Saber Hamimid

Messaoud Guellal

Madiha Bouafia

Numerical Simulation of Combined Natural Convection Surface Radiation for Large Temperature Gradients

I. Introduction

Coupled natural convection and radiation transport processes in cavities are interesting topics concerning thermal and mass transport processes involved in various types of engineering systems, such as solar energy collectors, cooling of electronic devices, multilayered walls (facades) and double windows, thermal behavior in nuclear reactors, space technology, and processes involving high temperatures.

In most of the work reported on enclosure (coupling natural convection with surface radiation), the Boussinesq approximation has been invoked. However, for higher overheat ratios, compressibility effects need to be taken into account, since the Boussinesq approximation can become inadequate. Such an approximation leads to an accurate description of convection flows if the temperature difference in the flow domain does not exceed a few degrees. However, in many physical and industrial applications such as thermal insulation systems, chemical vapor deposition reactors [START_REF] Ibrahim | The Modeling of Realistic Chemical Vapor Infiltration/Deposition Reactors[END_REF], atmospheric flows [START_REF] Klein | Multiple Spatial Scales in Engineering and Atmospheric Low Mach Number Flows[END_REF][START_REF] Klein | Asymptotic Analyses for Atmospheric Flows and the Construction of Asymptotically Adaptive Numerical Methods[END_REF], and combustion process [START_REF] Madja | The Derivation and Numerical Solution of the Equations for Zero Mach Number Combustion[END_REF][START_REF] Chen | A Novel Coupled Lattice Boltzmann Model for Low Mach Number Combustion Simulation[END_REF], the temperature differences reach tens and hundreds of degrees; all the assumptions used to justify the Boussinesq approximation fail; and a different, more complicated modeling approach is required that accounts for realistic nonlinear fluid properties variations. The low-Mach-number (LMN) approximation suggested by Paolucci [START_REF] Paolucci | On the Filtering of Sound from the Navier-Stokes Equations[END_REF], and used later by Hamimid et al. [START_REF] Hamimid | Numerical Study of Natural Convection in a Square Cavity Under Non-Boussinesq Conditions[END_REF] for pure natural convection, allows one to take into account arbitrary property variations for a general fluid.

This study illustrates a comparative investigation to analyze the physical differences between numerical simulations obtained with both the Boussinesq and LMN forms of the Navier-Stokes equations for natural convection flow coupled with surface radiation in simple geometries. The purpose of this Note is to study the combined effects of surface radiation and temperature-dependent viscosity and conductivity on the momentum and heat transfer in the enclosure, as well as to quantify how the incompressible flow assumption differs from the compressible form when solving flows driven by gravity acting upon density variations resulting from local temperature gradients.

A complete parametric study is made for different Rayleigh numbers, conductivities and viscosities of the fluid, Boussinesq parameter, and wall emissivity. From these simulations, the selected characterization parameters of the maximum convective and radiative Nusselt number, the average Nusselt number, and the normalized pressure reduction were calculated. Comparisons of these parameters were made with benchmark solutions for air with the ideal gas assumption. Additionally, we generated vertical and horizontal velocities at the midvertical plane and midhorizontal planes, respectively, streamlines, and isotherms distributions to provide a basis for further analysis.

II. Problem Formulation

We consider a bidimensional square cavity of side H (Fig. 1) filled with a variable-property Newtonian fluid of density ρ, molecular viscosity μ, thermal conductivity k, and temperature T. The natural convection problem is described by two vertically heated walls with *Assistant Professor, Laboratoire de Génie des Procédés Chimiques; sab_hamimid@yahoo.fr (Corresponding Author).

† Professor, Laboratoire de Génie des Procédés Chimiques; messaoud .guellal@gmail.com.

‡ Professor, Laboratoire de Mécanique et d'Energétique d'Evry; madiha .bouafia@ufrst.univ-evry.fr. prescribed temperatures T h and T c (T h ≻ T c ), which are the "hot" and "cold" wall temperatures, respectively. We assume that the horizontal surfaces are under the combined action of the convective and radiative contributions such as -k ∂T ∂y q r 0. All four walls have no-slip zero-mass flux boundary conditions (V 0) and have the same emissivity value, ε. A Cartesian x; y coordinate system is selected with y pointing vertical and x horizontal; thus, the gravitational vector g is applied on the y coordinate. Initial conditions are composed of constant distributions of reference pressure and temperature, p 0 and T 0 , and stationary flow (V 0). The reference temperature T 0 is defined as T 0 T h T c ∕2. Using the mean temperature and the temperature difference between the hot and cold walls (ΔT T h -T c ), we introduce the Boussinesq ratio ε b ΔT∕2T 0 . The inner surfaces, in contact with the fluid, are assumed to be gray, diffuse emitters and reflectors of radiation with identical emissivity.

In addition to assuming a Newtonian fluid, another assumption must be made in order to obtain the usual compressible Navier-Stokes equations. This assumption has to do with the relationship between the dynamic and bulk viscosity coefficients (μ and λ) that relate the strain rate and the stress: λ 2 3 μ 0. A low-Mach-number [START_REF] Suslov | Nonlinear Analysis of Convection Flow in a Tall Vertical Enclosure Under Non-Boussinesq Conditions[END_REF][START_REF] Suslov | Analysis of Coupling Between Hydrodynamic and Thermal Instabilities in Non-Boussinesq Convection[END_REF][START_REF] Codina | A Stabilised Finite Element Method for Generalised Stationary Incompressible Flows[END_REF][START_REF] Vierendeels | A Multigrid Method for Natural Convective Heat Transfer with Large Temperature Differences[END_REF][START_REF] Liu | An Implicit Finite Element Solution of Thermal Flows at Low Mach Number[END_REF][START_REF] Bouafia | Natural Convection for Large Temperature Gradients Around a Square Solid Body Within a Rectangular Cavity[END_REF][START_REF] Chenoweth | Natural Convection in an Enclosed Vertical Air Layer with Large Horizontal Temperature Differences[END_REF] flow model is used to describe the dynamics of the heated cavity. In this limit, the complete Navier-Stokes equations are expanded in powers of a small parameter M 2 (where M is the Mach number), and the total pressure P is decomposed into the mean thermodynamic pressure pt and the dynamic pressure P 0 .

A. Governing Equations

Since we are interested in the case of large temperature differences, the low-Mach-number equations are adopted in order to describe such a flow. The equations are made nondimensional by the use of the nondimensional variables:

x H ; y H ; uH α 0 ; vH α 0 ; t H 2 ∕α 0 ; pH 2 ρ 0 α 2 0 ; T -T 0 T h -T c ; and
Y π ρ 0 α 0 ∕H 2 p - p ρ 0 gy ρ 0 α 0 ∕H 2
The thermophysical properties (density, dynamic viscosity, thermal conductivity, and thermal diffusivity) are scaled by ρ 0 , μ 0 , k 0 , and α 0 , where the subscript 0 denotes values at the reference temperature T 0 . The influence of the temperature on the specific heat is assumed to be negligible so that C p ∕C p0 1 [START_REF] Suslov | Stability of Natural Convection Flow in a Tall Vertical Enclosure Under Non-Boussinesq Conditions[END_REF].

The system Navier-Stokes equations are expressed in nondimensional conservative form [START_REF] Hamimid | Numerical Study of Natural Convection in a Square Cavity Under Non-Boussinesq Conditions[END_REF] as

∂ρ ∂t ∂ρU ∂X ∂ρV ∂Y 0 (1) ρ ∂U ∂t U ∂U ∂X V ∂U ∂Y - ∂Π ∂X Pr ∂ ∂X μ ∂U ∂X ∂ ∂Y μ ∂U ∂Y 1 3 ∇μ∇:V (2) ρ ∂V ∂t U ∂V ∂X V ∂V ∂Y - ∂Π ∂Y -RaPr ρ -1 2ε b Pr ∂ ∂X μ ∂V ∂X ∂ ∂Y μ ∂V ∂Y 1 3 ∇μ∇ • V (3) ρc p ∂θ ∂t U ∂θ ∂X V ∂θ ∂Y ∂ ∂X k ∂θ ∂X ∂ ∂Y k ∂θ ∂Y γ -1 2ε b γ d P dt (4)
ρ P 2ε b T 1 (5)
Additional equations are needed to calculate the mean thermodynamic pressure pt and its derivate:

d P dt 2ε b γ • 1 R Ω dΩ • Z S k ∂T ∂n dS (6) P R Ω dΩ R Ω 1∕2ε b θ 1 dΩ (7)
In this study, the fluid (air) is considered as a perfect gas at reference temperature T o 300 K (we set in this case T h 480 K and T c 120 K) and the transport coefficients μT and kT are given by Sutherland'slaw:

μ 2ε b T 1 3∕2 1 S μ ∕T 0 2ε b T 1 S μ ∕T 0 ( 8
)
Where S μ 110.4 [START_REF] White | Viscous Fluid Flow[END_REF]. The conductivity is given by

kT μT Pr (9)

B. Radiative Analysis

When surface radiation transfer is considered in the analysis, the temperature boundary condition at the top and bottom adiabatic walls of the square cavity must be changed. Thus, one assumes that the solid surfaces are in thermal equilibrium under the combined action of the convective and radiative contributions, which give

∂θ ∂Y Y0;1 -NrQ r 0 ( 10
)
where Nr σT 4 0 H∕kΔT is the dimensionless parameter of conduction-radiation, and Q r q r ∕σT 4 0 is the dimensionless net radiative heat flux on the corresponding insulated wall expressed as

Q r;i R i - X N j1 R j F i-j (11)
where R i is the dimensionless radiosity of surface A i , obtained by resolving the following system: where the dimensionless radiative temperature Θ i is given by

X N j1 δ ij -1 -ε i F i-j R j ε i Θ 4 i (12) 0 r T q y λ ∂ -+ = ∂ 0, h uv TT == = 0, c uv TT == = 0 r T q y λ ∂ -+ = ∂
Θ i T i T 0 T h -T c θ i T 0 ∕T 0 θ i ΔT T 0 1 θ i θ 0 1 (13)

C. Heat Transfer Parameters

The nondimensional heat transfer rate in terms of convective and radiative Nusselt numbers, Nu c and Nu r , on the heated vertical surface are given by

Nu c - ∂θ ∂X Y0;1 (14) Nu r N r Q r j Y0;1 (15)
The average convective Nusselt number was calculated by integrating the temperature gradient over the vertical wall as

Nu c avg 1 A Z A 0 - ∂θ ∂X dX ( 16
)
The average radiative Nusselt number is obtained by integrating the net radiative flux

Nu r avg N r 1 A Z A 0 Q r dX (17)
The total average Nusselt number is calculated by summing the average values of convective and radiative Nusselt numbers [START_REF] Bouali | Radiation-Natural Convection Heat Transfer in an Inclined Rectangular Enclosure[END_REF]:

Nu avg 1 A Z A 0 - ∂θ ∂X 0;Y N r Q r 0;Y dY (18)

D. Numerical Modeling

The numerical solution of the governing differential equations for the velocity, pressure, and temperature fields is obtained by using a finite volume technique. A power scheme was also used in approximating advection-diffusion terms. The SIMPLER algorithm, for which the details can be found in work by Patankar [START_REF] Patankar | Numerical Heat Transfer and Fluid Flow[END_REF], with a staggered grid is employed to solve the coupling between pressure and velocity. The radiosities of the elemental wall surfaces are expressed as a function of elemental wall surface temperature, emissivity, and the view factors. The radiosity R i and temperature θ i are connected by a matrix of the type

A i;j fR i gfσθ 4 i g (19)
The inverse of the matrix A i;j is determined (only once) by the Gauss elimination method. The coefficients of A are constants and depend only on the emissivity and view factors.

In a two-dimensional formulation, the view factors are analytic [START_REF] Howell | A Catalog of Radiation Configuration Factors[END_REF]:

F ij -1 2x 2 -x 1 x 2 2 y 2 q y 2 y 1 - x 2 2 y 2 q y 2 y 1 ( 20
)
F i-k - 1 2x 2 -x 1 x 2 -x 2 H 2 q xx 2 xx 1 - x 1 -x 2 H 2 q xx 2 xx 1 ( 21
)
For the most computations reported in this study, 242 × 242 grid points were chosen to optimize the relation between the accuracy required and the computing time; nevertheless, in the case of variable properties, a 322 × 322 grid was used. The grid is nonuniform and very fine in the near-wall region.

III. Verification

The validity of the solution procedure described previously is demonstrated in this section by presenting a solution in the classical case of natural convection and surface thermal radiation in a Boussinesq approximation. A good agreement was found between the present calculations and those reported in the work of [START_REF] Wang | Étude Numérique du Couplage de la Convection Naturelle avec le Rayonnement de Surfaces en Cavité Carrée Remplie d'Air[END_REF] (Table 1).

IV. Results and Discussion

In this section, the low-Mach-number code is applied to analyze the effect of the Boussinesq parameter ε b , temperature-dependent fluid properties, and surface radiation on heat transfer and fluid flow in steady-state conditions.

The fixed parameters of simulation are Pr 0.71, T 0 300 K, A 1,a n dε. However, to examine the effect of the Boussinesq parameter and emissivity, we consider different values of ε b 0 :::0; 6 and ε0 :::1.

When the radiation exchange is taken into account, the characteristic dimension of the cavity may be calculated for a specific Rayleigh number (H 0.097 in the case Ra 10 6 ). For the Boussinesq solution, the difference temperature ΔT is fixed to [START_REF] Codina | A Stabilised Finite Element Method for Generalised Stationary Incompressible Flows[END_REF] Comparison with values published in [START_REF] Wang | Étude Numérique du Couplage de la Convection Naturelle avec le Rayonnement de Surfaces en Cavité Carrée Remplie d'Air[END_REF].

at Ra 10 6 in the presence of radiation with a wall emissivity of ε 0.2. The properties are assumed to be variable.

From a general point of view, it can be seen that, for a low temperature difference (ε 0.017), the solutions for compressible and incompressible models did not differ significantly. Isotherms and streamlines (Fig. 2) indicate a symmetric structure with respect to the cavity center with the presence of two secondary flows symmetrically arranged at the cavity center. At the horizontal walls, the isotherms show the existence of temperature gradients due to radiative exchange.

For a large temperature difference (ε b 0.6), the symmetric nature of the flow is broken. A rapid look in Fig. 2 allows us to note that the isotherms' slopes near the lower wall are different from those of the upper wall; moreover, the secondary rolls inside the primary flow are no longer symmetric.

Considering the convective heat transfer along the hot and cold walls, there is a remarkable change in their profiles according to the parameter ε b (Fig. 3). This behavior is especially observed at the bottom, where the Nusselt number for ε b 0.6 is larger than for ε b 0.017 on the hot wall. An opposed comportment is noticed on the cold wall.

Profiles of the local radiative Nusselt number on the vertical walls are depicted on Fig. 4. The quasi-linear character of these profiles reflects a uniform temperature on each side. Note, however, that the difference between Nu r h and Nu r c is even more important, as the parameter ε b is small. This remark is also observed on the horizontal walls (Fig. 5). The shape of these profiles reflects the temperature evolution on the top and bottom walls, which results from a balance between convective and radiative exchanges. Temperature profiles on the horizontal walls are plotted in Fig. 6. With increasing ε b , the bottom wall is cooling while the top wall is heating.

Horizontal profiles of air temperature at the midheight of the cavity are displayed in Fig. 7. One can see that, for ε b 0.6, the thermal boundary layer gets thicker near the hot wall and thinner near the cold wall, showing a deflection of peaks toward the cold wall. This behavior is due entirely to non-Boussinesq effects. In the center region, the fluid heats up with increasing ε b and the stratification decreases, as shown in Fig. 8. The effect of the parameter ε b on the flow is presented in Figs. 9 and 10, where horizontal and vertical velocity components are plotted at x 0.5 and y 0.5, respectively. Compared to the case with a low value (ε b 0.017), profiles of the U component show that the boundary layer contracts in the vicinity of the bottom wall and expands near the opposite wall. The same trend is observed for the V component near the vertical walls but not in the same proportions. It is interesting to note that, in the stratified region, the flow remains invariant for the component V, and the peak value near the cold vertical wall is slightly lower for ε b 0.6 than for ε b 0.017. In Fig. 11, the low variations of Nusselt number for the weak values of the Boussinesq parameterε b show that, under the condition ε b ≤ 0.05, the incompressible model based on the Boussinesq approximation can be used to simulate both pure natural convection (ε 0) and combined natural convection surface radiation (ε > 0). Furthermore, one may note that this interval of Boussinesq parameter corresponds to a temperature difference ΔT ≤ 30°C and maximal variations of thermophysical properties less than 9.5% for the density and 7.5% for the conductivity and viscosity (see Table 2). It is also interesting to note that Gray and Giorgini [START_REF] Gray | The Validity of the Boussinesq Approximation for Liquids and Gases[END_REF] showed in their study that, for air, the Boussinesq approximation is valid for ΔT ≤ 28.6 °C corresponding to the density variation of 10%.

B. Effect of Conductivity and Viscosity Variations

Another aim of this study is to examine the effect of fluid properties on the flow and heat transfer in the case of large temperature gradient. Simulation parameters for the compressible model are ε b 0.6, Ra 10 6 , ε 0.5, and a reference temperature T 0 300 K.

A comparison between results obtained with constant and variable properties is first illustrated by isotherms and streamlines plotted in Fig. 12. The most visible effect on isotherms occurs at the top wall. The center region of the cavity is marked by a sensible change in the fluid flow. Note that the symmetrical nature of the flow is strongly broken in the case of the constant. Distributions of convective and radiative Nusselt numbers along the vertical walls are displayed in Figs. 13 and14. By taking into account variable properties, the most noted change concerns the decrease of the convective Nusselt number on the cold wall at the upper part of the cavity. Unlike convection, Nu r profiles undergo a weak translation, showing a slight decrease of radiative heat transfer for variable properties. On the horizontal walls, the fluid properties weakly affect the radiative exchanges for which the effects are more visible on the upper wall (Fig. 15). Consideration of variable properties slightly enhances the radiative exchange at the top wall. The temperature profiles on the horizontal walls are higher for variable properties than for constant properties (Fig. 16).

The effect of fluid properties on the air temperature is evidenced in Fig. 17. By assuming variable properties, the temperature considerably increases in both boundary layers and at the core cavity. For the velocity components, the influence of fluid properties essentially appears near the bottom wall, at the cavity center for U, and near the hot wall for V. Variable properties lead to a decrease of the magnitude of both components, as seen in Figs. 18 and19. It is interesting to note that the boundary layers near the hot wall remain thicker for both cases (constant or variable properties), reflecting that this behavior is mainly due to the nonlinear density variation. 0,0 0,2 0,4

Y

Nu r (c), constant properties Nu r (h), constant properties Nu r (c), variable properties Nu r (h), variable properties

C. Effect of Surface Radiation

In this section, computations are carried out for different values of emissivity (0.1 ≤ ε ≤ 1.0), Ra 10 6 , ε b 0.6, and T 0 300 K. The properties are assumed to be variable according to Sutherland's law.

Typical isotherms and streamlines are plotted in Fig. 20 for different values of emissivity. Compared to the case without radiation, the temperature contours show the existence of temperature gradients near the top and bottom walls due to the radiative exchange. At the center region, the stratification is changed and the flow is modified but remains globally with the same structure: a primary flow along the walls and inside two asymmetrical secondary flows.

Figure 21 shows the variations of local convective Nusselt numbers on the vertical walls as a function of emissivity ε. Convective exchanges on the hot wall are greatly influenced by the wall radiation, especially on the lower part of the hot wall. Increasing the wall emissivity ε produces a decreasing of Nu h , whereas it is less significant for Nu c . For the local radiative exchange, an opposite trend is observed. Increasing ε leads to an increase of Nu r on the hot wall and a decrease on the cold wall (see Fig. 22). On the horizontal walls, it is clearly seen that an increase of the wall emissivity leads to the cooling of the top wall and the heating of the bottom wall (Fig. 23). This behavior is explained by the fact that the top wall loses heat while the bottom wall receives heat. The distribution of the radiative Nusselt number on the top and bottom walls, presented in Fig. 24, shows that the net radiative heat flux is essentially positive at the top and negative at the bottom. Vertical profiles of air temperature show that the emissivity reduces the stratification in the core (Fig. 25).

Figure 26 shows the variations of the average convective and radiative Nusselt numbers Nuc avg ;Nur avg according to the emissivity for Ra 10 5 , 10 6 , and 10 7 . With increasing of the wall emissivity ε, the radiative Nusselt number on the hot wall increases and the convective Nusselt number Nuc avg decreases. On the cold wall, an opposite trend is observed for the radiative Nusselt number, whereas the convective Nusselt number keeps the same shape of variation.

Velocity components are plotted in Figs. 27 and 28 across the midplanes. Unlike the vertical velocity where ε has almost no effect, the influence of the wall emissivity on the horizontal component indicates that the flow is intensified near the horizontal walls and the stratified region is almost at rest.

V. Conclusions

In this Note, computations have been made in the context of a low-Mach-number formulation to study the combined natural convection and surface radiation in a square enclosure using a control volume approach. The present study shows the effects of the temperature difference parameter, surface emissivity, and temperature-dependent fluid properties on the flow and heat transfer. In view of the results presented, the main points can be summarized as follows:

1) Under non-Boussinesq conditions, and when the temperature difference increases, a significant variation between incompressible and compressible solutions is observed. In this case, the compressible model based on a low-Mach-number approximation can be used with a sufficient accuracy to simulate the combined natural convective surface radiation.

2) The variation of thermophysical properties could put a significant influence on the convective and radiative heat transfer and on the fluid flow.

3) The radiation exchange reduces the stratification at the cavity center and intensifies the flow at the horizontal walls.

4) The presence of radiation increases the temperatures of the fluid, and it considerably modifies the fluid flow and temperature distribution.

  factor g = gravitational acceleration H = dimension of the enclosure k = thermal conductivity Nr = dimensionless parameter of conduction-radiation, σT 4 0 H∕kΔT Nu = convective Nusselt number Pr = Prandtl number, ν∕α p = dynamic pressure p 0 = dynamic perturbed pressure p = mean thermodynamic pressure Qr = dimensionless net radiative heat flux R i = radiosity Ra = Rayleigh number, gβΔTH 3 ∕να T = temperature t = dimensionless time U, V = dimensionless velocity components X, Y = dimensionless coordinates α = thermal diffusivity, k∕ρ • Cp β = thermal expansion coefficient ε b = temperature difference parameter (Boussinesq parameter), ΔT∕2T 0 Θ = dimensionless radiative temperature, T∕T 0 θ = dimensionless temperature, T -T 0 ∕T h -T c μ = dimensionless dynamic viscosity ν = kinematic viscosity Π = dimensionless pressure, pp ρ 0 gy∕ρ 0 α 0 ∕H 2 ρ

Fig. 1

 1 Fig. 1 Flow configuration and coordinate system.

Fig. 2 6 Nu h , 0. 6 Fig. 3 6 Fig. 4

 266364 Fig. 2 Streamlines (top) and temperature contours (bottom) at Ra 10 6 and ε 0.2. From left to right, Boussinesq model (BS), ε b 0.017, and ε b 0.6.

Fig. 6 Fig. 7 Fig. 8 Fig. 9

 6789 Fig. 6 Distributions of upper and lower wall temperatures for Ra 10 6 and ε 0.2.

Fig. 5 Radiative

 5 Fig. 5 Radiative Nusselt number distribution on the horizontal walls at Ra 10 6 and ε 0.2.

Fig. 11

 11 Fig. 11 Variation of the average convection Nusselt number on the hot wall as a function of Boussinesq parameter.

Fig. 12 Fig. 13

 1213 Fig. 12 Streamlines (top) and temperature contours (bottom) for Ra 10 6 and ε 0.5: a) constant properties, and b) Sutherland's law.

Fig. 14 Fig. 15 RadiativeFig. 16 Fig. 17 Fig. 18 Fig. 19

 141516171819 Fig. 14 Distributions of local radiative Nusselt numbers on the vertical walls for Ra 10 6 and ε 0.5.

Fig. 20 Fig. 21 Y

 2021 Fig. 20 Streamlines (top) and temperature contours (bottom) for Ra 10 6 and ε b 0.6.

Fig. 23 X

 23 Fig. 23 Distribution of upper and lower wall temperatures for Ra 10 6 and ε b 0.6.

Fig. 25 Fig. 26 Fig. 27 Fig. 28

 25262728 Fig. 25 Cross section of the temperature at x 0.5 for Ra 10 6 and ε b 0.6.

Table 1

 1 K. Substituting values of H and ΔT into the expression of Nr gives Nusselt number at active walls obtained with T 0 293.5Kand Δt 10 K a,b 0.097 0.2 8.381 2.355 10.736 8.417 2.319 10.736 8.379 2.355 10.734 8.416 2.318 10.734 10 6 0.097 0.8 7.815 11.265 19.080 7.930 11.150 19.078 7.861 11.265 19.126 7.971 11.151 19.126

											Nr	σT 4 0 kΔT	να gβΔT	1∕3	Ra 1∕3
									A. Effect of Boussinesq Parameter ε b
									Figures 2-10 allow comparison of the results obtained from
									Boussinesq, weak non-Boussinesq, and strong non-Boussinesq cases
						Wang et al. [20]					Present work
					Hot wall			Cold wall			Hot wall		Cold wall
	Ra	H	ε	Nu c	Nu r	Nu t	Nu c	Nu r	Nu t	Nu c	Nu r	Nu t	Nu c	Nu r	Nu t
	10 4 0.021 0	2.246 0	2.246 2.246 0	2.246 2.246 0	2.246 2.246 0	2.246
	10 4 0.021 0.2 2.260 0.507 2.767 2.268 0.499 2.767 2.262 0.507 2.769 2.271 0.498 2.769
	10 4 0.021 0.8 2.249 2.401 4.650 2.278 2.372 4.650 2.255 2.401 4.656 2.284 2.371 4.656
	10 5 0.045 0	4.540 0	4.540 4.540 0	4.540 4.532 0	4.532 4.532 0	4.532
	10 5 0.045 0.2 4.394 1.090 5.484 4.411 1.073 5.484 4.398 1.090 5.489 4.417 1.072 5.489
	10 5 0.045 0.8 4.189 5.196 9.385 4.247 5.137 9.384 4.200 5.196 9.397 4.261 5.136 9.397
	10 6 0.097 0	8.852 0	8.852 8.852 0	8.852 8.863 0	8.863 8.863 0	8.863
	10 6

a Nu t Nu c Nu r . b

Table 2

 2

			Variation of
	thermophysical properties as a
	function of Boussinesq parameter
	ε b	ΔT Δρ,% Δk,% Δμ,%
	0.0083	5	1.59	1.19	1.26
	0.017	10	3.24	2.57	2.50
	0.025	15	4.87	3.73	3.87
	0.033	20	6.47	4.98	4.95
	0.042	25	8.05	6.20	6.28
	0.05	30	9.59	7.42	7.46
	0.058	35 11.02	8.62	8.50
	0.083	50 15.48 12.04 12.05
	0.167 100 28.61 22.89 22.84
	0.25	150 40.00 32.74 32.78
	0.33	200 49.97 41.84 41.87
	0.417 250 58.81 50.39 50.44
	0.5	300 66.67 58.59 58.53
	0.6	360 75.01 67.95 67.97


domain: sdv chim

text:
Raoul Manuel
email: raoul.manuel@inserm.fr

Michelle De Souza Lima

Sébastien Dilly
email: sebastien.dilly@gmail.com

Sylvain Daunay
email: sylvain.daunay@innoverda.com

Patricia Abbe
email: patricia.abbe@unice.fr

Elodie Pramil
email: elodie.pramil@hotmail.com

Stéphanie Solier

Fabienne Guillaumond
email: fabienne.guillaumond@inserm.fr

Sarah-Simha Tubiana
email: sarah-simha.tubiana@inserm.fr

Alexandre Escargueil
email: alexandre.escargueil@inserm.fr

João Antonio

Pêgas Henriques
email: pegas.henriques@gmail.com

Nathalie Ferrand
email: nathalie.ferrand@inserm.fr

Irène Erdelmeier
email: irene.erdelmeier@innoverda.com

Jean-Luc Boucher
email: jean-luc.boucher@parisdescartes.fr

Gildas Bertho
email: gildas.bertho@parisdescartes.fr

Israel Agranat
email: isri.agranat@mail.huji.ac.il

Stéphane Rocchi
email: stephane.rocchi@univ-cotedazur.fr

Michèle Sabbah
email: michele.sabbah@inserm.fr

Anny Slama Schwok
email: anny.slama-schwok@inserm.fr

Rosario Ammendola

Fabio Cattaneo

Distinction between 2′-and 3′-Phosphate Isomers of a Fluorescent NADPH Analogue Led to Strong Inhibition of Cancer Cells Migration

Keywords: fluorescence, molecular modeling, cancer cell migration, NADPH oxidases, NO-synthases, in vivo imaging

Specific inhibition of NADPH oxidases (NOX) and NO-synthases (NOS), two enzymes associated with redox stress in tumor cells, has aroused great pharmacological interest. Here, we show how these enzymes distinguish between isomeric 2′-and 3′-phosphate derivatives, a difference used to improve the specificity of inhibition by isolated 2′-and 3′-phosphate isomers of our NADPH analogue NS1. Both isomers become fluorescent upon binding to their target proteins as observed by in vitro assay and in vivo imaging. The 2′-phosphate isomer of NS1 exerted more pronounced effects on NOS and NOX-dependent physiological responses than the 3′-phosphate isomer did. Docking and molecular dynamics simulations explain this specificity at the level of the NADPH site of NOX and NOS, where conserved arginine residues distinguished between the 2′-phosphate over the 3′-phosphate group, in favor of the 2′-phosphate.

Introduction

High levels of reactive oxygen and/or reactive nitrogen species (ROS or RNS, respectively) generated in tumor cells are crucial for their proliferation, invasion, and are associated with therapeutic resistance [START_REF] Cheng | Nitric oxide in cancer metastasis[END_REF][START_REF] Lahdenranta | Endothelial nitric oxide synthase mediates lymphangiogenesis and lymphatic metastasis[END_REF][START_REF] Liu-Smith | Updates of reactive oxygen species in melanoma etiology and progression[END_REF][START_REF] Parascandolo | Carcinogenesis and Reactive Oxygen Species Signaling: Interaction of the NADPH Oxidase NOX1-5 and Superoxide Dismutase 1-3 Signal Transduction Pathways[END_REF]. ROS are mainly produced by NADPH oxidases (NOX) and the mitochondrial respiratory chain while NO is generated by nitric oxide synthases (NOS). The production of ROS is also high in tumor cells as a consequence of increased metabolic rate [START_REF] Scharping | Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion[END_REF]. To enable better survival, tumor cells counterbalance the high ROS levels by overexpression of antioxidant enzymatic pathways. Consequently, any change in pro-oxidant (higher ROS) or antioxidant (lower ROS) level often modifies cancer cell proliferation as observed in melanoma cells [START_REF] Parascandolo | Carcinogenesis and Reactive Oxygen Species Signaling: Interaction of the NADPH Oxidase NOX1-5 and Superoxide Dismutase 1-3 Signal Transduction Pathways[END_REF][START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF][START_REF] Zheng | Ultrasound and Near-Infrared Light Dual-Triggered Upconversion Zeolite-Based Nanocomposite for Hyperthermia-Enhanced Multimodal Melanoma Therapy via a Precise Apoptotic Mechanism[END_REF].

Both NOX and NOS are often overexpressed enzymes in tumor cells [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF][START_REF] Panday | NADPH oxidases: An overview from structure to innate immunity-associated pathologies[END_REF][START_REF] Roy | NADPH oxidases and cancer[END_REF] and have different modes of regulation. However, they share the common cofactor NADPH as the source of electrons to initiate their catalysis. The NADPH binding sites of NOX and NOS comprise two subdomains, first, a chemically active one where reducing equivalents are injected into the primary electron acceptor FAD by the nicotinamide moiety of NADPH, and a second spatially separated subdomain, where recognition of conserved arginine and lysine residues of the protein binds to the 2′-phosphate group of NADPH [START_REF] Li | Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation[END_REF]. We have designed and synthesized a fluorescent NADPH analogue called nanoshutter NS1 (Scheme 1) in which the recognition motif of the adenosine monophosphate moiety of NADPH was conserved (red box) and the nicotinamide pyrophosphate moiety of NADPH was substituted with a chromophore (green box). This chromophore consists of an (E)-stilbene substituted by an acceptor para-nitro group at one end and by a para-tertiary amine donor group at the other end, making this chromophore a push-pull moiety unable to deliver the electrons required for NOX or NOS catalysis. NS1 was synthesized as a mixture of two constitutional isomers bearing the phosphate group at positions 2′ and 3′ of the ribose moiety [START_REF] Li | Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation[END_REF]. As expected from its design, NS1 is an inhibitor of the activity of constitutive recombinant NOS. It has been shown to reduce NO formed in aortic rings, and inhibit angiogenesis in a model of VEGFinduced endothelial tubes formation. Moreover, NS1 induces cell death by inhibition of NOX in melanoma cells without impeding the growth of healthy melanocytes [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF][START_REF] Rouaud | Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue[END_REF].

Moreover, NS1 has interesting fluorescent properties, being non-fluorescent in aqueous solutions and becoming fluorescent upon binding to e/nNOS [START_REF] Li | Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation[END_REF]. NS1 also inhibits NOX2 in activated macrophages [START_REF] Lejal | Turning off NADPH oxidase-2 by impeding p67(phox) activation in infected mouse macrophages reduced viral entry and inflammation[END_REF].

Here, we explore the specificity of NOX and NOS for each isolated isomer of NS1, carrying either a 2′-phosphate, NS1-2, or a 3′-phosphate, NS1-3. We aimed at testing whether NOS and/or NOX would distinguish between the 2′ from the 3′ isomer of NS1 in their inhibition of recombinant NOS and NOX2 C-terminal (cytosolic part, residues 300-580) activities, and their effects on the viability and migratory properties of melanoma and breast cancer cells. Additionally, we investigated the fluorescence properties of the two NS1 isomers upon binding to NOX2 in vitro and in vivo. Enhanced fluorescence properties were expected, therefore, this study aimed at providing a first of a kind fluorescent NOS/NOX inhibitor.

Experimental Procedures

General Methods

1 H and 31 P NMR spectra were recorded at room temperature on an Avance II 500 MHz spectrometer (Bruker, Wissembourg, France) using deuterated DMSO as solvent. Chemical shifts δ are reported in ppm relative to the non-deuterated solvent signal and coupling constants, J, are in Hz. We assessed the attribution of chemical shifts based on 1and 2-dimension (TOCSY, HSQC and HMBC) NMR experiments. High-resolution mass spectra (HRMS) were obtained by ESI in time-of-flight detection mode on a LCT (Waters-Micromass) spectrometer at ICSN (Gif sur Yvette, France).

Materials

Arginine, hemoglobin, superoxide dismutase (SOD), catalase, (6R)-5,6,7,8-tetrahydrobiopterin (H4B), NADPH, porcine brain calmodulin (CaM) and all common salts and buffers were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Antibodies against NOX1 (17772-1-AP), NOX2 (19013-1-AP), NOX3 (20065-1-AP) were purchased from Proteintech, (Euromedex, Souffelweyerheim, France) the antibody against NOX5 (ab191010) was obtained from Abcam (Paris, France).

Recombinant NOX2 C-Terminal Expression and Purification

The C-terminal of human NOX2 (residues 300-580) was purchased from MyBio-Source, San Diego, CA, USA. It was expressed in E. coli Rosetta (DE3) bacterial strain at 37 °C for 3 h. The expressed 5′ His-tagged protein had a molecular weight of 35.2 KDa. It was found in the non-soluble fraction and required inclusion body purification as detailed by the manufacturer.

Synthesis of NS1 and Separation of Isomers

NS1 (1 g) was synthesized by Innoverda (Villejuif, France) using a previously described protocol [START_REF] Rouaud | Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue[END_REF] and purified by chromatography over C18-cartridge to a single peak at (M-H)-= 653 by mass spectroscopy. The analytical separation of each NS1 isomer was performed on a HPLC system consisting of an Agilent pump connected to a Kromasil 5 µ C18 (250 × 46 mm) column and an Agilent detector set at λ = 445 nm. Data acquisition and treatment were performed with Agilent software. The solvent was a mixture of A: water + 0.1% formic acid, and B: acetonitrile + 0.1% formic acid with the following gradient: 0 to 22 min, 90% A; 22 to 27 min, 20% A; 27 to 38 min, 90% A. Flow rate was 1 mL/min and injection volumes were 20 µL. The preparative separation of the isomeric mixture was performed on a preparative pre-packed C18-grafted (50 µM, 30 g) column after preconditioning with a mixture of water/acetonitrile (95:5) containing 0.2% ammonium hydroxide solution (20%), followed by elution with a mixture of water/acetonitrile (85:15) containing 0.2% ammonium hydroxide solution (20%). Ten mL fractions were collected. Pure NS1-3 fractions were collected in fraction 11-15 and enriched NS1-2 isomer in fraction 21-23. The compounds were isolated after partial evaporation followed by lyophilization. NS1-2′phosphate isomer (NS1-2) and NS1-3′phosphate isomer (NS1-3) were recovered ~85% and >95% pure, according to analytical HPLC, 1 H and 31 P NMR spectroscopy (Supplementary Figures S1-S6). Due to the higher purity of NS1-3, full identification of H-atoms of its ribose moiety is shown in Figures S4 andS5.

Viability of Melanoma Cells

Human A375 (CRL-1619) melanoma cells were purchased from American Tissue Culture Collection (Molsheim, France). They were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO 2 . The effects of NS1 isomers on melanoma cells viability were measured by using the trypan blue dye exclusion method as previously described [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF][START_REF] Lejal | Turning off NADPH oxidase-2 by impeding p67(phox) activation in infected mouse macrophages reduced viral entry and inflammation[END_REF].

Viability of Breast Cells, Multiple Myeloma and Colon Cancer Cells

The human breast cancer cells-MDA-MB231 were obtained from American Type Culture Collection (ATCC, LGC Standards, Molsheim, France). U266 myeloma cells were a gift from Michel Renoir, Institut Gustave Roussy, Villejuif, France and were grown in RPMI medium supplemented with 1% FBS, HTC116 colon colorectal cancer cells were a gift from Bert Vogelstein (John Hopkins, Baltimore, MD, USA) and grown in McCoy medium supplemented with 1% FBS. The effects of NS1 isomers on these cancer cells' viability were measured by using the trypan blue dye exclusion method as previously described [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF][START_REF] Lejal | Turning off NADPH oxidase-2 by impeding p67(phox) activation in infected mouse macrophages reduced viral entry and inflammation[END_REF] or using the crystal violet method.

Melanoma Cell Migration in a Boyden Chamber

Cell migration was assessed by trans-well assay (Boyden chamber, Dutscher Issy les Moulineaux, France) according to the manufacturer's instructions. Briefly, 100,000 cells in serum-free media with or without drug were plated in the upper chamber on membranes with a pore size of 8 µm while the lower chamber contained culture media with FBS (10%) as chemoattractant. After 5 h, remaining cells were removed from the top-side of the inserts whereas migrating cells on the bottom of the inserts were stained with Diff-Quik (ThermoFischer Scientific, Waltham, MA, USA) and all migrating cells were counted. Results are expressed as means ± SEM and represent triplicate samples from at least two independent experiments.

Breast Cancer Cells Migration

Breast cancer SUM159PT cell line was provided by Dr P. Benaroch (Cellular Transport and Immunity Team, Curie Institute, Paris, France). SUM159PT cells were maintained in Ham's F12 medium supplemented with 5% FBS, 1 µg/mL hydrocortisone, 10 mM HEPES and 5 µg/mL of human insulin. Migration assays were performed using xCELLigence real-time cell analysis technology (Roche, Basel, Switzerland). The xCELLigence ® RTCA instrument uses noninvasive electrical impedance monitoring to quantify cell invasion and migration (CIM) using an electronically integrated Boyden chamber (CIM-Plate ® 16) in kinetic measurements. All data were recorded and analyzed by the TRCA software. The cell index (CI) value, which is directly influenced by cell spreading and/or proliferation, was used to measure the change in the electrical impedance divided by the background value. For cell migration, cells in serum-free medium were added to the upper well of the CIM plate, uncoated basement membrane matrix. SUM159PT cells were treated with 50 µM NS1 (mixture of isomers), NS1-2 or NS1-3 or L-NAME (100 µM) for 2 h in the absence of serum (see Figure S10). After treatment, cells were washed with DPBS, harvested with trypsin and added in serum-free media to the upper well of CIM plate. The plate was incubated for 24 h and the passage of cells through the porous membrane was measured as an impedance value, expressed as the cell index (CI).

Western Blots: SUM159 (aggressive breast cancer) were used to investigate the effect of NS1, NS1-2 and NS1-3 on the abundance of NOX isoforms. Cell extracts were obtained after lysis with RIPA buffer (0.5% sodium deoxycholate, 50 mM Tris-HCl; pH 8, 150 mM NaCl, 1% NP40, 0.1% SDS) supplemented with protease and a phosphatase inhibitor cocktail (Roche, Basel, Switzerland) and equal amounts of protein were loaded onto SDS-PAGE gels. After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with the appropriate antibody, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling, Danvers, MA, USA). Bands were visualized using the Clarity™ Western ECL substrate (BIO-RAD, Hercules, CA, USA). Protein expression was quantified by densitometric analysis of the immunoblots using Image Lab software developed by Bio-Rad.

Statistical Analysis

Data were expressed as mean ± SEM. Statistical analysis was performed using the Kruskal Wallis test with Dunn's post-test with GraphPad Prism 6 v6.01 (GraphPad Software, Inc., San Diego, CA, USA) or with a two-way ANOVA followed by Tukey's multiple comparisons test (GraphPad Prism version 8.4.2).

Effect of NS1-2 and NS1-3 on eNOS and nNOS Activity

Full length recombinant rat nNOS and bovine eNOS were expressed in Escherichia coli cell line BL21 (DE3) and purified using CaM-Sepharose column chromatography as described previously [START_REF] Moali | Substrate specificity of NO synthases: Detailed comparison of L-arginine, homo-L-arginine, their N omega-hydroxy derivatives, and N omega-hydroxynor-L-arginine[END_REF]. The initial rates of NO formation were determined at 37 °C in 1-cm pathlength cuvettes (total volume of 150 µL) using the oxyhemoglobin assay for NO [START_REF] Hevel | Nitric-oxide synthase assays[END_REF]. Usual incubation mixtures were performed in 50 mM Hepes buffer (pH 7.4) containing, 0.15 M NaCl, 5 mM DTT, 10-20 µM oxyhemoglobin, 100 U/mL each SOD and catalase, 10 µM H4B, 100 µM L-arginine, 1 mM CaCl2, 10 µg/mL CaM, 4-6 µg/mL n-or eNOS (in sample cuvette), and 0-100 µM NS1 isomers (dissolved at 10 mM in DMSO). The mixtures were preincubated for 2 min at 37 °C prior to initiation of the reactions by the addition of 10 µM NADPH to both cuvettes. The NO-mediated conversion of oxyhemoglobin to methemoglobin was monitored by repetitive scanning between 380 and 480 nm every 0.2 min on a Uvikon 941 spectrophotometer and quantitated using an extinction coefficient of 77 mM -1 •cm -1 between peak at 401 nm and valley at 420 nm. Control incubations were performed in the presence of similar amounts of DMSO but without NS1 isomers. All values are expressed relative to the control and are means + S.D. from 3-4 experiments.

Effect of NS1-2 or NS1-3 on Recombinant NOX2 C-Terminal Activity Determined by Fluorescence Measurements

The protein (5.5 µM) was incubated with 30 µM FAD for 1 h at room temperature and 1 µL of DMSO or NS1-2 or NS1-3 (5 mM in DMSO) 5 was added to the NOX2-FAD and put in a cuvette of a Uvikon 941 spectrophotometer using a reference cuvette containing 30 µM FAD in buffer. The reaction was initiated by the addition of NADPH (25 µM final concentration); 9 consecutive spectra were recorded during 40 min in the range of 500 to 250 nm. Typical results are shown in Figure 6 and correspond to the difference ΔOD monitored at the minima observed in the wavelengths 340-350 nm at time t minus recorded at time 0, immediately after addition of NADPH. The protein was stable for about 4 h.

Fluorescence spectra were measured at a fixed concentration of the compounds (1-2 µM) in 100 mM Tris buffer pH = 7.5, 150 mM NaCl with a PTI spectrophotometer set at T = 20 °C, with an excitation wavelength at 460 nm and emission wavelengths in the range 480-700 nm; slits of 10 nm were used at both excitation and emission. The protein was pre-incubated with FAD at an equimolar ratio for 15 min on ice just before the measurement.

Docking and MD Simulations

The ligands NS1-2 or NS1-3 were manually docked into the binding site of NADPH at the eNOS reductase domain previously obtained by homology modelling [START_REF] Lambry | Selective probing of a NADPH site controlled light-induced enzymatic catalysis[END_REF] with the rat nNOS-reductase domain as a template [START_REF] Garcin | Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase[END_REF]. The coordinates of the co-crystallized NADPH molecule were used to correctly superimpose the sugar-phosphate-adenine group of the isomers and NADPH. Moreover, the nitro group of the isomers was positioned in order to mimic the stacking of the nicotinamide group with the isoalloxazine moiety of the FAD molecule in the active site of eNOS.

Molecular Docking of the Ligand (NS1) to NADPH Oxidase-2 NOX2 (gp91 phox DH Domain) and Molecular Dynamics (MD) Simulations of the Resulting Docked Complex

The crystal structure of the C-terminal region (spanning the amino acid residues 385 to 570) of human gp91 phox (NOX2) protein was available from the Protein Data Bank (PDB 3A1F). First, the human sequence of the hNOX2 (code entry: P04839) was aligned with the sequence of the N-terminal part of CsNOX5 (code entry: Q96PH1) using the Align Sequences module of Discovery Studio 2020 v18 (Biovia, Dassault System, Paris, France). The sequence alignment indicated homology rates of 63.5%. The next step was to copy a set of constraints derived from the crystal structure of the CsNOX5 isoform (PDB code: 5o0X) to the corresponding residues of the sequences to be modeled using the Modeler module of Discovery Studio v18. The FAD molecule was conserved. Twenty models were generated. The model with the lower energy was selected for the next step. The selected structure was optimized according to the below procedure.

First, the compound NS1 was docked into the model using the flexible docking protocol of Discovery Studio v18. The resulting complex was then submitted to a 100 ns molecular dynamics simulation using the GROMACS 2018 package. A clustering (RMSD cutoff: 2 Å) was finally applied to extract the most representative structure of the simulation. The structure was validated using the Ramachandran plot generated by the RAMPAGE program. This plot indicates high quality stereochemical parameters with a very good distribution of φ and ψ angles. Indeed, more than 98% of the residues were located in the favored and allowed regions (Supplementary Figure S11). For the MD simulations of the system formed by the DH domain of NOX2, the cofactor FAD, and NS1-2 or NS1-3 bound to NOX2, each complex was centered in a cubic box with an edge length of 80 Å; the box was subsequently filled with water using the Simple Point Charge (SPC) model and the system was made neutral using the Genion tool of the GROMACS package [START_REF] Kumari | g_mmpbsa-A GROMACS tool for high-throughput MM-PBSA calculations[END_REF] (salt concentration of 0.15 mol L -1 ). The resulting system was then subjected to energy minimization (200 steps of steepest descent followed 200 steps of conjugate gradient until the maximum force was smaller than 1000.0 kJ mol -1 •nm -1 ) to remove steric clashes and to correct inappropriate geometry. Then, an equilibration step was applied to equilibrate the solvent and ions around the complex. Equilibration was carried out in two phases. The first was conducted in an NVT ensemble (N, constant number of particles, V, volume, and T, temperature) to reach a temperature of 310 °K using a velocity rescaling thermostat. The second phase was conducted in an NPT ensemble to equilibrate the system under 1 atm pressure using the v-rescale thermostat and the Parrinello-Rahman barostat. All equilibration phases were carried out for 2 ns. Upon completion of the equilibration step, the position restraints of the entire system were released for 20 ns production of molecular dynamic simulation (MDS) runs.

In Vivo Experiments

Female nude mice (Hsd Athymic Nude-Foxn1nu, Envigo, Gannat, France, 8 weeks old), were injected by intra-peritoneal (IP) route with NS1 (40 mg/kg) or with the vehicle (PBS 1X). The in vivo fluorescence was detected with an IVIS camera and monitored as a function of time. The peak of fluorescence was detected after 2-3 h and decreased afterwards, some signal was still detected after a day. Tissue fluorescence acquisitions were performed with the appropriate settings for NS1 (excitation: 465 nm, back-ground excitation: 415 nm and emission: 620 nm, acquisition time: 2 s). The fluorescence image was merged to a visible light image to delineate regions of interest (ROI) that perfectly fit to tissue shape. After subtraction of autofluorescence (time 0), ROI specific-NS1 fluorescence was expressed as ph/s/cm 2 /sr (M3 Vision software, version 1.1.2.26170, Biospace Lab, Nesles-la-Vallée, France).

Western Blots

Tissues were grounded in RIPA 1X buffer, supplemented with protease (cocktail of protease inhibitors (1/200, P8340, Sigma-Aldrich, Saint Quentin Fallavier, France) and phosphatase inhibitors (1% PMSF, 1 mM NaF, 100 µM NaVO3, 40 mM β-glycerophosphate), using Precellys homogenizer and ceramic beads (Bertin Technologies, Montignyle-Bretonneux, France). Total protein concentration was determined with Bio-Rad protein assay. The WB were performed as described above. The mouse anti-NOX2 antibody was used in the following conditions: 1/1000, (PA1667, Boster Bio, Pleasanton, CA, USA) and HRP secondary antibody 1/3000 (4030-05, Southern Biotech). NOX2 was detected with Immobilon Western Chemiluminescent HRP Substrate (Milipore, Guyancourt, France) and the PXi chemiluminescent imager (Syngene, Cambridge, UK). Relative NOX2 quantity was determined by densitometry using ImageJ software (NIH) and normalized to total transferred proteins stained with amido black reagent.

Results and Discussion

The synthesis of NS1 yielded two isomers in a 40:60 ratio. These isomers bear the phosphate group at positions 2′ and 3′ of the ribose moiety, respectively (Scheme 1) [START_REF] Li | Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation[END_REF]. Successive passages of NS1 on a preparative C18-Kromasil MPLC column enabled the separation of the two isomers (Scheme 1). Compounds NS1-2 and NS1-3 were recovered at a purity of ~85 and >90%, respectively, according to 1 H and 31 P NMR and HPLC analysis (see Supplementary Materials and Supplementary Figures S1-S6). They were found to be stable for weeks at room temperature in DMSO by 1 H NMR.

NS1-2 and NS1-3 as Fluorescence Probes In Vitro

We tested whether NS1 becomes fluorescent by binding to NOX. To do so, we used the recombinant human NOX2 C-terminal (residues 300-580) for a challenging expression and purification thereof (see Experimental section). This protein expressed in E. coli was found to be relatively stable over a period of 4 h at 20 °C (see below).

Interestingly, binding to the NOX2 C-terminal of both NS1-2 and NS1-3 increased their fluorescence (Figure 1) whereas both NS1-2 and its isomer were not fluorescent in buffer. Moreover, the apparent KD of NS1-2 for NOX2 was somewhat lower than that for its NS1-3 isomer, although the value should be taken with appropriate caution, due to the relative low stability of the protein. The large enhancement of fluorescence of these two isomers may serve as useful probes of (mainly) NOX and NOS, considering that an enhancement of NS1 fluorescence was observed upon binding to recombinant e/nNOS [START_REF] Li | Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation[END_REF].

The assumption is consistent with previous cellular studies [START_REF] Rouaud | Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue[END_REF][START_REF] Lejal | Turning off NADPH oxidase-2 by impeding p67(phox) activation in infected mouse macrophages reduced viral entry and inflammation[END_REF].

Fluorescence Properties of NS1 In Vivo

These experiments called for testing NS1 imaging properties in vivo. As both NS1-2 and NS1-3 cannot be distinguished in terms of fluorescence yield (Figure 1), we used NS1, the mixture of the two isomers. To do so, we administrated NS1 to nude mice by intraperitoneal injection. As it can be clearly seen in Figure 2, the fluorescence gradually built up with time and reached a plateau after 2-3 h, where the fluorescence intensity was about four-fold higher than the intensity monitored at t = 0. Further monitoring showed a slow decrease in the fluorescence up to 24 h. As the fluorescence of NS1 is due to the chromophore part of the molecule, these data indicated that NS1 chromophores remained relatively long-lived in vivo.

Moreover, after sacrificing the animals at 4 h or 24 h, Figure 3 shows that the fluorescence was concentrated in certain organs such as the kidney, the liver and the pancreas, with little fluorescence in the lung, heart or spleen. The abundance of NOX isoforms is not homogeneous, rather, it is concentrated in certain organs [START_REF] Panday | NADPH oxidases: An overview from structure to innate immunity-associated pathologies[END_REF]. We detected NOX2 by Western blots in the organs with enhanced fluorescence.

At 4 h post-injection, NS1 mainly accumulated in the kidney where NOX4 is expressed [START_REF] Gorin | Nox4 and diabetic nephropathy: With a friend like this, who needs enemies? Free Radic[END_REF]. This result is consistent with NS1 inhibition of overexpressed NOXs [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF]. We also observed some NS1 in the liver at 4 h, in agreement with NOX2 expressed in hepatocytes [START_REF] Panday | NADPH oxidases: An overview from structure to innate immunity-associated pathologies[END_REF]. It is difficult to explain why NS1 was not found in the lungs and in the spleen at 4 h as these organs express NOX isoforms, in particular NOX3 and NOX5. The most remarkable accumulation of NS1 was found in the pancreas at 24 h, an organ in which NOX2, NOX4, DuOX2 and e/nNOS are expressed. NOX2 was assessed as the major NOX isoform in human islet cells, with the contribution of NOX4 in glucose-stimulated insulin secretion [START_REF] Plecita-Hlavata | Glucose-Stimulated Insulin Secretion Fundamentally Requires H2O2 Signaling by NADPH Oxidase 4[END_REF], NOX2 exerts a negative modulator role of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation in pancreatic β-cells [START_REF] Li | NADPH oxidase NOX2 defines a new antagonistic role for reactive oxygen species and cAMP/PKA in the regulation of insulin secretion[END_REF]. Thus, the accumulation of NS1 in the kidney and pancreas may be due to increased levels of NOX and e/nNOS in these organs. As NS1-2 and NS1-3 were both fluorescent-bound to NOX2, we further explored their specificity for NOX and NOS in cellular and in vitro studies. S7. The staining with amido black of the total proteins transferred onto the nitrocellulose membrane is used to normalize the levels of the protein of interest (i.e., NOX2) in each tissue analyzed. This normalization was used because any housekeeping proteins, such as β-actin or β-tubulin, show variable levels between tissues. Statistical significance indicated by stars (** p-value < 0.01) was evaluated by a two-way ANOVA followed by Tukey's multiple comparisons test. A significant accumulation of NS1 was observed in the kidney after 4 h post-injection while at 24 h post-injection, NS1 fluorescence was significantly increased in the pancreas. No significant fluorescence of NS1 was noted in all other tissues analyzed.

Cellular Studies

The ability of the two separated isomers to induce melanoma cell death was tested. Previous studies have shown that the effects of NS1 on melanoma cell viability were mainly dependent on NS1 inhibition of NOX1/2/4 [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF][START_REF] Rouaud | Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue[END_REF]. Figure 4 shows that only 29 ± 2% of the melanoma cells survived treatment in the presence of 30 µM NS1-2 for 48 h. By contrast, NS1-3 was less efficient and 60 ± 2% melanoma cells survived. Consistently, NS1, added as a mixture of isomers, displayed a mean efficacy and reduced the cell viability to 48 ± 2% as previously reported. Kinetic studies showed that a decrease in ROS levels was observed in parallel with the inhibition of NOX1, NOX2 and NOX4 and with a cell viability decrease in multiple melanoma cell lines and cells from patients upon treatment with NS1 [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF]. In contrast to A375 and other melanoma cells [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF], NS1 did not reduce the viability of the other cancer cell lines assayed by trypan blue or crystal violet staining: U266 (myeloma), HCT116 (colon cancer) and aggressive breast cancer cells (MDA-MB-231), cells (see Supplementary Figure S8). Therefore, the decrease in melanoma cell viability, tested on different cell lines and primary cells from patients seems to be specific to melanoma cells among the four cancer cell lines tested [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF][START_REF] Rouaud | Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue[END_REF].

Overexpression of multiple NOX isoforms, including NOX2, NOX4 and NOX5, as well as e/nNOS and iNOS were reported to enhance breast cancer cells tumorigenesis and invasion [START_REF] Parascandolo | Carcinogenesis and Reactive Oxygen Species Signaling: Interaction of the NADPH Oxidase NOX1-5 and Superoxide Dismutase 1-3 Signal Transduction Pathways[END_REF][START_REF] Panday | NADPH oxidases: An overview from structure to innate immunity-associated pathologies[END_REF][START_REF] Roy | NADPH oxidases and cancer[END_REF][START_REF] Smeda | Nitric oxide deficiency and endothelial-mesenchymal transition of pulmonary endothelium in the progression of 4T1 metastatic breast cancer in mice[END_REF][START_REF] Zaoui | Breast-Associated Adipocytes Secretome Induce Fatty Acid Uptake and Invasiveness in Breast Cancer Cells via CD36 Independently of Body Mass Index, Menopausal Status and Mammary Density[END_REF]. Indeed, SUM159 cells and other aggressive cells such as MDA-MB-231 cell lines exhibited significant levels of NOX5, NOX4, NOX2 and nNOS mRNA (Supplementary Figure S9). Therefore, we tested the effect of the 2′-and 3′-phosphate isomers of NS1 in migration assays with both melanoma A375 (Figure 5) and breast SUM159 tumor cell lines (Figure 6), and analyzed the level of various NOX isoforms by Western blots. The migration assay showed a significant decrease in melanoma cells migration when treated with 30 µM NS1 for 24 h. The isomers 2′ or 3′ induce a similar decrease in migration to NS1 within experimental error. In these conditions of treatment, NS1 reduced the levels of ROS formed by multiple NOX isoforms found in melanoma cells, including NOX1/2 and NOX4 [START_REF] Rouaud | Mechanism of melanoma cells selective apoptosis induced by a photoactive NADPH analogue[END_REF].

In an additional assay using the xCELLigence ® system with an integrated electronic Boyden chamber (detailed in the Experimental section), the migration capacity of the aggressive breast SUM159 cancer cells was determined by a change in the impedance of a membrane through which the cells migrated. As shown in Figure 6, NS1-2 significantly decreased breast tumor cell migration by 71 ± 2% and 55 ± 3% after 12 h and 24 h, respectively. The 3′-isomer NS1-3 only reduced the SUM159 cell migration by 12 ± 3% and 20 ± 4% after 12 h and 24 h, respectively (see also Supplementary Figure S10 for raw data and details of the difference in migration of each isomer relative to the control as a function of time). Moreover, Western blot analysis showed a decrease of 40% in NOX2 levels in NS1treated SUM159 cells during 24 h (Figure 6B). Treatment of these breast cancer cells with 100 µM L-NAME, a NOS inhibitor, gave similar results as NS1-2 did (Supplementary Figure S10). We further tested whether NOX2 and other NOX abundance in SUM159 cells was affected by 48 h treatment with NS1 or its isomers. The level of NOX3 was most decreased by NS1-2 (50 µM), with smaller effects on NOX2 and NOX1 levels. The level of NOX5 was minimally modified by NS1-2, and NS1-3 seemed to be more effective.

Taken together, melanoma and breast cancer cells migration was inhibited by NS1 and its isomers. The efficacy of NS1-2 was greater than that of NS1 and NS1-3 in regard to inhibiting breast cancer cells migration. In contrast to NS1, which only decreased melanoma cells proliferation, NS1 and NS1-2 had no effect on the proliferation of breast MDA-MB231 cancer cells, HTC116 colon cancer and U266 myeloma cancer cell lines (Supplementary Figure S8). WB analysis of SUM159 breast cancer cells showed decreased levels of NOX isoforms, in particular NOX3, NOX2 and NOX1 by NS1-2 treatment. Both NOX1 and NOX2 are affected by NS1 inhibition in melanoma and breast cancer cells.

In Vitro Recombinant Enzymatic Activities Measurements

To further confirm that the difference between the two isomers was indeed due to a difference of binding to NOS present in SUM159 cells, we further compared the ability of NS1-2 and NS1-3 to inhibit recombinant e/nNOS and NOX2 activity in vitro [START_REF] Li | Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation[END_REF][START_REF] Rouaud | Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue[END_REF]. NS1-2 inhibited eNOS more efficiently than NS1-3 did (IC50 ~30 µM and >100 µM, respectively, Figure 7A). Similar results were obtained with recombinant nNOS (not shown). We further tested the effect of the two isomers on recombinant human NOX2 C-terminal. This protein expressed in E. coli was found to be relatively stable over a period of 4 h at 20 °C, but its activity was lost at more prolonged times. Nevertheless, freshly prepared NOX2 samples showed consumption of NADPH, evidenced by the decrease in the absorption of NADPH around 340 nm (Figure 7B). In the presence of NS1-2, the NADPH consumption markedly decreased while the opposite was observed in the presence of NS1-3, suggesting that NOX2 may distinguish between the two isomers, in favor of NS1-2.

Docking and MD Simulations

In order to get insight into the origin of the differences between the 2′-and 3′-phosphate isomers binding to their target proteins, NOX and NOS, we docked the two isomers in the simulated structure of NOX2, obtained by homology modelling (Supplementary Figure S11) based on PDB 3A1F (partial structure of human NOX2) and 5O0X [START_REF] Magnani | Crystal structures and atomic model of NADPH oxidase[END_REF] and on the structure of the reductase domain of eNOS, previously obtained by homology modelling based on PDB 1TLL [START_REF] Garcin | Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase[END_REF][START_REF] Magnani | Crystal structures and atomic model of NADPH oxidase[END_REF]. Isolated NS1-2 in solution adopted an extended conformation with its adenosine moiety away from the chromophore (Scheme 1). NS1-2 bound to NOX2 via its 2′ phosphate moiety, forming a strong H-bond with R446 (Figure 8A). The adenosine moiety was held extended by interactions with A409, L444 and A451. The chromophore group stacked with FAD isoalloxazine ring and experienced hydrophobic interactions with P539, F340, P415, T341 and F570. Isolated NS1-3 in the solvent was mainly folded due to adenine folding over the chromophore (Scheme 1). The same holds true when bound to NOX2 via its 3′ phosphate moiety to R446. NS1-3 adenine folding back was stabilized by I411, an H-bond between the nitro group and G412 and induced a deviation of the chromophore from planarity (Figure 8B). This folded structure likely created a steric hindrance for binding to the protein. Accordingly, the NS1-3-NOX2 complex exhibited a lower interaction energy as compared to the NS1-2-NOX2 complex (-44 versus -55 Kcal/mole, respectively).

Binding to eNOS was mediated by the strong interactions of the 2′-and 3′-phosphate groups of NS1-2 and NS1-3 with the conserved arginine residues R1078 and R1048 (Figure 9). Constrained by the strongest electrostatic arginine-3′-phosphate interaction, the adenine had rearranged. This rearrangement, at least in part, drove the chromophore orientation and further interaction of the chromophore with FAD. NS1-3 was oriented in a parallel conformation relative to the plane of FAD isoalloxazine rings rather than being perpendicular as NS1-2 was. Consequently, the adenine folded back onto the chromophore, held in place by hydrophobic interactions, a folded structure also observed in photoactive NADPH analogue [START_REF] Beaumont | Synchronous photoinitiation of endothelial NO synthase activity by a nanotrigger targeted at its NADPH site[END_REF] carrying a 3′-carboxylate instead of a 3′-phosphate group [START_REF] Dilly | Design of Light-Sensitive Triggers for Endothelial NO-Synthase Activation[END_REF]. It was reported that NOX2 binds a NADPH mimetic where a bulkier azidonitrophenyl-aminobutyryl group substituted its 3′-OH group with a decrease of only 1.9 folds as compared to NADPH itself [START_REF] Laporte | Characterization of multiple active forms of the NADPH dehydrogenase component of the oxidase complex from rabbit peritoneal neutrophils by photolabeling with an arylazido derivative of NADP+[END_REF], indicating little effect of steric hindrance at the 3′-position in the presence of a 2′-phosphate group linked to the flexible nicotinamide. The relative rigidity of the amide group linking the 2′-phosphate-AMP group to the chromophore also contributed to the good insertion of NS1-2 within the NADPH site of eNOS and NOX2.

Scheme 1 .

 1 Scheme 1. Structures of the NADPH analogues NS1 isomers, NS1-2 and NS1-3, which differ by the position of their phosphate moiety, with differences in their conformation (extended or folded) as shown by molecular modeling.

Figure 1 .

 1 Figure 1. Fluorescence enhancement of NS1-2 and NS1-3 upon binding to recombinant NOX2 Cterminal; the protein was complemented with FAD (at 1/1 ratio), Excitation at 460 nm, emission recorded in the range 480-700 nm. A broad maximum emission was detected at (560-580) nm. The data were corrected for the contribution of FAD by subtracting the signal obtained with FAD in buffer at each NOX2 concentration. The data are fitted to a dose-response curve embedded in Origin 8.1 software with the following parameters: NS1-2: A1 (lower asymptote) = 0, A2 (upper asymptote) = 212 ± 11, KD = 0.67 ± 0.11 µM, R 2 = 0.965; NS1-3: A1 = 0, A2 = 244 ± 14, KD = 1.14 ± 0.09 µM, R 2 = 0.984.

Figure 2 .

 2 Figure 2. NS1 monitoring in vivo by an IVIS in vivo imaging system after a single IP injection in nude mice (NS1 concentration was 40 mg/kg in PBS). (A,B) Time course of NS1 fluorescence was monitored by in vivo imaging (the excitation was at 465 nm and the emission at 620 nm) by IVIS for 24 h.

Figure 3 .

 3 Figure 3. (A) NS1 fluorescence (excitation at 465 nm, emission at 620 nm) in selected organs of the mice after sacrifice at 4 h and 24 h post-IP relative to vehicle alone and their quantification. (B) NOX2 levels at 4 h in these organs determined by Western blots and their quantification relative to amino black presented in Supplementary FigureS7. The staining with amido black of the total proteins transferred onto the nitrocellulose membrane is used to normalize the levels of the protein of interest (i.e., NOX2) in each tissue analyzed. This normalization was used because any housekeeping proteins, such as β-actin or β-tubulin, show variable levels between tissues. Statistical significance indicated by stars (** p-value < 0.01) was evaluated by a two-way ANOVA followed by Tukey's multiple comparisons test. A significant accumulation of NS1 was observed in the kidney after 4 h post-injection while at 24 h post-injection, NS1 fluorescence was significantly increased in the pancreas. No significant fluorescence of NS1 was noted in all other tissues analyzed.

Figure 4 .

 4 Figure 4. Effects of 30 µM NS1 (as a 40:60 mixture of isomers) and NS1-2 and NS1-3 on A375 melanoma cells viability after 48 h of culture. Representative data of 3 experiments in triplicate. The mean values of 5 experiments at 72 h are the following: NS1: 69 ± 4%, NS1-2: 59 ± 7%, NS1-3: 66 ± 11% (data not shown).

Figure 5 .

 5 Figure 5. Migration of A375 cells treated or not with NS1. Left panel: Serum-stimulated migration assays were performed in Boyden chambers. A total of 100,000 A375 melanoma cells were incubated with NS1 or its separated isomers (30 µM) or with control for 15 h or 24 h. Then, the pre-treated cells were plated in the upper chamber in serum-free medium, while the lower chamber contained culture media with 10% serum to stimulate migration for 5 h. Representative images of the lower surface of membranes are shown; the two incubation times were expected to yield similar results due to a fast penetration (within minutes) of NS1 in cells, and likely reflect the experimental variation. Right panel: the mean migration of the cells is shown in triplicates from two independent experiments; error bars, SEM. p-values indicate the difference between the migration of control cells and the treated cells and were calculated by the Kruskal Wallis test with Dunn's post-test (GraphPad Prism).

Figure 6 .

 6 Figure 6. (A) Effect of 50 µM NS1 (mixture of isomers), NS1-2, NS1-3 on the migration of SUM159 breast cancer cells, see also Supplementary Figure S10; (B) Western blot analysis of NOX2 in SUM159 cells treated or not with NS1 (50 µM) and their quantification after 48 h treatment; (C,D) Representative Western blots of NOX2, NOX1, NOX3 and NOX5 in SUM159 cells treated or not with NS1 or its isomers for 48 h and their quantification.

Figure 7 .

 7 Figure 7. Effect of NS1 and its isomers on (A) eNOS activity determined by the hemoglobin assay [19]; (B) Representative absorption changes around 340 nm due NOX2-induced NADPH consumption using NOX2 = 2.5 µM reconstituted with 50 µM FAD, NADPH = 50 µM with or without 25 µM of NS1-2 or NS1-3 in 20 mM Tris buffer pH = 7.5, 150 mM NaCl.

Figure 8 .

 8 Figure 8. Comparison of the binding of NS1-2 (A, shown in cyan) and NS1-3 (B, shown in yellow) to a model of NOX2. For clarity, only FAD, and residues involved in the ligands binding are shown. ADE stands for adenine. (C) Zoom-in of the phosphate interaction with lysine/arginine residues, driving the recognition of NOS and NOX.

Figure 9 .

 9 Figure 9. Binding of NS1-2 (left, shown in blue) and NS1-3 (right, shown in green) to eNOS reductase domain; the main residues involved in the interactions with eNOS are labeled. ADE stands for adenosine.

Acknowledgments:

We thank Yannick Boursin, Daniel Gautheret, and the Bioinformatics core of Gustave Roussy for granting access to the cluster Cigogne. We thank Julien Henri for access to the PTI fluorimeter at the Institut de Biologie Physico-Chimique, Paris.

Data Availability Statement: Data is contained within the article and supplementary material.

Conclusions

We showed that NOX2 and e/nNOS distinguished between NADPH analogues bearing a 2′-or a 3′-phosphate group. This distinction was observed in vitro by enhanced physiological responses depending on NOS or NOX2, inducing a specific cell death of melanoma cells. We observed a clear reduction in the migration capacity of breast cancer cells treated with NS1-2. The same holds true for melanoma cells, but NS1, NS1-2 and NS1-3 were equally potent in decreasing their migration capacity, within experimental error.

It is interesting to note that NS1-2 inhibition of SUM159 breast cancer cells invasion was most efficient between 6 and 12 h and declined afterwards by 2.5-fold (Figure 5 and Supplementary Figure S10). NS1-3 also decreased this activity but only by 1.5-fold. As the activity of NS1 towards NOX2 in a cell-free assay was lost when its phosphate moiety was substituted by an OH group [START_REF] Lejal | Turning off NADPH oxidase-2 by impeding p67(phox) activation in infected mouse macrophages reduced viral entry and inflammation[END_REF], this result suggests that the phosphatase(s) found in breast cancer cells, likely has some specificity for 2′ over 3′ phosphate. Indeed, phosphatase activity can be enhanced by the decreased ROS levels due to NOX inhibition [START_REF] Chen | Downstream targets and intracellular compartmentalization in Nox signaling[END_REF]. Moreover, recent work has shown that local NADP(H) levels are regulated by a phosphatase targeting the 2′-phosphate of NADPH called Nocturnin, linking metabolism, and redox status with the circadian clock [START_REF] Laothamatas | Spatiotemporal regulation of NADP(H) phosphatase Nocturnin and its role in oxidative stress response[END_REF]. Thus, the distinction of 2′-phosphate from 3′phosphate could be shared by enzymes such as NOX and NOS and possibly some phosphatases [START_REF] Fukuda | NADP(H) phosphatase activities of archaeal inositol monophosphatase and eubacterial 3'-phosphoadenosine 5'-phosphate phosphatase[END_REF] involved in a selective catalysis [START_REF] Cahn | A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases[END_REF]. Accordingly, we show here a clear inhibition of recombinant n-and eNOS-and NOX-dependent activities by NADPH analogues bearing a 2′-phosphate moiety. The calculated structures of NS1-2 bound to eNOS and NOX2 supported these conclusions.

In vivo studies showed significant accumulations of NS1 and NOX2 in the pancreas at 24 h. NOX2 [START_REF] Li | NADPH oxidase NOX2 defines a new antagonistic role for reactive oxygen species and cAMP/PKA in the regulation of insulin secretion[END_REF], DuOX2 [START_REF] Panday | NADPH oxidases: An overview from structure to innate immunity-associated pathologies[END_REF] and eNOS and nNOS [START_REF] Buchwalow | New Insight into the Role of Nitric Oxide Pathways in Pancreas[END_REF] are expressed in the pancreas, with NOX and NOS contributing to normal and impaired beta-cells functions. Further indepth analysis of NOX1/NOX2/NOX4 profiles in mouse pancreas may shed more light on the role of NOX in metabolism and test whether NS1 could act as a metabolic fluorescent marker.

Taken together, this work highlights the highly valuable properties of NS1-2, that is, it combines in vivo fluorescence in selected organs, presenting a relatively long-lived pharmaco-kinetics profile while being non-fluorescent in aqueous buffer, with improved targeting of NOS and NOX in a cellular context. Work is in progress to test whether the interesting pharmacological activity of NS1-2 in vitro can also be found in vivo.

Supplementary Materials:

The following are available online at www.mdpi.com/2076-3921/10/5/723/s1, Experimental procedures and Figures S1-S11 are gathered in the Supplementary Material. Figures S1-S6 describe the separation of the two isomers and their chacracterization by HPLC and NMR, Figure S7 illustrates illustrates the amido black staining, as loading control of the WB anti-NOX2 in Figure 3B, Figure S8 presents cell viabilities of cancer cells treated or not with NS1 or its separated isomers using trypan blue, Figure S9 presents NOX and NOS QPCR, Figure S10 cell migration raw data and Figure S11 shows a plot attesting the quality of our NOX2 model.

Author Contributions:

The manuscript was written through contributions from all authors. All authors have given approval to the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding: Michelle de Souza Lima was supported by a fellowship from CAPES (CAPES/COFECUB French-Brazilian Collaborative Research Grant no Me917/18). This work was granted access to the CINES resources Occigen made by GENCI (Grand Equipment National de Calcul Intensif) under Grant AP010710343 attributed to Sébastien Dilly.

Institutional Review Board Statement: Experimental imaging procedure was approved by the local ethics committee for animal experimentation (CEEA14, Marseille, France) (N°APAFIS #28834-2021010412218928).

Informed Consent Statement: Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest.

Abbreviations

FAD

Flavin adenine dinucleotide L-NAME N(ω)-nitro-L-arginine methyl ester MD molecular dynamics NOX NADPH oxidase NOS NO-synthase NS1 nanoshutter 1 [START_REF] Rouaud | Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue[END_REF]