (Fig. 4), its extrapolation to higher Ge amounts indicates that the DH structure must contain a significant amount of Ag, i.e. 0.46±0.09 ML. This is compatible with a Ag3Ge4 surface alloy. Due to the large error bars associated with the measurements, the DH structure is also compatible with AgGe or AgGe2 alloys. However, from this analysis as a function of the actual amount of Ge in these two structures, we can conclude with good confidence that, in both cases, they are not germanene layers, but rather Ag-Ge alloys with different concentrations in Ge.

Diffusion of Ge atoms

In the temperature range of our experiments, the inserted Ge atoms that are observed for very low coverage (i.e 001 . 0 Ge   ML) are quite mobile. At 380 K, we observe that more than half of the Ge atoms have moved, from one atomic site to another, during the time interval of 13 min between two consecutive images (see Fig. S8a-c). We have analyzed the displacements of Ge atoms between consecutive images obtained at this temperature. In the analysis, we have only taken into account Ge atoms far from the step edges, and having not too close neighbors.

We have also excluded Ge displacements higher than Several atomistic mechanisms may be involved in surface diffusion of atomic species [34]. As Ge atoms are inserted in the surface plane, either they diffuse in the surface plane, or above the surface. The first case corresponds to vacancy-assisted diffusion. It has been proposed for example for In atoms inserted in Cu(100) [35]. In that case, Ge atoms jump into a nearest neighbor site by exchange with the vacancy. Two possibilities can be considered. If the vacancy is mobile and does not remain in the vicinity of the Ge atom, the next jump will occur with another vacancy. Diffusion corresponds thus to a series of uncorrelated jumps over a distance 0 d . We have computed the histogram of displacements associated to such mechanism, using a basic Monte-Carlo simulation program of atomic diffusion (see Supplemental Material for a detailed description of the simulations). With this model, it is not possible to correctly reproduce the histogram of atomic displacements. The distribution, shown in Fig. 5b, is more peaked around the average value, and the fraction of atoms that have covered a distance larger than 0 5d is negligible.

On the contrary, if the vacancy remains in the neighborhood of the inserted atom, long correlated jumps may occur. This is generally the case for large atoms inserted in a surface, as

In in Cu(100), where the atom-vacancy interaction is attractive since the presence of the vacancy reduces the compressive stress associated with the insertion of the foreign atom. In this case, periods of long jumps, when the vacancy remains close to the inserted atom, are separated by periods where the inserted atoms keep still. In the present case, the atomic radius of Ge is smaller than the one of Ag, so we expect a repulsive interaction between an inserted Ge atom and a vacancy, which makes the vacancy-assisted diffusion mechanism improbable.

The second mechanism of diffusion involves a three-step process: exchange between an Ag adatom and a Ge inserted atom, diffusion of the Ge atom on the surface and reinsertion through exchange with a surface Ag atom. It has been for example shown to describe the diffusion mechanism of Pb/Cu(110) [36]. For this mechanism, the activation energy for deinsertion determines the frequency of diffusion events,

        kT E dis 0 dis dis exp  
, whereas the difference in activation energy between atomic jumps on the surface and reinsertion determines the average length of a diffusion event. We note for which the difference in activation energy between atomic jump and exchange was shown to be 0.17eV [37].

                         kT E kT
For an attempt frequency 3    s -1 and no atomic motion can be observed at the timescale of the experiment. On the contrary, at 415 K, we derive 03 . 0 dis   s -1 . We have followed the diffusion at this temperature on images scanned with the up and down mode, with a scan speed of 0.75s/line.

On the very top of the images, where similar lines on two consecutive images are scanned with a small time interval, some atoms are observed at the same position (Fig S8d-e [START_REF]STM movie and image analysis[END_REF]). As the time interval between similar lines increases, the proportion of atoms that have moved increases. After a time interval of 300 s, all atoms are observed at different position. This is in good agreement with the diffusion simulations performed at this temperature, that indicate that for these parameters, only 1% of the atoms should be found at the same position.

Conclusion

In summary, we have followed by real-time STM the evolution of the successive structures formed on Ag(111) upon Ge deposition in the [380 K -430 K] temperature range. Our results show that Ge atoms easily exchange with Ag atoms to form various Ag-Ge surface alloys. For 1/3 ML, the substitutional Ag2Ge alloy forming a striped phase is observed. For a coverage of 0.6 ML, the disordered hexagonal structure is obtained, which is also interpreted as an alloy with a larger proportion of Ge atoms. No structure was found compatible with a germanene layer. The quantitative analysis of diffusion of isolated Ge atoms shows that atoms move in a three-step process: exchange between an Ag adatom and a Ge inserted atom, diffusion on the surface of the Ge atom and reinsertion through exchange with a surface Ag atom. The activation energy for Ge/Ag exchange is small, which leads to a high mobility of Ge atoms in the [380 K -430 K] temperature range studied.

Monte-Carlo simulations

Single atom trajectories were computed using a Monte-Carlo program based on the Metropolis rejection algorithm. Two models were tested.

In the first one, Ge atoms diffuse in the surface plane into vacant sites. Ag vacancies are treated in a mean-field approach and the probability that one of six nearest neighbour sites is vacant is proportional to In the second one, the considered diffusion events were atom disinsertion, jumps and Tunneling conditions :

 S V 1.4 V- I 30pA.

Fig. 1 .

 1 Fig. 1. Evolution of the Ag(111) surface during Ge deposition at 380 K, for (a) 0 min, (b) 100 min (𝜃 Ge = 0.08 ML), (c) 190 min (𝜃 Ge = 0.16 ML), (d) 360 min (𝜃 Ge = 0.30 ML). In (b), the white arrows indicate the outgrowths that have grown during Ge deposition. Size of the images: 580 × 580 nm 2 . Tunneling conditions :  S V 1.4 V- I 30pA. See Movie in the

Fig 2 .

 2 Fig 2. Detailed view of the Ge/Ag(111) surface for increasing coverage. a) diluted phase for 0025 . 0 Ge  

3 3  3  3 3 

 3333 region (upper right part) is shown. The height profiles along the main axes of the different phases (Fig.S5b) indicate a lattice constant of 0.29 nm and 0.52 nm for the   reasonable agreement with the expected values for the   lattice constants (0.289 nm and 0.500 nm respectively). The comparison between successive images (see Fig.S2aand S2b[START_REF]STM movie and image analysis[END_REF]) shows that the dendrites form by the assembly of three-pointed hollow stars.As coverage increases, the network of twig-like dendrites becomes denser and transforms into a triangular network where the   1 1 domains of the diluted Ge phase have a triangular shape and are surrounded by the  3 regions. This can be seen in the bottom right corner of Fig.1dand in the detailed view shown in Fig.2d. The height profiles along the main axes of the  phase (Fig. S2d[START_REF]STM movie and image analysis[END_REF]) indicates a lattice constant of 0.50 nm. Note that these triangles adopt a more regular shape and form a more ordered network as the growth temperature increases (see Fig. S6[START_REF]STM movie and image analysis[END_REF]).

Fig. 3 .

 3 Fig. 3. Fraction of the different phases as a function of Ge coverage during growth at 380K.

Fig. 4 .

 4 Fig.4. Evolution of the Ag concentration in the surface structures, as a function of Ge coverage, during deposition at 380K. Red circles correspond to the experiment reported in Fig.1. The line is a linear fit with slope -0.9. The black square corresponds to another deposit at the same temperature, on an area not affected by shadowing effect.

Fig. 5 .

 5 Fig. 5. Histograms of Ge atomic displacements, normalized to the to the Ag-Ag interatomic distance. a) measured between successive images acquired at 13 min interval, and at T=380 K. b) best fit within the framework of single diffusion mechanism. c) best fit within the framework of diffusion through deinsertion, jumps and reinsertion.

  reinsertion of a Ge atom. We have simulated the diffusion of Ge atoms with such a mechanism. A good agreement is obtained with a characteristic frequency for deinsertion  dis  2 10 -3 s -1 , and a relative probability of reinsertion of 0.4, indicating that the difference in energy barrier between insertion and jump is small, of the order of 0.03 eV, 10. The corresponding histogram is shown in Fig.5cand is in excellent agreement with the experimental histogram of Fig.5a. This suggests that the exchange mechanism is easier for Ge/Ag(111) than for the similar system, Si on Ag(111),

1 ,

 1 we obtain an activation energy for deinsertion  dis E 1.1 eV. Note that this value includes the Ag adatom creation energy, which value has been calculated from DFT: ad E 0.67 eV[29]. Thus, the activation barrier for exchange strictly speaking is of the order of 0.4 eV only. At 300 K, with 



  is a characteristic frequency and bvac E is the energy barrier for Ge-vacancy exchange.

Fig. S1 :

 S1 Fig. S1: Evolution of the Ag(111) surface during Ge deposition at 380K. The image shows the formation of outgrowths. The Ge coverage (𝜃 Ge = 0.1 ML) is the same in the outgrowths and in the initial terraces. The position of step edges before evaporation is indicated by the dotted lines, and the motion of the step edges are given by the arrows. Size of the image: 70 × 70 nm 2 .

Fig. S2 :

 S2 Fig. S2: Evolution of the Ag(111) surface during Ge deposition at 380K. Consecutive images (65 × 65 nm 2 ) acquired at 5 min time interval. The images show the formation of dark twig-like dendrites, oriented along the <112> directions. Tunneling conditions :  S V 1.4 V- I 30pA.

Fig. S3 :

 S3 Fig. S3: Evolution of the Ag(111) surface during Ge deposition at 380K. (a) and (b): consecutive images (170 × 170 nm 2 ) acquired at 40 min time interval showing the motion of the outgrowth edge. The detail (c) show that the outgrowth is uniformly covered with the striped phase, as in the initial terrace. Tunneling conditions :  S V 1.5 V- I 30pA.

Fig. S4 :

 S4 Fig. S4: Ag(111) surface during Ge deposition at 416K for an average coverage 𝜃 Ge ≈ 0.4ML. (a) shows the formation of outgrowths at the right of the initial step edge position (marked by the presence of a line of clusters). The lower terrace (blue color) displays mainly the striped phase, while the upper terrace (green color) displays both the striped (at the left of the image) and disordered hexagonal phases (at the left of the initial step edge and in the outgrowths). Size of the image: 170x170 nm 2 . The detailed view (b) corresponds to the red rectangle in (a). Tunneling conditions:  S V 1.6 V- I 80pA.

Fig

  Fig. S5: a) Boundary between the diluted phase and a twig-like dendrite obtained after Ge deposition at 420 K. b) Height profiles along the lines drawn in a). c) Triangle phase obtained after Ge deposition at 400 K. d) Height profile along the line drawn in c). Tunneling conditions (a) -0.3 V  I 0.5 nA; (c)  S V 0.4 V- I 0.2 nA;

Fig. S8 .

 S8 Fig. S8. (a-c). Diffusion of Ge atoms on Ag(111) at 380 K. Consecutive images recorded at a time interval of 13 min. (d-e). Diffusion of Ge atoms on Ag(111) at 415 K. Successive images acquired from the bottom to the top and from the top to the bottom at a scan speed of 0.75s/line. Size of the images: 43 x 43 nm 2 . Tunneling conditions :  S V 1.5 V- I 30 pA.

  reinsertion. Disinsertion events occur through exchange between Ag adatoms and Ge inserted atoms, with an energy barrier bdis E . Ag adatoms are treated in a mean-field approach and the probability that a Ag adatom is next to a Ge inserted atom is proportional to kT

																		e		E	ad	where ad E is
	the Ag adatom formation energy. The probability of disinsertion per unit time is
		dis			0 dis	    exp	kT E dis	  	where	dis E		bdis E		ad E	. Disinserted atoms either diffuse by single atomic
	jumps with probability jump p , or reinsert in the surface with probability ins p . As no other atomic
	diffusion processes are considered,	ins p	 p	jump		1	and	ins p			ins	ins   	jump	with
		ins			0 ins	    kT E ins exp	  	and		jump			0 jump	     exp	kT E jump	   	. Moreover, we have considered that
	ins  		dis	and		jump			dis		since the disinsertion frequency dis  includes the extra Ag adatom
	concentration. Histograms of Ge atomic displacements were computed on 10000 individual
	trajectories.
	Movie 1.

Evolution of the Ag(111) surface during Ge deposition at 380K. Movie length: 7h25min. Size of the images: 500 × 500nm 2 .

Acknowledgments

This study is financially supported the French National Research Agency (Germanene project ANR-17-CE09-0021-03). K. Z. is supported by the Chinese Scholarship Council (CSC contract 201808070070).

Growth of germanium-silver surface alloys followed by in-situ scanning tunneling microscopy: absence of germanene formation. In this paragraph, we show that the fraction of Ag atoms in the observed structures is the complement of the advance of Ag step edges. For the demonstration, we can start with a planar surface. Before deposition, the whole surface is at z=0. The Ag surface coverage is the Ag content in the surface plane and is equal to 1. After deposition of a given Ge amount, expelled Ag atoms form outgrowths that occupy a fraction x of the surface. The surface plane of these outgrowths, with a Ag coverage 𝜃 Ag < 1, is now at z=d, where d is the Ag(111) interlayer spacing. Below these outgrowths, the plane at z=0 is a pure Ag plane, whereas in the rest of the surface, the plane at z=0 has a coverage 𝜃 Ag as it is covered with the same Ag-Ge structure as the outgrowths.

The conservation of Ag atoms leads to 1 = (1 -𝑥)𝜃 Ag + 𝑥 1 + 𝜃 Ag . Thus, 𝜃 Ag = 1 -𝑥.