Julien Hofmann
email: julien.hofmann@grenoble-inp.fr

Charles Thiébaut

Michel Riondet

Pierre Lhuissier

Sylvain Gaudion

Marc Fivel

Comparison of acoustic and hydrodynamic cavitation: material point of view

This study investigated the difference of mechanical response of the martensitic stainless steel X3CrNiMo13-4/S41500/CA6NM QT780 between hydrodynamic and acoustic cavitation erosion. Acoustic cavitation erosion tests have been performed using a 20 kHz ultrasonic horn located at 500 µm in front of a specimen. This experimental setup, known-as indirect method, is inspired from the ASTM G32 standard. Hydrodynamic cavitation erosion tests were conducted with classic experimental conditions of PREVERO device: a cavitation number of 0.87 corresponding to a flow velocity of 90 m.s -1 and an upstream pressure of 40 bars. Results show that acoustic cavitation erosion generates small pits at high temporal frequency on the material while hydrodynamic cavitation erosion produces larger pits at a lower frequency. In addition, for a given exposure time the percentage of surface covered by the pits is smaller for acoustic cavitation than for hydrodynamic cavitation. Three successive steps have been identified during the damage process: persistent slip bands (PSB) first appear on the surface, cracks initiate and propagate at the PSB locations and non-metallic interfaces and finally parts of matter are torn off. A careful time examination of the same small area of the exposed sample surface by scanning electron microscopy (SEM) reveals that acoustic cavitation is faster to initiate damage than hydrodynamic cavitation.

I. INTRODUCTION

Cavitation denotes the phenomenon in which vapor bubbles form in a liquid after a local pressure drop below the saturated vapor pressure [START_REF] Franc | enFundamentals of Cavitation[END_REF] . This phase transformation is common in hydraulic machinery: fuel injectors, valves, pumps and water turbines for example. The collapse of cavitation bubbles close to a solid surface can lead to harmful effects such as vibration, noise and erosion [START_REF] Karimi | Cavitation erosion of materials[END_REF] . Cavitation erosion is known as a significant cause of loss of efficiency of hydraulic machinery. Surface modification is attributed to microjet and shock waves occurring during the collapse of multiple cavitation bubbles when it happens close to a material surface [START_REF] Rayleigh | VIII. On the pressure developed in a liquid during the collapse of a spherical cavity[END_REF] . The collapse is a local and violent phenomenon: stress on the surface can reach few gigapascals 1 at a strain rate as high as 10 6 s -1 . If the stress resulting from the shock waves or micro jet impacts outreaches the yield stress of the material, a pit is created. Accumulation of these local plastic deformations on the surface increases the work hardening along the exposure time to cavitation. This will cause the material cracking and inescapably lead to mass loss [START_REF] Karimi | Cavitation erosion of materials[END_REF][START_REF] Caupin | Cavitation in water: a review[END_REF][START_REF] Allenstein | Improvement of the cavitation erosion resistance for low-temperature plasma nitrided Ca-6NM martensitic stainless steel[END_REF][START_REF] Krella | The resistance of S235JR steel to cavitation erosion[END_REF][START_REF] Gao | Cavitation erosion mechanism of 2Cr13 stainless steel[END_REF] . Cavitation erosion can be investigated in laboratories using different devices such as vibratory cavitation apparatus or hydrodynamic tunnels. Each device has its own characteristics in term of material solicitations including flow velocity, impact frequency and impact loads which allows to investigate a wide range of aggressiveness conditions. The objective of the present work is to compare the mechanical response of a lowcarbon martensitic stainless steel, commonly used in hydroelectric turbines, exposed to cavitation erosion from hydrodynamic and acoustic devices. Hydrodynamic cavitation is generated using a tunnel in which the flow velocity can be set to adjust the cavitation number [START_REF] Patella | Cavitation damage measurement by 3D laser profilometry[END_REF][START_REF] Dular | Relationship between cavitation structures and cavitation damage[END_REF][START_REF] Franc | en"Material and velocity effects on cavitation erosion pitting[END_REF] . Acoustic erosion cavitation testing, standardized by the ASTM G32, is one of the most popular laboratory technique [START_REF] Abouel-Kasem | Investigation Into Cavitation Erosion Pits[END_REF][START_REF] Dular | Pit clustering in cavitation erosion[END_REF][START_REF] Haosheng | Inelastic damages by stress wave on steel surface at the incubation stage of vibration cavitation erosion[END_REF] , generally used for convenience and availability of commercial solutions. In the present study, based on surface and microstructure analysis, we point out the similarities and differences in the material response when exposed to these two loading conditions: hydrodynamic and acoustic cavitation. These results are discussed from the material point of view.

II. MATERIALS AND METHODS

A. Materials

In this research, the low-carbon X3CrNiMo13-4 QT780 martensitic stainless steel provided by Ugitech was chosen as the tested material. It was austenitising at 1100 • C, quenched and then double tempered at 600 • C for 4 h. The resulting material is composed of a martensitic matrix α with lamellar reversed austenite γ rev and residual delta ferrite δ -Fe. It shows high corrosion resistance and high mechanical strength. This material is thus widely used for water turbine manufacturing. Its chemical composition identified by X-ray fluorescence spectrometry and inert gas fusion elemental analysis (Leco CS744, USA) is given in TABLE I.

B. Cavitation erosion testing

Two in-house devices were used for generating two types of cavitation: acoustic and hydrodynamic. A 20 kHz ultrasonic transducer (Sinaptec Lab750, France) was used for the acoustic cavitation erosion tests using indirect method. As shown in FIG. 1(a) stationary specimen is placed at 0.5 mm below the ultrasonic horn vibrating at 20 kHz with a peak-to-peak amplitude of 50 µm. The distance between specimen and ultrasonic horn was measured using a x100 optical camera (Dino-Lite AM4815ZTL, Taiwan) and adjusted using a micrometric motorized stage (OWIS LTM120, Germany). The erosion tests were performed in water at 23 ± 2 • C containing 4.2 mg.L -1 of dissolved oxygen (Hanna Instruments HI98199, United States). Specimen consisted of cylinders with a height of 5 mm and diameter of 25 mm. Hydrodynamic erosion tests were conducted using a 40 bars cavitation flow tunnel (PREVERO) located at LEGI laboratory [START_REF] Franc | Incubation Time and Cavitation Erosion Rate of Work-Hardening Materials[END_REF] . The water flow is created using a centrifugal pump rotated by an 80 kW electric motor. Pressurization of the liquid is performed using nitrogen gas on the free surface of a water tank. Tap water temperature was kept constant at 23 ± 2 • C using a heat exchanger. As illustrated in FIG. 1(b) on a cross section view, a gap of 2.5mm is defined between the nozzle wall and the specimen so that the radial outlet flow reach a maximum velocity of 90 m.s -1 leading to the formation of cavitation sheets. The dynamics of cavitation in PREVERO tunnel has been studied by Gavaises et al. [START_REF] Gavaises | Visualisation and les simulation of cavitation cloud formation and collapse in an axisymmetric geometry[END_REF] using high-speed visualisation and large eddy simulations. Cavitation firstly forms at the turn of the nozzle due to the rapid acceleration of the liquid. The toroidal cavitation cloud then grows as it is transported by convection until it reaches a maximum distance from the nozzle exit. The closure of the cavity, which is a saddle point, is known for generating instability [START_REF] Hunt | Kinematical studies of the flows around free or surface-mounted obstacles; applying topology to flow visualization[END_REF] . It hence creates a re-entrant liquid jet between the nozzle wall and the vapor cavity which separates the cavitation cloud from the wall. When the cavity is totally detached from the wall, a bubbles cloud is created. A significant vorticity forms due to opposite directions of liquid re-entrant jet and main flow, which makes bubbles cloud rotating and travelling downstream. When the centrifugal force, made by vorticity, is counterbalanced by the surrounding pressure, the edge of the bubble cloud starts to collapse. Specimen are inserted in the middle of the zone of cloud collapse. They consist of cylinders with a height of 6 mm and diameter of 20 mm. Hydrodynamic cavitation erosions tests were realized at a constant cavitation number σ = 0.870 ± 0.001. A complete description of this cavitation flow loop has been made by Franc et al. [START_REF] Franc | Incubation Time and Cavitation Erosion Rate of Work-Hardening Materials[END_REF] . Surface specimen exposed to acoustic and hydrodynamic cavitation have been polished using identical procedure. Specimen have been mechanically grounded using SiC abrasive papers from P400 to P1200 followed by polishing step using diamond suspension 9 µm, 3 µm and 1 µm. Finally, vibratory polishing (VibroMet, Buehler, United States) has been carried out using a 0.06 µm colloidal silica solution. After polishing and after each cavitation erosion tests, specimen were carefully cleaned using ethanol and soap.

C. Characterisation

Surface analysis

Morphology of the eroded surfaces was observed after each cavitation erosion tests using an optical profilometer (Zegage Pro HR, Zygo, United States) and a scanning electron microscope (Gemini ultra 55 SEM, Zeiss, Germany). For observing the same region for the different exposure time, the specimen has been marked using two Vickers indents with a force of 10 N (Presi MX7, France). For identifying the pits created by the collapse of cavitation bubble, a cut-off depth -0.2 µm has been applied below the original virgin material surface. The cut-off value has been chosen large enough to avoid the effect of the surface roughness and separate the pits and small enough for truthfully measure the pits' shape.

2. Phase analysis X-ray diffraction (XRD) was employed to identify and quantify the phases existing in the studied material X3CrNiMo13-4 QT780. Analysis was realized using a Cu-K α radiation (λ = 1.5406 Å) in 30-130 • interval using a scanning speed of 0.2 • .min -1 with a step size of 0.02 • (Malvern Panalytical, X'Pert Pro, England). The volume of fraction of reversed austenite γ rev was estimated using the semiquantitative method proposed by Tanaka and Choi 17 using the integrated intensities of main peak of reversed austenite (γ rev ) and martensite (α ). Equations 1 and 2 below were used to calculate the volume fraction of reversed austenite:

V γ rev +V α = 1 (1)
V γ rev = 1.4I γ rev (111) I α (110) + 1.4I γ rev (111) (2)
where V γ rev is the γ rev -phase volume fraction, V α is the αphase volume fraction, I γ rev (111) is the integrated intensity of the γ rev -phase (111) diffraction peak and I α (110) is the integrated intensity of the α -phase (110) diffraction peak. Microstructural investigation at submicrometer scale has been carried out using electron backscatter diffraction (EBSD). Experiment has been realized using a Zeiss Gemini SEM 500 FEG equipped with an EBSD detector (EDAX, Hikari Super, United States) with a step size of 100 nm. XRD and EBSD analysis were made on specimen prepared using the same methodology as described in II B.

III. RESULTS AND DISCUSSIONS

A. Pitting rate

At the very beginning of cavitation exposure, isolated pits appear on the material surface. It is hence possible to count the number of pits and measure their diameter. FIG. 2 presents the cumulative histograms of pitting rates versus pit equivalent diameter both for acoustic and hydrodynamic cavitation for an exposure time of 16 min. Cumulative pitting rate is defined as the number of pits per unit of time and per unit area counted with a equivalent diameter exceeding a given value. Pit can be more complex than circular shaped because of the appearance of re-entrant jet and the toroidal structure of the cavity which could create smaller bubbles which may collapse as well [START_REF] Franc | en"Material and velocity effects on cavitation erosion pitting[END_REF][START_REF] Philipp | Cavitation erosion by single laserproduced bubbles[END_REF] . As shown in FIG. 2, histograms are well fitted by straight lines in semi-logarithmic scales: the cumulative pitting rate can be approximated by an exponential decay law for pits from a few micrometers up to 250 µm. The slope of the straight lines is -9.59 10 -3 pits.cm -2 .s -1 .µm -1 for hydrodynamic cavitation and -9.56 10 -2 pits.cm -2 .s -1 .µm -1 for acoustic cavitation i.e. one order of magnitude higher.

FIG. 2 shows that the bigger the pit size, the smaller the pitting rate which expresses that vapor cavities produce few large pits and an important number of small pits whatever the type of cavitation.

Hydrodynamic cavitation erosion makes large pits at a small pitting rate and, conversely, acoustic cavitation erosion creates small pits at a high pitting rate. The difference of pit sizes could be attributed to the difference of the shape of the vapor cavity clouds: using ultrasonic horn, acoustic cavitation is known for generating small bubbles with a diameter between few microns up to hundreds of microns [START_REF] Makuta | Generation of microbubbles from hollow cylindrical ultrasonic horn[END_REF][START_REF] Lee | Determination of the Size Distribution of Sonoluminescence Bubbles in a Pulsed Acoustic Field[END_REF] . On the other hand, hydrodynamic cavitation cloud is made of bubbles with a wide dispersion of sizes and pressures as estimated by Roy [START_REF] Roy | Modeling and analysis of material behavior during cavitation erosion[END_REF] .

Moreover for hydrodynamic cavitation, the shedding frequency, i.e. the collapse frequency, has been approximately evaluated by Gavaises et al. [START_REF] Gavaises | Visualisation and les simulation of cavitation cloud formation and collapse in an axisymmetric geometry[END_REF] and Ylonën et al. [START_REF] Ylönen | Shedding frequency in cavitation erosion evolution tracking[END_REF] to 1,600 Hz for a downstream pressure of 19 bar and a cavitation number equal to 0.87 corresponding to the experimental conditions of our study. This low collapse frequency, compared to the 20 kHz resonance frequency of the ultrasonic horn, might explain the difference of pitting rate: high for acoustic cavitation and low for hydrodynamic cavitation.

FIG. 3 shows surface profiles for different cavitation erosion time (4, 16 and 40 min) for hydrodynamic and acoustic cavitation. After 4 min of cavitation, the roughness of the material was not changed drastically from the initial state when the roughness Ra was approximately equal to 60 nm. The difference of roughness between the two types of cavitation may be explained by the difference of bubble sizes. Difference of aggressiveness can be easily noticed between hydrodynamic and acoustic cavitation. For an identical cavitation erosion times, the roughness Ra is more than five times larger for hydrodynamic cavitation than acoustic cavitation.

B. Surface evolution

As the cavitation erosion testing time increases, more and more bubbles collapse so that the printed pits cover a bigger and bigger cumulated area. FIG. 4 shows the percentage of the surface covered by pits as a function of the cavitation exposure time. The covered surface is defined as the ratio of sum of pits area to total considered area. For hydrodynamic cavitation, the curve tends to a plateau. This could be attributed to the fact that the probability that a bubble collapses in a virgin surface decreases with the cavitation erosion time since the size of the virgin surface diminishes as well. For acoustic cavitation, the covered surface firstly slowly increases until 16 min due to the small sizes of the pits. A rapid growth is then observed, corresponding to the first matter loss. From FIG. 4, we observe a shift of the curves between hydrodynamic and acoustic cavitation: the covered surface is more important in the case of hydrodynamic cavitation than acoustic for an identical exposure time. It can be concluded that pits size has more influence than pitting rate on the coverage of the surface. For both types of cavitation, it was observed that bubbles could collapse in the vicinity of existing pits and hence the new pits could partly close the previously created pits. Thus part of solid material might move upward and not solely downward. This could affect the evolution of the covered surface. Observation of the surface morphology is not sufficient for understanding the damage mechanism. The material microstructure has to be studied in details.

FIG. 5(a) shows a phase map of the X3CrNiMo13-4 QT780 stainless steel with the two phases identified: martensite α and reversed austenite γ rev . Reversed austenite forms lamellas with a width approximately equal to 150 nm. Presence of these two phases was confirmed using XRD analysis as shown in FIG. 5(b). Using integrated peak area from XRD analysis, the volume fraction of reversed austenite has been estimated to 11.9 vol.%. This phase can transform to martensite when a plastic deformation happens: this is known as the transformation-induced plasticity (TRIP) effect. Due to the change of crystalline structure, this phase transformation causes a volumetric expansion, which produces compressive forces that delay crack initiation and propagation [START_REF] Olson | Transformation Plasticity and Toughening[END_REF][START_REF] Chiang | Effect of microstructure on retained austenite stability and work hardening of TRIP steels[END_REF] . Presence of reversed austenite is thus particularly relevant for the design of materials exposed to cavitation. At the very beginning of exposition to cavitation, isolated pits appear on the material surface. Pits rapidly overlap and hence totally cover the surface which harden the material. FIG. 6 shows the morphology of the eroded surface after being exposed 30 min to acoustic cavitation. First persistent slip bands (PSB) appear at the material surface oriented parallel to the primary activated slip plane. This is the first damage visible after the full coverage of the surface by the pits. PSB is the consequence of mechanical fatigue imposed by the bubble collapses.

SEM micrographs of the same region of X3CrNiMo13-4 QT780 stainless steel sample surface tested for exposure times of 30, 70 and 100 min are shown in FIG. 7 for hydrodynamic cavitation (a)-(c) and acoustic cavitation (d)-(f). The effect of the pit size on the material microstructure is different depending on the type of cavitation. Since acoustic cavitation generates small pits as shown in FIG. 2, the damage mechanisms induced by this type of cavitation could be more sensitive to the fine microstructure of the material, i.e. typically the martensitic laths. On the other hand, in the case of hydrodynamic cavitation, the observed area in the x3000 SEM observation (FIG. 7) is smaller than the average pits diameters for hydrodynamic cavitation. The damage observed on SEM micrographs for hydrodynamic cavitation are hence likely to be located within a pit as also evidenced by the wavelength plotted in FIG.

IV. CONCLUSION

Mechanical response of martensitic stainless steel X3CrNiMo13-4 QT780 exposed to hydrodynamic and acoustic cavitation were investigated. By analyzing and comparing the material surface, we might draw the following conclusions:

(i) Acoustic cavitation erosion generates small pits at a high frequency on the material while hydrodynamic cavitation erosion produces larger pits at a lower pitting rate.

(ii) For a given exposure time, the percentage of surface covered by the pits is smaller for acoustic cavitation than for hydrodynamic cavitation. It is concluded that pits' size have more influence than the pitting rate on the surface coverage for the X3CrNiMo13-4 QT780 stainless steel.

(iii) The damage mechanism of X3CrNiMo14-4 QT780 can be decomposed into three main stages. Firstly, persistent slip bands (PSB) appear on the surface. Secondly, cracks initiate and propagate at the PSB locations and non-metallic interfaces and lead to matter removal.

(iv) The steps enumerated at (iii) occur with different kinetics according to the type of cavitation. Surprisingly, despite a small covering rate, acoustic cavitation is faster to initiate cracks than hydrodynamic cavitation.

(v) Cracks initiation and propagation will be further studied in volume using in-situ X-ray tomography. These observations will help the construction of a damage model to be implemented in simulations of cavitation erosion.

FIG. 1 .

 1 FIG.1. Schematic of the devices used for the cavitation erosion experiments. (a) Acoustic cavitation apparatus: the specimen is located at 0.5 mm from the ultrasonic horn (indirect method). (b) Hydrodynamic tunnel: the samples are mounted in the zone of collapse of the cavitation cloud ; the cavitation number σ is here fixed at 0.870 corresponding to a flow velocity of 90 m.s -1 and upstream pressure of 40 bars.

FIG. 2 .

 2 FIG. 2. Cumulative pitting rate as a function of pit diameter onX3CrNiMo13-4 QT780 stainless steel. Straight lines correspond to exponential distributions. Acoustic cavitation tests have been carried out using an ultrasonic horn at 20 kHz on a sample located at 500 µm during 16 min. Hydrodynamic cavitation test has been carried out in the LEGI cavitation erosion facility at a upstream pressure of 40 bars and at a constant cavitation number of 0.870 during 16min. Pits have been counted and measured using a 0.2 µm cut-off.

FIG. 3 .

 3 FIG. 3. Surface profiles of X3CrNiMo13-4 QT780 stainless steel exposed to hydrodynamic cavitation (a)-(c) and acoustic cavitation (d)-(f) for different cavitation testing time: (a) and (d) 4 min, (b) and (e) 16 min, (c) and (f) 40 min.

FIG. 4 .

 4 FIG. 4. Percentage of the surface covered by pits as a function of cavitation testing time for X3CrNiMo13-4 QT780 stainless steel. Acoustic cavitation test has been carried out using an ultrasonic horn at 20 kHz on a sample located at 500 µm. Hydrodynamic cavitation test has been carried out in the LEGI cavitation erosion facility at a upstream pressure of 40 bars and at a constant cavitation number of 0.870. Pits have been measured using a 0.2 µm cut-off.

FIG. 5 .

 5 FIG. 5. (a) Phase map from EBSD scan of X3CrNiMo13-4 QT780 before being exposed to cavitation. (b) XRD pattern obtained using Cu-K α radiation.

3 .FIG. 7 .

 37 FIG. 7. X3000 SEM micrographs of eroded surfaces by hydrodynamic cavitation (a)-(c) and acoustic cavitation (d)-(f) of X3CrNiMo13-4 QT780 stainless steel for: (a) and (d) 30 min, (b) and (e) 70 min, (c) and (f) 100 min. Yellow circles show example of crack propagating.

TABLE I .

 I Chemical composition of main chemical elements of stainless steel X3CrNiMo13-4 QT780. Material has been analysed using X-ray fluorescence and inert gas fusion elemental analyser . Chemical compositions are given in wt.% if not indicated.

	Cr Ni Mo	C	Mn S [ppm]
	X3CrNiMo13-4 QT780 12.61 3.75 0.54 0.035 0.58	92

ACKNOWLEDGMENTS

This project was funded by the Hydro'like Chair supported by the Grenoble INP Foundation thanks to a donation from General Electric. Maxence Buttard is acknowledged for his help.


Eva Zahorska

Francesca Rosato

Kai Stober

Sakonwan Kuhaudomlarp

Joscha Meiers

Dirk Hauck

Dorina Reith

Emilie Gillon

Katharina Rox

Anne Imberty

Francesca -3+

Rosato

Winfried Römer
email: winfried.roemer@bioss.uni-freiburg.de

Alexander Titz
email: alexander.titz@helmholtz-hzi.de

Neutralizing the Impact of the Virulence Factor LecA from Pseudomonas aeruginosa on Human Cells with New Glycomimetic Inhibitors

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

Introduction

Carbohydrate-protein interactions are essential recognition codes in many biological processes, including bacterial and viral infections. Therefore, lectins of pathogenic origin involved in hostcell recognition, adhesion and/or biofilm formation are being recognized as new therapeutic targets. [1,2] Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that belongs to the group of highly drug resistant ESKAPE pathogens. [3] According to the World Health Organization (WHO), the pathogen ranks as critical priority 1 due to its high level of resistance to antibiotics and the lack of new treatment options to combat it. Consequently, several new therapeutic strategies aim at an anti-virulence therapy which circumvents the selection pressure of antibiotic treatment, thus preventing the emergence of new resistances. [4] Antimicrobial resistance of P. aeruginosa is further enhanced by the bacterium´s ability to grow in biofilmsthe causative mechanism for chronic infections. [5] To this end, P. aeruginosa adhesion, biofilm formation and virulence depend on the tetravalent lectins LecA and LecB, both of which are encoded in its conserved core genome and are functionally conserved across clinical isolates. [6- 9] Furthermore, it was shown that LecA acts as a lipid zipper upon binding to its cellular receptor, [START_REF] Eierhoff | Proc. Natl. Acad. Sci[END_REF][START_REF] Omidvar | [END_REF] the glycosphingolipid globotriaosylceramide (Gb3), and induces the formation of a plasma membrane nanodomain, [12,13] cellular signaling [14] and ultimately leads to bacterial invasion. [START_REF] Eierhoff | Proc. Natl. Acad. Sci[END_REF] Thus, the inhibition of LecA and LecB is a promising path forward to counteract its pathogenicity and break its antimicrobial resistance. [4] Encouragingly, it was also demonstrated that the inhalation of aerosols containing D-galactose and L-fucose, the monosaccharide ligands of LecA and LecB, respectively, reduced the bacterial burden and improved the clinical situation in cystic fibrosis patients. [15] Consequently, numerous glycomimetics have been developed based on D-galactose (Kd = 88 μM) [16] for LecA and L-fucose/D-mannose (Kd = 430 nM for methyl ɑ-L-fucoside, Kd = 71 µM methyl ɑ-D-mannoside) [17] for LecB. [2,4,18] Recently, we reported small molecules based on C-glycosides targeting LecB which showed potent inhibition of LecB in the low micromolar range, prevented biofilm formation and exhibited beneficial pharmacokinetic properties. [19][20][21] For LecA, numerous monovalent glycomimetic inhibitors have been reported, [22][23][24] demonstrating the importance of an aromatic aglycon attached to the β-galactoside to reach lower micromolar affinity. To alter the galactoside moiety itself, we have recently introduced the first covalent lectin inhibitor targeting a surface exposed cysteine residue in LecA. [25] Furthermore, we also identified the first non-carbohydrate lectin inhibitor for a bacterial lectin. [26] Despite numerous attempts to increase potency, all monovalent LecA inhibitors reached at best only moderate potencies in the 5 to 50 µM range.

In contrast to LecB, the quaternary structure of LecA [27] displays two adjacent binding sites that are optimally oriented in space for the simultaneous binding of two galactose moieties of a single divalent ligand, which can result in an increased binding affinity. Notably, Pieters and coworkers developed low nanomolar LecA inhibitors with Kds down to 12 nM by connecting two galactosides through a linker containing several copies of rigid glucose-triazole linkers. [28,29] Despite the efficient click chemistry applied for the final assembly of the divalent ligands, in total 17 synthetic steps were required to prepare the azide and alkyne building blocks and final assembly. Replacing one of the glucose-bistriazolyl spacers with a cyclohexyl bisthiourea moiety in a 9-step synthesis yielded one compound with 30 nM affinity. [30] Similarly, more complex divalent LecA ligands with peptide-based linkers were reported by Novoa et al. (Kd = 82 nM) and Huang et al. (Kd = 71 nM). [31,32] Inspired by the Pieters approach, we recently reported a series of highly active divalent LecA inhibitors with acylhydrazone based linkers (Kd = 11 -81 nM) that simplified the synthesis to only four chemical steps. [33] While these molecules showed the highest potency among reported divalent LecA inhibitors, they suffer from their intrinsic hydrolytic lability of the acylhydrazone bond and a very low aqueous solubility, despite the presence of the two hydrophilic carbohydrates. Furthermore, acylhydrazones undergo hydrolysis already at weak acidic pH resulting in toxic aldehyde and hydrazide degradation products that may also be formed in vivo.

Here, we report the optimization of these divalent molecules by replacing the acylhydrazone motif and varying the linker to balance potency, solubility and stability. The lead compound was then successfully analyzed in in vitro assays and showed excellent properties to block LecA-mediated host cell binding, wound healing and bacterial invasion into host cells.

Results and Discussion

We substituted the acylhydrazone motif present in our previous compounds with a more stable amide bond and varied linker identity and length in order to increase solubility and stability (Figure 1a). Two galactoside building blocks bearing coumaric acid (1, Figure 1b) or hydroxyphenyl propionic acid (2) as aglycons were selected to investigate the effect of the rigid olefin in 1, comparable to the parent acylhydrazones, versus the flexible alkyl motif in 2. These galactosylated carboxylic acids were then coupled to various bisanilines to yield the desired divalent LecA inhibitors. Since an optimal length and geometry of the divalent ligand is important to simultaneously bind to the two neighboring LecA sites, linker length was varied by stepwise introduction of methylene units. The aromatic moieties of the linker and its length were also varied and bisaniline linkers B-F and their monovalent control A (Figure 1c) were used to mimic the previously used bisbenzaldehyde structures; [33] furthermore, our rational solubility optimization included the introduction of hydrogen-bonding polar groups or ionizable moieties into the divalent ligands using bisaminopyridine linkers H-J or sulfonated linker L and their monovalent controls G and K, respectively. [33] (top) and new generation optimized bioisosters (bottom). Proposed chemical modifications are highlighted: amide linkage as acylhydrazone bioisoster in red and linker derivatizations in blue. (b) Galactoside building blocks with terminal α,β-unsaturated carboxylate 1 and its saturated analogue 2. (c) Linker moieties: anilines B-F, aminopyridines H-J and sulfonated linker L, and their monovalent controls A, G, and K.

Synthesis of the two galactoside building blocks 1 and 2 started with β-selective glycosylation of benzyl coumarate or methyl 3-(4-hydroxyphenyl)propanoate with β-ᴅ-galactose pentaacetate (3) under Lewis acid catalysis (Scheme 1). β-Glycosides 4 and 5 were obtained in 76% and 86% yields, respectively. Saponification of the esters with aqueous NaOH resulted in galactosides 1 and 2 in good yields. Synthesis of coumarate 1 was initially attempted using the methyl ester under identical glycosylation conditions as for compound 2, but this transformation was unsuccessful most probably due to its poor solubility in dichloromethane and only poor yields were achieved using more polar chloroform as a solvent instead. Changing from methyl to benzyl coumarate improved solubility in those solvents and the glycosylation gave compound 4 in good yield (76%). The linkers were synthesized or purchased: while anilines A and B, aminopyridine G and sulfonated linker K were commercially available, linkers C-F and bis-aminopyridine linkers H-J were prepared in two steps: a double nucleophilic substitution of the ɑ,𝜔alkyldihalides (6-9) with 4-nitrophenol or two nucleophilic aromatic substitutions using ɑ,𝜔alkyldiols (10-12) with 2-chloro-5-nitro-pyridine. In both cases a palladium-catalyzed hydrogenation followed to give the desired bis-anilines or bis-aminopyridines, respectively (Scheme 2). Ethyl-spaced bissulfonated linker L was obtained by reduction of 4,4'diaminostilbene-2,2'-disulfonic acid (13) with hydrogen on Raney nickel.

Final assembly of the divalent LecA inhibitors was achieved by coupling of the aminosubstituted linkers A-L with carboxylate-containing galactosides 1 or 2 using HBTU or PyBOP as peptide coupling reagents (Scheme 3). High turnovers were observed for all coupling reactions, but purification difficulties caused varying yields: the lower solubility was responsible for isolated yields in the benzene series (A1-F1 and A2-F2), whereas side product formation was observed in the pyridine series (H1-I1 and G2-J2). The sulfonated ligands K2 and L2 were obtained as ammonium salts since their chromatography required buffered eluents to avoid acid-catalyzed self-hydrolysis of the glyosidic linkage upon solvent removal and concentration.

Next, we quantified the solubility of at least one representative of each new class and of one parent bisacylhydrazone in aqueous media (Table 1). All tested new derivatives showed improved solubility compared to the previous bisacylhydrazone 14. The very low kinetic solubility of bisacylhydrazone 14 (S = 1.6 ± 0.4 µM) was increased twofold in its amide analogues D1 (S = 3.3 ± 1.3 µM) and D2 (S = 3.1 ± 2.1 µM). Substitution of the benzene ring with a pyridine moiety further increased solubility twofold in case of α,β-unsaturated amide analogue H1 (S = 8.1 ± 2.2 µM) and almost fivefold in the saturated analogue H2 (S = 14.8 ± 1.5 µM) compared to D1 and D2, respectively. Surprisingly, another fourfold increase in solubility was observed for I2 (S = 63.3 ± 4.2 µM) compared to H2, despite only a small structural difference of one additional methylene in the linker. The longest pyridine ligand J2 showed again almost identical solubility to H2. Thus, the linker has a strong impact on solubility in the pyridine amide series, which could result from the conformational preferences of an ethylene glycol compared to alkyl chains with different numbers of methylene units that might affect ligand self-aggregation and thus their solubility. However, excellent aqueous solubility was finally achieved with sulfonated divalent ligand L2, which was fully dissolved from its solid form in an aqueous buffer (S > 1.5 mM).

We then determined the in vitro metabolic stability in plasma and liver microsomes as well as plasma protein binding for a selected subset of the synthesized LecA inhibitors, i.e. D1, D2, H1, H2 and L2 and compared them to the parent bisacylhydrazone 14 (Table 2). Stability tests revealed a low intrinsic clearance (CLint) by mouse liver microsomes (MLM) for most tested compounds (CLint = 6.8 -23 µL/min/mg protein), with a slightly elevated clearance for D2 (CLint = 29.6 µL/min/mg protein) and sulfonated ligand L2 (CLint = 29.5 µL/min/mg protein). The metabolic stability of the compounds differed in presence of human liver microsomes (HLM). Introduction of a pyridine ring decreased stability (H1 CLint = 28.6 µL/min/mg protein, H2 CLint = 32.5 µL/min/mg protein) compared to the benzene analogues (D1 CLint = 21.9 µL/min/mg protein, D2 CLint = 21.0 µL/min/mg protein) and to the parent acylhydrazone 14 (CLint = 19.6 µL/min/mg protein). Finally, sulfonated L2 showed the highest stability in presence of human liver microsomes (CLint = 9.2 µL/min/mg protein). Then, we analyzed the stability of the compounds in plasma. Acylhydrazone 14 was degraded faster (t1/2 = 48.9 min) in mouse plasma whereas a higher stability was observed in human plasma (t1/2 = 123.5 min). The tested representatives of the new ligands exhibited high stability in both mouse and human plasma (t1/2  180 min), with exception of the coumarates: pyridine H2 was only moderately stable in mouse plasma (t1/2 = 81.1 min) and benzene D2 showed a slow degradation in human plasma (t1/2 = 132.5 min). The observed increase in plasma stability of the tested amide derivatives compared to bisacylhydrazone 14 supports our design hypothesis for the isosteric replacement of the bisacylhydrazone linking motif. In addition, all tested compounds showed very high mouse and human plasma protein binding (PPB) with lowest PPB of 97% for saturated pyridyl amide H2. In contrast, sulfonated L2 showed a remarkably low PPB in both species (mouse 14%, human 38%).

Table 1: Aqueous solubility of selected LecA inhibitors. Kinetic solubility determined in TBS/Ca 2+ buffer (pH 7.4) with 1% DMSO (N ≥ 3). *Thermodynamic solubility in TBS/Ca 2+ buffer (w/o DMSO, N = 1).

Table 2:

Plasma and metabolic stability in mouse and human liver microsomes (MLM, HLM) and plasma protein binding (PPB) of selected LecA inhibitors (N = 3).

All synthesized galactosides were then evaluated for LecA inhibition using a competitive binding assay based on fluorescence polarization (Figure S1). [24] Monovalent galactosides A1, A2, G2 and K2 showed similar IC50 values between 14 -19 µM. The monovalent acrylamide A1 (IC50 = 18.8 ± 6.6 µM) was equipotent to its saturated and more flexible propanamide analogue A2 (IC50 = 18.9 ± 5.5 µM). Replacement of the benzene ring with a pyridine in G2 (IC50 = 14.3 ± 7.2 µM) or addition of the sulfonate in K2 (IC50 = 14.4 ± 3.6 µM) had only minor effects on LecA inhibition. In contrast to the monovalent controls and similar to our previous observations for the bisacylhydrazones, titrations with all divalent LecA ligands exhibited very steep Hill slopes with fitted IC50 values between 3.2 -8.0 µM. These data are indicative for the high potency of divalent compounds and suggest that the lower assay limit was reached. [33] To overcome the assay limit for these highly potent inhibitors, direct binding to immobilized LecA was quantified using surface plasmon resonance (SPR, Figure 2, Figures S2-S4). In agreement with the competitive binding assay, the binding affinity determined by SPR for the monovalent acrylamide ligand A1 (Kd = 5.21 ± 0.60 µM) and propanamide ligand A2 (Kd = 5.38 ± 0.09 µM) were comparable to their IC50 values. Interestingly, a striking difference was observed among divalent inhibitors. Within the benzene series, the acrylamides B1-F1 showed Kd values in the nanomolar to micromolar range while their propanamide analogues B2-F2 were two-to threefold more active when attached to the shorter linkers B-D and 100-to 200-fold more potent for the longest linkers E and F. With respect to linker length, in the acrylamide series linker C containing one methylene unit exhibited strongest binding affinity (C1 Kd = 37.7 ± 11 nM,). Increasing to four methylene units in F1 led to a complete loss of divalent binding affinity boost (Kd = 2.25 ± 0.3 µM). In contrast, all divalent ligands carrying the propanamide motif (B2-F2) showed very high binding affinity to LecA between 15 and 23 nM. Kd values were oscillating with the number of methylene units present in the linker, presumably linked to the zig-zag geometry of the hydrocarbon chain. These observations could be explained by an increased rigidity of the acrylamides, that does not allow the ligand to simultaneously bind to two adjacent binding sites when the linker is too long. Monovalent ligands from the pyridine series (G2) and sulfonated K2 showed slightly enhanced binding affinity to LecA (G2 Kd = 2.97 ± 0.08 µM, K2 Kd = 3.78 ± 0.10 µM) when compared to A2. Substitution of the benzene ring with a pyridine was also favored for divalent ligands: three-and four-fold increase in binding affinity was observed for acrylamide-based ligand H1 (Kd = 13.7 ± 1.6 nM) and I1 (Kd = 40.2 nM) when compared to their benzene analogues D1 and E1, respectively. Among the propanamide derivatives, the increase in binding affinity was less pronounced: H2 (Kd = 9.9 ± 0.1 nM) and I2 (Kd = 30.8 ± 1.5 nM) were twice more active, while the longest pyridine ligand J2 (Kd = 25.1 ± 1.9 nM) was equipotent to its benzene analog F2. The observed affinity increase could result from additional interactions of the pyridine rings with the protein surface. Furthermore, divalent sulfonate L2 (Kd = 30.8 ± 1.5 nM) also reached low nanomolar affinity for LecA, indicating that an ether function in the linkers is not essential and that the negatively charged sulfonate solubility tags were tolerated. In general, divalent ligands containing propanamides (B2-F2, H2-J2 and L2) showed faster association kinetics (kon) compared to their acrylamide analogues (B1-F1, H2-I2), indicating the importance of a certain degree of flexibility to allow simultaneous binding to two LecA binding sites.

A2

Due to the high solubility of the propanamides in the pyridine series and the sulfonated ligand L2, we determined binding thermodynamics with LecA using isothermal microcalorimetry (ITC, Figure 3, Figures S5-S9). Kd values for monovalent G2 and K2 by ITC were in the low micromolar range (G2 Kd = 5.27 ± 0.03 µM, K2 Kd = 6.23 ± 0.44 µM), nearly twofold higher than those obtained by SPR. Divalent pyridine I2 (Kd = 35.1 ± 12.5 nM) and divalent sulfonated ligand L2 (Kd = 39.9 ± 3.6 nM) exhibited binding affinities in the low nanomolar range consistent with SPR. In both cases, the enthalpy of binding increased approximately twofold (I2: ∆H = -23.9 ± 1.2 kcal/mol, L2: ∆H = -19.5 ± 1.3 kcal/mol) compared to their monovalent analogues (G2: ∆H = -11.0 ± 0.2 kcal/mol, K2: ∆H = -10.3 ± 0.1 kcal/mol), while the binding entropy was increased approximately threefold (G2: -T∆S = 3.8 ± 0.2 kcal/mol vs. I2: -T∆S = 13.7 ± 1.3 kcal/mol, K2: -T∆S = 3.2 ± 0.2 kcal/mol vs. L2: -T∆S = 9.4 ± 1.3 kcal/mol). The divalent ligand with the longest linker, J2, was less potent, yet remaining in the nanomolar range (Kd = 79.5 ± 32.8 nM). However, this compound showed a decreased binding enthalpy (∆H = -13.5 ± 0.90 kcal/mol) but also lower entropy costs (-T∆S = 3.8 ± 0.5 kcal/mol), suggesting an alternative binding mode for J2. Next, we investigated the two most promising compounds H2 and L2 for their efficacy to inhibit LecA binding to H1299 human lung epithelial cells using flow cytometry and confocal microscopy. To this end, LecA was fluorescently labelled with Alexa Fluor 488 TM NHS ester (LecA-AF488), with varying concentrations of the inhibitors (100 nM to 10 µM) and subsequently added to the cells.

For flow cytometry analysis, inhibitors were preincubated with 0.16 µM LecA prior addition to the H1299 cells. After incubation for 30 min, fluorescence intensity was recorded at the flow cytometer. The sulfonated divalent inhibitor L2 decreased LecA binding to H1299 cells by more than 30% at 250 nM, whereas divalent pyridine H2 only showed 16% inhibition (Figure 4a,b). On the other hand, H2 was slightly more potent at the concentration range from 0.5 to 2.5 μM, consistent with its higher affinity measured by SPR. At 1 μM, H2 and L2 decreased LecA binding to the host cells by 50% and at 7.5 μM and above, a complete inhibition of lectin binding to the cells was achieved. In contrast, 10 µM of the corresponding monovalent ligands G2 and K2 were required for 50% inhibition (Figure S10). Thus, both divalent inhibitors, H2 and L2, showed high efficacy to prevent LecA binding to host cells and revealed a significant increase in potency compared to the monovalent inhibitors G2 and K2. When mean fluorescence intensity (MFI) of samples treated with LecA-AF488 and divalent inhibitors were normalized to the negative control and plotted as a function of inhibitor concentration (Figure S11), H2 and L2 demonstrated a dose-dependent inhibition of lectin binding to the host cells and determined IC50 values were 0.54 μM for H2 and 0.56 μM for L2 (Figure 4c). On the other hand, the IC50 values of the monovalent ligands were in micromolar range (G2 IC50 = 2.8 µM, K2 IC50 = 1.9 µM), thus further confirming the benefit of divalent ligands. The ability of H2 and L2 to diminish LecA binding to host receptors at the plasma membrane and its subsequent internalization into H1299 cells was further analyzed by confocal fluorescence microscopy. Here, the applied LecA concentration was increased to improve signal to noise and consequently image quality and inhibitors were preincubated with 0.5 µM LecA before addition to the cells. After 60 min at 37 °C, the cells were analyzed: in inhibitortreated cells, a remarkably low fluorescence signal for LecA was observed at 10 μM divalent ligands compared to those without inhibitor (Figure 4d). Sulfonated inhibitor L2 showed a stronger inhibition of binding than H2 from the pyridine series. A complete inhibition of LecA binding and cellular uptake in the presence of divalent ligands was achieved at 7.5 μM, corresponding to a 10-fold potency increase compared to the monovalent inhibitors G2 and K2, which required 75 μM to block LecA binding (Figure S11). Confocal microscopy images thus confirmed that the divalent inhibitors can prevent LecA binding to cell surface and reduce lectin uptake in a concentration-dependent manner. Overall, these data suggest that the divalent inhibitors H2 and L2 have a high potential to block LecA and prevent recognition of the glycosphingolipid Gb3 on host cells, possibly impairing the invasiveness of P. aeruginosa. The opportunistic bacterium P. aeruginosa depends on favorable circumstances to infect the host, such as a suppressed immunity or wounded skin. [34,35] Chronic infection of wounds with P. aeruginosa increases the risk of nosocomial spread, are difficult to treat and after systemic dissemination can result in a fatal outcome in patients. In a previous study, we investigated the effect of the fucose-binding P. aeruginosa lectin LecB on epithelial wound healing. [36] Cell migration was found to be strongly inhibited in the presence of LecB in a dosedependent manner, and could be fully blocked with 50 μg/mL (4.3 µM) LecB. Here, we investigated whether LecA also impairs the same physiological process. To this end, H1299 cell monolayers were grown, and after scratching with a pipette tip, wound closure was monitored microscopically (Figure 5a). Cells incubated with 3.9 µM LecA showed a visible decrease in cell migration, compared to full closure of the wound after 24 h in untreated cells. Furthermore, cell detachment in the close proximity of the wound was observed, suggesting that the lectin LecA can significantly hinder cell migration, adhesion and wound closure. The mode of action of LecA in the wound healing process remains elusive. However, in the presence of the monovalent inhibitor K2 (100 µM), wound healing was partially restored to about 50% after 24 h compared to samples treated solely with LecA (Figure S13). Importantly, when LecA was added to the cells together with L2 (10 µM and 100 µM, respectively), the wound healed at a similar rate to the control in absence of LecA. Remarkably, in presence of 10 µM L2, cell migration was restored to about 70% compared to the negative control, and the wound closed by 80% after 24 h of incubation with 100 µM L2 (Figure 5b). In conclusion, the detrimental effect of LecA in the wound-scratch assay could be reversed by addition of our inhibitors and wound healing was restored to a physiological closure rate. Thus, these data demonstrate that LecA binds to host cells to impair cell migration and, furthermore, unravel the potential of these inhibitors in restoring such a process, an important step towards the treatment and eradication of chronic infections.

P. aeruginosa is able to invade non-phagocytic cells via a lipid zipper mechanism. [START_REF] Eierhoff | Proc. Natl. Acad. Sci[END_REF] Binding of LecA to the glycosphingolipid Gb3 followed by LecA-induced receptor clustering plays a crucial role in this process. The LecA ligand 4-nitrophenyl α-D-galactopyranoside (PNPG) has been previously shown to interfere with these events at a high concentration of 10 mM leading to a reduction of bacterial invasiveness to 30-60% as compared to untreated bacteria (100% invasiveness). [START_REF] Eierhoff | Proc. Natl. Acad. Sci[END_REF]31] This assay relies on the addition of the antibiotic amikacin to kill all extracellular bacteria and allow subsequent quantification of the viable intracellular bacteria. To this end, H1299 cells were incubated with P. aeruginosa in presence and absence of LecA inhibitors, followed by amikacin treatment. Then, all intracellular bacteria were released by lysis of the human cells and viable bacteria were quantified by plating. To assess the impact of LecA inhibitors on the invasiveness, the influence of the divalent LecA inhibitor L2 on bacterial invasion into host cells was evaluated and compared to PNPG. Compared to untreated bacteria (100% invasion), invasiveness was slightly reduced to 89% in presence of 100 µM PNPG and to 44% with 10 mM PNPG, which is comparable to literature data. [START_REF] Eierhoff | Proc. Natl. Acad. Sci[END_REF]31] In contrast, the divalent LecA ligand L2 achieved an invasion decrease to 44% already at 100 µM (Figure 5c). L2 was thus able to reach a similar reduction of invasion at a 100-fold lower concentration than the one needed for PNPG.

Additionally, the cytotoxicity of sulfonated ligands K2 and L2 on H1299 cells was evaluated prior to the invasion assay (Figure S12). K2 and L2 did not exhibit significant toxicity during 24 h in ranges from 10 -100 µM and cellular viability was preserved. However, >50% inhibition of cell proliferation was observed for higher inhibitor concentrations (500 and 750 µM), indicating a potential toxicity window for the tested compounds. Encouraged by these promising in vitro results, an in vivo pharmacokinetic study was conducted for H2 and L2 in mice (1 mg/kg, i.v.) and in rats for L2 (10 mg/kg, i.v.). Compound concentrations were monitored in plasma and urine (Figure 6). H2 and L2 were cleared from mouse plasma within 1 h. Both compounds rapidly reached high concentrations in urine, several orders magnitude above their respective in vitro LecA binding affinity. In fact, concentrations above the KD's were detected over the entire observation period of 5 h. The overall lower urine exposure of L2 compared to H2 (𝑐 𝑚𝑎𝑥 𝑢𝑟𝑖𝑛𝑒 = 63.0 µM and 13.4 µM for H2 and L2, respectively) could result from first pass metabolism and lower hepatic stability of L2 in vitro (Table 2).

Interestingly, H2 showed higher renal elimination at 5 h than at 3 h, possibly a result of redistribution from another compartment. In rats, higher amounts of L2 were detected both in plasma and urine, not solely corresponding to the 10-fold increased dose. L2 showed a half-life of 37 ± 12 min, a clearance of 9.84 ± 5.0 mL/kg/min and a mean residence time 41 ± 18 min in rat plasma in vivo, exceeding the concentration of its on-target affinity for at least 6 h. As a result, the overall drug exposure in rat plasma was higher than in mice with an area under the curve (AUC) of 19.7 ± 12.8 µM*h. In agreement with its strong hydrophilicity, L2 showed a rather low distribution volume (VD = 0.46 L/Kg). Importantly, high levels of L2 were present in rat urine even 24 h after administration.

Conclusion

We previously reported highly potent LecA inhibitors which unfortunately suffered from very low aqueous solubility and limited stability, both precluding thermodynamic and biological analysis of these bisacylhydrazones. [33] Here, we replaced this disadvantageous linker motif using bioisosters, systematic linker variation and solubilization tags. We now succeeded to increase solubility over 5000-fold for the sulfonated ligand L2 and also increased stability while maintaining the nanomolar on target activity for LecA. The pyridine-based ligand H2 showed a further increased affinity of 9.9 nM by SPR. It is worth to note that low nanomolar binding affinities associated with a strong divalent potency boost were achieved for all synthesized compounds, with a single exception of the longest acrylamide-based ligand F1. The strongly increased solubility finally allowed to analyze the thermodynamic binding profile of selected ligands with LecA by microcalorimetry and complement the binding kinetics by SPR.

Importantly, we could now analyze divalent ligands H2 and L2 and their monovalent analogs G2 and K2 in cellular assays using flow cytometry and confocal microscopy to determine their efficacy to block LecA binding to host cell receptors. Binding of LecA to H1299 cells was remarkably decreased in presence of 10 µM H2 and L2. Moreover, we demonstrated for the first time that LecA impairs cell migration of human epithelial cells, similarly to the fucose-binding lectin LecB. [36] Divalent sulfonate L2 restored the migration of injured epithelial cells to a physiological rate in a scratch-wound assay. Finally, we also analyzed the effect of L2 on the invasion of live bacteria into human host cells, since P. aeruginosa can invade non-phagocytic cells. In these infection experiments, we found that L2 efficiently decreased LecA-mediated bacterial invasiveness, significantly better than galactoside PNPG.

Taken together, the novel divalent LecA inhibitor L2 is a promising lead molecule to combat P. aeruginosa virulence, such as host cell binding, bacterial invasion and the detrimental effects of LecA on wound healing. Thus, the inhibition of LecA-mediated virulence provides an alternative treatment option for the highly problematic infections with P. aeruginosa. The PK study revealed renal excretion and long-lasting presence of H2 and L2 in urine, opening further treatment options of urinary tract infections.

Figure 1 :

 1 Figure 1: Divalent precision LecA ligands: (a) parent bisacylhydrazone LecA inhibitors[33] (top) and new generation optimized bioisosters (bottom). Proposed chemical modifications are highlighted: amide linkage as acylhydrazone bioisoster in red and linker derivatizations in blue. (b) Galactoside building blocks with terminal α,β-unsaturated carboxylate 1 and its saturated analogue 2. (c) Linker moieties: anilines B-F, aminopyridines H-

Scheme 3 :

 3 Scheme 3: Synthesis of divalent LecA ligands and their monovalent analogues as controls. Reagents and conditions: (i) for A and G: HBTU, DIPEA, DMF, r.t., 1 hovernight, for K: PyBOP, N-methylmorpholine, DMF, r.t., overnight; (ii) galactoside 1 or 2, HBTU, DIPEA, DMF, r.t., 2 h -2 d, (iii) galactoside 2, PyBOP, N-methylmorpholine, DMF, r.t., overnight.

Figure 2 :

 2 Figure 2: Direct binding of LecA ligands determined by SPR (N = 3). Sensorgram of monovalent A2 (left) with affinity analysis (center) and sensorgram of divalent B2 (right) from single-cycle kinetics experiments (injections of 0, 10, 50, 100, 200 nM) are shown. Relative potencies (r.p.) were calculated compared to respective monovalent compound in each series and are valency-normalized. * N = 1 due to sample aggregation.

Figure 3 :

 3 Figure 3: Direct binding of selected ligands to LecA by ITC (N = 3). Titration of LecA (50 µM) with divalent sulfonated ligand L2 (250 µM) is depicted. Relative potencies (r.p.) were calculated compared to respective monovalent compound in each series and are valency-normalized.

Figure 4 :

 4 Figure 4: Divalent inhibitors H2 and L2 decreased LecA binding and uptake to lung cells (N = 3). Histograms of fluorescence intensity of gated live H1299 cells incubated with 0.16 µM of LecA-AF488 in presence of (a) H2 or (b) L2. H1299 cells (without LecA, H2 or L2) served as a negative control (grey). (c) IC50 values for inhibition of LecA binding to H1299 cells of monovalent inhibitors G2 and K2, divalent inhibitors H2 and L2 following titrations in flow cytometry assays. (d) Confocal imaging of H1299 cells incubated with 0.5 µM LecA-AF488 (in green) or 0.5 µM LecA-AF488 which was preincubated with 10 µM H2 or L2. Nuclei were counterstained with DAPI (blue). Scale bars = 10 μm.

Figure 5 :

 5 Figure 5: Divalent inhibitor L2 restored wound healing in LecA-treated cells and reduced P. aeruginosa PAO1 invasiveness into host cells. Wound healing (N = 3): (a) Light microscopy images of scratched H1299 cells at 0 h and 24 h post-treatment with (1) PBS, (2) 3.9 µM LecA, (3) 3.9 µM LecA preincubated with 10 µM L2, or (4) 3.9 µM LecA preincubated with 100 µM L2. Scale bars = 100 µm. (b) Quantification of wound closure after 24 h, control in absence of LecA. Bacterial Invasion (N ≥ 3): (c) Incubation of P. aeruginosa PAO1 with 100 µM PNPG, 10 mM PNPG or 100 µM L2 for 30 min reduced bacterial invasiveness into H1299 cells in comparison to the absence of inhibitors (positive control). For each experiment, conditions were normalized to invasion of untreated bacteria (positive control).

Figure 6 :

 6 Figure 6: In vivo pharmacokinetics of H2 and L2. Plasma and urine concentrations in (a) CD-1 mice (N = 2 per compound) after 1 mg/kg i.v. dose of H2 (1.15 µmol/kg) and L2 (0.97 µmol/kg) and in (b) Sprague-Dawley rat (N = 3) after 10 mg/kg i.v. dose of L2 (9.73 µmol/kg). Dashed lines represent the in vitro K d values.

Scheme 1: Synthesis of galactoside building blocks 1 and 2. Reagents and

	conditions: (i) benzyl
	p-coumarate/methyl 3-(4-hydroxyphenyl)propanoate, BF 3 . Et 2 O, CHCl 3 for 4 and CH 2 Cl 2 for 5, 0 °C -r.t.,
	overnight; (ii) NaOH, H 2 O/MeOH (1:1), 50 °C for 1 and r.t. for 2, 1 -2 h.
	Scheme 2: Synthesis of benzene, pyridine and phenylsulfonate linkers. Reagents and conditions: (i) 4-nitrophenol,
	K 2 CO 3 , DMF, 70 °C, microwave, 11 h -4 d (for C 10 d, no irradiation); (ii) H 2 , Pd/C, CH 2 Cl 2 /MeOH, r.t., 3h -
	o.n.; (iii) 2-chloro-5-nitropyridine, NaH, r.t., DMF, 1 h -2 d (for H K 2 CO 3 , 65 °C, DMF, 5 d); (iv) Raney Ni, H 2 ,
	r.t., H 2 O, 6 d.

Acknowledgments

The authors are grateful to J. Konstantinovic (HIPS, Saarbrücken) for assisting with HRMS. K.R. thanks J. Wolf and K. V. Sander for excellent technical assistance. K.R. receives support from the German Center for Infection Research (DZIF, TTU 09.710 and 09.719). A.T. acknowledges the European Research Council (ERC Starting Grant, Sweetbullets) and DZIF (TTU 09.718). A.I. thanks the French National Research Agency [ANR-17-CE11-0048] project 'Glycomime'for support and further acknowledges Glyco@Alps (ANR-15-IDEX-0002) and Labex Arcane/CBH-EUR-GS (ANR-17-EURE-0003). This research was also funded by the EU Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement synBIOcarb (No. 814029). This publication is partially based upon work from COST Action CA18103 (INNOGLY), supported by COST (European Coorperation in Science and Technology). W.R. acknowledges support by the Deutsche Forschungsgemeinschaft (DFG) under Germany's Excellence Strategy (EXC-294 and EXC-2189) and the DFG grant Major Research Instrumentation (project number: 438033605), by the Ministry for Science, Research and Arts of the State of Baden-Württemberg (Az: 33-7532.20) and by the Freiburg Institute for Advanced Studies.