Ananth Prasad Burada

Rajesh Vinnakota

Bertrand Lambolez

Ludovic Tricoire
email: ludovic.tricoire@upmc.fr

Janesh Kumar
email: janesh@nccs.res.in

Structural biology of ionotropic glutamate delta receptors and their crosstalk with metabotropic glutamate receptors

Keywords: Delta receptors, ionotropic, metabotropic, synaptic plasticity, glutamate receptors. Cryo- Delta receptors, ionotropic, metabotropic, synaptic plasticity, glutamate receptors. Cryo-

Introduction

Almost two decades after the excitatory amino acids first emerged as neurotransmitters in the vertebrate central nervous system, the three major subtypes of ionotropic glutamate receptors (iGluRs) were named N-methyl-D-aspartate (NMDA), quisqualate, and kainate in the seminal review by Watkins and[START_REF] Watkins | Excitatory amino acid transmitters[END_REF][START_REF] Watkins | Excitatory amino acid transmitters[END_REF]. Quisqualate receptors were later named α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [START_REF] Honoré | The Binding of [3H]AMPA, a Structural Analogue of Glutamic Acid, to Rat Brain Membranes[END_REF]. Delta receptors were, however, identified a decade later by low stringency hybridization screening using GluA1-GluA4 as probes on mouse forebrain library and named as Delta1 [START_REF] Yamazaki | Molecular cloning of a cDNA encoding a novel member of the mouse glutamate receptor channel family[END_REF] and Delta2 [START_REF] Lomeli | The rat delta-1 and delta-2 subunits extend the excitatory amino acid receptor family[END_REF] owing to their low but significant sequence similarity of ~17-28 % to other iGluR subfamilies. Despite similarities in sequence and structural topologies, glutamate, AMPA, and kainate did not bind to the Delta1 or Delta2 or evoked whole-cell currents from HEK293 cells expressing homomeric Delta1 or Delta2 receptors, attracting the label "orphan" [START_REF] Lomeli | The rat delta-1 and delta-2 subunits extend the excitatory amino acid receptor family[END_REF]. This discovery, however, necessitated the addition of the fourth class into the iGluR family resulting in NMDA, AMPA, kainate, and Delta receptors as is known today. While initial studies reported expression of delta receptors (GluD1 and GluD2) in the inner ear [START_REF] Gao | Orphan glutamate receptor delta1 subunit required for high-frequency hearing[END_REF], hippocampus [START_REF] Gao | Orphan glutamate receptor delta1 subunit required for high-frequency hearing[END_REF][START_REF] Tao | Postsynaptic δ1 glutamate receptor assembles and maintains hippocampal synapses via Cbln2 and neurexin[END_REF], and cerebellum [START_REF] Araki | Selective expression of the glutamate receptor channel delta 2 subunit in cerebellar Purkinje cells[END_REF], respectively, recent work has revealed much broader expression in several regions of the brain [START_REF] Hepp | Glutamate receptors of the delta family are widely expressed in the adult brain[END_REF][START_REF] Jakobs | Expression of mRNA for glutamate receptor subunits distinguishes the major classes of retinal neurons, but is less specific for individual cell types[END_REF][START_REF] Konno | Enriched expression of GluD1 in higher brain regions and its involvement in parallel fiber-interneuron synapse formation in the cerebellum[END_REF][START_REF] Nakamoto | Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain[END_REF]. GluD receptors mediate numerous behavioral and neurocognitive functions, insights into which primarily came from genetic and knockdown studies. Mice defective in the GluD2 receptors were ataxic and had defective Parallel Fiber (PF) -Purkinje cell (PC) synapse formation and cerebellar long term depression (LTD) [START_REF] Kashiwabuchi | Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluR delta 2 mutant mice[END_REF][START_REF] Kurihara | Impaired parallel fiber-->Purkinje cell synapse stabilization during cerebellar development of mutant mice lacking the glutamate receptor delta2 subunit[END_REF]. In addition, single-nucleotide polymorphism and copy number variation studies have linked GluD1 with autism, schizophrenia, bipolar disorder, aberrant social and cognitive behavior, and major depression [START_REF] Benamer | GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons[END_REF][START_REF] Liu | Striatal glutamate delta-1 receptor regulates behavioral flexibility and thalamostriatal connectivity[END_REF][START_REF] Yadav | Deletion of glutamate delta-1 receptor in mouse leads to enhanced working memory and deficit in fear conditioning[END_REF][START_REF] Yadav | Deletion of glutamate delta-1 receptor in mouse leads to aberrant emotional and social behaviors[END_REF][START_REF] Yuzaki | A GluD Coming-Of-Age Story[END_REF]. Similarly, de novo and inherited mutations in GluD2 have been associated with cerebellar symptoms, retarded speech, and cognitive impairment [START_REF] Maier | De novo partial deletion in GRID2 presenting with complicated spastic paraplegia[END_REF][START_REF] Utine | A homozygous deletion in GRID2 causes a human phenotype with cerebellar ataxia and atrophy[END_REF][START_REF] Yuzaki | A GluD Coming-Of-Age Story[END_REF].

GluD receptor function in synapse formation, maturation, and maintenance are primarily attributed to their role in mediating trans-synaptic, tripartite complex formation with cerebellin and neurexin at PF-PC synapses by GluD2 [START_REF] Cheng | Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex[END_REF][START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF][START_REF] Matsuda | Cbln1 is a ligand for an orphan glutamate receptor delta2, a bidirectional synapse organizer[END_REF][START_REF] Uemura | Trans-synaptic interaction of GluRdelta2 and Neurexin through Cbln1 mediates synapse formation in the cerebellum[END_REF] and at parallel fiber (PF)-molecular interneuron (MLI) synapses [START_REF] Konno | Enriched expression of GluD1 in higher brain regions and its involvement in parallel fiber-interneuron synapse formation in the cerebellum[END_REF], hippocampal synapses [START_REF] Tao | Postsynaptic δ1 glutamate receptor assembles and maintains hippocampal synapses via Cbln2 and neurexin[END_REF] and cortex [START_REF] Fossati | Trans-Synaptic Signaling through the Glutamate Receptor Delta-1 Mediates Inhibitory Synapse Formation in Cortical Pyramidal Neurons[END_REF] by GluD1.

The mechanisms underlying ionotropic functions of the GluD receptor are, however, much less clear. Extensive electrophysiological assays on the Lurcher mutant (Lc) and chimeric receptors generated by transplantation of LBD from AMPA and kainate receptors to GluDs had identified the presence of functional ion channels [START_REF] Kakegawa | D-serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor[END_REF][START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF][START_REF] Orth | The delta subfamily of glutamate receptors: characterization of receptor chimeras and mutants[END_REF][START_REF] Schmid | The glutamate receptor subunit delta2 is capable of gating its intrinsic ion channel as revealed by ligand binding domain transplantation[END_REF][START_REF] Schmid | To gate or not to gate: are the delta subunits in the glutamate receptor family functional ion channels?[END_REF][START_REF] Wollmuth | The Lurcher mutation identifies delta 2 as an AMPA/kainate receptor-like channel that is potentiated by Ca(2+)[END_REF]. Notably, the GluD2 Lc mutant has been instrumental in demonstrating the binding of D-ser and glycine. Both amino acids cause a reduction of GluD2 Lc ion current [START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF]. Recent studies have shed light on the role of group I metabotropic glutamate receptors (mGlu1/5) and adrenergic receptors in triggering the opening of GluD pore [START_REF] Ady | Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors[END_REF][START_REF] Benamer | GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons[END_REF][START_REF] Dadak | mGlu1 receptor canonical signaling pathway contributes to the opening of the orphan GluD2 receptor[END_REF][START_REF] Gantz | Delta glutamate receptor conductance drives excitation of mouse dorsal raphe neurons[END_REF][START_REF] Lemoine | Probing the ionotropic activity of glutamate GluD2 receptor in HEK cells with geneticallyengineered photopharmacology[END_REF]. Alike the Lc mutant, this current is reduced by D-ser and by NASPM, a blocker of GluD2 Lc, and was absent upon expression of dead pore mutant or in the absence of GluD1/2 [START_REF] Ady | Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors[END_REF][START_REF] Benamer | GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons[END_REF][START_REF] Gantz | Delta glutamate receptor conductance drives excitation of mouse dorsal raphe neurons[END_REF]. The recent development of an optogenetic pharmacological tool holds promise to probe further the elusive ionotropic contribution of GluD receptors to synaptic physiology [START_REF] Lemoine | Probing the ionotropic activity of glutamate GluD2 receptor in HEK cells with geneticallyengineered photopharmacology[END_REF].

Structural insights in GluD receptors first came from the crystal structures of ligand-binding binding domain (LBD) of GluD2 in apo and D-Ser bound states [START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF]. Subsequently, structures of the amino-terminal domain (ATD) of GluD1 and GluD2 [START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF] and extracellular domain (ATD-LBD) of GluD2 receptor [START_REF] Cheng | Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex[END_REF] gave initial insights into the structure and function of GluD receptors. These studies showed that the LBD cleft could not accommodate glutamate like GluN1 and shares structural similarities with other iGluRs [START_REF] Kristensen | Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors[END_REF][START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF]. Recent structures of the full-length GluD1 (Burada et al., 2020b) and GluD2 (Burada et al., 2020c) revealed a novel architecture distinct from other iGluR families (Burada et al., 2020a).

In this review, we summarize the insights gained from the structures of the isolated extracellular domains of GluD receptors and the recently determined cryoEM structures of full-length receptors.

In addition, we review the latest developments in the understanding of the crosstalk between metabotropic glutamate receptors and GluD receptors and their role in synaptic physiology.

Amino-terminal domains in Delta receptor function and stability

Amino-terminal domain (ATD) is the largest sub-domain of the iGluRs with an average of 400 amino acids, nearly one-third of the full-length receptor. They play a crucial role in tetrameric assembly by mediating inter-subunit interactions [START_REF] Clayton | Crystal structure of the GluR2 amino-terminal domain provides insights into the architecture and assembly of ionotropic glutamate receptors[END_REF][START_REF] Jin | Crystal structure and association behaviour of the GluR2 amino-terminal domain[END_REF][START_REF] Kumar | The N-terminal domain of GluR6-subtype glutamate receptor ion channels[END_REF][START_REF] Kumar | Crystal structures of the glutamate receptor ion channel GluK3 and GluK5 amino-terminal domains[END_REF] and receptor trafficking [START_REF] Qiu | An endoplasmic reticulum retention signal located in the extracellular amino-terminal domain of the NR2A subunit of N-Methyl-D-aspartate receptors[END_REF]. GluD-ATDs perform many functions, including maintenance of the receptor's tetrameric assembly (Burada et al., 2020b(Burada et al., , 2020a)); mediating interactions with the soluble cerebellin from presynaptic neurons, and neurexin from the presynaptic membrane for the formation of a trans-synaptic complex [START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF][START_REF] Tao | Postsynaptic δ1 glutamate receptor assembles and maintains hippocampal synapses via Cbln2 and neurexin[END_REF][START_REF] Yuzaki | The delta2 glutamate receptor: a key molecule controlling synaptic plasticity and structure in Purkinje cells[END_REF][START_REF] Yuzaki | A GluD Coming-Of-Age Story[END_REF]) essential for the proper apposition of the pre and postsynaptic neurons [START_REF] Cheng | Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex[END_REF][START_REF] Yamashita | Contribution of Postsynaptic GluD2 to Presynaptic R-type Ca2+ Channel Function, Glutamate Release and Long-term Potentiation at Parallel Fiber to Purkinje Cell Synapses[END_REF][START_REF] Yuzaki | A GluD Coming-Of-Age Story[END_REF]. Besides, ATD was found to be important for potentiating fast excitatory synaptic transmission via GluD1 [START_REF] Tao | Postsynaptic δ1 glutamate receptor assembles and maintains hippocampal synapses via Cbln2 and neurexin[END_REF]. Indeed, expression of GluD1 without ATD or GluD1 with GluA1 ATD or GluA1 alone failed to increase excitatory transmission, whereas chimeric GluA1 with GluD1 ATD could still carry out the functions of wild type GluD1 [START_REF] Tao | Postsynaptic δ1 glutamate receptor assembles and maintains hippocampal synapses via Cbln2 and neurexin[END_REF]. Moreover, the ATD mediates ATD-LBD coupling, which has been shown to be important for GluD gating [START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF]. Tinkering with the ATD-LBD linker by adding a glycan wedge impaired the inhibition of Lurcher currents requiring ~ 1.5 times more D-Ser than the wild type Lurcher mutant, highlighting the role of ATD-LBD coupling in the gating of GluD receptors [START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF][START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF].

The monomers of GluD family receptor ATDs have conserved bilobed (R1 and R2) architecture [START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF]. Interestingly, unlike other iGluRs, the cleft between the two lobes is occupied by a 12 amino acid long cleft loop that extends from the R1 to R2 lobe and is stabilized by a disulfide bridge, unique to GluDs [START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF]. Posterior to the cleft loop, another loop harbors an N-glycan and is stabilized by another GluD-specific disulfide bridge [START_REF] Cheng | Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex[END_REF][START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF] (Fig. 1a). This loop is well-extended and could be a hub for interacting sites of other proteins. Apart from these differences, a GluD-exclusive helix is also present and is hypothesized to be involved in ATD-LBD intra subunit interactions [START_REF] Cheng | Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex[END_REF][START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF]. These three exclusive structural components might play a role in the unique functionality and architecture of the GluD family of receptors.

Two GluD ATD monomers form 2-fold symmetric dimers similar to that observed in AMPA and kainate receptors (Fig. 1b). The dimers form with high affinity (nanomolar) as observed in other iGluRs and pack tightly with more interacting surface area when compared to other iGluRs (Burada et al., 2020b(Burada et al., , 2020c;;[START_REF] Cheng | Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex[END_REF][START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF]. The buried surface area [START_REF] Krissinel | Inference of macromolecular assemblies from crystalline state[END_REF] for the ATD dimer interface in GluD1 (5KC9) and GluD2 (5KC8) is ~1601 Å 2 and ~1588 Å 2 , respectively, which is slightly higher than ~1490 Å 2 for GluK2 (3H6G), and ~1152 Å 2 for GluA2 (6U5S). Further, ATD domains mediate the dimer-of-dimer interactions that contribute to iGluR assembly as tetramers. Usually, R2 lobes of proximal subunits are involved in tetramer formation at the ATD layer. The GluD1 receptor structure revealed a much lesser buried surface ~28Å 2 when compared to ~300 Å 2 interaction interface in AMPA, kainate receptors. The crystal structures of isolated GluD2 ATD fused with Cbln1 assembled as N-shaped tetrameric arrangement in unit cells as observed in GluA2 and kainate receptors [START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF]. However, in the full-length cryo-EM structures of GluD receptors (Burada et al., 2020b(Burada et al., , 2020c) ) and the crystal structures of the extracellular domain of the GluD2 receptor, it was not observed [START_REF] Cheng | Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex[END_REF]. In addition, unlike kainate and AMPA receptors where the two ATD dimer pairs are tilted at the dimer-of-dimer interface owing to a domain-swapped organization, the GluD1 ATD dimer pairs are arranged almost in the same plane. Due to this arrangement, a line connecting the center of masses (COMs) of the lower R2 lobes of subunits A and B makes a reflex angle of ~200 with COM of subunit D (from the BD dimer pair) (Fig. 2 a-b). However, in the case of AMPA (GluA2) and kainate (GluK3) receptors the R2 COMs of subunits A, B and D subtend an obtuse angle of ~120 (Fig. 2 c-d) owing to the tilt.

Insights from high-resolution structures of GluD receptor ligand-binding domains

LBDs undergo conformational changes on agonist binding and transduce the generated strain towards ATD and TM domains, resulting in ion channel opening in NMDA, AMPA, and KA receptors. LBDs are highly conserved with sequence identities of ~48% across the iGluR subfamilies and ~61% between GluD1 and GluD2 subunits (Burada et al., 2020a;[START_REF] Traynelis | Glutamate receptor ion channels: structure, regulation, and function[END_REF]. The first structure of an isolated domain of the GluD family was GluD2-LBD [START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF] and to date, only three crystal structures of isolated LBDs of GluD2 [START_REF] Kristensen | Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors[END_REF][START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF] have been reported. The LBD structures revealed structural features similar to other iGluRs having a venus flytrap-like architecture with two subdomains, D1 and D2 [START_REF] Armstrong | Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: Crystal structures of the GluR2 ligand binding core[END_REF][START_REF] Furukawa | Mechanisms of activation, Inhibition and Specificity: Crystal Structures of NR1 Ligand-Binding Core[END_REF][START_REF] Mayer | Crystal Structures of the GluR5 and GluR6 Ligand Binding Cores: Molecular Mechanisms Underlying Kainate Receptor Selectivity[END_REF][START_REF] Veran | Zinc potentiates GluK3 glutamate receptor function by stabilizing the ligand binding domain dimer interface[END_REF]. In addition, the apo form of the GluD2 LBD that crystallized as a dimer with calcium sitting at the dimer interface, possibly contributing to its stability, pointed towards a conserved mechanism of coupling at the LBD layer [START_REF] Hansen | Modulation of the dimer interface at ionotropic glutamate-like receptor delta2 by D-serine and extracellular calcium[END_REF][START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF]. The apo structure also gave insights into the binding pocket of the LBD domain. The LBD pocket of GluD2 is similar to GluN1 and cannot accommodate glutamate. Further, amino acids Val687 and Trp741 in GluD that corresponds to Val689 and Trp731 in GluN1 create a hydrophobic environment that hinders the binding of glutamate [START_REF] Furukawa | Mechanisms of activation, Inhibition and Specificity: Crystal Structures of NR1 Ligand-Binding Core[END_REF]. On the other hand, the LBD of GluD2 bound to D-Ser and 7-CKA (7-chloro Kynurenic acid) crystallized as a monomer [START_REF] Kristensen | Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors[END_REF][START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF] [START_REF] Kristensen | Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors[END_REF][START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF]. Structural studies of AMPA, KA, and NMDA LBDs have shown that agonist induces maximum cleft closure, while an antagonist on binding brings relatively small cleft closure compared to the apo conformation of the domain [START_REF] Kristensen | Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors[END_REF][START_REF] Pohlsgaard | Lessons from more than 80 structures of the GluA2 ligand-binding domain in complex with agonists, antagonists and allosteric modulators[END_REF][START_REF] Yu | Neurotransmitter Funneling Optimizes Glutamate Receptor Kinetics[END_REF]. Thus the D-Ser bound GluD2 LBD resembles the agonist-induced cleft closure, whereas the 7-CKA resembles an antagonist-induced cleft closure [START_REF] Kristensen | Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors[END_REF]. This is consistent with the lower inhibition of Lurcher currents by 7-CKA compared to D-Ser [START_REF] Kristensen | Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors[END_REF]. D-Ser induces complete cleft closure, which pulls the LBD-TM linkers with sufficient energy for closing the constitutively conducting pore. While D-Ser binds with much lower affinity to GluD2 LBD [START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF], attributed to the hinge region connecting the two lobes [START_REF] Tapken | The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region[END_REF]; recent computations of the conformational free energy landscapes remarkably revealed that D-Ser binding exhibited a significantly higher conformational free energy of agonist binding in comparison to GluA2, GluK2, GluN1, GluN2A and GluN3A LBDs [START_REF] Chin | D-Serine Potently Drives Ligand-Binding Domain Closure in the Ionotropic Glutamate Receptor GluD2[END_REF]. This is consistent with D-Ser-induced LTD (long-term depression) by GluD receptors that require the transmission of the signal from LBD to CTD. However, why the D-Ser binding is unable to gate the GluD receptors is still not understood as insufficient force generation on agonist binding may not the reason for the inactivity of GluD receptors and requires further exploration. The key could be the ATD-LBD and LBD-TM linkers that have been shown to significantly influence the receptor's functions [START_REF] Elegheert | Structural basis for integration of GluD receptors within synaptic organizer complexes[END_REF].

The unique arrangement of subunits in GluD receptor tetramer

All the iGluR full-length structures for AMPA, NMDA, and kainate receptors solved to date either via X-ray crystallography [START_REF] Lee | NMDA receptor structures reveal subunit arrangement and pore architecture[END_REF][START_REF] Sobolevsky | X-ray structure of AMPA-subtype glutamate receptor: symmetry and mechanism[END_REF] or cryoEM structures of recombinantly expressed receptors [START_REF] Kumari | Structural and Functional Insights into GluK3-kainate Receptor Desensitization and Recovery[END_REF][START_REF] Meyerson | Structural mechanism of glutamate receptor activation and desensitization[END_REF][START_REF] Romero-Hernandez | Molecular Basis for Subtype Specificity and High-Affinity Zinc Inhibition in the GluN1-GluN2A NMDA Receptor Amino-Terminal Domain[END_REF][START_REF] Twomey | Channel opening and gating mechanism in AMPA-subtype glutamate receptors[END_REF][START_REF] Zhu | Mechanism of NMDA Receptor Inhibition and Activation[END_REF] (Fig. 4 c) showed a lack of domain swapping at the LBD layer (Burada et al., 2020b(Burada et al., , 2020c)). Thus, GluDs maintain the same subunit partners at both the ATD and LBD layers. In the GluD1 receptor, the two-fold symmetry at the extracellular domain and 4-fold symmetry at the pore domains is, however, conserved. Also, the tetrameric dimer-of-dimer interface at the ATD layer was observed in "compact" conformation and confirmed by cysteine crosslinking experiments (Burada et al., 2020b). The lack of crossover imparts increased conformational freedom to the two extracellular dimer arms allowing the receptor to adopt an extended or splayed conformation whereby the tetrameric ATD dimer-of-dimer interface is disrupted (Burada et al., 2020b). While disrupted ATD dimer-of-dimer interface has been observed previously in AMPA [START_REF] Nakagawa | Structure and different conformational states of native AMPA receptor complexes[END_REF][START_REF] Dürr | Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states[END_REF][START_REF] Meyerson | Structural mechanism of glutamate receptor activation and desensitization[END_REF][START_REF] Zhao | Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM[END_REF], and NMDA receptors [START_REF] Jalali-Yazdi | Mechanisms for Zinc and Proton Inhibition of the GluN1/GluN2A NMDA Receptor[END_REF][START_REF] Zhu | Mechanism of NMDA Receptor Inhibition and Activation[END_REF], the non-swapped architecture is unique to GluD receptors. It seems likely that the extensive interactions at the ATD, LBD layer and ATD-LBD interface coupled with short ATD-LBD linkers contribute to the unique non-swapped architecture of GluD receptors. Due to the limited resolution of both the GluD1 and GluD2 structures, the ATD-LBD and LBD-TM linkers could not be modeled. However, non-crossover in GluD receptors would have an effect on the conformations of the linkers and the order in which the TM domains are arranged (Fig. 5).

Relevant to this, a non-swapped-like arrangement of LBD domains was observed recently in a study on the ATD-deleted NMDA receptors where a fraction of the receptor population adopted LBD packing similar to that in the GluD1, emphasizing the importance of ATDs in guiding the subunit arrangement of the LBD layer. The well-resolved LBD-TM linkers in this ΔATD NMDA receptor adopted a relaxed conformation, rendering the receptor inactive [START_REF] Song | Mechanism of NMDA receptor channel block by MK-801 and memantine[END_REF]. Apart from the non-swapped organization of the extracellular domains and the differences in the arrangement of TM domains (Fig. 5), the GluD1 structure also revealed a splayed assembly of the TM domains. In contrast to AMPA, kainate, and NMDA receptors, the TM domains appear to be more loosely packed (Fig. 6). The distances between the C atoms of the M3 helix residues L632 (Top) L622 (middle) and R611 (bottom) are ~13Å, 22Å, and 35Å in GluD1(Fig. 6 a), whereas the distances between corresponding residues in GluA2, GluK2, and GluN1/GluN2A are ~15Å, 16Å, 30Å (GluA2), 16Å, 16Å, 30Å (GluK2) and 15Å, 16Å, 28Å (GluN1/GluN2A) respectively (Fig. 6 b-d). Thus GluD1 M3 helix is more constricted at the top and splayed towards the bottom. This results in a much wider vestibule in the GluD1 receptor compared to other iGluRs.

It's also evident when the distances between M4 helices are analyzed (Fig. 6). Whether this TM domain arrangement also translates into requiring more energy to open the pore is an outstanding question. High-resolution structures of GluD receptors where the LBD-TM linkers are resolved coupled with molecular dynamics simulations might be able to provide some answers.

Several other questions remain to be answered, such as the driving force behind the domain swapping and its importance in the receptor's functionality. It could be hypothesized that domain swapping is required for the conductivity of the receptors. However, in the case of GluD receptors, the physiological relevance and implications of the unique non-swapped architecture are unclear and need further investigations. Moreover, biochemical and structural investigations in the emerging area of mGlu and GluD crosstalk and regulation of ionotropic activity of GluD receptors by mGlus (discussed below) are warranted to reveal mechanistic details into physiological functions of GluD receptors.

Overlap between mGlu1/5 and GluD1/2 molecular interactors

Both GluD2 and mGlu1 are strongly expressed in Purkinje cells (PC) of the cerebellum [START_REF] Araki | Selective expression of the glutamate receptor channel delta 2 subunit in cerebellar Purkinje cells[END_REF][START_REF] Baude | The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction[END_REF][START_REF] Hepp | Glutamate receptors of the delta family are widely expressed in the adult brain[END_REF][START_REF] Kano | Persistent Multiple Climbing Fiber Innervationof Cerebellar Purkinje Cellsin Mice Lacking mGluR1[END_REF][START_REF] Lomeli | The rat delta-1 and delta-2 subunits extend the excitatory amino acid receptor family[END_REF][START_REF] Nusser | Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization[END_REF]. While mGlu1 is present at both glutamatergic parallel fibers (PF) and climber fibers (CF)-to-PC synapse, GluD2 is only expressed at PF-PC synapses [START_REF] Baude | The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction[END_REF][START_REF] Landsend | Differential localization of delta glutamate receptors in the rat cerebellum: coexpression with AMPA receptors in parallel fiber-spine synapses and absence from climbing fiber-spine synapses[END_REF][START_REF] Nusser | Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization[END_REF]. In the cerebellar molecular layer, GluD1 is expressed in GABAergic interneurons, which also express mGlu1/5 [START_REF] Hámori | Immunocytochemical localization of mGluR1a metabotropic glutamate receptor in inhibitory interneurons of the cerebellar cortex[END_REF][START_REF] Konno | Enriched expression of GluD1 in higher brain regions and its involvement in parallel fiber-interneuron synapse formation in the cerebellum[END_REF]. GluD1 is selectively present at the PF-interneurons glutamatergic synapse [START_REF] Konno | Enriched expression of GluD1 in higher brain regions and its involvement in parallel fiber-interneuron synapse formation in the cerebellum[END_REF]. Similarly, both mGlu1/5 are expressed at glutamatergic synapses in striatal neurons but GluD1 is selectively expressed at synapses expressing type 2 vesicular glutamate transporter (VGluT2, putatively, thalamostriatal synapses) [START_REF] Liu | Striatal glutamate delta-1 receptor regulates behavioral flexibility and thalamostriatal connectivity[END_REF]. These data support the idea that GluD1/2 and mGlu1/5 are present at the same glutamatergic synapses. Biochemical assays further showed that they belong to the same protein complex or are very close to each other. In the cerebellum, GluD2 and mGlu1 have been reported to co-immunoprecipitate [START_REF] Kato | Glutamate receptor δ2 associates with metabotropic glutamate receptor 1 (mGluR1), protein kinase Cγ, and canonical transient receptor potential 3 and regulates mGluR1-mediated synaptic transmission in cerebellar Purkinje neurons[END_REF][START_REF] Uemura | Direct interaction of GluRdelta2 with Shank scaffold proteins in cerebellar Purkinje cells[END_REF]. Such protein-protein interaction has also been demonstrated between GluD1 and mGlu1/5, both in a HEK cell heterologous expression system [START_REF] Benamer | GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons[END_REF] and in native hippocampal tissue [START_REF] Suryavanshi | Glutamate Delta-1 Receptor Regulates Metabotropic Glutamate Receptor 5 Signaling in the Hippocampus[END_REF]. Further investigation is required to determine whether the interaction is direct, however, it is noteworthy that several interaction partners of GluD1/2 are also linked to mGlu1 signaling or membrane trafficking. This is the case of protein kinase C gamma, which is part of the canonical signaling cascade of mGlu1/5 [START_REF] Kato | Glutamate receptor δ2 associates with metabotropic glutamate receptor 1 (mGluR1), protein kinase Cγ, and canonical transient receptor potential 3 and regulates mGluR1-mediated synaptic transmission in cerebellar Purkinje neurons[END_REF] and of protein interacting with C kinase 1 (PICK1), which is necessary for mGlu1-dependent long term plasticity in PC [START_REF] Yawata | Membrane-Proximal Region of Glutamate Receptor δ2 Subunit Is Critical for Long-Term Depression and Interaction with Protein Interacting with C Kinase 1 in a Cerebellar Purkinje Neuron[END_REF]. Similarly, MRCKγ (CDC42-binding protein kinase gamma) and Itpr1 (inositol 1,4,5-triphosphate receptor 1) were detected in GluD2 immunoprecipitates from PC postsynaptic density, indicating that they are not only present at the PF-PC synapse but also participate in the same protein complex as GluD2. These proteins can bind to, respectively, DAG and IP3, which are the metabolites of PIP2 hydrolysis involved in mGlu1/5 signaling [START_REF] Selimi | Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse[END_REF]. Moreover, the alpha isoform of MRCK is found in GluD1 immunoprecipitate [START_REF] Fossati | Trans-Synaptic Signaling through the Glutamate Receptor Delta-1 Mediates Inhibitory Synapse Formation in Cortical Pyramidal Neurons[END_REF]. Both GluD1 and GluD2 were found to interact with the neuronal isoform of Golgi protein PIST (PDZ domain Protein Interacting Specifically with TC10), also known as CAL (Cystic fibrosis transmembrane conductance regulator (CFTR)-Associated Ligand) [START_REF] Yue | A novel protein complex linking the delta 2 glutamate receptor and autophagy: implications for neurodegeneration in lurcher mice[END_REF]. This protein regulates mGlu5 membrane targeting and mGlu1 activity [START_REF] Cheng | The PDZ domain protein CAL interacts with mGluR5a and modulates receptor expression[END_REF][START_REF] Zhang | A novel association of mGluR1a with the PDZ scaffold protein CAL modulates receptor activity[END_REF]. The family of scaffolding proteins Homer and Shank are crucial for regulating mGlu1/5 function. Interestingly, these two proteins were found in the same protein complex as GluD2 in the cerebellum [START_REF] Selimi | Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse[END_REF][START_REF] Uemura | Direct interaction of GluRdelta2 with Shank scaffold proteins in cerebellar Purkinje cells[END_REF]. Shank1 and Shank2 were shown to interact directly with GluD2 but not with GluD1. The Shank family also comprises a third isoform, Shank3, whose autism-associated mutations alter mGlu1/5 expression and mGlu1/5dependent synaptic plasticity [START_REF] Lee | Autism-associated Shank3 mutations alter mGluR expression and mGluR-dependent but not NMDA receptor-dependent long-term depression[END_REF]. Furthermore, Shank3 loss-of-function in the ventral tegmental area (VTA) causes a defect in excitatory transmission maturation and impairment of social interaction, which can be rescued by mGlu1 PAM [START_REF] Bariselli | SHANK3 controls maturation of social reward circuits in the VTA[END_REF]. However, whether Shank3 interacts with GluD1/2 is still unknown [START_REF] Uemura | Direct interaction of GluRdelta2 with Shank scaffold proteins in cerebellar Purkinje cells[END_REF]. The coexpression of mGlu1/5 and GluD1/2 at the same synapse combined with the physical interaction of GluD with mGlu receptors and mGlu-related signaling molecules suggests functional crosstalk between mGlu and GluD receptors signaling. The following sections will provide experimental evidence that GluD receptors regulate mGlu signaling and reciprocally that mGlu activation gate ionotropic function of GluD receptors.

mGlu1/5 gate the ion channels of GluD

Activation of mGlu1/5 induces a long-lasting excitation in several brain regions including the neocortex, thalamus, hippocampus, striatum, cerebellum, and midbrain [START_REF] Batchelor | Novel synaptic potentials in cerebellar Purkinje cells: probable mediation by metabotropic glutamate receptors[END_REF][START_REF] Camiré | Dendritic Signaling in Inhibitory Interneurons: Local Tuning via Group I Metabotropic Glutamate Receptors[END_REF][START_REF] Kaneda | Repetitive activation of glutamatergic inputs evokes a long-lasting excitation in rat globus pallidus neurons in vitro[END_REF][START_REF] Mo | Synaptic properties of the lemniscal and paralemniscal pathways to the mouse somatosensory thalamus[END_REF][START_REF] Viaene | Activation requirements for metabotropic glutamate receptors[END_REF]. Electrical stimulation of PF-PC synapse induced a slow excitatory postsynaptic current (sEPSC) mediated by mGlu1 activation in PC. It has been reported that this current (mGlu1-sEPSC) could be the result of the opening of canonical transient receptor (TRPC) channels [START_REF] Hartmann | TRPC3 channels are required for synaptic transmission and motor coordination[END_REF][START_REF] Kim | Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1[END_REF]. Similar observations have been made regarding the role of TRPC channels in mGlu1gated slow excitation in cerebellar interneurons, in the hippocampus, and the midbrain [START_REF] Bengtson | Transient receptor potential-like channels mediate metabotropic glutamate receptor EPSCs in rat dopamine neurones[END_REF][START_REF] Kougioumoutzakis | TRPC1 mediates slow excitatory synaptic transmission in hippocampal oriens/alveus interneurons[END_REF][START_REF] Kubota | mGluR1-mediated excitation of cerebellar GABAergic interneurons requires both G protein-dependent and Src-ERK1/2-dependent signaling pathways[END_REF]. However, in PC, although mGlu1-mediated excitation is absent in TRPC3-KO, it seems that the TRPC3 channel accounts for only a fraction of the mGlu1-sEPSC since selective TRPC3 blocker does not abolish the mGlu1-sEPSC [START_REF] Ady | Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors[END_REF][START_REF] Kubota | mGluR1-mediated excitation of cerebellar GABAergic interneurons requires both G protein-dependent and Src-ERK1/2-dependent signaling pathways[END_REF]. Indeed, in cerebellar PC and midbrain dopaminergic (DA) neurons, it has been shown that this excitation also relies on the opening of the GluD1/2 ion channel [START_REF] Ady | Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors[END_REF][START_REF] Benamer | GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons[END_REF]. Inward currents elicited either pharmacologically with mGlu1/5 agonist or synaptically by high-frequency electrical stimulation are strongly dampened in loss-of-function GluD1/2 mice or upon expression of GluD1/2 dead-pore mutants. This is reproduced in HEK cells upon coexpression of mGlu1/5 and GluD1/2 [START_REF] Ady | Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors[END_REF][START_REF] Benamer | GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons[END_REF]. These results indicate that both TRPC and GluD channels contribute to the mGlu1/5-gated current, but their relative contribution still needs to be determined. Interestingly, DA neurons exhibit two types of firing activity, phasing and bursting, the latter being associated with high DA release in target regions. The absence of GluD1 or expression of dead-pore GluD1 mutant specifically in DA neurons results in the absence of bursting activity [START_REF] Benamer | GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons[END_REF]. These data provide the first evidence of the physiological opening of the GluD1/2 ion channel.

Previous characterizations of the GluD ion channel were solely based on the analysis of GluD point mutants called Lurcher [START_REF] Wollmuth | The Lurcher mutation identifies delta 2 as an AMPA/kainate receptor-like channel that is potentiated by Ca(2+)[END_REF][START_REF] Yadav | Mutations in the transmembrane domain M3 generate spontaneously open orphan glutamate δ1 receptor[END_REF][START_REF] Zuo | Neurodegeneration in Lurcher mice caused by mutation in delta2 glutamate receptor gene[END_REF]. For example, the mutation A654C in GluD1 or A654T in GluD2 make the ion channel constitutively open. These mutants were used to identify some molecules inhibiting ion flow such as 1-Naphthyl acetyl spermine (NASPM), pentamidine, 7-chloro kynurenic acid (7-CKA), D-Ser/glycine [START_REF] Hansen | Modulation of the dimer interface at ionotropic glutamate-like receptor delta2 by D-serine and extracellular calcium[END_REF][START_REF] Kohda | Mutation of a glutamate receptor motif reveals its role in gating and delta2 receptor channel properties[END_REF][START_REF] Kristensen | Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors[END_REF][START_REF] Naur | Ionotropic glutamate-like receptor delta2 binds D-serine and glycine[END_REF][START_REF] Williams | Pharmacology of δ2 Glutamate Receptors: Effects of Pentamidine and Protons[END_REF]. Although these ligands can reduce mGlu1/5-dependent excitation [START_REF] Ady | Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors[END_REF][START_REF] Benamer | GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons[END_REF], they are not specific to GluD subunits since NASPM also antagonizes calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and 7-CKA, D-Ser, and glycine also bind NMDA receptors [START_REF] Traynelis | Glutamate receptor ion channels: structure, regulation, and function[END_REF]. To circumvent this issue of selectivity, an optopharmacological approach has been implemented to regulate the ion flow of the GluD2 subunit specifically. This approach consists of incorporating a cysteine mutation in the cavity located above the putative ion channel pore for site-specific conjugation with a photoswitchable ligand (PTL [START_REF] Mondoloni | Cell-Specific Neuropharmacology[END_REF][START_REF] Mondoloni | Cell-Specific Neuropharmacology[END_REF][START_REF] Paoletti | Optical control of neuronal ion channels and receptors[END_REF]. In this new subunit called LiGluD2 (GluD2 I677C), the PTL, called MAGu, adopts two different configurations upon light excitation at 380 and 535 nm, and can rapidly and reversibly inhibit GluD2 ion flow [START_REF] Lemoine | Probing the ionotropic activity of glutamate GluD2 receptor in HEK cells with geneticallyengineered photopharmacology[END_REF] (Fig. 7). This photo-inhibitable GluD2 subunit effectively regulates mGlu-gated GluD2 inward current, thereby confirming that mGlu signaling triggers the opening of the GluD2 ion channel [START_REF] Lemoine | Probing the ionotropic activity of glutamate GluD2 receptor in HEK cells with geneticallyengineered photopharmacology[END_REF]. Such modulation of iGluR ion flow by mGlu1/5 activation has also been reported for NMDA receptors and involves the Pyk2/Src-family kinase pathway and tyrosine phosphorylation of GluN2 subunits [START_REF] Heidinger | Metabotropic glutamate receptor 1-induced upregulation of NMDA receptor current: mediation through the Pyk2/Src-family kinase pathway in cortical neurons[END_REF].

However, in the case of GluD1/2, mGlu1/5 signaling seems the only way for glutamate to trigger the opening of the GluD ion channel since glutamate does not bind GluD directly. The molecular mechanism linking mGlu1/5 activation and pore opening of GluD is still under investigation but, in the case of GluD2, it has been reported in HEK cells and PC that it requires activation of the Gq/PLC/PKC canonical pathway [START_REF] Dadak | mGlu1 receptor canonical signaling pathway contributes to the opening of the orphan GluD2 receptor[END_REF] (Fig. 8). However, the mechanism of the mGlu1-sEPSC is still a matter of debate since previous reports provide conflicting evidence.

Several studies in PC indicate that the mGlu1-sEPSC is G-protein-dependent but insensitive to PKC and PLC inhibitors [START_REF] Canepari | Ca2+ ion permeability and single-channel properties of the metabotropic slow EPSC of rat Purkinje neurons[END_REF][START_REF] Canepari | The conductance underlying the parallel fibre slow EPSP in rat cerebellar Purkinje neurones studied with photolytic release of L-glutamate[END_REF][START_REF] Canepari | Evidence for protein tyrosine phosphatase, tyrosine kinase, and Gprotein regulation of the parallel fiber metabotropic slow EPSC of rat cerebellar Purkinje neurons[END_REF][START_REF] Hartmann | Distinct roles of Galpha(q) and Galpha11 for Purkinje cell signaling and motor behavior[END_REF][START_REF] Nelson | Lack of kinase regulation of canonical transient receptor potential 3 (TRPC3) channel-dependent currents in cerebellar Purkinje cells[END_REF], which is surprising since TRPC currents require PLC activity and production of diacylglycerol (DAG) [START_REF] Ramsey | An introduction to TRP channels[END_REF]. The mGlu1-sEPSC in PC further appears to be dependent on the balance between tyrosine kinase and tyrosine phosphatase. In PC, tyrosine kinase inhibitors and more specifically Src kinase inhibitors potentiate the mGlu1-sEPSC [START_REF] Auger | AMPA receptor activation controls type I metabotropic glutamate receptor signalling via a tyrosine kinase at parallel fibre-Purkinje cell synapses[END_REF][START_REF] Canepari | Evidence for protein tyrosine phosphatase, tyrosine kinase, and Gprotein regulation of the parallel fiber metabotropic slow EPSC of rat cerebellar Purkinje neurons[END_REF], while tyrosine phosphatase inhibitors cause a reduction of the mGlu1-sEPSC amplitude [START_REF] Canepari | Evidence for protein tyrosine phosphatase, tyrosine kinase, and Gprotein regulation of the parallel fiber metabotropic slow EPSC of rat cerebellar Purkinje neurons[END_REF]. This is in stark contrast with what is observed in other cell types or brain structures where the mGlu1-sEPSC has been described. In DA neurons and hippocampal neurons, the regulation by the balance tyrosine kinasephosphatase is the opposite of what is observed in PC [START_REF] Heuss | G-protein-independent signaling mediated by metabotropic glutamate receptors[END_REF][START_REF] Tozzi | Group I mGluRs coupled to G proteins are regulated by tyrosine kinase in dopamine neurons of the rat midbrain[END_REF]. In cerebellar interneurons of the molecular layer, inhibition of Src kinase leads to the reduction of the mGlu1-mediated excitation in contrast with what is observed in PC [START_REF] Kubota | mGluR1-mediated excitation of cerebellar GABAergic interneurons requires both G protein-dependent and Src-ERK1/2-dependent signaling pathways[END_REF].

Regarding the mechanism of GluD channel opening, it is unclear whether GluD2 phosphorylation by PKC is the triggering event. While PKC activity is necessary for the process of mGlu1-induced GluD2 opening, direct stimulation of PKC activity failed to mimic the effect of pharmacological activation of mGlu1 [START_REF] Dadak | mGlu1 receptor canonical signaling pathway contributes to the opening of the orphan GluD2 receptor[END_REF]. Regardless of the target of PKC, the Gq/PLC/PKC signaling pathway seems important for the opening of the GluD ion channel. Recently, the modulation of GluD1 ion function upon activation of Gq-coupled α1-adrenergic receptors (α1-AR) in serotonergic dorsal raphe neurons has been reported [START_REF] Gantz | Delta glutamate receptor conductance drives excitation of mouse dorsal raphe neurons[END_REF]. GluD1 ion channels appear to regulate the excitability of these neurons and underlie the slow excitatory current observed upon high-frequency stimulation of noradrenergic fibers. Furthermore, this slow excitation is reduced in the presence of NASPM and D-Ser/glycine and absent after the targeted deletion of GluD1 [START_REF] Gantz | Delta glutamate receptor conductance drives excitation of mouse dorsal raphe neurons[END_REF]. Noteworthy, the loss of GluD1, specifically in dorsal raphe neurons, causes increased anxiety in mice [START_REF] Gantz | Delta glutamate receptor conductance drives excitation of mouse dorsal raphe neurons[END_REF]. Interestingly, it has been demonstrated in the same neurons that activation of Gq protein-coupled histamine H1 and orexin OX2 receptors also produces an excitatory response that is occluded by the α1-AR-dependent response [START_REF] Brown | Convergent excitation of dorsal raphe serotonin neurons by multiple arousal systems (orexin/hypocretin, histamine and noradrenaline)[END_REF]. Whether the GluD1 channel is a common effector of these receptors, and possibly of yet, other Gq coupled receptors remains to be elucidated as well as the underlying mechanisms.

GluD regulate mGlu signaling

Group I mGlu activation drives several intracellular signaling pathways, including Gq/PLC/PKC, AkT/mTOR, MAPK/ERK cascades, and regulates trafficking of ionotropic glutamate receptors and local translation [START_REF] Niswender | Metabotropic glutamate receptors: physiology, pharmacology, and disease[END_REF]. Among multiple functions, mGlu signaling is crucial for regulating synaptic transmission via its role in long-term depression (mGlu-LTD [START_REF] Bellone | Mechanisms of synaptic depression triggered by metabotropic glutamate receptors[END_REF][START_REF] Gladding | Metabotropic glutamate receptor-mediated long-term depression: molecular mechanisms[END_REF][START_REF] Lüscher | Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease[END_REF]. The mGlu-LTD at parallel fiber to PC synapse has been the subject of intense investigation as it is believed to underlie motor learning, and GluD2 has been shown to be instrumental in the gating and expression of cerebellar mGlu-LTD [START_REF] Kashiwabuchi | Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluR delta 2 mutant mice[END_REF][START_REF] Yuzaki | A GluD Coming-Of-Age Story[END_REF]. Upon LTD induction and mGlu1 activation, PKC phosphorylates GluA2 subunits, which results in the internalization of GluA2-containing AMPA receptors and reduction of EPSC amplitude. The C-terminal end of GluD2 binds the tyrosine phosphatase PTPMEG, which regulates the phosphorylation state of GluA2 and its propensity to be internalized upon PKC phosphorylation [START_REF] Hironaka | The protein-tyrosine phosphatase PTPMEG interacts with glutamate receptor delta 2 and epsilon subunits[END_REF][START_REF] Kohda | The δ2 glutamate receptor gates long-term depression by coordinating interactions between two AMPA receptor phosphorylation sites[END_REF]. Interestingly, the ion function of GluD2 is not necessary for mGlu-LTD since LTD is absent in GluD2-KO mice but is restored upon re-expression of the dead pore GluD2 mutant [START_REF] Kakegawa | The delta2 "ionotropic" glutamate receptor functions as a non-ionotropic receptor to control cerebellar synaptic plasticity[END_REF][START_REF] Kohda | The extreme Cterminus of GluRdelta2 is essential for induction of long-term depression in cerebellar slices[END_REF]. Furthermore, it has been reported that the binding of D-Ser to GluD2 LBD induces a conformational change and produces a GluD2dependent LTD at immature PF to PC synapses [START_REF] Kakegawa | D-serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor[END_REF]. Not only this, D-Serdependent LTD occludes the regular mGlu-dependent LTD at the same synapse, but also, GluD2 mutant mice lacking D-Ser binding exhibit impaired LTD and motor dyscoordination during development [START_REF] Kakegawa | D-serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor[END_REF], suggesting that GluD2-dependent and mGlu-dependent LTD share common molecular mechanisms.

Regarding a similar role of GluD1 in LTD, several forms of postsynaptic mGlu1/5-LTD have been described in brain regions where GluD1 predominates over GluD2 [START_REF] Hepp | Glutamate receptors of the delta family are widely expressed in the adult brain[END_REF] such as the striatum, VTA, and hippocampus. They rely on the presence of calcium-permeable AMPA receptors, on the trafficking of GluA2 containing receptors, and regulation of local translation as the postsynaptic mechanism of LTD expression [START_REF] Bellone | Mechanisms of synaptic depression triggered by metabotropic glutamate receptors[END_REF][START_REF] Gladding | Metabotropic glutamate receptor-mediated long-term depression: molecular mechanisms[END_REF][START_REF] Lüscher | Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease[END_REF]. However, the role of GluD1 is still unclear. For example, in the striatum, agonist-mediated activation of mGlu1/5 or D-Ser application in the presence of NMDA receptor antagonist induces a decrease of EPSC amplitude without a change in release probability, suggesting a postsynaptic origin of the LTD. However, this LTD persists after the selective loss of GluD1 in the striatum [START_REF] Liu | Striatal glutamate delta-1 receptor regulates behavioral flexibility and thalamostriatal connectivity[END_REF]. Nonetheless, GluD1 appears to regulate mGlu1/5 signaling in the hippocampus, as shown by [START_REF] Suryavanshi | Glutamate Delta-1 Receptor Regulates Metabotropic Glutamate Receptor 5 Signaling in the Hippocampus[END_REF]. This study showed that the interaction of mGlu5 with Homer was abnormal in GluD1-KO mice. In the same mutant mice, the basal levels of phosphorylated mTOR and Akt were higher, and no increase was observed upon application of the mGlu5 agonist DHPG. They observed higher basal protein translation and an absence of DHPG-induced increase in protein synthesis. In accordance with the role of mGlu5mediated mTOR signaling in synaptic plasticity, DHPG-induced internalization of surface AMPA receptor subunits was impaired in GluD1 knockout mice.

These data clearly point to a bidirectional interaction between mGlu1/5 and GluD1/2 signaling.

Several studies have reported interaction between GPCR and iGluRs, especially between mGlu5 and other iGluRs such as NMDA receptors [START_REF] Reiner | Glutamatergic Signaling in the Central Nervous System: Ionotropic and Metabotropic Receptors in Concert[END_REF]. Using bioluminescence resonance energy transfer (BRET), a dynamic interaction between the C-terminal end of mGlu5 and GluN1/2B receptors has been reported that results in G protein-independent bidirectional inhibition [START_REF] Moutin | Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability[END_REF][START_REF] Perroy | Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors[END_REF]. The existence of such direct interaction between mGlu1/5 and GluD1/2 is still unclear and deserves further investigation.

Conclusions

In the nineties, ionotropic glutamate receptors were essentially viewed as a ligand-gated ion channel whose function was to convey chemical transmission from one neuron to another. However, over the past years, the idea emerged that they fulfill much more complex functions at the synapse. GluD subfamily is an excellent example since they not only demonstrate ionotropic function but are crucial for synaptogenesis, synaptic plasticity and also regulate metabotropic intracellular signaling. Based on studies of GluDs, ionotropic glutamate receptors start to be considered more than just ligand-gated channels. Recent structural and functional insights have profoundly changed our understanding of the GluD receptors and their role in synaptic physiology.

Knowing that, it is not surprising that mutations in the gene encoding for GluD receptors are associated with several neurological disorders. But the molecular link between GluD and its synaptic partners are still unclear and deserves a lot of attention. Several genetic and pharmacological tools have been developed to tackle the different functions of GluDs, and they will be instrumental in the understanding of GluD in neuronal brain circuitry. The crosstalk between GluD and metabotropic glutamate receptor and other GPCR seems to be unique to GluD receptors, and further investigation is required to better understand the mechanism linking these two receptor families and to apprehend how this relationship shapes synaptic transmission.

  and, by comparison with apo structure, gave insights into LBD domain movements. The degree of cleft closure varied between the apo, D-Ser, and 7-CKA bound structures (Fig 3). The apo state was most relaxed having an extended cleft with a 29.9º angle compared to D-Ser bound form that induced a closed cleft conformation. LBD clam-shell in the 7-CKA bound state showed an intermediate cleft closure of ~19.5º

  or isolated from native source (Yu et al., 2021; Zhao et al., 2019) showed a conserved domain architecture. The extracellular domains are arranged as 2-fold symmetric dimers with a 4-fold symmetric pore domain. A hallmark of the full-length structures has been domain crossover at the ATD and LBD layer between the proximal and distal subunits such that the dimer partners at the ATD and LBD layers are swapped (Fig. 4 a). However, the full-length structures of both GluD1 (Fig. 4 b) and GluD2

Figure 1 .

 1 Figure 1. Unique structural features of GluD Amino-terminal domains (ATD). Panel (a) Superimposition of GluD1 (orange) and GluD2 (green) ATD monomers (a) and dimers (b) are shown, highlighting the structural similarities between the two GluD subtypes. The unique loops and the extra helix found in GluD ATDs are shown. Panels c and d respectively show the superimposition of GluD1 ATD monomers and dimers with that of GluA2 ATD. Key differences between the GluA2 and GluD ATDs are highlighted.

Figure 2 .

 2 Figure 2. The arrangement of ATDs in the context of receptor tetramer. Panel (a) shows the COMs (spheres) for the R1 and R2 domains mapped onto the GluD1-ATD tetramer (6KSS). Panels (b-e) show the distances and the angles subtended between the COMs of GluD1, GluA2, GluK3, and GluN1/N2B ATD domains. The distances, angles are shown along with the PDB IDs of the coordinates.

Figure 3 .

 3 Figure 3. GluD Ligand-binding domains undergo ligand-induced conformational changes. A superposition of the crystal structures of GluD2 LBD on the S1 lobe in apo, D-Ser, and 7-CKA bound state is shown. The degree of cleft closure with respect to the D-Ser bound state is shown, highlighting the ligand-induced conformational changes in GluD-LBD. GluD2 in the apo state (2V3T), or complexed with D-Ser (2V3U), 7-CKA (5CC2), and LBD from the ATD-LBD ectodomain structure (5L2E) are shown.

Figure 4 .

 4 Figure 4. Full-length GluD receptors reveal a distinct architecture. (a) CryoEM structure of homomeric GluA2 (PDB ID: 5KBV) in complex with antagonist shows domain swapping at the LBD layer. Panel (b) and (d) show cryoEM structures of the homomeric GluD1 (PDB ID: 6KSS) and GluD2 (PDB ID: 6LU9) receptors, respectively in the presence of 7-CKA and calcium ions, highlight a non-swapped arrangement of the domains. The four subunits of receptor tetramer are colored uniquely subunit A (orange), subunit B (green), subunit C (yellow), and subunit D (cyan). The TM domains were not resolved in the GluD2 structures and hence are not shown and are depicted by a disc.

Figure 5 .

 5 Figure 5. Non-swapped architecture is unique to GluD receptors. Shown are the domain arrangement in GluD1, GluD2, GluA2, GluK2, GluN1/GluN2B, and GluN1/GluN2A (super splayed) receptors. Views from the extracellular side (top views) are shown for the ATD, LBD,

Figure 6 .

 6 Figure 6. Assembly at the TM domains. The packing of helices of GluD1 TMD is more compact at the top and expanded towards the cytosolic side when compared with other iGluRs. Distances between residues L632 (top), L622 (middle), and R611 (bottom) of the M3 helices of subunits B and D of GluD1 (a) and the corresponding residues in GluA2 (b), GluK2 (c), and GluN1/GluN2A (d) are shown. Similarly, the distances between M4 helices of subunits C and D are shown in the left panel. Note that due to domain swapping in GluD1 receptors, the position of the TM domains for subunits C and D are interchanged with respect to other iGluRs shown.

Figure 7 .

 7 Figure 7. GluD receptor activation by mGlu. A schematic of GluD receptor activation by Gq-PLC-PKC is depicted. Also shown are the potential sites for direct interaction between mGlu and GluD receptors. The mGlu5 cryoEM structures in apo (PDB ID: 6N52) and active (PDB ID: 6N51) state and GluD1 receptor (PDB ID: 6KSS) are depicted in a lipid bilayer. Apo mGlu1/5 on the binding of agonist glutamate switches to an active state, leading to a cascade of cytosolic events that might trigger the opening of the GluD pore. Another tantalizing possibility is GluD receptors'activation by direct physical interaction between the mGlu and GluD receptors. However, this poorly understood aspect of mGlu-GluD interactions is highlighted by question marks.

Figure 8 .

 8 Figure 8. Principle of photoswitchable GluD subunit (a). Approach to block ion channel of GluD2 as described in Lemoine et al. 2020. GluD2 is genetically modified to incorporate a cysteine residue (yellow ball) at the entrance to the pore, which serves as a handle for the covalent attachment of a synthetic, photoswitchable tethered ligand called MAGu. Under green light (525 nm), MAGu adopts an elongated state and places its cationic head group in the lumen, resulting in ion channel blockade. Under violet light (380 nm), MAGu switches to a twisted, shorter form and unblocks the channel. (b) Molecular modeling of GluD2 structure (orange and green) showing the ion channel (grey) in presence of MAGu (with courtesy of Antoine Taly -Université de Paris, UPR 9080, Laboratoire de Biochimie Théorique)

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Acknowledgments

This work was supported by the Wellcome Trust DBT India Alliance fellowship (grant number IA/I/13/2/501023) awarded to JK and by the French Agency for Research (ANR-16-CE16-0014-01) to LT. APB thanks ICMR, New Delhi, India, for the senior research fellowship (3/1/3/JRF-2014/HRD). RV thanks the Department of Biotechnology, New Delhi, India for a Research Associate fellowship. LT thanks the fondation Lejeune for its support (grants #1693 and 1959).

Author contributions

The manuscript was conceptualized, designed, and written by APB, RV, BL, LT, and JK. All authors reviewed and approved the manuscript.

Conflict of Interest

The authors declare no conflicts of interest.


Arnaud Hochard

Luc Reininger

Sandrine Ruchaud

Stéphane Bach
email: bach@sb-roscoff.fr

High-Throughput Screening of marine resources

Keywords: Screening, marine natural products, bioactivity, therapeutic targets bioluminescence resonance energy transfer, CDKs: cyclin-dependent kinases, CK1: casein kinase 1, CytP450: cytochrome P450, Da: daltons, DMSO: dimethyl sulfoxide, EC 50 : half maximal effective concentration, ELISA: enzyme-linked immunosorbent assay, FRET: fluorescence resonance energy transfer, GPCR: G-protein coupled receptor, GSK3 β: glycogen synthase kinase 3 β, HCS: high-content screening, HMD: Hymenialdisine, HTRF: homogeneous time resolved fluorescence, HTS: high-throughput screening, IC 50 : half maximal inhibitory concentration, NMR: nuclear magnetic resonance, PK: pharmacokinetics, PKC: protein kinase C, PKIs: inhibitors of protein kinase, PPIs: protein-protein interactions, RNAi: ribonucleic acid interference, S/B: signal-to-background, SAR: structure activity relationship, SPA: scintillation proximity assay, TR-FRET: time resolved-FRET, SW: signal window, Y2H: yeast two-hybrid

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Introduction

Oceans cover 70% of our planet and contain the greatest diversity of living organisms on earth. Considering the critical importance of natural products derived from living organisms in the discovery and development of bioactive agents of medical interest, the marine environment represents a vast resource to discover novel chemical structures with novel modes of action. For instance, 47% of drugs used in anticancer treatments are natural products and/or derivatives [START_REF] Skropeta | Kinase inhibitors from marine sponges[END_REF]. In recent years, a growing number of new chemical entities from marine organisms with potent pharmacological properties have been described [START_REF] Vinothkumar | Recent advances in marine drug research[END_REF][START_REF] Hu | Statistical research on marine natural products based on data obtained between 1985 and 2008[END_REF][START_REF] Molinski | Drug development from marine natural products[END_REF] (Figure 1). Noteworthy, bryostatin-like compounds are under current investigations for anticancer therapies (for details see part 4.4). It is worth mentioning a growing interest for the development of marine natural products and chemical derivatives with applications in agrochemical and cosmetics industries bearing great potential of economical impact.

The stagnation of the market in terms of newly approved drugs along with an increase in research and development expenses (so called "big pharma crisis"), creates an urgent need to speed-up the discovery of new natural chemical scaffolds (5) (Figure 2). To be effective, the exploration of marine natural products should be developed in parallel to high-rate analysis of bioactivity.

One of drug discovery process critical step is the determination of a therapeutic target, which can be defined as a cellular component (protein, DNA) or a "biological pathway" clearly involved in a specific pathology. Importantly, the therapeutic target must be "druggable", compatible with the treatment of patients (e.g. small chemical compounds, antibodies.) in order to inhibit/activate its cellular function. According to Hugues et al., a "druggable" target is accessible to putative drug molecules and elicits a biological response which may be measured both in vitro and in vivo [START_REF] Hughes | Principles of early drug discovery[END_REF].

High-throughput screening (HTS) consists in testing compounds in high numbers in order to determine if they can modulate a given molecular pathway. HTS relies on two major classes of assays: in vitro biochemical assays which are developed to select compounds with activities on purified molecular targets (e.g. disease-related enzymes as protein kinases, ligand-receptor interactions); and ex vivo cell-based assays developed in mammalian cells, yeast or bacteria engineered to overexpress the target protein (GPCRs, ion-channels and others membrane receptors…) or express a reporter gene (for review see [START_REF] Michelini | Cell-based assays: fuelling drug discovery[END_REF]). High-Content Screening (HCS), in comparison with HTS, is based on the analysis of specific cellular phenotypes through automated image capture and analysis by heavy computing systems (8; 9). This chapter will focus on HTS applied to the identification and selection of marine natural products and derivatives with biological activities of medical interest.

High-Throughput Screening and Drug Development

HTS is based on the development of automated and reproducible assays sufficiently robust to be miniaturized and fit different microtiter plate formats (96 to 3456 wells). The HTS approach is fully integrated in a drug development strategy (Figure 3). The central role of HTS and notably in basic research is to allow pharmaceutical companies to save time and money. The purpose of HTS assay is to select the optimal chemical structures that will be analyzed during pre-and clinical studies.

Screening assay development and validation

As mentioned above and as indicated on Figure 3, identification of therapeutic targets is an essential step prior to screening development. Several targets can be identified and validated for a single disease [START_REF] Westby | Cell-based and biochemical screening approaches for the discovery of novel HIV-1 inhibitors[END_REF]. Tools available to identify therapeutic targets include mRNA/protein levels analyses, genetic associations, bioinformatics (data mining) [START_REF] Hughes | Principles of early drug discovery[END_REF] or RNAi technology [START_REF] Tyner | RNAi screen for rapid therapeutic target identification in leukemia patients[END_REF]. The next step is to determine whether the molecular pathway requires inhibition or activation in order to produce significant beneficial effects. whereas homogeneous assays require only additions and incubations followed by reading.

Multistep assays prevent the tested compounds to interfere with the readout and usually produce a higher signal-to-background ratio (S/B ratio) whereas homogeneous assays are more amenable to high-throughput analysis. Sensitivity, cost, automation, speed, robustness, reliability are important criterions to be taken into consideration. It is to be noted that downscaling of assays, reducing the volumes of material used, can significantly reduce total cost of HTS screens (Table 1).

Statistical tools for quality assessment of HTS assays

Generally, screening evaluation is based on the percentage of inhibition/activation obtained by the tested molecule on a specific target. Experimental data analysis requires their normalization in order to remove systematic plate-to-plate variation and standardize measurements across plates. Quality indicators such as reproducibility and accuracy are essential to assess the suitability of the assay for HTS. Many factors can affect the reproducibility of microtiter plate assays, such as "edge-effect" in cell-based assays. A widely used way to determine the suitability of an assay, prior to launch a full-scale high-throughput screen, is the calculation of the Z'-factor coefficient. The Z'-factor is a statistical parameter calculated as follow (originally described in ( 27)):

µ and σ are the means and standard deviations of the positive (p) and negative (n) sets of data obtained with dedicated controls, respectively. A proper Z'-factor should be higher than 0.5 as described in Table 2. In the context of an enzyme inhibition-based assay, the positive control is defined as the signal obtained when the enzyme is incubated with its substrate and a known inhibitor, while the negative control is defined as the enzyme incubated with its substrate in the absence of inhibitor. Since most chemical compounds are dissolved in dimethyl sulfoxyde (DMSO), controls should contain the same amount of DMSO, and its potential effect evaluated. The use of more than 1% DMSO (final concentration) is not recommended for cellular-based assay. Z'-factor is totally dependent on the HTS-assay conditions. Therefore, a new Z'-factor must be determined whenever changes are implemented in the assay (including reagents variability),. Statistical tools are available to achieve quality assessment including percentage of control, median absolute deviation (MAD), signal-to-background ratio (S/B) and signal window (SW) (for review see ( 28) and ( 29)).

Choice of screening strategy

In a typical HTS assay, a primary screen is performed at a single dose for all compounds. Results are then validated in a second round of screening. Other strategies can be used, namely molecular pool and replicate screening. The first one consists in testing a pool of compounds in one reaction. The advantage of this technique is to increase the screening output but it requires sorting steps for every positive reaction in order to extract and identify the interesting molecule. This strategy is somehow reminiscent of natural extracts screening where several rounds of fractionation/screening are necessary before approaching the positive compound. Replicate screening strategy (number of independent experiments, n>3) allows direct elimination of false positives, the probability of reproducing experimental error decreasing with the number of replicates (see below for false positive definition). Inevitably, the chosen strategy will affect directly the screening cost and speed and should be carefully evaluated.

Whatever the strategy chosen, it is necessary to determine an activity threshold to identify "hits". The threshold can be set using various methods such as percent inhibition cutoff or Bayesian methods (described in ( 29)).

Data analysis: from hits to leads 2.4.1. Hits

The products tested during screening and having a positive action on the therapeutic target are called "Hits" (Figure 3). Valuable hits are potent and specific; they have a strong effect on the target of interest and minor off-target effects. However, even if the screening is based on a reliable technique and all quality controls validate the assay, the possibility of having identified a false positive or negative compound remains. False positive can be identified by preliminary analysis of the compounds to be screened. For instance, it is well known that some dyes can interfere with activity readout or that alkylating agents can alter the target irreversibly and have no specific inhibitory activity. Reproducible hits without activity on the therapeutic target can be safely regarded as false positives and eliminated. When required (e.g. targeted pathology), cell mortality can be determined in parallel using cellbased assay to discard cytotoxic compounds.

Finally, remaining false positive compounds can be identified in subsequent screening rounds performing a dose effect (serial dilutions of the compound) where no effect will be observed whereas, for a "confirmed" hit, an IC 50 (concentration needed to inhibit half of the maximum biological response, Figure 4) or EC 50 (compound concentration where 50% of its maximal effect is observed, commonly used for agonist/stimulator assays) value can be determined. It is important to indicate here that various factors may influence IC 50 value. For instance, there is an interdependency between the IC 50 value and adenosine tri-phosphate (ATP) concentration for ATP-dependent enzymes such as kinases. IC 50 values can be used to compare antagonists' efficiency and the most potent hits will be identified and selected.

However, it is important to select molecules with chemical structures representative of each family identified in order to keep the highest degree of chemical diversity during the screening process.

To continue hit validation, selected structures can be analyzed using various other bioactivity assays to evaluate the relative selectivity of the molecules to their primary targets.

In the case of protein kinases inhibitors (PKIs), large panels of kinases (>89% of the whole kinome), can be tested to obtain a comprehensive view of the molecule's selectivity (KINOMEscan® program, DISCOVEREx, Fremont, USA).

A chemical scaffold has to be analyzed according to the Lipinski's Rules or Rule of 5 (RO5, all numbers are multiples of five) before further studying its mechanism of action.

These rules are used to evaluate if a chemical compound has properties that would make it a likely orally active drug in humans. They were formulated by Christopher A. Lipinski in 1997 [START_REF] Lipinski | Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings[END_REF] and are based on the observation that most medication drugs are relatively small and lipophilic molecules. These characteristics are associated with 90% of orally active drugs that have achieved Phase II clinical status and states that, in general, an orally active drug has no more than one violation of the following criteria: (i) molecular weight < 500 daltons (Da); (ii) hydrogen bond donor groups < 5 (nitrogen or oxygen atoms with one or more hydrogen atoms); (iii) hydrogen bond acceptor groups < 10 (nitrogen or oxygen atoms); (iv) log P < 5

(octanol-water partition coefficient).

However, these rules are not always applicable to every drug. Indeed, Abbott/Genentech's oral Phase II candidate Navitoclax ® , violates three of Lipinski's Rules for oral-drug-likeness (it has a molecular mass of 975 Da, a log P of 12, and 11 hydrogen-bond acceptors) [START_REF] Mullard | Protein-protein interaction inhibitors get into the groove[END_REF]. Therefore, one should keep an open mind when screening complex and highly diverse marine natural chemical scaffolds [START_REF] Colas | High-throughput screening assays to discover small-molecule inhibitors of protein interactions[END_REF].

The next step in hit validation consists to evaluate its molecular properties such as solubility and stability and, using a panel of in vitro assays, testing intestinal and hematoencephalic permeability, metabolic stability, activity on cytochrome P450 (CytP450), or cell permeability,... [START_REF] Hughes | Principles of early drug discovery[END_REF].

Leads

Following the screening campaign, evaluation of selectivity and pharmacokinetics properties of the selected hits, the more potent molecules, will reach "leads" status (Figure 3).

Medicinal chemistry is then applied in order to obtain derivative structures exhibiting optimized bioactivities. Thus, the lead structure is a starting point for improvement by chemical modification. In this perspective, dose response curves are generated using primary screen assays for each pharmaco-modulation to determine activity through IC 50 or EC 50 evaluation to achieve Structure Activity Relationship (SAR: relationship between the chemical or tridimensional structure of a molecule and its bioactivity). Additional screens can be done to measure selectivity and pharmacokinetics properties. Overall the drug candidate will be a compromise between all these parameters. It will modulate the target efficiently and specifically, have good metabolic stability and bioavailability, be soluble and slightly lipophilic [START_REF] Hefti | Requirements for a lead compound to become a clinical candidate[END_REF].

At this stage, further experiments might help to characterize the leads and especially their mechanism of action. Different affinity-based analysis [START_REF] Guiffant | Identification of intracellular targets of small molecular weight chemical compounds using affinity chromatography[END_REF] or other "omics"-based methods can be used to carefully and comprehensively determine the cellular targets of the new chemical scaffold. Unexpected cellular targets can explain potential side effects that could appear during preclinical or clinical phases.

From HTS assay to market: the drug development process

After selection, drug candidates are submitted for clinical trials (Figure 3). The first step of these trials is pre-clinical stage, which consists in studying on animals various parameters such as genotoxicity, drug absorption, metabolism and metabolites elimination, as well as toxicity of the drug and its metabolites (ADME-tox). The clinical stage is divided in four phases: (i) Phase I is carried out on a small number of healthy human volunteers. During this phase, metabolic and pharmacological effects of drugs are determined along with associated side effect. The purpose of phase I studies is mainly to determine safety profiles;

(ii) Phase II consists in further evaluation of safety together with efficiency in patients, and in the selection of dose regimen; (iii) Phase III is intended to gather additional information about efficiency and safety to evaluate the overall benefit-risk relationship of the drug; (iv)

Phase IV is long term monitoring for adverse reactions reported by pharmacists and doctors (35).

Overall, drug development is a very expensive and lengthy process. It may cost up to US $ 1.7 billion and take between 12 and 15 years. The efficiency of drug development from lead compounds selected by screening is estimated at 1/5,000 [START_REF] Collier | Drug development cost estimates hard to swallow[END_REF].

Examples of High-Throughput Screening

Many HTS assays have been developed depending on the therapeutic targets, some of which helped to highlight potential of marine products for pharmaceutical.

Chemical libraries: the fuel of HTS

Along with HTS development, many molecules libraries have been compiled: e.g.

ICSN-CNRS' (Institut de Chimie des Substances Naturelles, Gif-sur-Yvette, France) library constituted of +4000 compounds from synthetic and natural origin and Greenpharma Natural

Compound library constituted of 240 known phytochemicals (Greenpharma SAS, Orléans, France). Development of these libraries is a long and tedious process. Indeed, each compound has to be controlled (mass spectrometry and/or NMR), diluted to specific concentration, archived and stored. Thus, screening requires organization, storage capacity and sometime laboratory information management system (LIMS). In addition, "working" microtiter-plates (also known as "daughter" plates) will be generated to limit freeze-thaw cycles and manipulation of "mother" plates. A LIMS solution will allow inventory tracking, especially if multiple users are involved on the screening platform.

Biochemical assay, the example of protein kinases

A protein kinase is an enzyme that transfers a phosphate group from ATP to a specific substrate; this process is called "phosphorylation". The 518 human protein kinases play an essential role in regulating cellular growth and survival, differentiation or membrane Screening campaigns have identified several marine compounds as protein kinase inhibitors [START_REF] Leclerc | Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors?[END_REF][START_REF] Bettayeb | Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases[END_REF][START_REF] Bharate | Meridianins: marinederived potent kinase inhibitors[END_REF][START_REF] Debdab | Leucettines, a class of potent inhibitors of cdc2-like kinases and dual specificity, tyrosine phosphorylation regulated kinases derived from the marine sponge leucettamine B: modulation of alternative pre-RNA splicing[END_REF]. One of them, Hymenialdisine (HMD) (Figure 5), originally identified based on its antiproliferative effects on cultured lymphocytic leukemia cells, was characterized using Scintillation Proximity Assay (SPA), a homogeneous method to measure kinase activity where the amount of ATP left after a kinase reaction is determined. The luminescent signal is proportional to the amount of ATP and inversely proportional to the amount of kinase activity [START_REF] Meijer | Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by hymenialdisine, a marine sponge constituent[END_REF].

HMD is a bromopyrrole alkaloid isolated from a variety of marine sponges including Hymeniacidon aldis (or Stylissa massa), Axinella verrucosa (Figure 5 Mek1 (IC 50 = 6 nM) [START_REF] White | Synthesis and evaluation of novel anti-proliferative pyrroloazepinone and indoloazepinone oximes derived from the marine natural product hymenialdisine[END_REF]. Kinetic analysis showed that HMD acts in an ATP-competitive fashion, and the co-crystal structure with CDK2 revealed that HMD occupies the ATP site and shows many of the hydrogen bonding interactions seen in other CDK-inhibitor complexes [START_REF] Wan | Synthesis and target identification of hymenialdisine analogs[END_REF].

Protein-protein interactions, PPIs

Interactions between proteins are at the center of the interactomic of any living cell. This makes the PPI modulation one of the most challenging tasks in drug discovery. Despite their structural diversity, protein-protein interfaces are in many cases large and compact hydrophobic and relatively flat surfaces and thus can be considered as poorly druggable (46; 47). Nevertheless, only few residues on protein-protein interfaces show a dominant contribution to the binding free energy. These residues are known as "hot spots" and their discovery by crystallographic analysis and directed mutagenesis is a major breakthrough in the development of small molecule inhibitors for PPIs [START_REF] Mullard | Protein-protein interaction inhibitors get into the groove[END_REF].

Extreme complementarities of protein-protein interfaces allows development of specific inhibitors and offers some advantages such as: lower drug resistance emergence due to binding of the inhibitor outside of the active site of the targeted proteins compare for example to the ATP-mimetic inhibitors of protein kinases described above) known to decrease side effects observed on human patients.

Despite these advantages, identification of small molecules that modulate protein-protein interactions remains largely untapped by pharmacological research [START_REF] Thiel | Small-molecule stabilization of protein-protein interactions: an underestimated concept in drug discovery?[END_REF]. Few natural compounds were nonetheless identified such as microtubule-stabilizing agents (e.g., taxanes, and the marine compounds discodermolide, eleutherobin or laulimalide) [START_REF] Bergstralh | Microtubule stabilizing agents: their molecular signaling consequences and the potential for enhancement by drug combination[END_REF] anddestabilizing molecules (e.g., vinca alkaloids, and the marine compounds halichondrin B, dolastatin 10 or hemiasterlin) with important applications notably in cancer treatment [START_REF] Kingston | Tubulin-interactive natural products as anticancer agents[END_REF].

In the last decade, following spectacular development of genomics and proteomics, a growing number of potentially druggable protein-protein interactions have been identified Finally, as an emergent and promising concept, study of PPI is still at an early stage and

has not yet allowed the discovery of numerous interesting drugs. However, developing HTS in this application field will probably allow identification of potentially interesting new molecules and offer innovative treatment opportunities. In this respect, and due to their high chemical diversity, marine molecules will certainly play a major role.

Cell-based assay, the example of Bryostatins

In 1965-1966, Georges Pettit and his collaborators began the first systematic study of marine invertebrates and vertebrates as potential sources of new and potentially useful cancer chemotherapeutic drugs using the NCI's P388 ex vivo lymphocytic leukemia screening system to measure antineoplastic activity [START_REF] Pettit | Chapter 12 -Biosynthetic products for anticancer drug design and treatment: The bryostatins[END_REF]. In 1981, fractionation and purification of California

Bugula neritina extracts led to characterization of a macrocyclic lactone, the Bryostatin-1 (Figure 5) showing cell growth inhibition properties at subnanomolar concentrations [START_REF] Pettit | Chapter 12 -Biosynthetic products for anticancer drug design and treatment: The bryostatins[END_REF].

Subsequently, additional researches led to the isolation of 20 new bryostatins from B. neritina collections from the Gulf of Mexico, Gulf and coast of California and Japan (Gulf of Sagami) [START_REF] Pettit | Chapter 12 -Biosynthetic products for anticancer drug design and treatment: The bryostatins[END_REF]. Interestingly, a recent study has reported that bryostatins are produced by symbiotic bacteria from B. neritina [START_REF] Wender | Design, synthesis, and evaluation of potent bryostatin analogs that modulate PKC translocation selectivity[END_REF]. Bryostatin-1 is one of the most abundant and best-studied compounds of this structural family. It was found to target protein kinase C (PKC) through high-affinity binding to the diacylglycerol-binding site of the C-1 regulatory domain of PKC.

While it was originally described to inhibit cell growth (58), Bryostatin 1 has subsequently shown a wide range of properties including anticancer activity, synergetic effect with other anticancer agents, reversal of multidrug resistance, stimulation of the immune system, improvement of learning and memory, neuroprotection after stroke, reduction of amyloid plaque formation, and activity against HIV [START_REF] Trindade-Silva | Bryostatins: biological context and biotechnological prospects[END_REF]. After more than thirty phase I and II clinical trials (61) in a variety of cancers, alone and in combination with other chemotherapy agents, bryostatin 1 failed to reach phase III due to a lack of efficiency on patients.

Bryostatin-1 is still in clinical trial phase II for treatment against Alzheimer's disease (62).

Conclusions and Perspectives

HTS assay development has supported an increased rate of discovery of marine bioactive compounds with various medical applications [START_REF] Singh | Clinical status of anti-cancer agents derived from marine sources[END_REF]. Recent methodological screening developments, allowing the identification of protein-protein interaction modulators or the development of cell-based high-content screening assays will certainly lead to the discovery of new marine-derived medicines. One of the current challenges in pharmaceutical is to increase the access to new chemical scaffolds in order to widen the diversity of chemical libraries: the key for success in drug discovery. Nature is an amazing medicinal chemist providing surprisingly innovative chemical scaffolds of which marine biodiversity is a vast, yet under-explored, supplier with many great discoveries to come, bearing hope for new potent human therapies.

6.

Plate Format Under 0 There is too much overlap between the positive and negative controls for the assay to be useful.

Nb of tested compounds per plates

Nb of plates

Table 2: Interpretation of the Z'-factor value: one of the crucial step of the preparatory phase of a HTS campaign [START_REF] Zhang | A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays[END_REF].

  transport. Deregulations of kinases have been notably described in cancer (since 1978 and the discovery of the first oncogene, Src, a tyrosine kinase), inflammatory or Alzheimer's disease and related neuronal disorders. This led to a growing interest in protein kinases and the development of specific and potent small molecule inhibitors of kinase activity, historically targeting ATP binding pocket. This class of enzymes has emerged as the most important class of targets in oncology drug discovery. More than 20 compounds are already on the market, such as Imatinib (Gleevec ® , Glivec ® ) targeting Bcr-Abl (37). The market for kinase inhibitors is expected to reach $11.6 billion in US and $40.3 billion globally by 2016 (38) and so far only a slight part of the kinome is targeted by already approved-drugs. Kinase inhibitors have great potential. In light of this, HTS assays have been developed using multiple technologies including radioactivity-based assays, luminescent technology, TR-FRET, fluorescent polarization and scintillation proximity assay (SPA).

  ), Acanthella aurantiaca in the early 1980s. It is a potent inhibitor of several closely related Cyclin Dependent Kinases (CDKs), such as CDK1/cyclin B (IC 50 = 22 nM), CDK2/cyclin A (IC 50 = 70 nM), CDK2/cyclin E (IC 50 = 40 nM), and CDK5/p25 (IC 50 = 28 nM), as well as against more distantly related kinases such as GSK3 β (IC 50 = 10 nM), Casein Kinase 1 (IC 50 = 35 nM) or

  pressing the development of HTS assays in this area. Several technologies have been developed such as: yeast two-hybrid (Y2H), reverse yeast two-hybrid[START_REF] Rezwan | Yeast "N"-hybrid systems for protein-protein and drugprotein interaction discovery[END_REF], bacteria twohybrid[START_REF] Stynen | Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system[END_REF], Mammalian Protein-Protein Interaction Trap (MAPPIT)[START_REF] Lievens | Array MAPPIT: high-throughput interactome analysis in mammalian cells[END_REF], reverse MAPPIT[START_REF] Lemmens | Reverse MAPPIT detects disruptors of protein-protein interactions in human cells[END_REF], Bimolecular Fluorescence Complementation (BiFC), FRET-and BRET-based methods[START_REF] Ciruela | Fluorescence-based methods in the study of protein-protein interactions in living cells[END_REF];[START_REF] Corbel | First BRET-based screening assay performed in budding yeast leads to the discovery of CDK5/p25 interaction inhibitors[END_REF].One of the most promising research fields, in the design and development of PPI new therapeutics, is related to the human immunodeficiency virus (HIV) propagation. Indeed, the viral infection relies heavily on protein-protein interactions, especially between virus and host proteins, in almost every step of the virus lifecycle. A Y2H screening assay lead to the discovery of a hexapeptide from the marine worm, Eunicidae gen. sp., showing potent inhibition properties on the 3'-processing activity of HIV-1 integrase through an unusual homoserine residue[START_REF] Maes | Peptides that inhibit HIV-1 integrase by blocking its protein-protein interactions[END_REF].

  '-factors can never exceed 1. 0.5> Z' < 1.0 An excellent assay. 0> Z' <0.5A marginal assay.

Figure 1 :

 1 Figure 1: Temporal evolution in the number of novel chemical entities characterized from marine organisms (adapted from (3)).

Figure 2 :

 2 Figure 2:The "R&D productivity crisis" in pharmaceutical. NME, New Molecular Entities; FDA, US Food and Drug Administration[START_REF] Esteban | Unbiased insight on biopharma's innovation crisis[END_REF]. The curve in light grey (top left) represents the ratio NME/R&D expenses per year.

Figure 3 :

 3 Figure 3: Drug development: from bench to the market, a long adventure. Drug development is used to define the process of bringing a new drug to the market. The screening is one of the major initial steps.

Figure 4 :

 4 Figure 4: Determination of an IC 50 value on a dose-response curve.

Figure 5 :

 5 Figure 5: Images of marine producers of bioactive molecules. Left, Bugula neritina (© V. Lamare for the whole organism, © T.H. Ermak for the picture inset) for Bryostatin-1 and right, Axinella verrucosa (©, Oceanopolis, Brest, France) for Hymenialdisine. The corresponding chemical structures are depicted on the pictures.

iv) based on the use of radioactive elements

  Strategies for in vivoand ex vivo drug target validation include notably gene knock-out/in, dominant negative mutant or RNAi[START_REF] Hughes | Principles of early drug discovery[END_REF].

	Biochemical assays are well adapted to assess effects on enzymatic reactions or
	binding interactions, whereas cell-based assays are preferred for molecular pathways, multiple
	targets or cell membrane compartments analysis. Current available methods are (i)
	Colorimetric (12) (e.g. Enzyme-Linked ImmunoSorbent Assay (ELISA) ((13)); (ii)
	Fluorescent, (e.g Fluorescence Resonance Energy Transfer, (FRET) (14; 15), Dissociation-
	Enhanced Lanthanide Fluorescent Immunoassay, (DELFIA) (16)), fluorescence polarization
	(17), Time Resolved-FRET (TR-FRET) or Homogeneous-TRF, (HTRF) (18; 19), fluorogenic
	substrates (20), calcium flux (21), membrane translocation (22)); (iii) Luminescent (e.g.
	Bioluminescence Resonance Energy Transfer, BRET (23), bioluminescent substrates (24) or
	Luciferase reporter gene (25)); ((e.g
	Scintillation Proximity Assay, SPA (26)).
	Screening assays are considered as heterogeneous (e.g. ELISA) or homogenous (e.g.
	HTRF). Heterogeneous assays (multistep) require steps that go beyond simple reagent
	additions, incubations, and reading (e.g. filtration, centrifugation, and plate washing steps),

volume of target added/well (µl) Assay volume (µl) Total volume of target added (ml)

							Cost ($)
	96	88	2273	10	100	2182	436 400
	384	352	568	2	20	436	87 200
	1536	1408	142	0,4	4	87	17 400
	3456	3168	36	0,08	0,8	17,4	3 480

Table 1 :

 1 Impact of miniaturization on the cost of HTS assays. A library containing 200,000 compounds is evaluated in this example.

Acknowledgments Dr. S. La Barre (CNRS, GDR BioChiMar) is warmly acknowledged for helpful discussions; Dr. X. Fant for critical reading of the manuscript; we thank Cancéropôle Grand-Ouest and notably Prof. S. Routier (axis: Natural sea products in cancer treatment) for continuing support. We thank E. Combot and C. Liret (Oceanopolis, Brest, France), V. Lamare and the DORIS website (http://doris.ffessm.fr) and Dr. T. H. Ermak (Sea Peach Bio Science, Newton, MA, USA) for the images of marine organisms. FUNDING: Screening of marine biodiversity by USR3151-CNRS is notably funded by the ANR/Investissement d'Avenir program by means of the Oceanomics project (grant n°. ANR-11-BTBR-0008).