Epitope Identification of an mGlu5 Receptor Nanobody Using Physics-Based Molecular Modeling and Deep Learning Techniques.

The world has witnessed a revolution in therapeutics with the development of biological medicines such as antibodies and antibody fragments, notably nanobodies. These nanobodies possess unique characteristics including high specificity and modulatory activity, making them promising candidates for therapeutic applications. Identifying their binding mode is essential for their development. Experimental structural techniques are effective to get such information, but they are expensive and time-consuming. Here, we propose a computational approach, aiming to identify the epitope of a nanobody that acts as an agonist and a positive allosteric modulator at the rat metabotropic glutamate receptor 5. We employed multiple structure modeling tools, including various artificial intelligence algorithms for epitope mapping. The computationally identified epitope was experimentally validated, confirming the success of our approach. Additional dynamics studies provided further insights on the modulatory activity of the nanobody. The employed methodologies and approaches initiate a discussion on the efficacy of diverse techniques for epitope mapping and later nanobody engineering.

Determination of the absolute configuration of phosphinic analogues of glutamate.

A series of phosphinic glutamate derivatives (e.g.LSP1-2111) have been proven to be potent agonists of metabotropic glutamate (mGlu) receptors and shown promising in vivo activity. However, so far all were synthesized and tested as a mixture of two diastereomers whose absolute and relative configurations are not known. In this study, the stereomers were separated on a Crownpack CR(+) column and their absolute configuration was assessed by means of a diastereoselective synthesis. Both separated L-stereomers activated the mGlu4 receptor with EC50's of 0.72 and 4.4 µM for (1S,1'S)-and (1S,1'R)-LSP1-2111, respectively.

Design, Synthesis, Pharmacology, and In Silico Studies of (1S,2S,3S)-2-((S)-Amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic Acid (LBG30300): A Picomolar Potency Subtype-Selective mGlu2 Receptor Agonist.

Metabotropic glutamate (Glu) receptors (mGlu receptors) play a key role in modulating excitatory neurotransmission in the central nervous system (CNS). In this study, we report the structure-based design and pharmacological evaluation of densely functionalized, conformationally restricted glutamate analogue (1S,2S,3S)-2-((S)-amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic acid (LBG30300). LBG30300 was synthesized in a stereocontrolled fashion in nine steps from a commercially available optically active epoxide. Functional characterization of all eight mGlu receptor subtypes showed that LBG30300 is a picomolar agonist at mGlu2 with excellent selectivity over mGlu3 and the other six mGlu receptor subtypes. Bioavailability studies on mice (IV administration) confirm CNS exposure, and an in silico study predicts a binding mode of LBG30300 which induces a flipping of Tyr144 to allow for a salt bridge interaction of the acetate group with Arg271. The Tyr144 residue now prevents Arg271 from interacting with Asp146, which is a residue of differentiation between mGlu2 and mGlu3 and thus could explain the observed subtype selectivity.

Novel Inhibitory Site Revealed by XAP044 Mode of Action on the Metabotropic Glutamate 7 Receptor Venus Flytrap Domain.

Metabotropic glutamate (mGlu) receptors play a key role in modulating most synapses in the brain. The mGlu7 receptors inhibit presynaptic neurotransmitter release and offer therapeutic possibilities for post-traumatic stress disorders or epilepsy. Screening campaigns provided mGlu7-specific allosteric modulators as the inhibitor XAP044 (Gee et al. J. Biol. Chem. 2014). In contrast to other mGlu receptor allosteric modulators, XAP044 does not bind in the transmembrane domain but to the extracellular domain of the mGlu7 receptor and not at the orthosteric site. Here, we identified the mode of action of XAP044, combining synthesis of derivatives, modeling and docking experiments, and mutagenesis. We propose a unique mode of action of these inhibitors, preventing the closure of the Venus flytrap agonist binding domain. While acting as a noncompetitive antagonist of L-AP4, XAP044 and derivatives act as apparent competitive antagonists of LSP4-2022. These data revealed more potent XAP044 analogues and new possibilities to target mGluRs.

Synthesis of quinoline dicarboxylic esters as biocompatible fluorescent tags.

A series of dicarboxylic quinoline derivatives bearing electron-releasing or -withdrawing substituents have been synthesized using mono- or/and biphasic methodologies. By controlling the regioselectivity of addition into our electrophilic intermediate, we also characterized by which mechanism the Doebner-Miller cyclization step occurred. As anticipated, electron-releasing substituents induce a red shift of the low-energy absorption allowing excitation in the visible region. In addition, by playing on the strength and position of the electron-releasing substituents, chromophore having interesting fluorescent properties such as large Stoke shifts, good fluorescent quantum yields, emission in the visible green-yellow region and reasonable two-photon absorption in the NIR region have been obtained. These small-size fluorophores, which can be made water-soluble and have been shown to be non-toxic, can be hetero- and/or polyfunctionalized and thus represent promising key units for fluorescence-based physiological experiments with low background interactions.

Metabotropic glutamate receptor orthosteric ligands and their binding sites.

Metabotropic glutamate receptors (mGluRs) have been discovered almost four decades ago. Since then, their pharmacology has been largely developed as well as their structural organization. Indeed mGluRs are attractive therapeutic targets for numerous psychiatric and neurological disorders because of their modulating role of synaptic transmission. The more recent drug discovery programs have mostly concentrated on allosteric modulators. However, orthosteric agonists and antagonists have remained unavoidable pharmacological tools as, although not expected, many of them can reach the brain, or can be modified to reach the brain. This review focuses on the most common orthosteric ligands as well as on the few allosteric modulators interacting with the glutamate binding domain. The 3D-structures of these ligands at their binding sites are reported. For most of them, X-Ray structures or docked homology models are available. Because of the high conservation of the binding site, subtype selective agonists were not easy to find. Yet, some were discovered when extending their chemical structures in order to reach selective sites of the receptors.

Identification of a novel NR2B-selective NMDA receptor antagonist using a virtual screening approach.

We report the identification of a novel NR2B-selective NMDAR antagonist with an original scaffold, LSP10-0500. This compound was identified by a virtual high-throughput screening approach on the basis of a quantitative pharmacophore model of NR2B-specific NMDAR antagonists. A SAR study around LSP10-0500 is also described.

Rose Bengal analogs and vesicular glutamate transporters (VGLUTs).

Vesicular glutamate transporters (VGLUTs) allow the loading of presynaptic glutamate vesicles and thus play a critical role in glutamatergic synaptic transmission. Rose Bengal (RB) is the most potent known VGLUT inhibitor (Ki 25 nM); therefore we designed, synthesized and tested in brain preparations, a series of analogs based on this scaffold. We showed that among the two tautomers of RB, the carboxylic and not the lactonic form is active against VGLUTs and generated a pharmacophore model to determine the minimal structure requirements. We also tested RB specificity in other neurotransmitter uptake systems. RB proved to potently inhibit VMAT (Ki 64 nM) but weakly VACHT (Ki>9.7 microM) and may be a useful tool in glutamate/acetylcholine co-transmission studies.

Asc-1 Transporter (SLC7A10): Homology Models And Molecular Dynamics Insights Into The First Steps Of The Transport Mechanism.

The alanine-serine-cysteine transporter Asc-1 regulates the synaptic availability of D-serine and glycine (the two co-agonists of the NMDA receptor) and is regarded as an important drug target. To shuttle the substrate from the extracellular space to the cytoplasm, this transporter undergoes multiple distinct conformational states. In this work, homology modeling, substrate docking and molecular dynamics simulations were carried out to learn more about the transition between the "outward-open" and "outward-open occluded" states. We identified a transition state involving the highly-conserved unwound TM6 region in which the Phe243 flips close to the D-serine substrate without major movements of TM6. This feature and those of other key residues are proposed to control the binding site and substrate translocation. Competitive inhibitors ACPP, LuAE00527 and SMLC were docked and their binding modes at the substrate binding site corroborated the key role played by Phe243 of TM6. For ACPP and LuAE00527, strong hydrophobic interactions with this residue hinder its mobility and prevent the uptake and the efflux of substrates. As for SMLC, the weaker interactions maintain the flexibility of Phe243 and the efflux process. Overall, we propose a molecular basis for the inhibition of substrate translocation of the Asc-1 transporter that should be valuable for rational drug design.

Amino Acids Bearing Aromatic or Heteroaromatic Substituents as a New Class of Ligands for the Lysosomal Sialic Acid Transporter Sialin.

Sialin, encoded by the SLC17A5 gene, is a lysosomal sialic acid transporter defective in Salla disease, a rare inherited leukodystrophy. It also enables metabolic incorporation of exogenous sialic acids, leading to autoantibodies against N-glycolylneuraminic acid in humans. Here, we identified a novel class of human sialin ligands by virtual screening and structure-activity relationship studies. The ligand scaffold is characterized by an amino acid backbone with a free carboxylate, an N-linked aromatic or heteroaromatic substituent, and a hydrophobic side chain. The most potent compound, 45 (LSP12-3129), inhibited N-acetylneuraminic acid 1 (Neu5Ac) transport in a non-competitive manner with IC50 ≈ 2.5 µM, a value 400-fold lower than the KM for Neu5Ac. In vitro and molecular docking studies attributed the non-competitive character to selective inhibitor binding to the Neu5Ac site in a cytosol-facing conformation. Moreover, compound 45 rescued the trafficking defect of the pathogenic mutant (R39C) causing Salla disease. This new class of cell-permeant inhibitors provides tools to investigate the physiological roles of sialin and help develop pharmacological chaperones for Salla disease.

Increased Potency and Selectivity for Group III Metabotropic Glutamate Receptor Agonists Binding at Dual sites.

A group III metabotropic glutamate (mGlu) receptor agonist (PCEP) was identified by virtual HTS. This orthosteric ligand is composed by an l-AP4-derived fragment that mimics glutamate and a chain that binds into a neighboring pocket, offering possibilities to improve affinity and selectivity. Herein we describe a series of derivatives where the distal chain is replaced by an aromatic or heteroaromatic group. Potent agonists were identified, including some with a mGlu4 subtype preference, e.g., 17m (LSP1-2111) and 16g (LSP4-2022). Molecular modeling suggests that aromatic functional groups may bind at either one of the two chloride regulatory sites. These agonists may thus be considered as particular bitopic/dualsteric ligands. 17m was shown to reduce GABAergic synaptic transmission at striatopallidal synapses. We now demonstrate its inhibitory effect at glutamatergic parallel fiber-Purkinje cell synapses in the cerebellar cortex. Although these ligands have physicochemical properties that are markedly different from typical CNS drugs, they hold significant therapeutic potential.

L-(+)-2-Amino-4-thiophosphonobutyric acid (L-thioAP4), a new potent agonist of group III metabotropic glutamate receptors: increased distal acidity affords enhanced potency.

L-2-Amino-4-phosphonobutyric acid (l-AP4), l-2-amino-4-thiophosphonobutyric acid (l-thioAP4), and l-2-amino-4-(hydroxy)phosphinylbutyric acid (desmethylphosphinothricin, DMPT) were synthesized from protected vinylglycine. They were tested as agonists at group III metabotropic glutamate receptors (mGluR) along with phosphinothricin (PT). DMPT and PT display a much lower potency at mGlu4 receptor (EC50 = 4.0 and 1100 microM, respectively) in comparison to l-AP4 (EC50 = 0.08 microM), whereas l-thioAP4 has a 2-fold higher potency (EC50 = 0.039 microM). Similar rank orders of potency were observed at mGlu6,7 and mGlu8 receptors. The higher potency of l-thioAP4 is due to its stronger second acidity compared to l-AP4. These pKa values of 5.56 and 6.88, respectively, were determined using 31P NMR chemical shift variations. The second distal negative charge of l-AP4/l-thioAP4 probably provides stronger binding to specific basic residues of the binding sites of group III mGluRs, which stabilizes the active conformation of the receptor.

Synthesis and biological evaluation of 1-amino-2-phosphonomethylcyclopropanecarboxylic acids, new group III metabotropic glutamate receptor agonists.

Stereoisomers of 1-amino-2-phosphonomethylcyclopropanecarboxylic acid (APCPr), conformationally restricted analogues of L-AP4 (2-amino-4-phosphonobutyric acid), have been prepared and evaluated at recombinant group III metabotropic glutamate receptors. They activate these receptors over a broad range of potencies. The most potent isomer (1S,2R)-APCPr displays a similar pharmacological profile as that of L-AP4 (EC50 0.72, 1.95, >500, 0.34 microM at mGlu4, 6, 7, 8 receptors, respectively, and no effect at group I/II mGluRs). It was characterized on native receptors located in the basal ganglia (BG) where it induced a robust and reversible inhibition of synaptic transmission. It was tested in vivo in haloperidol-induced catalepsy, a model of Parkinsonian akinesia, by direct infusion in the globus pallidus of the BG. At a dose of 0.5 nmol/microL, catalepsy was significantly antagonized. This study reveals that (1S,2R)-APCPr is a potent group III mGluR agonist and confirms that these receptors may be considered as a therapeutic target in the Parkinson's disease.

Synthesis, binding affinity at glutamic acid receptors, neuroprotective effects, and molecular modeling investigation of novel dihydroisoxazole amino acids.

The four stereoisomers of 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid(+)-4, (-)-4, (+)-5, and (-)-5 were prepared by stereoselective synthesis of two pairs of enantiomers, which were subsequently resolved by enzymatic procedures. These four stereoisomers and the four stereoisomers of the bicyclic analogue 5-amino-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-3,5-dicarboxylic acid (+)-2, (-)-2, (+)-3, and (-)-3 were tested at ionotropic and metabotropic glutamate receptor subtypes. The most potent NMDA receptor antagonists [(+)-2, (-)-4, and (+)-5] showed a significant neuroprotective effect when tested in an oxygen glucose deprivation (OGD) cell culture test. The same compounds were preliminarily assayed using Xenopus oocytes expressing cloned rat NMDA receptors containing the NR1 subunit in combination with either NR2A, NR2B, NR2C, or NR2D subunit. In this assay, all three derivatives showed high antagonist potency with preference for the NR2A and NR2B subtypes, with derivative (-)-4 behaving as the most potent antagonist. The biological data are discussed on the basis of homology models reported in the literature for NMDA receptors and mGluRs.

Two-photon "caging" groups: effect of position isomery on the photorelease properties of aminoquinoline-derived photolabile protecting groups.

High two-photon photolysis cross sections and water solubility of probes are important to avoid toxicity in biomedical applications of photolysis. Systematic variation of the position of a carboxyl electron-withdrawing group (EWG) on photolysis of 8-dimethylaminoquinoline protecting groups identified the C5-substituted isomer as a privileged dipole. The 5-benzoyl-8-DMAQ substitution yields a caging group with an enhanced two-photon uncaging cross section (δu = 2.0 GM) and good water solubility (c ≤ 50 mM, pH 7.4).

Common and selective molecular determinants involved in metabotopic glutamate receptor agonist activity.

Several potent and group selective agonists of metabotropic glutamate receptors (mGluRs) have been docked at mGlu1,2,4R binding sites in the closed conformation of the bilobate extracellular domain. Quisqualic acid and (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) were selected for mGlu1R, dicarboxycyclopropylglycine (DCG-IV), LY354740, (S)-4-carboxyphenylglycine (4CPG) for mGlu2R, and (S)-2-amino-4-phosphonobutyric acid (AP4), 1-aminocyclopentane-1,3,4-tricarboxylic acid (ACPT-I), (S)-4-phosphonophenylglycine (PPG) for mGlu4R. The models show a conserved binding pattern for the glycine moiety (alpha-amino and alpha-acidic functions) and group specific bindings for the distal acidic function. The best agonists allow optimized interaction with both lobes of the binding domain. Interlobe connections around the ligand are also described and participate in stabilizing the closed form of the amino-terminal domain. Altogether, the docking models support the proposal that the stabilization of a closed state represents a key step in agonist activation of mGluRs.

Molecular determinants of agonist selectivity in glutamate-gated chloride channels which likely explain the agonist selectivity of the vertebrate glycine and GABAA-ρ receptors.

Orthologous Cys-loop glutamate-gated chloride channels (GluClR's) have been cloned and described electrophysiologically and pharmacologically in arthropods and nematodes (both members of the invertebrate ecdysozoan superphylum). Recently, GluClR's from Aplysia californica (a mollusc from the lophotrochozoan superphylum) have been cloned and similarly studied. In spite of sharing a common function, the ecdysozoan and lophotrochozoan receptors have been shown by phylogenetic analyses to have evolved independently. The recent crystallization of the GluClR from C. elegans revealed the binding pocket of the nematode receptor. An alignment of the protein sequences of the nematode and molluscan GluClRs showed that the Aplysia receptor does not contain all of the residues defining the binding mode of the ecdysozoan receptor. That the two receptors have slightly different binding modes is not surprising since earlier electrophysiological and pharmacological experiments had suggested that they were differentially responsive to certain agonists. Knowledge of the structure of the C. elegans GluClR has permitted us to generate a homology model of the binding pocket of the Aplysia receptor. We have analyzed the differences between the two binding modes and evaluated the relative significance of their non-common residues. We have compared the GluClRs electrophysiologically and pharmacologically and we have used site-directed mutagenesis on both receptor types to test predictions made from the model. Finally, we propose an explanation derived from the model for why the nematode receptors are gated only by glutamate, whereas the molluscan receptors can also be activated by ß-alanine, GABA and taurine. Like the Aplysia receptor, the vertebrate glycine and GABAA-ρ receptors also respond to these other agonists. An alignment of the sequences of the molluscan and vertebrate receptors shows that the reasons we have given for the ability of the other agonists to activate the Aplysia receptor also explain the agonist profile seen in the glycine and GABAA-ρ receptors.

Design, synthesis and biological evaluation of small-azo-dyes as potent Vesicular Glutamate Transporters inhibitors.

Vesicular Glutamate Transporters (VGLUTs) allow the loading of presynapic glutamate vesicles and thus play a critical role in glutamatergic synaptic transmission. VGLUTs have proved to be involved in several major neuropathologies and directly correlated to clinical dementia in Alzheimer and Parkinson's disease. Accordingly VGLUT represent a key biological target or biomarker for neuropathology treatment or diagnostic. Yet, despite the pivotal role of VGLUTs, their pharmacology appears quite limited. Known competitive inhibitors are restricted to some dyes as Trypan Blue (TB) and glutamate mimics. This lack of pharmacological tools has heavily hampered VGLUT investigations. Here we report a rapid access to small molecules that combine benefits of TB and dicarboxylic quinolines (DCQs). Their ability to block vesicular glutamate uptake was evaluated. Several compounds displayed low micromolar inhibitory potency when size related compounds are thirty to forty times less potent (i.e. DCQ). We then confirmed the VGLUT selectivity by measuring the effect of the series on vesicular monoamine transport and on metabotropic glutamate receptor activity. These inhibitors are synthesized in only two steps and count among the best pharmacological tools for VGLUTs studies.

Variations on glutamate binding in channel-gated receptors.

In this issue of Structure, Lomash and colleagues report on the crystal structure and ligand binding properties of a primitive eukaryote glutamate-gated ion channel: AvGluR1.

The antipsychotic-like effects of the mGlu group III orthosteric agonist, LSP1-2111, involves 5-HT1A signalling.

RATIONALE: Several studies have suggested that modulation of the glutamatergic system via metabotropic glutamate receptors (mGlu) could be a new way to achieve antipsychotic-like activity. LSP1-2111, the group III mGlu receptor orthosteric agonist, with a high affinity towards mGlu4 receptors, was previously shown to exhibit antipsychotic-like action in animal models displaying positive symptoms of schizophrenia. OBJECTIVES: Here, we decided to investigate the possible role of LSP1-2111 in models of negative (social interaction) and cognitive (NOR) symptoms of psychosis. We also investigated the involvement of 5-HT1A receptors in the LSP1-2111-induced antipsychotic effects. Apart from the above-mentioned models of negative and cognitive symptoms, MK-801 and amphetamine-induced hyperactivity tests, plus the DOI-induced head twitches in mice as models for positive symptoms of psychosis, were used in this part of the investigations. RESULTS: LSP1-2111 (0.5, 2, and 5 mg/ kg) dose-dependently inhibited MK-801-induced deficits in social interaction and NOR tests. The effects of the drug were antagonized by 5-HT1A antagonist, WAY100635 (0.1 mg/kg). A similar inhibition of LSP1-2111-induced effects was observed in models of positive symptoms of schizophrenia. Moreover, the concomitant administration of subeffective doses of LSP1-2111 (0.3-0.5 mg/kg) with a subeffective dose of 5-HT1A agonist, (R)-(+)-8-Hydroxy-DPAT (0.01 mg/kg), induced a clear antipsychotic-like effect in all of the procedures used. CONCLUSIONS: Altogether, we propose that the activation of group III mGlu receptors may be a promising target for the development of novel antipsychotic drugs, towards not only positive but also negative and cognitive symptoms. The action of the compound is 5-HT1A-dependent.