The determination of very low doping levels in solid materials is an important issue for many applications. When considering paramagnetic dopants, NMR relaxation technique appears to be much more accurate than classical techniques such as the Vegard's law resulting from X-ray diffraction (XRD) measurement or chemical analysis that cannot provide information of appropriate dopant spatial distribution. In a recent report, the linear variation of 1/T1, i.e. the nuclear relaxation rate, as a function of Nd 3+ content has been used to determine doping levels with a good dispersion homogeneity in the monazite LaPO4 matrix down to 0.1 mol. %. We here extend this study to more complex compounds doped with Nd 3+ , such as YPO4, the solid solution Y0.8Sc0.2PO4, Ba5(PO4)3Cl and a phosphate glass. For all considered compounds except Ba5(PO4)3Cl:Nd, 1/T1 is found to be linearly proportional to the nominal Nd concentration, confirming the ability of the method to investigate dopant concentration and spatial homogeneity. The results on different compounds open the discussion on the parameters influencing the nuclear relaxation rate, among which the orbital overlap and the average P-P distances.

Introduction

In many cases, the interesting physical properties of inorganic compounds are not intrinsic to the materials themselves but result from the addition of extra ions, called doping ions, allowing to control electrical, optical or magnetic properties. Trivalent rare-earth (RE) ions are widely used as doping ions in order to elaborate optically-active materials with various technological applications (lasing [START_REF]Rare-Earth Doped III-Nitrides for Optoelectronic and Spintronic Applications[END_REF] , data memory 2 , phosphors etc…). These interesting optical properties are conferred by the 4f orbitals of RE, presenting the particularity to be shielded from the environment by the outer 5s and 5p orbitals. In term of material synthesis, a key challenge is to obtain well-dispersed RE ions within their host matrix. Indeed, the formation of RE pairs, small aggregates or clusters usually decreases optical performances through RE-RE interactions. In optics, a well-known phenomenon is the concentration quenching for which, above a given RE concentration, the RE luminescence decreases as energy transfers between RE ions are likely to favor the RE de-excitation in a non-radiative manner (through defects, surface traps, etc), at the expense of the radiative de-excitation. 3,[START_REF] Blasse | Luminescent Materials[END_REF] In this context and for the mentioned applications, the doping concentration remains low, usually below 10 mol. %. [START_REF] Lyberis | [END_REF]6 Between the nominal and the real effective RE doping concentration, there can be a nonnegligible discrepancy due to dopant segregation during the synthesis (often observed in singlecrystals grown by Czochralski method 7 ) or poor incorporation due to size or charge mismatch between the RE and the substituted ion. 8 Heterogeneity of the RE distribution in the matrix can also result from unappropriate synthesis conditions, especially in the case of a bad mixing of the precursors or too low calcination temperatures (observed in ceramics prepared by solidstate reaction) or of a non-homogeneous melt in the case of glasses. It is thus very important to analyze the obtained compound in order to determine with accuracy its doping concentration and the good dispersion of the elements.

There are not many techniques to evaluate exactly a doping concentration, especially if this concentration is of the order of a few mol.%. The commonly-used method is based on the position of Bragg diffraction peaks. Indeed, according to Vegard's law, the homogeneous incorporation of doping ions inside a matrix, corresponding the formation of a solid solution induces a shift of the X-ray diffraction peaks with respect to pure phases. This shift varies linearly with the concentration of substituting elements. The limitation of Vegard's law lies in the fact that the shift of the diffraction peaks is only visible when the insertion of the substituent is of a few mol.%. 9 In addition, this technique is only available for crystalline materials as it is based on the position of Bragg diffraction peaks. The crystallinity is also a restrictive factor for the use of the Electron Spin Resonance (ESR) for doping concentration determination. Indeed, ESR can be used to quantify the number of paramagnetic doping ions through the double integration of the ESR signal, but only if the samples are single-crystals. [START_REF] Abragam | Electron Paramagnetic Resonance of Transitions Ions[END_REF][START_REF] Guillot-Noël | [END_REF] For other types of samples (amorphous or polycrystalline), the broadening of the ESR signal hides some characteristic features, such as ion pair signatures, and prevent any accurate quantitative measurements. 12 Other techniques, such as Energy Dispersive X-ray analysis or Atomic Absorption spectrometry, turn out to be inappropriate for the detection of elements present at low concentration (typically below 1 mol.%) and are inadequate to provide information on element spatial dispersions. Finally, for optically-active materials, optical absorption spectroscopy can be used; but, for the absolute measurement of the doping concentration, the knowledge of the absorption cross-section is mandatory. 7 Some other techniques could be used for determining very low doping concentrations (from 100's of ppm to sub-ppm) such as SIMS, electron microbeam with WDX and nanoSIMS but with a spatial resolution of 10 to 1 m.

In this context, we focus on an original method based on Solid-State Nuclear Magnetic Resonance (SSNMR) relaxation to determine the doping concentration in some solid state materials, whether they are crystalline or amorphous. In a previous paper, we studied the LaPO4 crystalline compound doped by two different RE ions, Gd 3+ and Nd 3+ , and measured their 31 P NMR relaxation time T1 in static and MAS conditions. 13 The key result of this study is the linear variation of 1/T1 as a function of Nd 3+ (or Gd 3+ ) concentration in the 0 -10 mol.% range, allowing the determination of low doping concentration (< 0.1 mol.%) with a high accuracy (error < 5%). This result was used to determine the homogeneity of the doping ions with the LaPO4 matrix, similarly to the results obtained by Levin et . [14][15][16][17] This method is highly powerful as long as the doping ions are paramagnetic and embedded within a matrix containing an element that can be analyzed easily by NMR ( 31 P in our case).

In the present paper, we extend our work to other 31 P-containing compounds doped with Nd 3+ ions and study the influence of different structural factors on the T1 variation. In addition to LaPO4, YPO4, the solid solution Y0.8Sc0.2PO4, the chloroalforsite Ba5(PO4)3Cl and a phosphate glass are studied with Nd 3+ concentration varying between 0 and 5.6 mol.%. These compounds were chosen as they do not contain any paramagnetic element, so that only the Nd 3+ -doping will affect the relaxation time T1. Moreover they present different crystallographic structures (summarized in Table 1) that will help us to understand how the distances and the orbital overlap influence T1 with the Nd concentration variation. Our overall goal is to determine whether it is possible to predict the Nd concentration in an arbitrary P-containing compound. This paper is divided into two parts: in a first part, we present the synthesis and thorough characterizations of the structure of the different compounds; in a second part we present the results concerning their relaxation time T1 as a function of the Nd 3+ doping concentration and discuss them in the light of the material structure. These glasses are obtained by mixing NH4H2PO4, MgO, Na2CO3 and Nd2O3 in a planetary mill.

The powders are heated in a quartz crucible according to the following program: 2 hours at 250°C, 30 min at 800°C and 1 hour at 950°C, with slow heating ramps (around 1°C/min). These different steps allow the progressive calcination of the reagents and the slow departure of CO2 and NH3. After the last step, the melted glass is quenched at room temperature.

Structural characterization

Powder X-ray diffraction diagrams of the samples are systematically recorded using an X'Pert Philips diffractometer (40 kV, 40 mA) with CuK radiation ( = 1.54056 Å). The unit cell parameters are obtained by fitting the diagrams with Fullprof software. 22,23 In crystals, distances between two phosphorus atoms are determined by Vesta software. 24 The concentration of neodymium ions in the glass samples are determined by ICP-AES at Crealins (Lyon, France).

UV/Vis Absorbance

To control the homogeneity of the incorporation of the dopant into phosphate glasses, different UV/Vis spectra of a same sample are made on a Cary 50 Scan spectrophotometer from 200 to 1100 nm.

Results and discussion

To understand the mechanism governing the 31 P relaxation in a paramagnetic environment, we synthesized different compounds and compared their relaxation rates with respect to the LaPO4 model system that we first investigated. 13 First, yttrium phosphate, whose structure (xenotime) is different from LaPO4 (monazite) but which presents similar unit cell parameter, was directly compared to LaPO4 to determine the influence of the crystalline structure, and thus the orbital overlap, onto the 31 P relaxation rate. Second, the relaxation rates of two isomorphs (YPO4 and Y0.8Sc0.2PO4) were compared to evaluate the influence of the unit cell parameters and consequently of the P-P interatomic distances. This study was extended to two other compounds: the alforsite, presenting a different crystalline structure and even more distinct P-P distances and a phosphate glass, presenting an amorphous structure and a larger P-P distance distribution.

Structural characterization

Nd-doped LaPO4

A characteristic X-ray Powder Diffraction of La1-xNdxPO4 is given in Figure 1a. It corresponds to the monazite structure. 25 The unit cell parameter, calculated by Fullprof, 22,23 decreases with the incorporation of Neodymium ion but its variation is scattered as we saw it in our previous paper (Figure 1b). 13 The SSNMR of LaPO4:Nd has been previously described 13,26 and is here reported in SI The doping concentration of our samples calculated from SS-NMR spectra, using the following law , x:

(
Ik = m * x k * (1-x) n-k (Eq. 1)
where Ik is the intensity of paramagnetic peak at +16 ppm m is the number of configurations that can give rise to a specific peak, k is the number of Nd 3+ interacting with the 31 P nucleus for the specific peak, n is the total number of sites into which Nd 3+ can enter and cause a specific paramagnetically shifted peak (equal to 7 here for the 7 distinct first-shell 31 P -Nd 3+ configurations), and x is the probability of Nd 3+ occupying an RE-O9 site and is equal to the concentration of Nd 3+ (out of total Nd + La). 27,28 As shown on 25 The different Y1-xNdxPO4 compounds, for 0 ≤ x ≤ 0.2, crystallize in the same tetragonal crystalline structure. 29 The typical unit cell parameter a is determined in the whole range of doping concentration x (Figure 2b). Its variation is scattered again, as seen in Maron et al. for LaPO4 13 , evidencing the resolution limit of diffraction in the determination of doping concentration.

Above x = 8 mol. %, the unit cell parameters appears constant, showing a limit of solubility. The SSNMR spectrum of Y0.9975Nd0.0025PO4 is reported on Figure 3 with a long repetition delay. A single peak at -9.8 ppm is observed, equivalent to Palke and Stebbins at -12 ppm or Briche et al. at around -10.5 ppm. 26,30 No paramagnetically shifted peaks, corresponding to 31 P interacting with paramagnetic Nd 3+ ions in the vicinity, are observed. In order to observe such peaks, very short repetition delay should be used 13,26,31 The spectrum of Y0.925Nd0.075PO4 evidences a peak at the same chemical shift, but its Full Width at High Maximum (FWHM) appears much broader due to the strong anisotropy of the chemical shift.

- As in LaPO4, paramagnetically shifted peaks can be observed in YPO4 and are labelled A to C, in reference to the LaPO4 spectrum (Figure SI-4). 26,27 The evolution of the area and the FWHM as a function of Nd doping concentration in YPO4 is presented in SI (Figure SI-5 a and b). The area of a paramagnetic peak (δ = +36 ppm for YPO4) varies linearly, as in LaPO4, indicating that Nd is well-incorporated inside YPO4.

As the doping concentration of Nd increases, the probability of having P with two Nd ions in its vicinity increases. One should then observe paramagnetic peaks that are even more shifted: for instance, in YPO4, the peak at -72 ppm should be observed. However, in our case, our interest lies in low Nd doping concentration. So, for further characterizations, and especially relaxation measurements, only the region between +60 to -40 ppm will be considered.

As for LaPO4, the doping level of our samples is checked by the application of the The Ba5(PO4)3Cl alforsite belongs to the apatite family. It crystallizes in the hexagonal system (P63/m) with typical unit cell parameters of 10.2840 Å for a and 7.6510 Å for c, i.e. very different than the ones of LaPO4. Ba5(1-x)Nd5x(PO4)3ClO5x/2 was doped with different Nd concentrations: x = 0, 0.01 and 0.04. These compounds are called CAl-U, CAl-LD and CAl-HD, respectively. Our XRD results exhibit the formation of a pure alforsite phase for all doping concentrations (Figure 6). No impurity phase is detected. The three doping composition of alforsite do not respect the Vegard's law, but at this step we are unable to know if it arise from uncertainty of measurements or a weakness in Nd incorporation (Figure 6b).

Figure 7 presents two solid-state NMR spectra of chloroalforsite (undoped and with 4% of neodymium ion). On the undoped spectra, the first peak at around 0.6 ppm corresponds to Ba5(PO4)3Cl, whereas the minor peak at around 1.6 ppm is attributed to -tribaryum phosphate. 33 The spectrum of CAl-HD exhibits a broader peak, centered at 2.5 ppm. The increase of the FWHM, as well as the chemical shift, indicates a change in the P environment.

-10 -5 0 5 10

 (ppm/H 3 PO 4 85 %)
CAl-0 CAl-HD NMR spectroscopy exhibits a broad and intense peak at around -22 ppm (Figure 8), attributed to 31 P in Q 2 sites. 34 A second peak at around -9 ppm representing less than 10 % is attributed to the presence of Q 1 sites, probably resulting to some impurities in our precursors. Due to the broad peak at -22 ppm, we could not see paramagnetic peak even with a repetition time of 50 ms (Figure SI-7).

- Absorption spectra were recorded for the different Nd concentrations, evidencing the different optical transitions of Nd 3+ (Figure 9a). %, as observed by elementary analysis, and the linear variation of the absorption coefficient in the whole doping range, one can state that Nd 3+ ions are introduced in the expected proportion into the different glasses for concentrations ranging from 0 to 5.6 mol. %. Measurements were repeated on different points of the samples to confirm their spatial homogeneity.

Relaxation measurements

Relaxation mechanism

The relaxation rate of 31 P atoms is plotted on Figure 10 as a function of the Nd 3+ concentration for the different compounds: LaPO4 crystal, YPO4 crystal, the Y0.8Sc0.2PO4 compound, Ba5(PO4)3Cl crystal and the Na2O:MgO:P2O5 glass. The more paramagnetic Nd 3+ ions in the matrix, the faster the relaxation of 31 P atoms. In all compounds except the alforsite, the variation of 31 P relaxation rate is significant and linear with the Nd content, evidencing a homogeneous distribution of the doping ions within the various matrices. 13,14,36 Note that the x-axis, corresponding to the Nd concentration C has been plotted in mol/cm 3 in order to take into account the density of the different compounds and thus to compare them. There are two main channels of desexcitation for a nucleus in the vicinity of a paramagnetic impurity: the direct relaxation and the relaxation via spin diffusion. [START_REF] Abragam | The Principles of Nuclear Magnetism[END_REF] In dilute materials, the spin diffusion mechanism is predominant; whereas, in highly-doped systems, the direct relaxation should be considered. The latter corresponds to a dipolar coupling between the 31 P (I = 1/2, with a Larmor frequency I) and a paramagnetic ion characterized by a momentum J, at a Larmor frequeny J and situated at a distance d from the Phosphor. The nucleus relaxation time T1 can be written as: [START_REF] Abragam | The Principles of Nuclear Magnetism[END_REF] 1

𝑇 1 = ( 𝜇 0 4𝜋 ) 2 1 15 𝐽(𝐽 + 1) ℏ 2 𝛾 I 2 𝛾 J 2 𝑑 6 [ 6𝜏 1 1 + 𝜔 I 2 𝜏 1 2 + 14𝜏 2 1 + 𝜔 J 2 𝜏 2 2 ], (Eq.2)
where I (resp. J) is the gyromagnetic ratio of Phosphor (resp. the electron), and 1 (resp. 2) is the electronic spin-lattice (resp. spin-spin) relaxation time.

The second regime is the relaxation through spin diffusion. It can be separated into two regimes:

one, predominant at low temperature, is known as the diffusion limited regime; and the other, occurring at higher temperature, is called the fast spin diffusion (FSD) regime. In the latter case, the relaxation time 𝑇 1 FSD can be written as:

1 𝑇 1 FSD = 𝐴 𝐶𝜏 1 1 + 𝜔 I 2 𝜏 1 2 (Eq.3)
with x the molar fraction of paramagnetic ions, and where A is

𝐴 = 8 15 𝜋 ( 𝜇 0 4𝜋 ) 2 𝑁ℏ 2 𝛾 I 2 𝛾 J 2 𝐽(𝐽 + 1) 𝑉𝑏 2 3 (Eq.4)
and only depends on the parameter b2 , described in Goudemond et al. [START_REF] Goudemond | [END_REF] and which corresponds to the distance below which the nuclear spin diffusion is ineffective. At room temperature τ1(Nd 3+ ) < 1 ns, one can assume 𝜔 I 2 𝜏 1 2 << 1 13 , so:

1 𝑇 1 FSD = 𝐴𝐶𝜏 1 (Eq.5)
As the experimental 1/T1 variation is linear with the Nd molar concentration (Figure 10), it can be determined that the relaxation mechanism is a fast spin diffusion regime, as already found in Maron et al. 13 Taking into account the slopes of the linear fits shown on Figure 10, as well as Eq.3 and Eq.4

and the structural characteristics indicated on Table 1, it is possible to calculate the electronic signature should be looked for at low temperature (usually below 40 K). [START_REF] Guillot-Noël | [END_REF]13 The value obtained for LaPO4:Nd is identical to the one calculated at low concentration and reported in Maron et al. 13 The question that we now want to address is the one of the factor governing the slope of the line i.e. the A coefficient, and how it could be correlated with the material structure.

Influence of the crystal structure

To determine whether the crystalline structure, i.e. the Nd-P distances and the orbital overlap, has an influence of the relaxation rates, one can first compare the slopes of LaPO4 and YPO4 compounds in Regarding the variation of the relaxation rate as a function of the Nd concentration, it seems that this parameter has an impact on the 31 P nuclear relaxation. Indeed the relaxation rate is faster for LaPO4:Nd than for YPO4:Nd.

Regarding the direct influence of the Nd-P distances, the comparison between YPO4 and the solid solution Y0.8Sc0.2PO4 is more relevant. Indeed, YPO4 and Y0.8Sc0.2PO4 crystallize in the same crystallographic group but they present an absolute variation of 2% of the unit cell parameters. In YPO4, there are two types of Nd-P distances: 3.014 Å (2 distances) and 3.765 Å (4 distances) for an average distance of 3.515 Å, as calculated with Gretep software. [START_REF] Laugier | LMGP-Suite Suite of Programs for the interpretation of X-ray Experiments[END_REF] In Y0.8Sc0.2PO4, these distances are 2.985 Å and 3.720 Å, giving an average distance of 3.475 Å.

The relaxation rates of YPO4 andY0.8Sc0.2PO4 are similar for the same Nd concentration. A Nd-P distance variation of around 1% does not influence the relaxation mechanism but it is true that this variation is very small.

To analyze the variations shown in Figure 10, one could also have considered and compared LaPO4 with Ba5(PO4)3Cl as their crystalline structure and their average Nd-P distance are different (average Nd-P distance in alforsite = 3.71 Å, considering that the Nd substitutes the Ba 2+ ions). However, the constant value of 1/T1 with the Nd concentration (in the 0 -10 - 4 mol/cm 3 range) tends to prove that the Nd 3+ ions are not incorporated into the alforsite structure.

Influence of the P-P distances

In Eq. 4, b2 is defined as the distance below the shift of the nuclear resonance frequency due to their dipolar coupling with the electronic magnetic moment makes the nuclear spin diffusion ineffective. A mathematical expression is given by Goudemond, [START_REF] Goudemond | [END_REF] and b2 is only function of the P-P distance dP-P. As 𝑏 2 ∝ (𝑑 P-P ) 1/4 , 1/T1 can be written as:

1 𝑇 1 ∝ 𝐶𝜏 1 1 (𝑑 P-P ) 3/4 (Eq.6)

The different values of dP-P, calculated for LaPO4 and YPO4 crystals with Vesta 24 when considering the 6 P in the first coordination sphere, are reported in Table 3 for each compound.

General description of the variation of 1/T1

Our different measurements show two main parameters involved in the nuclear relaxation of 31 P in the presence of Nd 3+ ions in their vicinity:

(1) the molar concentration of Nd, C, as shown on Figure 10;

(2) the P-P distances, expressed by the law:

1 𝑇 1 = 𝛼 (𝑑 P-P ) 3/4 + 𝛽 (Eq.7)

established for a given Nd concentration (Figure 11).

With the aim of determining a universal law allowing the calculation of T1 for an unknown material and Nd concentration, these two laws can be gathered and we thus propose to consider the variation of 1/T1 as:

1 𝑇 1 = 𝛿 𝐶 (𝑑 P-P ) 3/4 + 𝜀𝐶 (Eq.8)

where  and  are two parameters that can be determined using the fits obtained on Figure 11 for the different concentrations. We find a remarkable linear dependence for our three compounds of interest, with characteristic P-P distances that are quite different, and although they exhibit a very different structure when comparing crystals and glass. This clearly shows that the two parameters and  do not depend on the material and are thus intrinsic to the dopant element (Neodymium here) and to the Phosphor atom and their respective interactions independently from their arrangement at the microscopic scale.

Hence, a result of this study is to propose an empiric law that links three different parameters of Nd 3+ doped phosphates: the concentration C (in mol/cm 3 ) of paramagnetic ions, the nuclear relaxation time T1 (in s) and the Phosphor-Phosphor distance dP-P, in Å, characteristic of each material:

1 𝑇 1 𝐶 = 5077 (𝑑 P-P ) 3/4 -1173 (Eq.9)

Conclusion

In this study different 31 P-based compounds were considered in order to evaluate the possibility to measure low paramagnetic Nd 3+ doping concentration using a technique based on solid-state NMR relaxation. We show that the relaxation rate 1/T1 varies linearly with the Nd 3+ concentration for a given compound and that, for a given Nd concentration, it varies linearly with 1/dP-P 3/4 , where dP-P is the average distance between 31 P closest neighbors. By considering these two variations, we further derived a simple empiric law describing the variation of 1/T1 as a function of C and dP-P. It would now be interesting to check for the validity of this law for any other phosphate compounds expecting to show some universal behavior, and to get a theoretical description of the physical phenomena driving the values of the two fitted constant  and . Moreover, this kind of approach could probably be extended to any other material hosting paramagnetic ions inside an NMR sensitive matrix (fluorides, silicates…).

21

 21

1. 2

 2 Characterizations 1.2.1 NMR Magic-angle spinning (MAS) Solid-State NMR (SSNMR) experiments is performed using a Tecmag Apollo360 spectrometer and a 4 mm Bruker probe spinning at 15 kHz. The 31 P Larmor frequency (SF) is 145 MHz. 85% H3PO4 is used as external reference for chemical shift.Fully relaxed conditions (repetition time equal to 5*T1) are employed for all spectra, except mentioned. The spin-lattice relaxation time T1 is measured by using a saturation-recovery method, using a sequence of 24 /2 pulses (equal to 2.1 s) spaced by 100 ms. In this sequence, the repetition time is equal to 1 s. FIDs are processed using NTNMR (Tecmag) software, applying a line broadening of 50 Hz. The whole spectrum, including spinning sidebands, is considered for the T1 determination. A stretched exponential, with a coefficient  = 0.83, is used for adjusting the relaxation recovery curves. As explained in our previous paper, since the best fitting  values are close to each other for samples with different Nd concentrations, we decided to use a common  = 0.83 to determine T1 in order to make the comparison of the results for the different samples more consistent.

Figure 1 :

 1 Figure 1: a) X-ray Powder Diffraction diagram of LaPO4 and as a reference the positions of the diffraction peaks of LaPO4 (JCPDF n°01-083-0651) and (b) variation of the unit cell parameter a in La1-xNdxPO4 as a function of the Nd 3+ concentration x.

Figure SI- 1 )

 1 . Briefly, with a short repetition time, paramagnetic peaks, associated to P in a vicinity of one Nd ions, can be observed between +40 and -40 ppm. The evolution of the FWHM is shown as a function of the Nd concentration (Figure SI-2a). The FWHM of a paramagnetic peak increases linearly with Nd concentration, evidencing an increase of the disorder around P. The area of a paramagnetic peak (at  = +16 ppm for LaPO4) also varies linearly with the increase of Nd doping, evidencing a good incorporation of Nd in the crystal structure (Figure SI-2b).

Figure SI- 3 ,

 3 Figure2ashows a X-ray Powder Diffraction diagram of Y0.999Nd0.001PO4. It is characteristic of the tetragonal xenotime structure with the I41/amd space group.25 The different

Figure 2 :

 2 Figure 2: a) X-ray Powder Diffraction of Y0.999Nd0.001PO4 and as a reference the positions of the diffraction peaks of YPO4 (JCPDF n°01-084-0335) and (b) variation of the unit cell parameter a in Y1-xNdxPO4 as a function of the Nd 3+ concentration x.

Figure 3 :

 3 Figure 3: Solid-State NMR spectra at 15 kHz of Y0.9975Nd0.0025PO4 (blue) and Y0.925Nd0.075PO4 (light blue).

6 ,Figure 4 :On Figure 5 ,Figure 5 :

 6455 Figure 4: X-ray Powder Diffraction diagram of Y0.8Sc0.2PO4 (cyan, noted YScPO4), ScPO4 (green) and YPO4 (blue). Whole diagram (a) and zoom on the (2,0,0) peak (b). On a), the star corresponds to an amorphous unidentified phase.On Figure5, the NMR spectrum of ScPO4 presents two peaks: the first one at around -12 ppm attributed to our product and another one, much broader, at around -40 ppm which confirms the presence of an amorphous phase that we have not been able to remove playing on stoichiometry and calcination temperature. The solid-state NMR spectrum of Y0.8Sc0.2PO4 exhibits two peaks: the first at -9.8 ppm corresponding to the chemical shift of31 P in the vicinity of Y atoms, as found in YPO4, and the second peak at -11.6 ppm corresponding to the chemical shift of31 P in the vicinity of Sc atoms, as found in ScPO4. The deconvolution processed by DMFit 32 confirms the good relative proportion between Yttrium and Scandium (not shown).The interpretation of such an observation of two separate contributions corresponding to the two phases (YPO4 and ScPO4 respectively) plays in favor of a phase segregation. Nevertheless, this is contradictory with the result from XRD characterization that follows the Vegard's law and thus tends to demonstrate the solid solution. An interpretation could be that some segregation of the Sc ions occurs leading to P(Sc)6 units homogeneously dispersed in the host YPO4 phase. Nevertheless, confirmation of this hypothesis goes beyond the scope of this paper, and the Y0.8Sc0.2PO4 compound should thus be considered with precautions in the further discussion.

Figure 6 :

 6 Figure 6: a): X-ray Powder Diffraction diagram of the HD sample and JCPDF file 01-070-2318 of alforsite. b): Variation of the unit cell parameters a = b as a function of the nominal Neodymium concentration in Ba5(PO4)3Cl.

Figure 7 :

 7 Figure 7: SSNMR (r = 15 kHz) of two alforsite compounds: CAl-U (purple) and CAl-HD (light purple).

2. 1 . 5

 15 Influence of structure: Phosphate glasses A series of metaphosphate glasses (characterized by a theoretical Q 2 proportion of 100 %) were synthesized according to Pukhaya et al. 21 X-ray Powder Diffraction confirms the amorphous nature of the different compounds, by the absence of Bragg peaks (not shown).

Figure 9 :

 9 Figure 9: a): Absorbance of a phosphate glass doped by 4.2 mol. % of Neodymium. The main absorption peaks at 590 nm, 750 nm and 800 nm correspond to the 4 I9/2→( 2 G5/2, 2 G7/2), 4 I9/2→ ( 4 F7/2, 4 S3/2) and 4 I9/2→( 4 F5/2, 2 H9/2) transitions, respectively. 35 b): Absorption coefficient for different Neodymium concentrations in the phosphate glass.

Figure 10 :

 10 Figure 10: Comparison of the 31 P relaxation rate for LaPO4 (red squares), YPO4 (blue circles), the Y0.8Sc0.2PO4 compound (green triangles), Ba5(PO4)3Cl (purple diamonds) and phosphate glass (orange triangles) in function of Neodymium concentration C (in mol/cm 3 ). The different linear regressions are respectively: 1/T1 = 4.9*10 2 *C for LaPO4, 1.9*10 2 *C for YPO4, 3.5*10 2 *C for Y0.8Sc0.2PO4, 2.7*10 1 *C for Ba5(PO4)3Cl and 1.1*10 3 *C for the metaphosphate glass with T1 in s -1 and C in mol/cm 3 . A zoom for 0 ≤ x ≤ 2.5*10 -4 mol/cm 3 of this figure is shown in SI-8.

  relaxation times τ1 for the crystalline compounds: τ1(LaPO4:Nd) = 11 ps with b2 = 13.6 Å and τ1(YPO4:Nd) = 10 ps with b2 = 18.8 Å. τ1(LaPO4:Nd) and τ1(YPO4:Nd) are very short, due to spin-orbit coupling. This explains why, in Electron Spin Resonance (ESR) experiments, Nd 3+

Figure 10 .

 10 LaPO4:Nd and YPO4:Nd present similar unit cell parameters (aLaPO4 = 6.8367 Å, bLaPO4 = 7.0746 Å, cLaPO4 = 6.5074 Å and aYPO4 = 6.8817 Å, cYPO4 = 6.0177 Å), inducing similar Nd-P distances (average Nd-P distance in LaPO4 = 3.59 Å and in YPO4 = 3.51 Å, calculated using the Gretep software 39 ); however, they crystallize in different structures (monazite and xenotime, resp.), showing different orbital overlap.

Figure 11

 11 Figure11shows the variation of 1/T1 as a function of 1/(dP-P)3/4 for fixed Nd concentrations: C = 1.0*10 -4 , 1.8*10 -4 , 2.0*10 -4 , 3.0*10 -4 , 4.0*10 -4 , 5.0*10 -4 and 6.0*10 -4 mol/cm 3 . Those concentrations have been chosen arbitrarily in the 0-10 -3 mol/cm 3 range, which is the range under consideration in this study. The values of 1/T1 for each concentration and each compound

Figure 11 :

 11 Figure 11: Values of 1/T1 at a neodymium concentration of 6.0*10 -4 mol/cm 3 (circles), 5.0*10 -4 mol/cm 3 (top triangles), 4.0*10 -4 mol/cm 3 (squares), 3.0*10 -4 mol/cm 3 (diamonds), 1.8*10 -4 mol/cm 3 (bottom triangles) and 1.0*10 -4 mol/cm 3 (bottom left triangles)for our three different structures (monazite LaPO4, xenotime YPO4 and phosphate glass) in function of the average distance Phosphor -Phosphor dP-P -0.75 . The different linear laws are respectively: 1/T1 = -0.71184 + 3.0759 dP-P -0.75 , 1/T1 = -0.58813 + 2.5517 dP-P -0.75 , 1/T1 = -0.47090 + 2.0380 dP-P -0.75 , 1/T1 = -0.35197 + 1.5230 dP-P -0.75 1/T1 = -0.22355 + 0.95727 dP-P -0.75 , and 1/T1 = -0.10499 + 0.44710 dP-P -0.75 ,with T1 in s and dP-P in Å.

Figure 12 4 Figure 12 :

 12412 Figure12displays the variation of 1/T1C as a function of the corresponding dP-P -3/4 for six different concentrations and for three of our compounds.

  al. in PbTe and GeTe or BiTe, by Sen et al. in silicate glasses, and by Li et al. in SrH2:Eu

  2+

Table 1 : Summary of the studied compounds, with their structural parameters. (  : calculated values).

 1

		Space Group	JCPDF number	Unit cell parameters	Density (g/cm 3 )	Molecular (g/mol) weight	Z
				a = 6.8313 Å,
	LaPO4	P21/n	01-083-0651	b = 7.0705 Å,  = 103.270°, c = 6.5034 Å,	5.33	234	4
				V = 306 Å 3
	YPO4	I41/amd	01-084-0335	a = 6.8817 Å, V = 285 Å 3 c = 6.0177 Å,	4.28	184	4
				a  = 6.8190 Å,
	Y0.8Sc0.2PO4	I41/amd	-	c  = 5.9742 Å,	-	175	4
				V  = 278 Å 3
	Ba5(PO4)3Cl	P63/m	01-070-2318	a = 10.2840 Å, V = 700 Å 3 c = 7.6510 Å,	4.81	975	2
	50P2O5:17Mg O:33Na2O	amorphous	-	-	2.5

18 98 1 Synthesis and Characterization 1.1 Synthesis

	mixture is placed for 3 hours at 90 °C in an oven. After filtration, the obtained powder is
	annealed at 1000 °C during 4 hours. For this study, the Nd doping concentration x is comprised
	between 0 and 10 mol.%. In the following, the doped compounds will be labeled La1-xNdxPO4
	and Y1-xNdxPO4. Nd-doped Y0.8Sc0.2PO4 solid-solution is synthesized by a solid-state route:
	yttrium, scandium and neodymium oxides are mixed together with NH4H2PO4 in stoichiometric
	proportions. The mixture, previously homogenized by a planetary milling (250 rpm, 20 min),
	is heated at 1000 °C during 2 hours and then at 1550 °C during 8 hours. 19 The nominal Nd
	concentration is set at x = 0, 0.5 and 1 mol.%. The alforsite Ba5(PO4)3Cl doped with Nd 3+ is
	synthesized according to Ju et al 20 : an excess of barium chloride is mixed together with BaCO3,
	NH4H2PO4 and Nd2O3 in a planetary mill (2 cycles, corresponding to 10 min grinding at 250
	rpm spaced by 30 s pauses). The mixture is annealed at 950 °C for 3 hrs. As the doping induces
	a substitution of Ba 2+ by Nd 3+ , the nominal Nd concentration x is calculated taking into account
	the following chemical formula: Ba5(1-x)Nd5x(PO4)3ClO5x/2 and x is set to 0, 1 and 4 mol.%. The
	obtained compounds will be labeled CAl-U (U for undoped), CAl-LD (LD for low doping) and
	CAl-HD (HD for high doping) respectively. Note that the charge compensation can occur
	through different mechanisms (such the creation of a Ba vacancy), as discussed in Ju et al. 20
	Regarding the Nd-doped metaphosphate glasses (Q 2 type), the different compositions are
	summarized in
	All precursors are purchased from Sigma-Aldrich. Except for pentasodium
	tripolyphosphate (TPP) and NH4H2PO4, they present purity over 99.9 % to minimize the level
	of paramagnetic impurities (such as Fe 3+ ions). The co-precipitation synthesis of the monazite
	phase LaPO4 doped with Nd 3+ ions and of the xenotime phase YPO4 doped with Nd 3+ are
	described in Maron et al. 13 Briefly, La(NO3)3.6H2O (or Y(NO3)3.6H2O), Nd(NO3)3.6H2O and
	an excess of pentasodium tripolyphosphate (TPP) are mixed together in a Teflon vessel. The

Table 2: Table 2: Compositions of the different glasses synthesized and concentrations of Neodymium ion in mol. % and mol/cm 3 .

	Nominal composition	x (mol. %)	C (mol/cm 3 )
	500P2O5:170MgO:330Na2O	0	0
	499P2O5:167MgO:333Na2O:1Nd2O3	0.20	5.1*10 -5
	498P2O5:167MgO:331Na2O:4Nd2O3	0.80	2.0*10 -4
	492P2O5:166MgO:328Na2O:14Nd2O3	2.8	7.1*10 -4
	489P2O5:165MgO:325Na2O:21Nd2O3	4.2	1.1*10 -3
	485P2O5:164MgO:323Na2O:28Nd2O3	5.6	1.4*10 -3

SSNMR spectra of metaphosphate glasses at r = 15 kHz. From top to bottom, with 5.6, 4.2, 2.8, 0.8 and 0 mol. % of Nd 3+ .

		5.6 at. %
		4.2 at. %
		2.8 at. %
		0.8 at. %
		no doped
	20	10	0	-10		-20	-30	-40	50
		 (ppm/H	PO	85 %)
				3	4
	Figure 8:

Table 3 : Distances between P and P atoms in the different studied structures. These distances were obtained using Vesta software and JCPDF files 01-084-0335 for YPO4 and 01-083-0651 for LaPO4 and by Whetherall et al. for the glass. 40

 3

	Compounds	between phosphorus atoms Mean distances dP-P (in Å)	(dP-P) -3/4 (in Å -3/4 )
	YPO4			5.96	0.262
	LaPO4			4.27	0.337
	Glass			2.95	0.444
			0.8	Glass
			0.6
		(s -1 )	0.4	LaPO 4
		1 1/T	0.2	YPO 4
			0
				0.25 0.3 0.35 0.4 0.45
				d	P-P -3/4 ( Å	-3 / 4 )

Sébastien Maron&,* , Nadège Ollier ǂ , Thierry Gacoin & , Géraldine Dantelle † &

Acknowledgements

The authors would like to thank François Devreux and Vera Pukhkaya for their help.


Pierre Mounkoro

Thomas Michel

Brigitte Meunier
email: brigitte.meunier@i2bc.paris-saclay.fr

Revisiting the mode of action of the antimalarial proguanil using the yeast model

Keywords: antimalarial drug, mitochondrial function, drug mode of action, drug resistance, yeast model

Proguanil in combination with its synergistic partner atovaquone has been used for malaria treatment and prophylaxis for decades. However its mode of action is not fully understood.

Here we used yeast to investigate its activity. Proguanil inhibits yeast growth, causes cell death and acts in synergy with atovaquone. It was previously proposed that the drug would target the system that maintains the mitochondrial membrane potential when the respiratory chain is inhibited. However our data did not seem to validate that hypothesis. We proposed that proguanil would not have a specific target but accumulate in the mitochondrial to concentrations that impair multiple mitochondrial functions leading to cell death. Selection and study of proguanil resistant mutants pointed towards an unexpected resistance mechanism: the decrease of CoQ level, which possibly alters the mitochondrial membrane properties and lowers proguanil intramitochondrial level.

Introduction

The biguanide proguanil, in combination with its synergistic partner atovaquone, has been used for malaria treatment and prophylaxis for decades. Atovaquone is a well-known inhibitor of the mitochondrial respiratory chain cytochrome bc1 and binds in the Qo-site of the complex [START_REF] Birth | Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action[END_REF]. The drug is highly potent against the malaria agents Plasmodium. However, its inhibitory action is compromised by resistant mutations located in its binding site, such as Y268C and Y278S found in atovaquone resistant P. falciparum parasites (see [START_REF] Cottrell | Emergence of resistance to atovaquone-proguanil in malaria parasites: insights from computational modeling and clinical case reports[END_REF]

and refs within).

A potent fast-acting activity of proguanil is attributed to its metabolite cycloguanil, which acts as an inhibitor of the dihydrofolate reductase (DHFR). DHFR mutations causing resistance to cycloguanil were reported [START_REF] Peterson | Molecular basis of differential resistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria[END_REF]. Proguanil itself (and a cyclization-blocked analogue) shows slow-acting anti-parasitic activity [START_REF] Skinner-Adams | Cyclization-blocked proguanil as a strategy to improve the antimalarial activity of atovaquone[END_REF]. The efficiency of the combined atovaquone-proguanil therapy is not due to cycloguanil but rather to proguanil [START_REF] Skinner-Adams | Cyclization-blocked proguanil as a strategy to improve the antimalarial activity of atovaquone[END_REF] [START_REF] Vaidya | A mechanism for the synergistic antimalarial action of atovaquone and proguanil[END_REF]. Although the synergistic effect of the two compounds is well documented [START_REF] Vaidya | A mechanism for the synergistic antimalarial action of atovaquone and proguanil[END_REF] [6] [START_REF] Painter | Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum[END_REF], the mode of action of proguanil is still poorly understood. The drug has no effect on the respiration of the parasite [START_REF] Vaidya | A mechanism for the synergistic antimalarial action of atovaquone and proguanil[END_REF]. It was hypothesized that, in the parasite, proguanil target the alternative system of mitochondrial membrane potential generation, comprising the F1 domain of the ATP synthase with the adenine nucleotide translocator and the phosphate carrier, which is essential for the cell in absence of an active respiratory chain [START_REF] Painter | Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum[END_REF]. This could explain why proguanil potentiates the activity of atovaquone. The combination of the two drugs would impair the generation of membrane potential by both the respiratory chain and the alternative system. It was reported that proguanil inhibited ATP hydrolysis by the ATP synthase prepared from animal source. However this effect did not seem specific to proguanil as it was also observed with cycloguanil and other biguanides [START_REF] Bridges | Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria[END_REF].

Here we used the yeast model to study the mode of action of proguanil. Yeast, as P.falciparum, does not possess P450 cytochromes that could metabolise proguanil in cycloguanil, as it occurs in human liver. Therefore a growth inhibition observed upon addition of proguanil in the culture medium would be caused by that compound itself and not its metabolite.

As observed with the parasite, we showed that proguanil acted in synergy with atovaquone to inhibit yeast growth and that the inhibition of the bc1 complex by atovaquone was required for the synergy. We then compared the wt strain and its rho° derivative that exclusively relies on the alternative system for the generation of membrane potential. We observed the same sensitivity to proguanil, thus suggesting that the drug would not act via the inhibition of that system. To obtain clues on the drug mode of action, proguanil resistant mutants were selected and studied.

Materials and Methods

Growth media and yeast strains

The following growth media were used: YPD (1% yeast extract, 2% peptone, 3% glucose), YPEth (1% yeast extract, 2% peptone, 2% ethanol), YPG (1% yeast extract, 2% peptone, 2% glycerol),YPGal (1% yeast extract, 2% peptone, 0.2% glucose, 2% galactose). Cells were cultured at 28°C with agitation (150 rpm) for a good aeration.

The strain AD1-9 and its derived mutants were used for the growth assays. AD1-9, lacking several membrane transporters (α, ura3, his1, Δyor1, Δsnq2, Δpdr5, Δpdr10, Δpdr11, Δycf1, Δpdr3, Δpdr15, Δpdr1) was kindly provided by M. Ghislain, UCL, Belgium. coq5 mutant was constructed by PCR-based deletion.

Growth assays and drug sensibility tests

Drug sensitivity was assessed by monitoring the inhibition of yeast cell proliferation. Yeast were grown in YPEth or YPGal with increasing concentrations of drugs. Cultures were inoculated at an OD600nm of 0.2 and incubated at 28°C with vigorous shaking for three days. OD600 nm were then measured. Data were plotted for each strain as the percentage of growth relative to control, i.e. untreated. The experiments were repeated at least twice and the data averaged. IC50 (drug concentration required to obtained 50% growth inhibition) were estimated from inhibitor titration plots.

Drug combination

In vitro drug-drug interactions were investigated using the fixed-ratio isobologram method established by Fivelman et al. [START_REF] Fivelman | Modified fixed-ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug-resistant strains of Plasmodium falciparum[END_REF]. This approach is based on determining the IC50 values of each individual drug alone and the IC50 values of a mixture of both drugs at fixed concentration ratios. Drugs were applied alone or in combination at fixed concentration ratios (A:B ratios [vol/vol] of 1:4, 2:3, 3:2, and 4:1). The FIC50 of each drug in a given combination was estimated as follows: FIC50 (A) = IC50 of A in combination / IC50 of A alone; FIC50 (B) = IC50 of B in combination / IC50 of B alone. All FIC50s were confirmed by independent experiments. The isobolograms were drawn by plotting pairs of FIC50 values of drug A and drug B for each combination. The straight line indicated an indifferent interaction (no interaction or additive action), a concave curve towards the origin of the axes represented synergy and a convex curve towards the opposite indicated antagonism.

Measurement of O2 consumption of mitochondria and of intact cells

Oxygen consumption activities were measured in a Clarke-type oxygen electrode. Mitochondria, prepared as described in [START_REF] Lemaire | Preparation of respiratory chain complexes from Saccharomyces cerevisiae wild-type and mutant mitochondria: activity measurement and subunit composition analysis[END_REF], were added at around 50 g of protein in 1 mL of 0.7 M sorbitol, 50 mM potassium phosphate pH 7.5 and 0.2 mM EDTA. The reaction was initiated by the addition of of 0.8 mM NADH.

Sequencing analysis

Total genomic DNA was isolated from the WT strain AD1-9 and from the derived mutants. The genomic libraries, the sequencing and bioinformatics analysis were performed by the Next Generation Sequencing Core Facility of I2BC (www.i2bc.paris-saclay.fr). The presence of the identified mutations was confirmed by PCR and sequencing of the mutated genes, using independent DNA preparations.

Results and discussion

Effect of proguanil and atovaquone combination on yeast respiratory growth

Before investigating the mode of action of proguanil in yeast, we addressed the question of the relevance of the yeast model. To that end, we checked whether yeast showed the same behaviour as the parasite in regards to the sensitivity to atovaquone-proguanil combination. For the growth assays, we used the respiratory growth medium YPEth and the strain AD1-9 (and its derivatives) that lacks several membrane transporters, which renders the cells more sensitive to compounds as previously shown for atovaquone and other anti-malaria drug [START_REF] Hill | Recapitulation in Saccharomyces cerevisiae of cytochrome b mutations conferring resistance to atovaquone in Pneumocystis jiroveci[END_REF].

First, we checked whether proguanil and atovaquone acted synergistically to inhibit yeast growth, as observed in P. falciparum. We also tested the combination of proguanil with three others drugs: the antimalarial plasmodione, a potent yeast respiratory growth inhibitor acting via oxidative stress [START_REF] Mounkoro | Investigating the mode of action of the redox-active antimalarial drug plasmodione using the yeast model[END_REF], and two bc1 complex inhibitors, the antifungal azoxystrobin and the anti-parasitic ELQ-271 [13] [START_REF] Doggett | Endochin-like quinolones are highly efficacious against acute and latent experimental toxoplasmosis[END_REF]. Atovaquone and azoxystrobin bind at the Qo-site of the complex while ELQ-271 targets its Qi-site.

As shown in fig. 1A, proguanil acts synergistically with atovaquone, azoxystrobin and ELQ-271 while it antagonizes plasmodione in growth inhibition assays. The antagonistic effect of plasmodione was previously observed with the malaria parasite, but not yet explained [START_REF] Ehrhardt | The redox cycler plasmodione is a fast-acting antimalarial lead compound with pronounced activity against sexual and early asexual blood-stage parasites[END_REF]. The synergistic effect of the bc1 complex inhibitor ELQ-300, structurally similar to ELQ-271 was reported in P.falciparum [START_REF] Skinner-Adams | Cyclization-blocked proguanil as a strategy to improve the antimalarial activity of atovaquone[END_REF].

Secondly, we asked whether atovaquone potentiates proguanil by inhibiting the bc1 complex or whether another drug-drug interaction, independent of bc1 complex inhibition, should be proposed. To that end, we tested the effect of the drug combination on the atovaquone resistant mutant Y279C, derived from the control strain AD1-9. The cytochrome b mutation Y279C (corresponding toY268C in P. falciparum) confers a high level of atovaquone resistance in yeast as in the parasite [START_REF] Song | Saccharomyce cerevisiaebased mutational analysis of the bc1 complex Qo site residue 279 to study the trade-off between atovaquone resistance and function[END_REF]. In yeast growth assays, the IC50 of the control strain was around 0.1 µM atovaquone whereas it was >10 µM for Y279C. Fig. 1B top panel shows the proguanil and combined proguanil-atovaquone inhibition curves of the control. A dramatically increased inhibition was observed with the drug combination. By contrast, as shown in the bottom panel, the proguanil and proganil-atovaquone inhibition curves were identical for Y279C. Thus atovaquone did not potentiate proguanil activity in the mutant. This confirmed that, as previously reported for the malaria parasite [START_REF] Srivastava | Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites[END_REF], the atovaquone-proguanil synergistic effect observed in the control (Fig. 1B top panel) required the inhibition of the bc1 complex by atovaquone. Another drug-drug interaction independent of atovaquone inhibitory action on the respiratory chain complex seems excluded.

Then, in order to pursue the comparison with data obtained with the malaria parasite, we tested the effect of proguanil and atovaquone-proguanil combination on the O2 consumption of mitochondria isolated from yeast control strain (data not shown). Proguanil alone had not effect and did not potentiate the inhibitory effect of atovaquone on the respiratory chain actvity, which was also in agreement with results obtained with the parasites [START_REF] Vaidya | A mechanism for the synergistic antimalarial action of atovaquone and proguanil[END_REF].

Thus based on these data, yeast seems an appropriate model to decipher the mode of action of proguanil.

Consequence of proguanil treatment on cell viability

We tested whether proguanil had a cytostatic or a cytotoxic effect on yeast. Cells were grown in YPEth to an OD600nm of 2 and then treated with different concentrations of proguanil. After 24h treatment, the OD600nm were recorded. The cells were then harvested, resuspended in water, counted, serially diluted and plated on YPD medium. After four days of incubation at 28°C, the colonies were counted and their number was reported to the number of plated cells (fig. 2). Proguanil treatment at concentrations inhibiting cell proliferation severely lowered cell viability By contrast, treatment with bc1 complex inhibitors had no effect on cell viability (not shown).

A role for the respiratory chain and for the alternative system for mitochondrial membrane potential generation

We then addressed the question of the role of the respiratory chain and the alternative mechanism for the generation of mitochondrial membrane potential in proguanil activity.

To that end, we compared the sensitivity to proguanil of the strain AD1-9 and its derived rho° mutant without mtDNA. Rho° cells lack the respiratory chain complexes III and IV and the FOpart of complex V (or ATP synthase), as part of their components are mitochondrially encoded. The cells were grown in YPGal with a vigorous agitation for a good aeration of the cultures. In these conditions, the WT cells (rho + ) use mainly the respiratory function while the rho° cells exclusively produce their energy by glycolysis.

If the inhibitory activity of proguanil requires an active respiratory chain, the WT cells would be sensitive to the drug while the rho° cells would be unaffected. This is observed for plasmodione and primaquine [START_REF] Mounkoro | Investigating the mode of action of the redox-active antimalarial drug plasmodione using the yeast model[END_REF] [START_REF] Lalève | The antimalarial drug primaquine targets Fe-S cluster proteins and yeast respiratory growth[END_REF], and of course for atovaquone. By contrast, if the drug interferes with the alternative mechanism for mitochondrial membrane potential generation, the rho° cells should be more sensitive than WT as rho° cells rely exclusively on that mechanism.

In WT cells, the mitochondrial membrane potential is generated by the activity of the respiratory chain. In rho° cells, the mitochondrial membrane potential (indispensable even when ATP derived from glycolysis) is maintained by an alternative mechanism, by the electrogenic exchange of ATP 4-/ ATP 3-via the adenine nucleotide translocator in concert with the ATP hydrolysis activity of the F1 domain of complex V and the phosphate carrier. Inhibition of the adenine nucleotide translocator by mutation or by bongkrekic acid, or inhibition of the F1 domain of complex V is lethal for the rho° cells [START_REF] Giraud | The absence of the mitochondrial ATP synthase δ subunit promotes a slow growth phenotype of rhoyeast cells by a lack of assembly of the catalytic sector F1[END_REF] [START_REF] Dupont | The role of adenine nucleotide translocation in the energization of the inner membrane of mitochondria isolated from ϱ + and ϱ o strains of Saccharomyces cerevisiae[END_REF].

As shown in fig. 3, rho° cells were slightly less sensitive to proguanil, suggesting that the respiratory chain plays only a minor role, if any, in proguanil sensitivity. It could also be inferred that the activities of the adenine nucleotide translocator, the F1-domain of complex V and the phosphate carrier were not impaired by proguanil.

It thus seems that the drug would not act (mainly) via the inhibition of the alternative system for membrane potential generation, and that other possible mechanisms need to be investigated. Its synergy with atovaquone (and other bc1 complex inhibitors) strongly argues for mitochondrial targets. Its effect on cell viability points towards essential mitochondrial functions.

Cell strategy to decrease proguanil inhibitory effect

Uncovering the cell strategies to decrease a drug inhibitory effect could give clues about its mode of action. We thus selected resistant mutants.

Several subclones from AD1-9 were grown on YPD, and then incubated on YPG containing 150 M of proguanil, a concentration that fully inhibited cell growth. Only a few resistant colonies appeared after two months of incubation at a frequency of around 10 -10 , which was lower than the frequency we observed in other screens. Four mutants were studied, issued from different subclones, and analysed. They presented a weak resistance to proguanil with an IC50 (determined in YPEth medium) three-fold higher than the IC50 of the parental AD1-9 strain and unaffected growth competence.

Whole genomic sequencing was performed and revealed the same mutation: a T to G nucleotide substitution at position 41 downstream PUF3 coding sequence, in the terminator region [START_REF] Wei | Characterization of terminators in Saccharomyces cerevisiae and an exploration of factors affecting their strength[END_REF]. The mRNA-binding protein Puf3 was reported to have several mitochondrial targets. In particular it was shown to regulate Coq5 abundance and Co-enzyme Q (CoQ) biosynthesis [START_REF] Lapointe | Multi-omics reveal specific targets of the RNA-binding protein Puf3p and its orchestration of mitochondrial biogenesis[END_REF]. Therefore we tested whether the deletion of COQ5 could affect the sensitivity to proguanil. COQ5 encodes the 2-hexaprenyl-6-methoxy-1,4-benzoquinone methyltransferase, forming a large complex with other CoQ biosynthetic enzymes.

We compared ∆coq5 with mutant rho°, both deriving form AD1-9 and both defective in respiratory function, and presenting same growth behaviour (fig. 4A). The rho°, as shown in fig. 3, presented a proguanil sensitivity very similar to WT. Interestingly, the absence of Coq5 and thus of CoQ synthesis, resulted in a decreased sensitivity to proguanil.

We then tested the effect of adding the CoQ analogue decylubiquinone in the growth medium, using the WT strain (AD1-9) (fig. 4B). Addition of decylubiquinone increased proguanil inhibitory effect.

CoQ plays a key and well-known role in electron transfer and has been shown to act as an antioxidant that protects the membrane lipids against peroxidation (see for instance, [START_REF] Turunen | Metabolism and function of coenzyme Q[END_REF] and references within). Yeast synthesize only saturated and monounsaturated fatty acids that are not prone to peroxidation but CoQ-deficient yeast cells were shown to be hypersensitive to the autooxidation of added polyunsaturated fatty acids [START_REF] Do | Enhanced sensitivity of ubiquinone-deficient mutants of Saccharomyces cerevisiae to products of polyunsaturated fatty acids[END_REF].

CoQ was also reported to stabilize the phospholipid membrane. In presence of CoQ-10 the membranes are less permeable to hydrophilic solutes [START_REF] Hernández | Ubiquinone-10 alters mechanical properties and increases stability of phospholipid membranes[END_REF]. In absence of CoQ, one could thus expect that the membrane might be more permeable. The effect of decylubiquinone addition and of ∆coq5 mutation that abolishes CoQ biosynthesis on proguanil sensitivity might be related to that property. In the mutant lacking CoQ, a more permeable mitochondrial membrane might facilitate the diffusion of proguanil out of the mitochondria while adding decylubiquinol might increase proguanil retention, which could explain the lower or enhanced sensibility of the yeast growth to the drug. We could also hypothesize that proguanil would be transported into the mitochondria by a transporter whose activity is modulated by CoQ level.

Possible mode of action

Proguanil and other biguanides are positively charged molecules. It was suggested that biguanides could accumulate in the mitochondrial matrix at concentrations 1,000-fold higher than extracellular concentrations in response to cell and mitochondrial membrane potentials and depending on the presence of transporters [START_REF] Bridges | Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria[END_REF].

Little is known about the transport of proguanil into the mitochondria. In mammalian cells, the drug seems unable to access the mitochondrial matrix contrarily to the related biguanide phenformin whose uptake would involve the organic cation/carnitine transporter (OCTN1) [START_REF] Bridges | Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria[END_REF] [26] [START_REF] Shitara | Role of organic cation/carnitine transporter 1 in uptake of phenformin and inhibitory effect on complex i respiration in mitochondria[END_REF]. By contrast, in yeast as in the malaria parasite, proguanil could most likely access, and thus possibly accumulate into, the mitochondria as the synergy with atovaquone that specifically targets the bc1 complex points toward a mitochondrial location of the drug activity.

We propose that proguanil would not have a specific target but accumulate at concentrations that impair multiple mitochondrial functions and/or block transporters, which would be lethal for the cells. The only resistance strategy would be to lower the mitochondrial level of proguanil.

The search for and study of proguanil resistant mutants seem to support this proposal. The low frequency of resistant mutants found would be in favour of a multi-target mode of action for proguanil. The sole mechanism of resistance (decrease in CoQ level) likely works by lowering the drug concentration. One could have expected that the resistance screens would identify mitochondrial transporter(s) possibly required for the transport of proguanil into the mitochondria. Our selection assays may not have fished all the possible resistance mutants or proguanil might have multiple transporters. The possible proguanil transporter(s) might also be essential and their activity could not be impaired.

If proguanil toxicity is due to its mitochondrial accumulation, resulting in multiple function defect, what could be the mechanism of the synergy with atovaquone (and other bc1 complex inhibitors)? It should be noted that the synergy with atovaquone is not specific to proguanil but observed, in the parasite, with other biguanides [START_REF] Jones | Biguanide-atovaquone synergy against Plasmodium falciparum in vitro[END_REF].

A decrease of the mitochondrial membrane potential caused by the inhibition of the respiratory chain by atovaquone could not explain the synergy. Indeed, a rho° mutant (lacking a respiratory chain) is not more sensitive to proguanil than its parent rho + (fig. 3). On the contrary, a slightly decreased sensitivity was observed. Likewise, when the WT strain was grown in fermentation conditions (in glucose rich medium without aeration), its sensibility to proguanil was two-to three-fold decreased compared to its sensitivity in respiration conditions (not shown). An accumulation of proguanil depending on the mitochondrial membrane potential (as reported for other biguanides [START_REF] Bridges | Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria[END_REF]) could explain that lower sensitivity.

More work would thus be needed to unravel the mechanism of synergy. showing the effect of proguanil combination with atovaquone (ATV), azoxystrubin (AZO), ELQ271 and plasmodione (PD) on yeast growth. The dotted line shows a theoretical indifferent interaction. (B) Respiratory growth sensitivity of the atovaquone resistant strain Y279C and WT to proguanil alone (PG, straight line) and proguanil-atovaquone combination (PG+ATV, dotted line). Cells were grown in YPEth with increasing concentration of drugs. OD 600nm were measured after three days culture at 28°C. The results are presented as % of growth yield without treatment. Fig. 2 Effect of proguanil treatment in cell growth and viability. WT cells were grown for 24h in YPEth to an OD 600nm of around 2, and then incubated for another 24h in YPEth without or with drugs. The OD 600nm of cultures were then recorded. The cells were then harvested, plated on YPD and incubated four days. The number of colonies observed was reported on the number of plated cells to determine the percentage of viability. Each measurement was repeated at least three times and averaged. Error bars represent standard deviation.

Fig. 1

 1 Fig. 1 Effect of proguanil combination with respiratory growth inhibitors. (A) Isobologramshowing the effect of proguanil combination with atovaquone (ATV), azoxystrubin (AZO), ELQ271 and plasmodione (PD) on yeast growth. The dotted line shows a theoretical indifferent interaction. (B) Respiratory growth sensitivity of the atovaquone resistant strain Y279C and WT to proguanil alone (PG, straight line) and proguanil-atovaquone combination (PG+ATV, dotted line). Cells were grown in YPEth with increasing concentration of drugs. OD 600nm were measured after three days culture at 28°C. The results are presented as % of growth yield without treatment.

Fig. 3 Fig. 4

 34 Fig.[START_REF] Peterson | Molecular basis of differential resistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria[END_REF] Effect of respiratory chain on cell sensitivity to proguanil. WT (rho + ) and rho° strains were grown in YPGal with various concentrations of proguanil. After two days growth at 28°C, OD 600nm were measured. For each strain, the data are presented as percentage of the OD 600nm of the untreated culture. The experiments were repeated at least twice and the data averaged. Error bars represent standard deviation

Acknowledgements

We acknowledge the High-throughput sequencing facility of I2BC for its sequencing and bioinformatics expertise. The work was supported by the French National Research Agency (ANR) (PlasmoPrim project to B.M). We are grateful to J. Clain and R. Duval, University Paris-Sorbonne for their comments on the data and on the manuscript.