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.

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.

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.

LSP5-2157 a new inhibitor of vesicular glutamate transporters.

Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

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.

Allosteric modulation of metabotropic glutamate receptors by chloride ions.

Metabotropic glutamate receptors (mGluRs) play key roles in the modulation of many synapses. Chloride (Cl(-)) is known to directly bind and regulate the function of different actors of neuronal activity, and several studies have pointed to the possible modulation of mGluRs by Cl(-). Herein, we demonstrate that Cl(-) behaves as a positive allosteric modulator of mGluRs. For example, whereas glutamate potency was 3.08 ± 0.33 µM on metabotropic glutamate (mGlu) 4 receptors in high-Cl(-) buffer, signaling activity was almost abolished in low Cl(-) in cell-based assays. Cl(-) potency was 78.6 ± 3.5 mM. Cl(-) possesses a high positive cooperativity with glutamate (Hill slope ≈6 on mGlu4), meaning that small variations in [Cl(-)] lead to large variations in glutamate action. Using molecular modeling and mutagenesis, we have identified 2 well-conserved Cl(-) binding pockets in the extracellular domain of mGluRs. Moreover, modeling of activity-dependent Cl(-) variations at GABAergic synapses suggests that these variations may be compatible with a dynamic modulation of the most sensitive mGluRs present in these synapses. Taken together, these data reveal a necessary role of Cl(-) for the glutamate activation of many mGluRs. Exploiting Cl(-) binding pockets may yield to the development of innovative regulators of mGluR activity.

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.

Protein-ligand-based pharmacophores: generation and utility assessment in computational ligand profiling.

Ligand profiling is an emerging computational method for predicting the most likely targets of a bioactive compound and therefore anticipating adverse reactions, side effects and drug repurposing. A few encouraging successes have already been reported using ligand 2-D similarity searches and protein-ligand docking. The current study describes the use of receptor-ligand-derived pharmacophore searches as a tool to link ligands to putative targets. A database of 68,056 pharmacophores was first derived from 8,166 high-resolution protein-ligand complexes. In order to limit the number of queries, a maximum of 10 pharmacophores was generated for each complex according to their predicted selectivity. Pharmacophore search was compared to ligand-centric (2-D and 3-D similarity searches) and docking methods in profiling a set of 157 diverse ligands against a panel of 2,556 unique targets of known X-ray structure. As expected, ligand-based methods outperformed, in most of the cases, structure-based approaches in ranking the true targets among the top 1% scoring entries. However, we could identify ligands for which only a single method was successful. Receptor-ligand-based pharmacophore search is notably a fast and reliable alternative to docking when few ligand information is available for some targets. Overall, the present study suggests that a workflow using the best profiling method according to the protein-ligand context is the best strategy to follow. We notably present concrete guidelines for selecting the optimal computational method according to simple ligand and binding site properties.

Successful prediction of substrate-binding pocket in SLC17 transporter sialin.

Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.

A novel selective metabotropic glutamate receptor 4 agonist reveals new possibilities for developing subtype selective ligands with therapeutic potential.

Metabotropic glutamate (mGlu) receptors are promising targets to treat numerous brain disorders. So far, allosteric modulators are the only subtype selective ligands, but pure agonists still have strong therapeutic potential. Here, we aimed at investigating the possibility of developing subtype-selective agonists by extending the glutamate-like structure to hit a nonconsensus binding area. We report the properties of the first mGlu4-selective orthosteric agonist, derived from a virtual screening hit, LSP4-2022 using cell-based assays with recombinant mGlu receptors [EC(50): 0.11 ± 0.02, 11.6 ± 1.9, 29.2 ± 4.2 µM (n>19) in calcium assays on mGlu4, mGlu7, and mGlu8 receptors, respectively, with no activity at the group I and -II mGlu receptors at 100 µM]. LSP4-2022 inhibits neurotransmission in cerebellar slices from wild-type but not mGlu4 receptor-knockout mice. In vivo, it possesses antiparkinsonian properties after central or systemic administration in a haloperidol-induced catalepsy test, revealing its ability to cross the blood-brain barrier. Site-directed mutagenesis and molecular modeling was used to identify the LSP4-2022 binding site, revealing interaction with both the glutamate binding site and a variable pocket responsible for selectivity. These data reveal new approaches for developing selective, hydrophilic, and brain-penetrant mGlu receptor agonists, offering new possibilities to design original bioactive compounds with therapeutic potential.

Predicting fragment binding poses using a combined MCSS MM-GBSA approach.

Improved methods are required to predict the position and orientation (pose) of binding to the target protein of low molecular weight compounds identified in fragment screening campaigns. This is particularly important to guide initial chemistry to generate structure-activity relationships for the cases where a high resolution structure cannot be obtained. We have assessed the benefit of an implicit solvent method for assessment of fragment binding poses generated by the Multiple Copy Simultaneous Search (MCSS) method in CHARMm. Additionally, the effect of using multiple receptor structures for a flexible receptor is investigated. The original MCSS performance -50% of fragment positions accurately predicted and scored - was increased up to 67% by scoring MCSS energy minima with a Molecular Mechanics Generalized Born approach with molecular volume integration and Surface Area model (MM-GBSA). The same increase in performance (but occasionally for different targets) was observed when using the docking program GOLD followed by MM-GBSA rescoring. The combined results from both methods resulted in a higher success rate emphasizing that a comparison of different docking methods can increase the correct identification of binding poses. For a receptor where multiple structures are available, Hsp90, the average performance on randomly adding receptor structures was also investigated. The results suggest that predictions using these docking methods can be used with some confidence to guide chemical optimization, if the structure of the target either remains relatively fixed on ligand binding, or if a number of crystal structures are available with diverse ligands bound and there is information on the positions of key water molecules in the binding site.

A critical pocket close to the glutamate binding site of mGlu receptors opens new possibilities for agonist design.

A recent publication from Ogawa et al. suggested a possible allosteric chloride binding site in the extracellular domain of metabotropic glutamate receptors (mGluRs) by comparison with a similar site found in atrial natriuretic peptide receptor. We simultaneously reported about (S)-PCEP an agonist of subtype 4 mGluR that would bind to a similar pocket, adjacent to the glutamate binding site. Here we disclose LSP1-2093, a new derivative of (S)-PCEP that holds a nitrophenyl substituent. Docking experiments predict that the nitro group binds to the receptor at the putative chloride ion site. It is thus possible to take advantage of this putative chloride binding site to develop new types of mGluR agonists. This pocket is present in the structural family of Leucine Isoleucine Valine Binding Protein that includes class C GPCRs, suggesting that extended agonists may be identified at receptors bearing such a structural domain.

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.

A virtual screening hit reveals new possibilities for developing group III metabotropic glutamate receptor agonists.

(R)-PCEP (3-amino-3-carboxypropyl-2'-carboxyethyl phosphinic acid, 1), a new metabotropic glutamate receptor 4 (mGlu4R) agonist, was discovered in a previously reported virtual screening. The (S)-enantiomer and a series of derivatives were synthesized and tested on recombinant mGlu4 receptors. A large number of derivatives activated this receptor but was not able to discriminate between mGlu4 and mGlu8 receptors. The most potent ones 6 and 12 displayed an EC(50) of 1.0 +/- 0.2 microM at mGlu4R. Interestingly these agonists with longer alkyl chains revealed a new binding pocket adjacent to the glutamate binding site, which is lined with residues that differ among the mGluR subtypes and that will allow the design of more selective compounds. Additionally 6 was able to activate mGlu7 receptor with an EC(50) of 43 +/- 16 microM and is thus significantly more potent than L-AP4 (EC(50) of 249 +/- 106 microM).

High-potency olfactory receptor agonists discovered by virtual high-throughput screening: molecular probes for receptor structure and olfactory function.

The detection of diverse chemical structures by the vertebrate olfactory system is accomplished by the recognition of odorous ligands by their cognate receptors. In the present study, we used computational screening to discover novel high-affinity agonists of an olfactory G protein-coupled receptor that recognizes amino acid ligands. Functional testing of the top candidates validated several agonists with potencies higher than any of the receptor's known natural ligands. Computational modeling revealed molecular interactions involved in ligand binding and further highlighted interactions that have been conserved in evolutionarily divergent amino acid receptors. Significantly, the top compounds display robust activities as odorants in vivo and include a natural product that may be used to signal the presence of bacteria in the environment. Our virtual screening approach should be applicable to the identification of new bioactive molecules for probing the structure of chemosensory receptors and the function of chemosensory systems in vivo.

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.

Amino-pyrrolidine tricarboxylic acids give new insight into group III metabotropic glutamate receptor activation mechanism.

Like most class C G-protein-coupled receptors, metabotropic glutamate (mGlu) receptors possess a large extracellular domain where orthosteric ligands bind. Crystal structures revealed that this domain, called Venus FlyTrap (VFT), adopts a closed or open conformation upon agonist or antagonist binding, respectively. We have described amino-pyrrolidine tricarboxylic acids (APTCs), including (2S,4S)-4-amino-1-[(E)-3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid (FP0429), as new selective group III mGlu agonists. Whereas FP0429 is an almost full mGlu4 agonist, it is a weak and partial agonist of the closely related mGlu8 subtype. To get more insight into the activation mechanism of mGlu receptors, we aimed to elucidate why FP0429 behaves differently at these two highly homologous receptors by focusing on two residues within the binding site that differ between mGlu4 and mGlu8. Site-directed mutagenesis of Ser157 and Gly158 of mGlu4 into their mGlu8 homologs (Ala) turned FP0429 into a weak partial agonist. Conversely, introduction of Ser and Gly residues into mGlu8 increased FP0429 efficacy. Docking of FP0429 in mGlu4 VFT 3D model helped us characterize the role of each residue. Indeed, mGlu4 Ser157 seems to have an important role in FP0429 binding, whereas Gly158 may allow a deeper positioning of this agonist in the cavity of lobe I, thereby ensuring optimal interactions with lobe II residues in the fully closed state of the VFT. In contrast, the presence of a methyl group in mGlu8 (Ala instead of Gly) weakens the interactions with the lobe II residues. This probably results in a less stable or a partially closed form of the mGlu8 VFT, leading to partial receptor activation.

Design and synthesis of APTCs (aminopyrrolidinetricarboxylic acids): identification of a new group III metabotropic glutamate receptor selective agonist.

A new family of mGlu receptor orthosteric ligands called APTCs was designed and synthesized using a parallel chemistry approach. Amongst 65 molecules tested on mGlu4, mGlu6 and mGlu8 subtypes, (2S,4S)-4-amino-1-[(E)-3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid (8a06-FP0429) has been shown to be a full mGlu4 agonist and a partial mGlu8 agonist. In addition, 8a06 was shown to be selective versus group I and II mGlu subtypes. A possible explanation for this efficacy difference is proposed by docking experiment performed with molecular model of the receptor.

Amino acid recognition by Venus flytrap domains is encoded in an 8-residue motif.

A motif foramino acid recognition by proteins or domains of the periplasmic binding protein-like I superfamily has been identified. An initial pattern of 5 residues was based on a multiple sequence alignment of selected proteins of that fold family and on common structural features observed in the crystal structure of some members of the family [leucine isoleucine valine binding protein (LIVBP), leucine binding protein (LBP), and metabotropic glutamate receptor type 1 (mGlu1R) amino terminal domain)]. This pattern was used against the PIR-NREF sequence database and further refined to retrieve all sequences of proteins that belong to the family and eliminate those that do not belong to it. A motif of 8 residues was finally selected to build up the general signature. A total of 232 sequences were retrieved. They were found to belong to only three families of proteins: bacterial periplasmic binding proteins (PBP, 71 sequences), family 3 (or C) of G-protein coupled receptor (GPCR) (146 sequences), and plant putative ionotropic glutamate receptors (iGluR, 15 sequences). PBPs are known to adopt a bilobate structure also named Venus flytrap domain, or LIVBP domain in the present case. Family 3/C GPCRs are also known to hold such a domain. However, for plant iGluRs, it was previously detected by classical similarity searches but not specifically described. Thus plant iGluRs carry two Venus flytrap domains, one that binds glutamate and an additional one that would be a modulatory LIVBP domain. In some cases, the modulator binding to that domain would be an amino acid.

Virtual screening workflow development guided by the "receiver operating characteristic" curve approach. Application to high-throughput docking on metabotropic glutamate receptor subtype 4.

The "receiver operating characteristic" (ROC) curve method is a well-recognized metric used as an objective way to evaluate the ability of a given test to discriminate between two populations. This facilitates decision-making in a plethora of fields in which a wrong judgment may have serious consequences including clinical diagnosis, public safety, travel security, and economic strategies. When virtual screening is used to speed-up the drug discovery process in pharmaceutical research, taking the right decision upon selecting or discarding a molecule prior to in vitro evaluation is of paramount importance. Characterizing both the ability of a virtual screening workflow to select active molecules and the ability to discard inactive ones, the ROC curve approach is well suited for this critical decision gate. As a case study, the first virtual screening workflow focused on metabotropic glutamate receptor subtype 4 (mGlu4R) agonists is reported here. Six compounds out of 38 selected and tested in vitro were shown to have agonist activity on this target of therapeutic interest.