Charline Kieffer

Jean Pierre

Marie Jouanne

Anne Sophie Voisin-Chiret

Jean-Pierre Jourdan

Anne Sophie
email: anne-sophie.voisin@unicaen.fr

Voisin-Chiret Anne

Sophie Voisin-Chiret

Noncellular screening for the discovery of protein-protein interaction modulators

Keywords: protein-protein interaction modulators, noncellular assays, screening, high and low throughput, drug discovery

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

Introduction

PPIs (see Glossary) have an important role in the regulation of biological pathways, and PPI disorders are involved in the progression of many diseases [START_REF] Ryan | Protein-protein interactions in human disease[END_REF][START_REF] Li | The OncoPPi network of cancer-focused protein-protein interactions to inform biological insights and therapeutic strategies[END_REF]. According to Azzarito et al. [START_REF] Azzarito | Inhibition of alpha-helix-mediated protein-protein interactions using designed molecules[END_REF], the PPI network is estimated to involve >650 000 interactions, but only a small part of this complex network has been characterized so far (www.interactome-atlas.org). The interactome constitutes many potential therapeutic targets to discover new drugs with original mechanisms of action [START_REF] Scott | Small molecules, big targets: drug discovery faces the protein-protein interaction challenge[END_REF][START_REF] Fry | Targeting protein-protein interactions for cancer therapy[END_REF][START_REF] Arkin | Small-molecule inhibitors of protein-protein interactions: progressing toward the reality[END_REF][START_REF] Ran | Inhibitors of protein-protein interactions (PPIs): an analysis of scaffold choices and buried surface area[END_REF].

Targeting PPIs is a challenge: the surfaces involved are large and flat [START_REF] Hwang | Protein-protein docking benchmark version 4.0[END_REF], featureless, and often lacking in suitable pockets for small molecules to bind to [START_REF] Arkin | Small-molecule inhibitors of protein-protein interactions: progressing toward the reality[END_REF]. Recent progress was made to target challenging PPIs with synthetic molecules [START_REF] Chung | Targeting protein-protein interactions perspective[END_REF][10][START_REF] Wuo | Engineered protein scaffolds as leads for synthetic inhibitors of protein-protein interactions[END_REF], and some inhibitors are now in oncology clinical trials. For examples of molecules in clinical trials, see [START_REF] Zhao | Small-molecule inhibitors of the MDM2-p53 protein-protein interaction (MDM2 Inhibitors) in clinical trials for cancer treatment[END_REF][START_REF] Wang | Targeting the MDM2-p53 protein-protein interaction for new cancer therapy: progress and Challenges[END_REF] and https://clinicaltrials.gov/ct2/show/NCT02503423.

To access new PPI modulators (mPPIs), several drug discovery strategies have been used, and can be mixed [14]. HTS is the most common methodology that can rapidly identify small molecules as potential mPPIs [START_REF] Sharma | Protein-protein interactions: lessons learned[END_REF]. HTS includes the screening of not only classical molecule libraries, but also fragment libraries, and the fragment-based drug design (FBDD) strategy is being increasingly used to identify new mPPIs [START_REF] Magee | Progress in discovery of small-molecule modulators of protein-protein interactions via fragment screening[END_REF][START_REF] Valenti | Clinical candidates modulating protein-protein interactions: the fragment-based experience[END_REF]. However, the success of a HTS depends on whether the library contains putative hits, and it is well established that the chemical space of mPPIs differs from that of other ligands: many mPPIs do not fit with the Lipinski's rule of five and have higher molecular weight (MW) and higher hydrophobicity [18][19][START_REF] Ran | Inhibitors of protein-protein interactions (PPIs): an analysis of scaffold choices and buried surface area[END_REF]. Therefore, mPPIs libraries are more diversified. Villoutreix et al. [21] showed that mPPIs have a cumulated proportion of pan assay interference compounds (PAINS) equal to 23%, whereas it is only 5% for enzyme inhibitors. This leads to potentially finding more false positives in screening. The complexity of the screening process is also increased if the protein function is unknown or if no direct activity readout is accessible. Moreover, with the expansion of the FBDD method in the design of mPPIs, the screening of fragments, which have a low affinity for the target, requires the use of sensitive methods to detect and measure the interactions [START_REF] Nero | Oncogenic protein interfaces: small molecules, big challenges[END_REF].

The screening of mPPIs involves the use of noncellular in vitro assays using purified proteins, to determine the affinity of molecules. The hits are then confirmed by a screening on a cellular model, to check the disruption of the PPI in a cellular environment. In this review, we particularly focus on noncell-based in vitro screening assays, used for first-line screening (Figure 1 and Table 1). Cell-based assays are reviewed elsewhere [START_REF] Wade | Inhibition of protein-protein interactions: cell-based assays[END_REF]. Given that most mPPIs are developed against cancer, we provide examples of mPPIs in oncology to determine how to choose the most suitable noncell-based assay for the screening of small molecules targeting PPIs.

High-throughput assays

Mix-and-read assays

Fluorescence polarization assay First described in 1926 [24], the fluorescence polarization assay (FPA) is based on the ability of a small fluorescent molecule (ligand A), excited by a polarized light, to emit a depolarized light depending on the rotational diffusion. Free in solution, ligand A rotates rapidly, resulting in low polarization of the emitted light. When ligand A interacts with protein B, the heavier AB complex rotates more slowly and results in high polarization of the emitted light (Figure 2a). The FPA for PPI inhibitor screening tends to be carried out in a competitive inhibition mode. The presence of a mPPI releases the fluorescent ligand A from its binding site and a decrease in the emitted light depolarization is then recorded. This technique, based on rotational diffusion, is closely linked to the mass difference between the free fluorescent ligand A and the AB complex. Therefore, it requires the use of a fluorescent ligand A, small enough for the recorded polarization difference to be significant. For this, ligand A, which is fluorophore labeled, is often a truncated protein containing 'hot-spots', capable of interacting with B. FPA can be implemented using a microplate reader that includes polarizing filters.

The use of FPA in drug discovery targeting PPIs has been widely developed since the 2000s (e.g., see [START_REF] Knight | A fluorescence polarization assay for the identification of inhibitors of the p53-DM2 protein-protein interaction[END_REF][START_REF] Du | Monitoring 14-3-3 protein interactions with a homogeneous fluorescence polarization assay[END_REF][START_REF] Zhang | Development of a high-throughput fluorescence polarization assay for Bcl-xL[END_REF]). Nikolovska-Coleska et al. [START_REF] Nikolovska-Coleska | Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization[END_REF] have participated extensively in FPA deployment in PPI screening [START_REF] Rossi | Analysis of protein-ligand interactions by fluorescence polarization[END_REF]. Given that it is readily versatile to low volume and requires simple procedures without separation or washing steps, FPA is particularly suitable for HTS [START_REF] Jolley | Fluorescence polarization assays for the detection of proteases and their inhibitors[END_REF][START_REF] Lea | Fluorescence polarization assays in small molecule screening[END_REF][START_REF] Zhai | High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1[END_REF][START_REF] Du | Fluorescence polarization assay to quantify protein-protein interactions in an HTS format[END_REF]. Inexpensive, FPA is also mostly used by academic labs. Recently, in addition to existing methods, Zhou et al. [START_REF] Zhou | Fluorescence polarization assay for the identification and evaluation of inhibitors at YAP-TEAD protein-protein interface 3[END_REF] developed a FPA to screen YAP-TEAD interaction small inhibitors, as potential anticancer drugs targeting the Hippo pathway, specifically binding to the Ω-loop region on TEAD.

Among its drawbacks, FPA needs the introduction of fluorophore tags on the ligand, which can also induce nonnative binding properties. FPA is also limited by the aggregation of tested compounds, especially of compounds with hydrophobic moieties [START_REF] Shoichet | Screening in a spirit haunted world[END_REF]. The biggest hurdle to FPA is fluorescence interference [START_REF] Owicki | Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer[END_REF]. Using longer wavelength fluorophores are pertinent options for reducing fluorescence interference and light scattering [START_REF] Turek-Etienne | Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease, and 1 phosphatase[END_REF]. Amplified luminescent proximity homogeneous assay screen Amplified luminescent proximity homogeneous assay screen (AlphaScreen®) is a chemiluminescent technology, first licensed by PerkinElmer® for drug discovery applications in 1999 [START_REF] Eglen | The use of AlphaScreen technology in HTS: current status[END_REF]. AlphaScreen® uses a donor bead (DB) and an acceptor bead (AB). Excited at 680 nm, DB releases a singlet oxygen ( 1 O2). If AB and DB are close enough (<200 nm), this singlet oxygen excites AB, which emits a signal between 520 and 620 nm. Protein A is captured on the DB and protein B is captured on the AB. When A interacts with B, DB transfers the singlet oxygen to AB and the luminescent signal is registered (Figure 2b). At the 'hook' point, either the donor or the acceptor component is saturated with the target molecule and a maximum signal is detected. For screening, AlphaScreen® is used as a competitive binding assay: the presence of a mPPI prevents the interaction and induces a decrease in the signal.

Compared with the fluorescence (or Förster) resonance energy transfer (FRET) assay, AlphaScreen® detects advantageously the interaction between two proteins at a longer distance (200 versus 10 nm). Technically, this sensitive assay is easily performed in a microplate format without any wash or filtration steps, adaptable to many assay types. Therefore, it is well suited for HTS-compatible assays [START_REF] Yi | An AlphaScreen-based high-throughput screen to identify inhibitors of Hsp90-cochaperone interaction[END_REF][START_REF] Gul | Protein-protein interaction modulator drug discovery: past efforts and future opportunities using a rich source of low-and high-throughput screening assays[END_REF]. In a recent example [START_REF] Miller | Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors[END_REF], AlphaScreen® was used in a screening campaign of a chemical library containing ~90 000 compounds (associated with TR-FRET), to discover novel small molecules that disrupt the CD47-SIRPα interaction, involved in cancer as an innate immune checkpoint. Given that AlphaScreen® is an easy and adaptable method, it is also widely used in academic research, even for low throughput screening. For the discovery of XIAP inhibitors, Seigal et al. [42] used AlphaScreen® to assess the affinity of compounds for the XIAP-BIR2 domain, as an alternative to FPA, which is less sensitive [START_REF] Sun | Design, synthesis, and characterization of a potent, nonpeptide, cell-permeable, bivalent Smac mimetic that concurrently targets both the BIR2 and BIR3 domains in XIAP[END_REF] and requires larger protein amounts [START_REF] Flygare | Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152)[END_REF].

From a practical point of view, the technique needs a specialized plate reader, and the signal is temperature sensitive. DMSO (usually used to solubilize the molecules) also disturbs the signal, limiting its use [START_REF] Yasgar | AlphaScreen-based assays: ultra-high-throughput screening for small-molecule inhibitors of challenging enzymes and protein-protein interactions[END_REF]. Moreover, the signal is produced by a chemical reaction, leading to false positive, called 'hitters'. Frequent AlphaScreen® hitters were identified by Schropp et al. [46] and are listed in a specific open-access database (http://ochem.eu/alerts). After a structural analysis of >100 000 compounds from four HTSs using AlphaScreen®, the authors showed that hitters were often chelating agents, which contain elements that might quench excitation/emitted radiation or singlet oxygen, such as 8-hydroxyquinolines, picolylamines, or pyridine moieties.

Even though this technique is relatively expensive, mainly because of the cost of the beads, such costs can be greatly reduced after optimization of their concentration [START_REF] Veloria | Development of a cost effective and robust AlphaScreen® platform for application[END_REF]. Fluorescence (or Förster) resonance energy transfer The basic principle of resonance energy transfer (RET) was first described by Förster in 1948 [START_REF] Förster | Zwischenmolekulare Energiewanderung und Fluoreszenz[END_REF], and comprises nonradiative energy transfer between an excited probe, called the 'donor' and an accepted probe, called the 'acceptor'. This energy transfer, called FRET, occurs when the donor and the acceptor are <10 nm from each other, which results in a reduction in donor emissions and an increase in acceptor emissions. FRET can occur if the emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore. In a screening assay, each protein partner, A and B, is fused with a fluorescent protein [i.e., Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP), respectively]. If A interacts with B, CFP and YFP are close enough for FRET to occur, leading to an emission in the YFP wavelength (530 nm). The presence of a mPPI abolishes the FRET signal by dissociating the AB complex, resulting in an emission at the CFP wavelength (480 nm) (Figure 3a). A and B can be directly labeled with a FRET donor and acceptor, using reactive functional groups on the fluorophore (i.e., amine, carboxylic acid, or thiol), or by using specific fluorescent conjugated antibodies or fluorescent fusion proteins [START_REF] Arkin | Inhibition of protein-protein interactions: non-cellular assay formats[END_REF]. Similar to other mix-and-read assays, FRET methods are miniaturized and automated assays [50]. Many FRET pairs are available and, in contrast to FPA, FRET can be used with a range of protein sizes [51].

For example, Schaarp et al. [START_REF] Schaap | Development of a steady-state FRET-based assay to identify inhibitors of the Keap1-Nrf2 protein-protein interaction[END_REF][START_REF] Hancock | Peptide inhibitors of the Keap1-Nrf2 protein-protein interaction with improved binding and cellular activity[END_REF] developed a FRET-based assay to identify inhibitors of the Keap1-Nrf2 interaction involved in oxidative-stress defence, using a CFP-Nrf2-derived peptide and a YFP-Keap1 domain. The FRET assay was adapted to a multiwell plate format, and the competition assay conditions were validated by screening a series of previously described inhibitors. The authors monitored the decrease in the donor emission to minimize fluorescence interferences. Compared with isothermal titration calorimetry (ITC; see later), FRET has higher sensitivity and throughput and does not require sample immobilization, which is needed for surface plasmon resonance (SP; see later). Compared to FPA, FRET assays can be applied using less sophisticated plate readers with simpler optics. However, both FRET and FPA can be confounded by fluorescence interference from inhibitors and, thus, a second confirmation of hits is required in the context of HTS.

Frequently, because of fluorescence interference (i.e., strong background autofluorescence of biological media or tested compounds), the FRET signal can be significantly contaminated and lead to false positive readouts. Donor fluorophores with long emission half-lives, such as cryptates of lanthanides ions (europium or terbium) can be used to reduce FRET signal contamination. This modification of FRET is known as time-resolved (TR) FRET (also called HTRF). This long emission results in a time delay between the donor excitation and the recording of the acceptor emission [START_REF] Degorce | HTRF: a technology tailored for drug discovery -a review of theoretical aspects and recent applications[END_REF].

TR-FRET is largely used as a mPPI screening method. For instance, in the search for new Mcl-1 inhibitors, Du et al. designed and developed a HTS technology that allows the generation of two HTS readouts from one reaction by combining FRET and FPA technologies into one platform [START_REF] Du | A dual-readout F2 assay that combines fluorescence resonance energy transfer and fluorescence polarization for monitoring bimolecular interactions[END_REF]. The test, called a 'dual-readout F2 assay', was validated in an HTS campaign of 102 255 compounds [START_REF] Mady | Discovery of Mcl-1 inhibitors from integrated high throughput and virtual screening[END_REF]. Of these, 36 molecules were selected and characterized using FP-and SPR-based competitive binding assays. Guided by chemoinformatics, the authors highlighted a new family of Mcl-1 inhibitors, and performed a structure-activity relationship (SAR) study.

Another variant of FRET is BRET, which substitutes the initial excitation of the system by an enzymatic reaction, using bioluminescence as the energy source. Although BRET is widely used for the study of PPIs [START_REF] Dimri | Use of BRET to study protein-protein interactions in vitro and in vivo[END_REF], it is rarely used for the screening of molecules. A model was recently developed by Corbel et al. to screen inhibitors of the p53-MDM2 interaction, involved in apoptosis evasion [START_REF] Corbel | Screening for protein-protein interaction inhibitors using a bioluminescence resonance energy transfer (BRET)-based assay in yeast[END_REF]. Differential scanning fluorimetry Differential scanning fluorimetry (DSF), also called a thermal shift assay (TSA) or Thermofluor®, was first described in 2001 as a method for drug discovery [START_REF] Pantoliano | High-density miniaturized thermal shift assays as a general strategy for drug discovery[END_REF]. The thermal denaturation of a folded protein B leads to an unfolded protein B, which exposes some hydrophobic parts, which are usually unreachable. A hydrophobic fluorescent dye (the most popular being Sypro Orange®) [START_REF] Niesen | The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability[END_REF], is able to bind to the hydrophobic parts of proteins. The more the temperature increases (usually from 25°C to 95°C), the more the protein B is unfolded, the more the Sypro Orange® binds, and the higher the fluorescence is important. The melting temperature (Tm) is then recorded. The presence in the medium of a ligand L of the protein B stabilizes the LB complex and increases the melting temperature (Tm′). The calculated thermal shift ∆Tm (Tm′-Tm) is proportional to the ligand affinity (Figure 3b).

DSF does not require specific equipment, and can be performed in real-time PCR machines. It can also be adapted to a large number of label-free proteins, and requires no specific knowledge of the protein. Thus, it is particularly suitable for HTS [START_REF] Simeonov | Recent developments in the use of differential scanning fluorometry in protein and small molecule discovery and characterization[END_REF][START_REF] Vivoli | Determination of protein-ligand interactions using differential scanning fluorimetry[END_REF][START_REF] Scott | Differential scanning fluorimetry as part of a biophysical screening cascade[END_REF][64] dedicated to targeting PPIs [START_REF] Madhumalar | Design of a novel MDM2 binding peptide based on the p53 family[END_REF] and is widely used to screen fragment libraries in FBDD.

However, DSF uses a fluorescent dye. As a consequence, because of a high fluorescence background, some proteins might not be amenable to DSF screening by using Sypro Orange® [START_REF] Senisterra | Thermal denaturation assays in chemical biology[END_REF]. Moreover, the PPI binding sites are particularly hydrophobic [START_REF] Keskin | Principles of protein-protein interactions: what are the preferred ways for proteins to interact?[END_REF], leading to potential competition between the compounds and the dye. However, binding of small molecules to the protein surface is usually so weak that no significant Tm change can be obtained. As a consequence, the compounds are screened at relatively high concentrations (1-10 mM), which is incompatible with low-solubility compounds [START_REF] Niesen | The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability[END_REF][START_REF] Ciulli | Biophysical screening for the discovery of small-molecule ligands[END_REF].

Complementarily to classical FPA, Wan et al. used DSF as a secondary tool to screen small-molecule inhibitors of Bcl-xL, which is involved in apoptosis evasion [START_REF] Wan | Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-xL[END_REF]. In this study, the binding profile results of peptide ligands were found to be consistent with results obtained through SPR and FPA. Nevertheless, results obtained with abiotic ligands, such as ABT-737, were not concordant with those obtained by FPA. The advanced hypothesis was that the chemical inhibitors could induce a global change in the protein structure, unlike peptides, which induce a moderate change. This highlights the importance of coupling two different screening techniques in the drug discovery of mPPIs.

Recently, Tran et al. used TSA to study modulators of the Nrf2-Keap1 interaction [START_REF] Tran | A comparative assessment study of known small-molecule Keap1-Nrf2 protein-protein interaction inhibitors: chemical synthesis, binding properties, and cellular activity[END_REF], a promising target to combat oxidative stress implicated in cancers [START_REF] Taguchi | The KEAP1-NRF2 System in Cancer[END_REF]. The authors compared 21 known small inhibitors using a three orthogonal assay (FPA, TSA, and SPR). Results were confirmed for the half of the compounds. The others were false positive compounds, with PAINS moieties or fluorescence interference. This study also highlighted the advantage of combining several screening methods to obtain relevant results.

Nuclear magnetic resonance

In the mPPI drug discovery process, NMR assays are usually the most adapted for hit validation and binding studies [START_REF] Zartler | 1D NMR Methods in Ligand-Receptor Interactions[END_REF]. Recently, NMR-based approaches have also found application in mPPI screening processes. These approaches, termed in general 'HTS by NMR', are particularly suitable for fragment screening. Highly valuable, effective, and sensitive, NMR spectrometry directly identifies and characterizes the protein-ligand binding. This method is less prone to artefacts compared with biochemical HTS (reviewed in [START_REF] Wu | High-throughput screening by nuclear magnetic resonance (HTS by NMR) for the identification of PPIs antagonists[END_REF][START_REF] Barile | NMR-based approaches for the identification and optimization of inhibitors of protein-protein interactions[END_REF]).

Two main strategies stand out regarding screening by NMR to detect mPPIs, 1D 1 H-NMR and 2D 1 H-NMR (Figure 4). The first and simplest is the measurement of 1D 1 H-aliphatic NMR shift spectra. This method represents the portion of the protein 1D 1 H-NMR spectrum having a chemical shift <0.7 ppm. This spectral region is typical of the chemical shift of methyl groups from several proteins because protons of small peptides or molecules generally do not provide signals in this area. Hence, the comparison of 1D 1 H-aliph NMR spectra of the target protein in the absence and presence of the ligands allows the detection of ligand binding (Figure 4). This label-free method mainly concerns small to medium-sized proteins (<30 kDa) and needs relatively low protein concentrations (1-10 µM). As illustration, a 1 H-NMR experiment was used by Astex Pharmaceuticals as the first step to identify new hits targeting the BIR3 domain of IAP family proteins [START_REF] Chessari | Fragment-based drug discovery targeting inhibitor of apoptosis proteins: discovery of a non-alanine lead series with dual activity against cIAP1 and XIAP[END_REF]. They screened a fragment library containing 100 fragments selected by virtual screening. In this study, other ligand-detected NMR methods, such as LOGSY or STD-NMR commonly used to detect fragment binding, demonstrated lower sensitivity because of the small size of the protein.

Without labeled proteins or ligands, the intermolecular environment using homonuclear bi-dimensional proton [ 1 H, 1 H] Nuclear Overhauser Effect SpectroscopY (NOESY) can be detected [START_REF] Becattini | Structure-activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid[END_REF]. The assay time is longer (12-48 h), depending on the ligand concentration (500 mM-2 mM). However, coupled with the 13 C spectra, this approach is useful to detect intermolecular distances (via NOE) and to study the docking of ligands [START_REF] Fesik | Heteronuclear three-dimensional NMR spectroscopy. A strategy for the simplification of homonuclear twodimensional NMR spectra[END_REF][START_REF] Pellecchia | NMR-based structural characterization of large protein-ligand interactions[END_REF]. A faster method using [ 1 H, 1 H] NOE spectroscopy combined with Heteronuclear Single Quantum Correlation (HSQC) is also available. Increasing the analysis time, the 2D band-Selective Optimized-Flip-Angle Short-Transient-HSQC (2D SOFAST-HSQC) is also promising [START_REF] Schanda | Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds[END_REF]. This fast process was used to identify, among 24 scaffolds, a novel inhibitor of the CXCL12-CXCR4 interaction, involved in tumor progression [START_REF] Schanda | SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds[END_REF][START_REF] Smith | Structure-based identification of novel ligands targeting multiple sites within a chemokine-G-protein-coupledreceptor interface[END_REF].

mPPI studies can also rely on the detection of 2D heteronuclear experiments by using uniformly or selective 15 N and/or 13 C-labeled proteins. These 2D NMR methods are not suitable for HTS, but those combinatory experiments, called chemical-shift mapping, are widely used for the characterization of protein-ligand binding. The target proteins have to be 13 C or 15 N labeled and the acquisition is developed with and without ligands. As described for aliphatic assays, the protein size has to be small or medium (<25-30 kDa) and the protein concentration has to be increased (10-50 mM). Pfizer® [START_REF] Stockman | Identification of allosteric PIF-pocket ligands for PDK1 using NMR-based fragment screening and 1H-15N TROSY experiments[END_REF] used 2D [ 1 H, 15 N] NMR to develop disruptive PDK1-downstream kinase interactions for cancer research. This method was also applied by Wyeth® [START_REF] Wu | HTS by NMR of combinatorial libraries: A fragment-based approach to ligand discovery[END_REF], combined with 1D 1 H-aliph NMR assay, to identify new ligands against the binding domain of EphA, a tyrosine kinase receptor involved in some cancers, among more than 10 5 compounds.

One general limit of NMR spectroscopy is the solubility of proteins and ligands. Given that fragments present generally a better water solubility, NMR spectroscopy is particularly suitable for fragment screening, enabling the detection of fragment hits of weak binding affinity (Kd values up to mM) without ambiguity [84].

Concluding remarks on high-throughput assays

Fast, relatively simple, and miniaturizable, high-throughput methods are widely preferred in HTS campaigns. Less costly than the techniques described later. they also make it possible to conduct low-and medium-throughput screening campaigns at a lower cost. The major drawback of these techniques (outside of NMR), remains the high rate of false positive results, especially with methods using fluorescence.

Low-throughput assays

Methods with immobilization

Surface plasmon resonance The optical technique SPR detects the interaction between two partners: one (protein A) is fixed on a sensor chip and the other one is mobile (ligand L). The sensor chip comprises a gold thin film, and a reaction section enables the immobilization of A (via noncovalent immobilization using carboxymethylated dextran matrix, or a covalent immobilization using a specific matrix). Upon binding of L to A, the mass fluctuation directly changes the refractive index of polarized light close to the gold surface, which is then measured (Figure 5a). The variation of the refractive index is proportional to the ligand concentration and enables the Kd value to be determined (for a guide to the set-up of SPR experiments in PPI drug discovery, see [85]).

Among its advantages, SPR is a fast and highly sensitive technique requiring minimal sample quantities. SPR provides access to kinetic, affinity, and thermodynamic data for analyzing biomolecular interactions without using any labels. For these reasons, SPR is widely used to characterize PPIs and mPPIs and, therefore, is considered to be a 'gold standard'. SPR can also be used for screening of mPPIs, including fragment screening [START_REF] Chavanieu | Developments in SPR fragment screening[END_REF]. Given the low fragment affinity (in the millimolar range), higher fragment concentrations (up to five to tenfold of the KD) should be used.

In a recent example, a SPR-based fragment screening assay against BCL6 was performed by Kamada et al. [START_REF] Kamada | Discovery of a B-cell lymphoma 6 protein-protein interaction inhibitor by a biophysics-driven fragment-based approach[END_REF] A selection of 64 fragments was made from a library (1494 compounds) using a primary SPR-based screening. For SPR assay development, mutated BCL6 was used, which contained fewer cysteine residues, to prevent both protein aggregation and false positives. A second screening using STD-NMR highlighted one fragment as a starting point for a medicinal chemistry study using FBDD with fragment growing.

However, SPR needs the immobilization of one of the binding partners, which can change the native configuration of the protein and access to the active binding site [START_REF] Renaud | Biophysics in drug discovery: impact, challenges and opportunities[END_REF]. SPR assay conditions have to be adapted according to the chemical series to which the ligands belong. Furthermore, to decrease the rate of false negatives, a selectivity test should be performed in parallel on the main target, with a protein bearing the binding site mutated or blocked with a low off-rate ligand. SPR also requires the use of a dedicated and expensive device and sensor chips [START_REF] Korshunova | Modern approaches to analysis of protein-ligand interactions[END_REF].

Advances in technology, including miniaturization of devices and the ability to perform measurements in parallel in the same device, increased the screening throughput even though the cost remains a significant brake to their use. Nevertheless, SPR is widely used as a secondary assay to confirm and profile results from biochemical or biophysical screening. For example, Jiang et al. [START_REF] Jiang | Identification of a novel small-molecule Keap1-Nrf2 PPI inhibitor with cytoprotective effects on LPS-induced cardiomyopathy[END_REF] used SPR as a second screening method to identify new Keap1-Nrf2 PPI inhibitors. A library of 569 compounds was first screened in a FPA, and a SPR competition assay was then used to confirm the interaction of the hits with Keap1. Bio-layer interferometry First described in 2005 by ForteBio® (www.fortebio.com/applications/bli-technology), biolayer interferometry (BLI) is a label-free optical technology that analyzes the interference pattern of white light reverberated from two surfaces: an internal reference layer, and a layer of immobilized protein A on the biosensor surface. The binding between a ligand L and A induces an increase in optical thickness at the biosensor tip. The length of the reflected light path increases, leading to a shift in the interference pattern [START_REF] Concepcion | Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization[END_REF][START_REF] Sultana | Measuring protein-protein and protein-nucleic acid interactions by biolayer interferometry[END_REF]. A sensorgram is registered, which enables the kinetic parameters of the interaction to be determined (Figure 5b).

Compared with SPR, BLI is compatible with a 96-plate format and has a better throughput capacity. Wartchow et al. [START_REF] Wartchow | Biosensor-based small molecule fragment screening with biolayer interferometry[END_REF] used BLI to screen a fragment library of 6500 fragments targeting Bcl-2 and eIF4E proteins, modulators of disease progression in oncology. The results were compared with those obtained in a FRET-HTS. The overlap between the hits identified in the two assays was 52%. In addition to the hits observed in the biochemical screening, 68% of the hits from the BLI screening were unique and were not found in the biochemical screening. These results are encouraging and show the advantage that BLI can represent in the screening of mPPIs, although additional investigations are required to better understand the differences observed. In parallel, BLI can also be used in complex media, because refractive index changes in the medium do not affect shifts in the interference pattern. The BLI technique is also more tolerant of samples containing a high level of solvent, in particular DMSO.

However, BLI is less sensitive than SPR. Given the limited temperature control of the samples, a thermodynamic characterization of ligand binding is impossible. In addition, because SPR uses a continuous flow system, it is more repeatable than BLI. Yet, because BLI can be used in mix-and-read format, this makes it a promising technology in future mPPI screening.

Immobilization-free methods

Isothermal titration calorimetry ITC measures quantitative energy changes that are associated with the binding reaction of two components. An adiabatic system is used, containing two cells: a reference cell and a cell with the protein A, maintained at strictly identical temperatures. The ligand L in the syringe is introduced in several portions into the sample cell. When L binds to A, a heat change occurs, resulting in a temperature difference between the two cells. The energy (DP for differential power in µcal.s -1 ) necessary to maintain a zero temperature difference is then recorded (Figure 6a) [START_REF] Korshunova | Modern approaches to analysis of protein-ligand interactions[END_REF].

Both an immobilization-free and label-free method, ITC is the only technique that determines directly all the thermodynamic parameters of ligand binding: affinity (Kd), stoichiometry, enthalpy change (∆H), and calculation of both free energy change (∆G) and entropy change (∆S). Given that all these parameters can be determined in a single experiment [46], ITC is a suitable tool to both characterize mPPI binding mode [START_REF] Linkuvienė | Isothermal titration calorimetry for drug design: precision of the enthalpy and binding constant measurements and comparison of the instruments[END_REF][START_REF] Peters | Recent developments in isothermal titration calorimetry label free screening[END_REF] and guide molecular design [START_REF] Garbett | Thermodynamic studies for drug design and screening[END_REF][START_REF] Damian | Isothermal titration calorimetry for studying protein-ligand interactions[END_REF].

However, ITC method dedicated and expensive equipment. Sample consumption (protein and ligand) can be relatively high and limits its application for proteins that are difficult to produce. As a second consequence, a large quantity of ligands and high ligand solubility are required. Thus, ITC can be challenging for weak binding of mPPIs. Given that the affinities are weak, ITC is also challenging for fragment screening [START_REF] Renaud | Biophysics in drug discovery: impact, challenges and opportunities[END_REF]. During optimization, it is also important to coordinate buffers in the syringe and in the sample cell to eliminate heat of dilution (for a guide to data interpretation, see [START_REF] Falconer | Applications of isothermal titration calorimetry -the research and technical developments from 2011 to 2015[END_REF]).

Recently, Gal et al. [START_REF] Gal | Efficient isothermal titration calorimetry technique identifies direct interaction of small molecule inhibitors with the target protein[END_REF] used ITC to identify small-molecule inhibitors of the p53-MDM2 interaction. First, a virtual screening extracted 80 hits from a library of 15 million compounds. ITC was then used to test these 80 molecules to identify a hit, confirmed by FPA and NMR. The authors developed an original ITC protocol assay, where the targeted protein MDM2 was injected into a mixture of four compounds placed in the ITC cell, limiting the number of injections required and making a binary decision concerning the change in released or absorbed heat. Finally, a FPA measurement and NMR experiments confirmed that the protein competed with p53. Microscale thermophoresis Developed by NanoTemper®, microscale thermophoresis (MST) monitors fluorescence. In a typical MST experiment, protein A is covalently labeled with a fluorescent dye. A solution of labeled-protein A is placed in a transparent capillary tube, part of which is illuminated with an infrared (IR) laser, thus establishing a stable thermal gradient. Given 'thermophoresis' ('Soret effect'), A migrates along this gradient, and its movements can be monitored using a fluorescence microscope focused on the IR-illuminated region. The thermophoretic movement of A leads to a decrease in the fluorescence signal. In the presence of a ligand L, the thermophoretic movement induced by IR irradiation of the LA complex differs from the movement of A alone. The decrease in registered fluorescence is less important, enabling the affinity of the interaction to be estimated. However, MST can be performed in a label-free manner, in which the intrinsic ultraviolet (UV)-fluorescent signal of the aromatic amino acid residues is detected (Figure 6b) [100,101].

Easy to handle, MST is a fast and precise way to measure binding affinity. Compared with ITC, it is highly sensitive, has low sample consumption, and measures interactions with no limitation on molecule size or weight. Moreover, MST is able to analyze interactions of small molecules with proteins in complex media (i.e., cell lysates or biological liquids) [102] (for a guide to data interpretation, see [103]).

Currently, MST is mainly used to characterize the binding mode of mPPIs. Gibaud et al.

[104] used MST to characterize the binding mode of inhibitors of the YAP-TEAD interaction (implicated in the Hippo pathway, which is deregulated in cancer processes). The experiments were successfully carried out on cell lysates. For another example, Rainard et al. [105,106] used MST for the triage and characterization of small-molecule inhibitors of the Nrf2-Keap1 interaction. Among 318 132 compounds tested by a FPA, eight compounds were selected for further characterization using a label-free MST assay.

Although MST is not yet used for HTS in drug discovery of PPIs, its automation will push this technology toward a higher throughput, rendering it more promising as a drug discovery technology.

Concluding remarks on low-throughput assays

Compared with high-throughput assays, the techniques described generally require more expensive equipment. More sophisticated, they allow access to a large amount of data, which is particularly interesting for characterizing protein-ligand interactions, but often require dedicated know-how to interpret the data. Recent technological advances in equipment, particularly in terms of automation, will make it possible to consider the use of these methods for higher-throughput campaigns.

Concluding remarks

Targeting PPIs in drug discovery is specific and challenging. As a consequence, finding the best screening processes can be difficult. Moreover, PPIs often have no directly accessible activity readout. Thus, it can be unclear which test to choose for the screening of mPPIs. Putting throughput considerations aside, to answer this question, several parameters must be taken into account: (i) laboratory experience and available budget. Some technologies, such as SPR and ITC, require dedicated and expensive equipment and trained staff. By contrast, DSF, for example, is one of the most accessible techniques because it can be performed with a simple PCR device. Other experiments giving rapid results, such as FPA are also suitable with a small budget; (ii) nature of the library. On the one hand, autofluorescent molecules can create interference with fluorescent-based assays, particularly FPA or FRET. On the other hand, because hydrophobic molecules tend to precipitate or aggregate especially in aqueous buffers, DSF, ITC, or NMR are poorly suited for such uses. Conversely, with libraries of fragments that are smaller and more soluble in aqueous media, NMR will be particularly suitable, including for high throughput; and (iii) nature of the protein partners involved in the interaction. If the targeted protein is difficult to purify, MST and BLI will be particularly suitable, because they allow measurements in complex media. With unstable proteins, SPR or BLI, which require immobilization, should be avoided. If two protein partners are not available, competitive methods, such as FPA, FRET, or AlphaScreen®, cannot be used. Likewise, if a label partner is not available, it will be necessary to use label-free methods. If the proteins are poorly soluble, NMR and ITC will be poorly adapted. Finally, if the interaction is weak, sensitive techniques, such as SPR or AlphaScreen®, are preferred.

The optimal approach is to use two complementary techniques in an orthogonal screening test. With a limited screening capacity, it is also possible to use in silico screening to reduce the size of the library. Janzen showed that the less complex the test, the higher the rate of hitters [107]. This is a parameter to take into account when choosing the test. Having the appropriate screening process is the key to success, because the more appropriate the process is, the more relevant the hit selected, and the shorter the hit-to-lead process. Chemist-biologist collaborations in both the choice of the screening test and the triage process are essential for a successful drug discovery campaign. An advantage of noncellular-based assays is that they can be used on isolated proteins to characterize the protein-ligand interaction without cellular interferences. Nevertheless, the subsequent validation of the identified hit compounds on a cellular model remains a key step in the hit-to-lead process.

Figure 1. Use of screening assays in a hit-to-lead strategy applied to small molecules targeting protein-protein interactions (PPIs). Abbreviations: AlphaScreen, amplified luminescent proximity homogeneous assay screen; BLI, biolayer interferometry; CETSA, cellular thermal shift assay; FPA, fluorescence polarization assay; FRET, fluorescence (or Förster) resonance energy transfer; ITC, isothermal titration calorimetry; NMR, nuclear magnetic resonance; SPR, surface plasmon resonance; TSA, thermal shift assay; X-ray, X-ray crystallography.

Glossary

Fragment: small organic molecules that are small in size, low in molecular weight (MW), and have a weak affinity for the targeted protein. Sometimes, in analogy to the 'rule of five', it has been proposed that an ideal fragment should follow the 'rule of three' (MW <300, logP <3, and number of hydrogen bonds <3). Fragment-based drug design (FBDD): in a hit to lead strategy, the screening of a fragment library enables the identification of hit fragments presenting a weak affinity for the targeted protein (in the micromolar range). Pharmacomodulation studies are then conducted to produce a lead with an affinity in the nanomolar range. In FBDD, three main strategies are used: fragment growing, fragment merging, and fragment linking. Hot-spots: in some cases, PPI surfaces contain subsets of a few amino acids, which are called hot-spots, where most of the PPI binding energy is localized [108]. Sometimes, the design of mPPIs is possible by targeting these hot-spots. Mix-and-read assay: simple procedure without separation, filtration, or washing steps; often used in HTS. Modulators of PPIs (mPPIs): compounds that disrupt a PPI by inhibition (with a PPI inhibitor) or stabilization (with a PPI stabilizer) of the interaction. Orthogonal assay: experiment combining two or more assays that use different technologies to limit false positive screening results (i.e., FPA and SPR, or AlphaScreen® and NMR). Pan assay interference compounds (PAINS): also called 'hitters'; designate compounds that generally interact nonspecifically with numerous biological targets rather than specifically affecting one desired target [109], leading to false positive readouts in many different biological assays Protein-protein Interactions (PPIs): physical interaction between two proteins in a living organism implicated in a biological process. The PPI network of an organism constitutes its interactome.

Figure 2 .

 2 Figure 2. High-throughput assays in a mix-and-read format (Part 1). Principles of (a) florescence polarization assays (FPA) and (b) amplified luminescent proximity homogeneous assay screen (AlphaScreen) assays.

Figure 3 .

 3 Figure 3. High-throughput assays in a mix-and-read format (Part 2). (a) Fluorescence resonance energy transfer (FRET) assays and (b) differential scanning fluorimetry (DSF) assays.

Figure 4 .

 4 Figure 4. Principle of nuclear magnetic resonance (NMR) screening assays.

Figure 5 .

 5 Figure 5. Principles of low-throughput assays: Methods requiring immobilization of a partner: (a) surface plasmon resonance (SPR) assays and (b) biolayer interferometry (BLI) assays.

Figure 6 .

 6 Figure 6. Principles of low-throughput assays: Immobilization-free methods: (a) isothermal titration calorimetry (ITC) and (b) microscale thermophoresis (MST).

Table 1 . Noncellular-based assays for targeting PPIs used in drug discovery Assay Throughput Mix and read Biochemical/biophysical Advantages Drawbacks

 1

	FPA	High	Yes	Biochemical	Very simple; low cost; adaptable to	Needs fluorescent-tagged proteins;
					low volumes	interferences in fluorescence
	AlphaScreen	High	Yes	Biochemical	Detection of interaction until 200	High cost (beads); sophisticated plate
					nm; very sensitive; label-free	reader; temperature sensitive; high
					proteins	rate of 'hitters'
	FRET/TR-FRET High	Yes	Biochemical	Sensitive; adaptable to low	Interaction <10 nm; needs two
					volumes; low-cost plate reader	fluorescent-tagged proteins (acceptor
						and donor); fluorescence interferences

After completing a postdoctoral period in medicinal chemistry in CERMN (Caen,

Tm' Tm


Jean-Louis Schlienger
email: jean-louis.schlienger@wanadoo.fr

Louis Monnier

une trop belle histoire encore en quête de preuves N-3 fatty acids: A too beautiful story still in quest of evidence

Keywords: Acides gras oméga 3 -Nutrition -Histoire de la médecine, -------------- n-3 polyunsaturated fatty acids -Past and present -Medicine history -Nutrition, -----

C'est il y a un demi-siècle que les danois Dyerberg et Bang, intrigués par une incidence des événements cardiovasculaires significativement plus basse chez les Esquimaux du Groenland que chez les Danois, ont conçu le concept protecteur des acides gras (AG) polyinsaturés en omega 3 (AG n-3). Ils constatèrent que le profil lipidique des Esquimaux était bien plus satisfaisant que celui des Danois, en dépit d'une alimentation particulièrement riche en graisses. Ils attribuèrent ce phénomène paradoxal à la forte consommation d'AG n-3 contenus dans les produits marins, avec une mention particulière pour l'acide eicosapentaénoïque (EPA ; C20:5 n-3) et l'acide docosahexaénoïque (DHA ; C22:6 n-3). Ce concept novateur a été conforté par diverses études expérimentales et chez l'homme montrant que les AG n-3 avaient des effets bénéfiques sur les lipides plasmatiques, la pression artérielle, l'agrégation plaquettaire, la thrombose, le rythme cardiaque, et le développement de l'athérosclérose. Il s'en suivit un engouement des médecins et du grand public pour cette famille d'AG dont l'intérêt est bien documenté en physiologie. Des médicaments à base d'huile de chair de poisson ont été mis sur le marché dans le but de diminuer la triglycéridémie et de prévenir les événements coronariens en prevention cardiovasculaire primaire et secondaire. Des compléments alimentaires et des produits alimentaires supplémentés ou « fortifiés » en AG n-3, comme la margarine, ont été commercialisés. Longtemps, le bien fondé de ces pratiques a été admis en dépit de l'absence de preuves en termes d'evidence-based medicine. Ce n'est que récemment que trois études méthodologiquement convaincantes ont montré que la supplémentation en AG n-3 à des doses légèrement supra-nutritionnelles n'avait aucun effet bénéfique en termes de maladies cardiovasculaires ou de cancer. Au terme d'une histoire extraordinaire née dans le grand froid polaire, force est de conclure qu'une supplémentation en AG n-3 n'est pas nécessaire chez les adultes en bonne santé consommant 2 portions de poisson par semaine, ce qui suffit à couvrir les besoins physiologiques en EPA and DHA de l'ordre de 250 mg par jour.

Introduction

* Le rôle crucial des acides gras (AG) en physiologie et en pathologie a été établi à la suite d'un long processus d'identification [START_REF] Schlienger | Histoire chimique, sociale, et clinique des graisses[END_REF]. La hiérarchisation des AG proposée à partir de données observationnelles et expérimentales a permis de préciser leur place en nutrition humaine. Les AG polyinsaturés à longue chaîne dont la première double liaison est située entre le 3 e et le 4 e atome de carbone (AG en n-3, ou oméga-3) se singularisent des innombrables autres AG par leur caractère « essentiel » et par leur implication dans la synthèse de prostaglandines, dont certaines ont des propriétés anti-thrombogènes et anti-inflammatoires. Contenus tout particulièrement dans les huiles de chair de poisson, les « oméga-3 » ont été considérés comme de véritables héros nutritionnels, et placés rapidement -sans doute trop -au coeur (sic) de la prévention cardiovasculaire primaire et secondaire par les épidémiologistes, les cardiologues et … le grand public, sans compter l'extraordinaire appétence de l'industrie, aussi bien agro-alimentaire que pharmaceutique, qui s'est saisie sans trop de scrupules de cette nouvelle manne. * L'histoire singulière des oméga-3 n'a que 50 ans, mais elle en dit long sur l'histoire des hommes et des sciences de la nutrition. De surcroit, elle illustre merveilleusement le biais de confirmation, péché mignon de tant de chercheurs qui les conduit à privilégier l'illusion de corrélations confirmant une idée préconçue ou une opinion aux données factuelles n'allant pas dans le sens souhaité.

Singularité nutritionnelle et sanitaire des Esquimaux

Tout est parti d'une étude observationnelle effectuée entre 1950 et 1974 auprès des 1 800 habitants du district d'Upernavik, au Groenland (figure 1). Son but était d'étudier les indicateurs de santé en fonction du mode de vie de cette population ethniquement homogène et stable (moins de 10 % d'étrangers, principalement danois). Chez les Esquimaux Inuits ayant conservé un mode de vie traditionnelvivant de la pêche à la baleine, de la pêche et de la chasse (figure 2) -l'incidence de l'infarctus du myocarde (IDM) et du diabète est remarquablement faible, alors que celle de l'épilepsie et de « l'apoplexie » (hémorragie cérébrale) est très élevée par rapport à celles observées chez les Esquimaux convertis à la vie moderne et aux Danois résidents [START_REF] Kromann | Epidemiological studies in the Upernavik district, Greenland. Incidence of some chronic diseases 1950-1974[END_REF]. Ces données, colligées à une époque où l'épidémiologie ne disposait pas d'outils statistiques élaborés, semblaient confirmer les constatations publiées en 1940 par un médecin du district, Alfred Léopold Bertelsen (1877Bertelsen ( -1950)). D'origine danoise, il participa en 1903 avec Knud Rasmussen et Ludvig Mylius-Erichsen à une expédition arctique pour étudier la langue et des coutumes des Esquimaux (« Expédition littéraire du Groenland »). Ornithologue à ses heures perdues, il abandonna l'expédition lorsqu'il parvint à Upernavik où il s'installa comme médecin généraliste jusqu'en 1927. Amateur de chiffres, il ne se contenta pas de recenser les oiseaux, et tint également une comptabilité des maladies de ses patients [START_REF] Bertelsen | Grønlandsk medicinsk Statistik og Nosografi Undersøgelser og Efaringer fra 30 Aars grønlandsk Laegevirksomhed. III. Det saedvanlige grønlandske sygdomsbillede [Medical statistics and nosography in Greenland: the usual disease pattern in Greenland[END_REF]. Il fut frappé par la rareté des pathologies « artério-scléreuses » et métaboliques. Il attribua cette singularité au mode de vie et à l'alimentation traditionnelle des Esquimaux de cette région du nord-ouest du Groenland (figure 1). À cette époque, les cétacés, les poissons et les grands mammifères nordiques, constituaient l'essentiel, sinon la totalité de l'ordinaire, à en croire Auguste Krogh (1874Krogh ( -1949) ) et son épouse Marie . Ce dernier, physiologiste danois, distingué par le prix Nobel de physiologie en 1920 pour ses travaux sur la régulation de la circulation capillaire, et qui joua un rôle décisif pour la commercialisation de l'insuline en Europe, avait fait partie d'une expédition polaire en 1902. Bien que son sujet d'étude se limitât officiellement à la mesure de la teneur en oxygène et en CO2 des cours d'eau et de la mer, il fit une enquête alimentaire auprès des populations indigènes. Au début du XX e siècle, il estimait les apports quotidiens en protéines à 280 grammes (!), à 135 g pour les lipides, et à 54 g pour les glucides [START_REF] Krogh | A study of the diet and metabolism of Eskimos undertaken in 1908 on an expedition to Greenland[END_REF].

Naissance du concept des oméga-3

* En 1971, le mode de vie et l'alimentation des Esquimaux avaient bien changé lorsque deux autres Danois, Jorn Dyerberg (1937-) (figure 3) et Olaf Hans Bang (1913Bang ( -1994) ) [START_REF] Bang | Plasma lipid and lipoprotein patterns in Greenlandic West-coast Eskimos[END_REF]. Pour Dyerberg et Bang, cette relation pour le moins paradoxale à une époque où la doxa anti-AG saturés s'imposait déjà à la communauté médicale, pouvait s'expliquer par la teneur élevée en AG polyinsaturés à chaine longue caractéristiques des graisses d'origine marine, en l'occurrence les AG oméga-3 [START_REF] Dyerberg | Fatty acid composition of the plasma lipids in Greenland Eskimos[END_REF]. Convaincus que c'est à ce type d'AG que les Inuits devaient leur état de santé paradoxal, ils entreprirent six autres expéditions au Groenland en s'intéressant plus étroitement à l'alimentation des Esquimaux. Comparée à l'alimentation danoise, l'alimentation locale était plus riche en certains AG oméga-3, avec une mention particulière pour l'acide eicosapentaénoïque (C20:5n-3) [START_REF] Dyerberg | Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis?[END_REF]. Toutefois, la somme totale des AG polyinsaturés englobant à la fois ceux de la série oméga-3 et oméga-6 est moindre que chez les Danois ce qui, selon les conceptions en vigueur à cette époque dominée par l'équation de Keys, ne pouvait expliquer la faible concentration de cholestérol plasmatique. * Au cours de l'hiver 1976, ils réalisèrent une enquête alimentaire auprès de 25 couples de Groenlandais pendant 5 jours, par la technique de duplication des ingesta pour réaliser une analyse qualitative. Ils démontrèrent que la nourriture traditionnelle comportant plus de 400 g de poisson ou d'équivalents marins par jour était riche en AG polyinsaturés de la série oméga-3 ou n-3. Le ratio AG polyinsaturés/saturés est de 0,84 chez les Inuits, contre 0,24 chez les Danois. Par ailleurs, les AG n-3 sont prépondérants chez les Inuits, alors que ce sont les AG n-6 qui sont dominants chez les Danois [START_REF] Bang | The composition of the Eskimo food in north western Greenland[END_REF]. Forts de ces résultats, Dyerberg et Bang soulignent l'existence d'une relation entre la faible incidence des maladies coronariennes et la forte concentration sérique en AG n-3 dans les populations Inuits, bien qu'ils n'avaient entrepris aucune étude d'incidence ou de prévalence. Ils s'évertuent dès lors à démontrer que la rareté de l'incidence des maladies cardiaques pourrait être la conséquence d'un effet anti-thrombotique de l'acide eicosapenténoïque présent dans les huiles de poissons et de mammifères marins. La folle histoire des oméga-3 venait de débuter. * Au même moment, à l'autre bout du monde, le groupe de Aizan Hirai, Tomohito Hamazaki et Takashi Terano qui s'intéressent au régime des habitants de l'archipel d'Okinawa (Japon) attribuent également la faible prévalence des maladies cardiovasculaires dans ce coin du monde à l'importance de la consommation de poisson (de l'ordre de 250 g par jour) [START_REF] Hirai | Eicosapentaenoic acid and platelet function in Japanese[END_REF].

Métabolisme et fonction des AG oméga-3

* Les AG polyinsaturés regroupent deux grandes familles, dont les chefs de file sont l'acide linoléique (LA ; C18:2 n-6), dont la première double liaison se situe entre le 6 e et le 7 e atome de carbone, et l'acide alpha-linolénique (ALA ; C18:3 n-3) dont la première double liaison se situe entre le 3 e et le 4 e atome de carbone. La numérotation s'effectue de manière rétrograde en partant du radical méthyl (CH3) situé en n ème position de la chaine carbonée, ce qui explique l'écriture simplifiée n-6 ou n-3. Ces deux AG sont indispensables, car l'espèce humaine ne possède pas l'appareil enzymatique permettant leur synthèse, l'alimentation étant leur seule source. Après ingestion, ils sont oxydés, intégrés dans les membranes (figure 4), stockés ou métabolisés, et forment d'autres AG et diverses substances impliquées dans les mécanismes de l'agrégation plaquettaire et de l'inflammation, comme les leucotriènes, les prostaglandines, les prostacyclines et les thromboxanes (figure 5). L'ALA (C18:3 n-3) est le précurseur de l'acide eicosapentaénoïque (EPA ; C20:5 n-3) et de l'acide docosahexaénoïque (DHA ; C22:6 n-3). La conversion de l'ALA en EPA se fait après deux désaturations et une élongation. Une nouvelle élongation suivie d'une désaturation supplémentaire transforme l'EPA en DHA. Ces deux acides gras sont des constituants de la chair des poissons et des mammifères marins qui se nourrissent d'algues où ces deux acides gras sont présents à l'état naturel. La conversion se fait en compétition avec le métabolisme du LA, les enzymes de la cascade métabolique étant les mêmes pour les deux familles (figure 5). Les recommandations nutritionnelles visent à privilégier la synthèse de DHA grâce à un apport journalier d'au moins 2 grammes d'AG n-3 contre 10 g d'AG n-6, en veillant à maintenir un rapport n-6/n-3 de l'ordre de 5/1, puisqu'un afflux de substrat en AG n-6 est à même de compromettre, par compétition de substrat, la génération d'EPA et de DHA. * Les recommandations nutritionnelles ont été établies à la suite d'études tendant à prouver que les AG n-3 étaient bénéfiques en prévention cardiovasculaire. La consommation quotidienne souhaitable d'ALA fixée à 2 g est réalisable en consommant des huiles de soja, colza et noix, et des fruits à coque ; celle d'EPA et de DHA a été fixée à 500 mg au total (à part égale) fournis par 2 portions hebdomadaires de poisson gras. * Dans les années 1960, il fut démontré par John O'Brien et Loïs Sampson [START_REF] O'brien | Fatty acids and fatty aldehyde composition of the major brain lipids in normal gray matter, white matter, and myelin[END_REF], à partir d'analyses biochimiques pratiquées sur des cerveaux de cadavres, que les AG polyinsaturés -deux issus de la famille n-6 (l'acide arachidonique ou C20:4 n-6 et son dérivé supérieur élongué, le C22:4 n-6) et un autre issu de la famille n-3 (le DHA) -représentaient plus de 50 % des AG totaux (figure 4). * Quelques années plus tard, en 1982, Holman et al. [START_REF] Holman | A case of human linolenic acid deficiency involving neurological abnormalities[END_REF] rapportèrent le cas d'une fillette de 6 ans ayant subi une résection de 3 mètres d'intestin grêle et maintenue sous nutrition parentérale avec une préparation contenant de l'acide linoléique, mais dépourvue d'AG oméga-3. Au bout de quelques semaines, cette fillette présenta des signes neurologiques, et l'analyse des AG plasmatiques montra un déficit majeur en acide alpha-linolénique. Après supplémentation avec cet AG et correction du déficit, les symptômes disparurent. Cette observation, et des travaux ultérieurs, conduisirent à proposer un apport de 2 grammes d'acide alpha-linolénique pour tout apport énergétique égal à 2 000 kcalories. * Actuellement, les laits infantiles et de croissance sont supplémentés en AG essentiels, en particulier en oméga-3. Dans les mois à venir, une directive Européenne devrait préciser que les laits du 1 er et 2 e âge devront avoir une teneur en DHA comprise entre 14 et 35 mg/100 g, permettant ainsi de couvrir les besoins de l'enfant en AG oméga-3.

Un concept en quête de preuves

L'hypothèse du rôle cardio-protecteur des huiles de poissons suggérée par le groupe de Dyerberg a suscité d'innombrables études in vitro, chez l'animal de laboratoire et chez l'homme. * Les études observationnelles ont achevé de convaincre de l'intérêt de la consommation de poisson à hauteur de 2 portions par semaine, ou d'un complément à base d'huile de chair de poisson.

-Rappelons l'étude longitudinale menée pendant 10 ans à Zutphen, petite ville néerlandaise, où le risque relatif de la mortalité cardiovasculaire était réduit de moitié chez les hommes qui consommaient du poisson par rapport à ceux qui n'en consommaient pas [START_REF] Kromhout | The inverse relation between fish consumption and 20-year mortality from coronary heart disease[END_REF]. -D'autres études longitudinales, comme la « Chicago Western Study », d'une durée de 30 ans, où les hommes consommant plus de 35 grammes de poisson ont 3 fois moins de risque de faire une mort subite après un IDM que ceux qui n'en consomment pas, parviennent aux mêmes conclusions [START_REF] Kris-Etherton | Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease[END_REF]. Dans la « Nurse's Health Study », les accidents vasculaires cérébraux sont plus fréquents chez les femmes consommant moins d'une portion mensuelle de poisson, et, chez les hommes de la « Physicians Health Study », un taux plasmatique élevé d'AG n-3 est associé à une diminution du risque d'IDM mortel … -Une méta-analyse portant sur 11 études de cohorte confirme que la consommation de poisson est associée à une réduction des décès par maladie coronarienne et permet d'affirmer, pour frapper les esprits, que chaque consommation supplémentaire de 20 g de poisson par jour réduit la mortalité de 7 % ! [START_REF] He | Accumulated evidence on fish consumption and coronary heart disease mortality: a meta-analysis of cohort studies[END_REF]. * Les premières études d'intervention ne vont pas à l'encontre du concept cardioprotecteur des AG n-3 en prévention secondaire.

-Dans l'étude « Diet and Reinfarction Trial » (DART), réalisée chez plus de 2 000 sujets, le risque de décès diminue de 29 % après l'augmentation de la consommation de poisson [START_REF] Burr | Effects of change in fat, fish, and fiber intakes on death and myocardial reinfarction: Diet And Reinfarction Trial (DART)[END_REF]. -Dans l'étude « Gruppo Italiano per la Studio della Sopravvivenza nell'Infarto Miocardico » (GISSI), une supplémentation en EPA/DHA et ALA réduit le risque de décès et d'évènements cardio-cérébro-vasculaires de 10 %, et celui de mort subite de 26 %, chez des sujets ayant présenté un IDM moins de 3 mois auparavant [START_REF] Marchioli | Early protection against sudden death by n-3 polyunsaturated fatty acids after myocardial infarction: time-course analysis of the results of the Gruppo Italiano per la Studio della Sopravvivenza nell'Infarto Miocardico (GISSI)-Prevenzione[END_REF].

-La revue systématique de 46 études d'intervention ou de cohorte suggère que la supplémentation en EPA et DHA a un effet bénéfique sur la mortalité globale et cardiaque, et plus encore sur la mort subite, alors que la supplémentation en ALA est sans effet positif [START_REF] Wang | n-3 Fatty acids from fish or fish-oil supplements, but not alpha-linolenic acid, benefit cardiovascular disease outcomes in primary-and secondary-prevention studies: a systematic review[END_REF]. * À ce stade, l'intuition de Dyerberg et ses collègues semble vérifiée et conduit à envisager les graisses de poisson comme un moyen de prévention cardiovasculaire, voire même comme un moyen thérapeutique. Pourtant, plusieurs études récentes méthodologiquement irréprochables viennent obscurcir l'horizon des AG n-3.

-Le premier, publié par Kromhout et al. est une étude qui ne parvient pas à montrer de diminution significative du risque de récidive post-IDM après l'administration de doses nutritionnelles d'EPA, DHA, et ALA [START_REF] Kromhout | n-3 fatty acids and cardiovascular events after myocardial infarction[END_REF], alors que son auteur principal était l'un des artisans de la bonne réputation du poisson gras.

-Les trois autres études [START_REF] Aung | Omega-3 Treatment Trialists' Collaboration. Associations of omega-3 fatty acid supplement use with cardiovascular disease risks: meta-analysis of 10 trials involving 77 917 individuals[END_REF][START_REF] Bowman | Effects of n-3 fatty acid supplements in diabetes mellitus[END_REF][START_REF] Manson | Marine n-3 fatty acids and prevention of cardiovascular disease and cancer[END_REF] ont été publiées au cours des 2 dernières années. Leur analyse globale, rapportée dans une revue générale publiée dans l'un des derniers numéros de Médecine des maladies Métaboliques [START_REF] Monnier | Le double visage des études nutritionnelles : entre « fake news » et véritables informations[END_REF], montre que les supplémentations en AG oméga-3 n'apportent aucun bénéfice particulier en termes de protection cardiovasculaire. * Toutefois, ceci ne signifie pas que les AG oméga-3 sont inutiles. La recommandation de consommer 250 mg d'EPA et 250 mg de DHA reste valable, car cette quantité peut être atteinte lors d'une alimentation normale et équilibrée. En revanche, le recours à des corps gras enrichis en oméga-3 paraît peu pertinent chez les sujets qui ne souffrent d'aucune pathologie particulière. Ces résultats et conclusions ne peuvent que décevoir ceux qui avaient soutenu l'action bénéfique des AG n-3, en se basant sur les résultats d'études courtes ou sur des considérations physiopathologiques. L'administration de 2 à 3 grammes d'AG n-3 entraine une diminution de la triglycéridémie et une augmentation modérée des lipoprotéines HDL et LDL, et réduit discrètement la pression artérielle. En théorie et en pratique, les AG n-3 interviennent sur la fonction plaquettaire en déplaçant l'équilibre des prostaglandines issues du métabolisme des AG n-6 et n-3 vers le sens antiagrégant. L'administration d'huile de poisson est associée également à une diminution de la fréquence cardiaque chez l'homme, mais ses effets sur la variabilité du rythme cardiaque et sur les troubles du rythme sont incertains, bien qu'un effet antiarythmique spécifique ait été démontré in vitro et chez l'animal de laboratoire. Par ailleurs, les effets d'une supplémentation en AG n-3 sur l'insuffisance cardiaque sont ambigus et peu probants.

Une prescription précipitée

* Les apports alimentaires en oméga-3, et tout particulièrement en EPA et DHA, sont globalement insuffisants sous nos latitudes. Aussi, la possibilité de réduire le risque cardiovasculaire suggérée par les études observationnelles a conduit rapidement à la mise au point d'une supplémentation thérapeutique sous la forme de capsules de chair de poisson gras. En France, le Maxepa ® à la dose de 3x2 capsules/j est pris en charge par l'Assurance Maladie dès 1988, sur la seule foi des données de la littérature, avec l'indication « traitement de l'hypertriglycéridémie en complément d'un régime adapté ». Le médicament connait un engouement d'autant plus grand qu'il est « naturel », une fois n'est pas coutume, et parce qu'il peut être utilisé, non seulement comme en hypolipémiant en complément des fibrates, mais également comme agent anti-athéromateux et anti-agrégant, sans compter d'hypothétiques effets pléiotropes bénéfiques. En effet, pour beaucoup, l'intérêt des oméga-3 ne se limiterait pas au seul domaine cardiovasculaire, mais se manifesterait également dans le cancer, les troubles cognitifs, les atteintes neurologiques ou cutanées. Peu à peu, les AG oméga-3 deviennent une panacée, dont l'essor est interrompu par son déremboursement en 1998 au motif d'un service médical rendu incertain, et d'une place marginale dans la stratégie du traitement des dyslipidémies au moment où les statines sont enfin disponibles sur le marché français. * Il n'empêche que cette décision, tout compte fait logique, n'a pas manqué de faire des mécontents, puisqu'elle est intervenue peu avant la publication des « fabuleux » résultats de l'étude italienne GISSI [START_REF] Marchioli | Early protection against sudden death by n-3 polyunsaturated fatty acids after myocardial infarction: time-course analysis of the results of the Gruppo Italiano per la Studio della Sopravvivenza nell'Infarto Miocardico (GISSI)-Prevenzione[END_REF]. Cette décision administrative n'entama en rien la réputation des oméga-3. Bien au contraire, ils sont considérés comme des auxiliaires thérapeutiques « tout terrain » et jouissent d'une belle image auprès du grand public qui en redemande sous toutes les formes possibles. Libérés du corset de l'Autorisation de mise sur le marché, les oméga-3 sont désormais disponibles comme « médicament-conseil », « over-the-counter », compléments alimentaires, ou aliments fortifiés, comme la margarine enrichie en oméga-3. Les filières d'enrichissement de l'alimentation animale en graines de lin ou de lupin permettant d'augmenter la teneur des produits d'origine animale en C18:3 n-3 sont appréciés des consommateurs préoccupés par leur santé, conscients qu'il vaut mieux prévenir que guérir. * L'impact réel sur la santé de ces pratiques est évidemment difficile à démontrer. Néanmoins, compte tenu d'une alimentation d'usage globalement déficitaire en ALA et insuffisante en EPA et DHA, on peut admettre que ces mesures participent au rééquilibrage des apports en AG et à l'amélioration du rapport n-6/n-3 qui devrait être de 5/1 et non de 10 ou 15/1.

La réputation des oméga-3 est-elle surfaite ?

* L'attention portée aux AG n-3 n'a pas faibli au fil des ans, mais les preuves du bienfondé de la capacité de ces AG à réduire le risque cardiovasculaire ne sont pas convaincantes en l'état actuel des exigences, qui ont beaucoup évolué en un demisiècle. L'accumulation d'études et de méta-analyses aux conclusions incertaines ou négatives a quelque peu échaudé les enthousiasmes de la première heure, bien que personne ne songe à contester le rôle physiologique des AG n-3. Les propositions formulées par Dyerberg et ses collègues laissent dubitatifs. Comme indiqué plus haut, elles ont même été éreintées par plusieurs études récentes [START_REF] Aung | Omega-3 Treatment Trialists' Collaboration. Associations of omega-3 fatty acid supplement use with cardiovascular disease risks: meta-analysis of 10 trials involving 77 917 individuals[END_REF][START_REF] Bowman | Effects of n-3 fatty acid supplements in diabetes mellitus[END_REF][START_REF] Manson | Marine n-3 fatty acids and prevention of cardiovascular disease and cancer[END_REF]. Les observations initiales à la base du concept ont été taillées en pièce au motif premier que l'équipe de Dyerberg n'avait pas réalisé d'enquête épidémiologique de mortalité, mais utilisé des statistiques officielles pour le moins contestables dans une région où l'habitat éloigné et l'offre de soins minimaliste ne permettait pas de connaitre les vraies causes de décès. * En 2014, George J. Fodor, cardiologue à l'Université d'Ottawa (Canada), avait déjà exécuté littéralement les travaux fondateurs en affirmant que les données de mortalité à la base du concept étaient vraisemblablement erronées et spéculatives [START_REF] Fodor | Fishing" for the origins of the ''Eskimos and heart disease'' story: facts or wishful thinking? A review[END_REF], pour la bonne raison que les données de mortalité actuelles font état d'une prévalence des maladies cardiovasculaires et d'un taux de mortalité 2 fois plus élevé dans les populations inuites que dans les populations non-inuites [START_REF] Jørgensen | High prevalence of markers of coronary heart disease among Greenland Inuit[END_REF]. Pour cet auteur, croyance et allégation se sont alliées pour construire une théorie démentie par les faits.

Conclusion

* En un demi-siècle, l'histoire des AG n-3 a été marquée par bien des rebondissements. Née d'une intuition qui tarde à être confirmée, l'allégation prêtant à ces AG essentiels un rôle préventif et curatif en pathologie cardiovasculaire est mise à mal par les études les plus récentes. Pourtant, l'engouement du grand public ne se dément pas, et les AG n-3 font partie des compléments alimentaires vedettes, avec des indications dépassant largement le cadre cardiovasculaire. Les sociétés savantes et les agences de santé, plus mesurées après avoir été échaudées par l'échec des traitements par capsule de chair de poisson, recommandent avec conviction la consommation de portions de poisson gras par semaine et des huiles riches en acide alpha-linolénique. Cette attitude mesurée témoigne d'une sagesse certaine, puisque l'alimentation occidentale est une faible pourvoyeuse d'AG n-3 en regard de l'excédent d'apport en AG n-6, réalisant un déséquilibre favorisant thrombose et inflammation. * En l'état de nos connaissances, la chair de poisson n'est pas la panacée annoncée, mais vaut certainement mieux que la poudre de corne de rhinocéros dont on connait la place éminente dans la médecine traditionnelle chinoise …

Les points essentiels

• Le concept protecteur des acides gras oméga3

Déclaration d'intérêt

Les auteurs déclarent ne pas avoir de liens d'intérêts.

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-Figure 1 .

 1 Figure 1. Localisation du district d'Upernavik au Groenland.

Figure 2 .

 2 Figure 2. Le repas Inuit.

Figure 3 .

 3 Figure 3. Jorn Dyerberg (1937-), en 2011.

Figure 4 .Figure 5 .

 45 Figure 4. Itinéraire métabolique des acides gras (AG), de leur apport alimentaire jusqu'à

  Si les données épidémiologiques sur lesquelles Dyerberg et Bang ont fondé le concept des AG n-3 apparaissent aujourd'hui contestables, le rôle physiologique des AG n-3 ne l'est pas.• L'engouement des médecins et du grand public pour les AG n-3 a tout lieu d'être échaudé par trois études récentes méthodologiquement satisfaisantes montrant que la supplémentation en AG n-3 n'avait aucun effet bénéfique en terme de maladies cardiovasculaires ou de cancer. • La supplémentation en AG n-3 n'est pas nécessaire chez les adultes en bonne santé consommant 2 portions de poisson par semaine, ce qui suffit à couvrir les besoins en EPA et DHA.

	(AG n-3) a été élaboré à partir de données statistiques indiquant que l'incidence des maladies cardiovasculaires était moindre et que le bilan lipidique était plus satisfaisant chez les Esquimaux du Groenland que chez les Danois, en dépit d'une alimentation hyperlipidique comparable. • L'effet potentiellement bénéfique attribué à la forte teneur en acides eicosapentaénoïque (EPA ; C20:5 n-3) et docosahexaénoïque (DHA ; C22:6 n-3) de l'alimentation à base de produits marins a été partiellement conforté par des études expérimentales et chez l'homme. •

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