Design and synthesis of a novel water-soluble NMDA receptor antagonist with a 1,4,7,10-tetraazacyclododecane group

Polyamines, especially spermine, inhibit N-methyl-D-aspartate (NMDA) receptors as open channel blockers. Two types of water-soluble NMDA receptor antagonist, ACCn (1) and TGCn (2), with a 1,4,7,10-tetraazacyclododecane cyclic polyamine group, were synthesized and the effects of both compounds on NMDA receptors were studied using voltage-clamp recordings of recombinant NMDA receptors expressed in Xenopus oocytes. These compounds inhibited macroscopic currents in both NR1/NR2A and NR1/NR2B receptor subtypes in oocytes voltage-clamped at -70 mV. Inhibition by the compounds of NR1/NR2A receptors were more prominent than that of NR1/NR2B receptors. The inhibitory effects of ACCn (1) on both NMDA receptors were more potent than those of TGCn (2).     

Synthesis and pharmacology of glutamate receptor ligands: new isothiazole analogues of ibotenic acid

The naturally occurring heterocyclic amino acid ibotenic acid (Ibo) and the synthetic analogue thioibotenic acid (Thio-Ibo) possess interesting but dissimilar pharmacological activity at ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). Therefore, a series of Thio-Ibo analogues was synthesized. The synthesis included introduction of substituents by Suzuki and Grignard reactions on 4-halogenated 3-benzyloxyisothiazolols, reduction of the obtained alcohols, followed by introduction of the amino acid moiety by use of 2-(N-tert-butoxycarbonylimino)malonic acid diethyl ester. The obtained Thio-Ibo analogues (1, 2a-g) were characterized in functional assays on recombinant mGluRs and in receptor binding assays on native iGluRs. At mGluRs, the activity at Group II was retained for compounds with small substituents (2a-2d), whereas the Group I and Group III receptor activities for all new compounds were lost. Detection of NMDA receptor affinity prompted further characterization, and two-electrode voltage-clamp recordings at recombinant NMDA receptor subtypes NR1/NR2A-D expressed in Xenopus oocytes were carried out for compounds with small substituents (chloro, bromo, methyl or ethyl, compounds 2a-d). This series of Thio-Ibo analogues defines a structural threshold for NMDA receptor activation and reveals that the individual subtypes have different steric requirements for receptor activation. The compounds 2a and 2c are the first examples of agonists discriminating individual NMDA subtypes.     

Synthesis of a novel water‐soluble NMDA receptor antagonist

A novel water‐soluble N‐methyl‐D‐aspartate (NMDA) receptor antagonist ATGDMAP 1 as synthesized and the effect of 1 on NMDA receptors was studied using voltage‐clamp recordings of recombinant NMDA receptors expressed in Xenopus oocytes. The compound 1 inhibited macroscopic currents in NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptor subtypes in oocytes voltage‐clamped at ‐70 mV. Mutant NR1/NR2B receptors containing NR1(T648A) or NR1(T648S) had very large holding currents when voltage‐clamped at ‐70 mV compared to that of wild‐type NMDA receptors, because these mutants generate constitutively open channels. ATGDMAP 1 and Mg²⁺, a channel blocker of the NMDA receptor, reduced the large holding currents needed for mutant NMDA receptors. These data indicate that ATGDMAP 1 directly acts on the channel pores of the NMDA receptor.     

Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay

We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors.     

Development of selective high affinity antagonists, agonists, and radioligands for the P2Y1 receptor

The P2Y(1) receptor is a member of the P2Y family of nucleotide-activated G protein-coupled receptors, and it is an important therapeutic target based on its broad tissue distribution and essential role in platelet aggregation. We have designed a set of highly selective and diverse pharmacological tools for studying the P2Y(1) receptor using a rational approach to ligand design. Based on the discovery that bisphosphate analogues of the P2Y(1) receptor agonist, ADP, are partial agonists/competitive antagonists of this receptor, an iterative approach was used to develop competitive antagonists with enhanced affinity and selectivity. Halogen substitutions of the 2-position of the adenine ring provided increased affinity while an N(6) methyl substitution eliminated partial agonist activity. Furthermore, various replacements of the ribose ring with symmetrically branched, phosphorylated acyclic structures revealed that the ribose is not necessary for recognition at the P2Y(1) receptor. Finally, replacement of the ribose ring with a five member methanocarba ring constrained in the Northern conformation conferred dramatic increases in affinity to both P2Y(1) receptor antagonists as well as agonists. These combined structural modifications have resulted in a series of selective high affinity antagonists of the P2Y(1) receptor, two broadly applicable radioligands, and a high affinity agonist capable of selectively activating the P2Y(1) receptor in human platelets. Complementary receptor modeling and computational ligand docking have provided a putative structural framework for the drug-receptor interactions. A similar rational approach is being applied to develop selective ligands for other subtypes of P2Y receptors.     

Specific GABA(A) agonists and partial agonists

The GABA(A) receptor system is implicated in a number of neurological and psychiatric diseases, making GABA(A) receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the hetero-pentameric GABA(A) receptor complex, reflecting the very strict structural requirements for GABA(A) receptor recognition and activation. Within the series of compounds showing agonist activity at the GABA(A) receptor site that have been developed, most of the ligands are structurally derived from the GABA(A) agonists muscimol, THIP, or isoguvacine, which we developed in the initial stages of the project. Using recombinant GABA(A) receptors, functional selectivity was demonstrated for a number of compounds, including THIP, showing highly subunit-dependent potency and maximal response. In light of the interest in partial GABA(A) receptor agonists as potential therapeutics, structure-activity studies of a number of analogs of 4-PIOL, a low-efficacy partial GABA(A) agonist derived from THIP, have been performed. In this connection, a series of GABA(A) ligands has been developed that exhibit pharmacological profiles from moderately potent low-efficacy partial GABA(A) agonist activity to potent and selective antagonist effects. Very little information is available on direct-acting GABA(A) receptor agonists in clinical studies. However, the results of clinical studies on the effect of the partial GABA(A) agonist THIP on human sleep patterns show that the functional consequences of a direct-acting agonist are different from those seen after the administration of GABA(A) receptor modulators, such as benzodiazepines and barbiturates.     

3-Fluoro-N-methyl-D-aspartic acid (3F-NMDA) stereoisomers as conformational probes for exploring agonist binding at NMDA receptors

N-Methyl-D-aspartate (NMDA) is the prototypical agonist of the NMDA receptor subtype of ionotropic glutamate receptors. Stereogenic placement of a C-F bond at the 3-position of (S)-NMDA generates either the (2S,3S)- or (2S,3R)- diastereoisomers of 3F-NMDA. The individual diastereoisomers were prepared by synthesis in enantiomerically pure forms and it was found that (2S,3S)-3F-NMDA is an agonist with a comparable potency to NMDA itself, whereas the (2S,3R)-diastereoisomer has negligible potency. The difference in potency of these stereoisomers is attributed to a preference of the C-F bond (2S,3S)-3F-NMDA to adopt a gauche conformation to the C-N(+) bond in the binding conformation, whereas the (2S,3R)-3F-NMDA forces these bonds anti, losing electrostatic stabilisation, to achieve the required binding conformation. These observations illustrate the utility of stereoselective fluorination in influencing the molecular conformation of β-fluorinated amino acids and thus probing the active conformations of bioactive compounds at receptors.     

Environment-Sensitive Fluorescence of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-Labeled Ligands for Serotonin Receptors

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin_1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin_1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.     

Identification and characterization of mGlu3 ligands using a high throughput FLIPR assay for detection of agonists, antagonists, and allosteric modulators

When targeting G-protein coupled receptors (GPCRs) in early stage drug discovery, or for novel targets, the type of ligand most likely to produce the desired therapeutic effect may be unknown. Therefore, it can be desirable to identify potential lead compounds from multiple categories: agonists, antagonists, and allosteric modulators. In this study, we developed a triple addition calcium flux assay using FLIPR Tetra to identify multiple ligand classes for the metabotropic glutamate receptor 3 (mGlu3), using a cell line stably co-expressing the human G-protein-coupled mGlu3 receptor, a promiscuous G-protein (G(α16)), and rat Glast, a glutamate transporter. Compounds were added to the cells followed by stimulation with EC(10) and then EC(80) concentration of glutamate, the physiological agonist for mGlu receptors. This format produced a robust assay, facilitating the identification of agonists, positive allosteric modulators and antagonists/negative allosteric modulators. Follow up experiments were conducted to exclude false positives. Using this approach, we screened a library of approximately 800,000 compounds using FLIPR Tetra and identified viable leads for all three ligand classes. Further characterization revealed the selectivity of individual ligands.     

Synthesis and Investigation of Mixed μ‐Opioid and δ‐Opioid Agonists as Possible Bivalent Ligands for Treatment of Pain

Several studies have suggested functional association between μ‐opioid and δ‐opioid receptors and showed that μ‐activity could be modulated by δ‐ligands. The general conclusion is that agonists for the δ‐receptor can enhance the analgesic potency and efficacy of μ‐agonists. Our preliminary investigations demonstrate that new bivalent ligands constructed from the μ‐agonist fentanyl and the δ‐agonist enkephalin‐like peptides are promising entities for creation of new analgesics with reduced side effects for treatment of neuropathic pain. A new superposition of the mentioned pharmacophores led to novel μ‐bivalent/δ‐bivalent compounds that demonstrate both μ‐opioid and δ‐opioid receptor agonist activity and high efficacy in anti‐inflammatory and neuropathic pain models with the potential of reduced unwanted side effects.     

Real time differentiation of G-protein coupled receptor (GPCR) agonist and antagonist by two photon fluorescence laser microscopy

Receptor-based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors (GPCR) is the largest and most ubiquitous of the receptor-mediated processes. Desensitization of G-protein-coupled receptors is a fundamental mechanism regulating the cellular response to agonists. We have recently studied the agonist and antagonist of the human melanocortin receptors (hMC1, hMC3, hMC4, and hMC5 receptors), the human delta opioid receptor, and the human gluacagon receptor with the help of synthetic fluorescent labeled ligands and fluorescent protein-labeled beta-arrestin-receptors that shed new insight on cellular signaling and rapid screening of drugs in real time. It was demonstrated that stimulation of these receptors by the cognate agonist triggers the rapid internalization of ligand-receptor complexes, while the interaction of the receptor with antagonists does not follow this pathway. Furthermore, receptor internalization is dependent upon beta-arrestin, which has been shown to be responsible for the rapid desensitization of cAMP-signaling processes.     

3-D QSAR for intrinsic activity of 5-HT1A receptor ligands by the method of comparative molecular field analysis

The affinity of a ligand for a receptor is usually expressed in terms of the dissociation constant (K_i) of the drug-receptor complex, conveniently measured by the inhibition of radioligand binding. However, a ligand can be an antagonist, a partial agonist, or a full agonist, a property largely independent of its receptor affinity. This property can be quantitated as intrinsic activity (1A), which can range from 0 for a full antagonist to 1 for a full agonist. Although quantitative structure–activity relationship (QSAR) methods have been applied to the prediction of receptor affinity with considerable success, the prediction of IA, even qualitatively, has rarely been attempted. Because most traditional QSAR methods are limited to congeneric series, and there are often major structural differences between agonists and antagonists, this lack of success in predicting IA is understandable. To overcome this limitation, we used the method of comparative molecular field analysis (CoMFA), which, unlike traditional Hansch analysis, permits the inclusion of structurally dissimilar compounds in a single QSAR model. A structurally diverse set of 5-hydroxytryptamine_1A (5-HT_1A) receptor ligands, with literature IA data (determined by the inhibition of 5-HT sensitive forskolin-stimulated adenylate cyclase), was used to develop a 3-D QSAR model correlating intrinsic activity with molecular structure properties of 5HT_1A receptor ligands. This CoMFA model had a crossvalidated r² of 0.481, five components and final conventional r² of 0.943. The receptor model suggests that agonist and antagonist ligands can share parts of a common binding site on the receptor, with a primary agonist binding region that is also occupied by antagonists and a secondary binding site accommodating the excess bulk present in the sidechains of many antagonists and partial agonists. The CoMFA steric field graph clearly shows that agonists tend to be “flatter” (more coplanar) than antagonists, consistent with the difference between the 5-HT_1A agonist and antagonist pharmacophores proposed by Hibert and coworkers. The CoMFA electrostatic field graph suggests that, in the region surrounding the essential protonated aliphatic amino group, the positive molecular electrostatic potential may be weaker in antagonists as compared to agonists. Together, the steric and electrostatic maps suggest that in the secondary binding site region increased hydrophobic binding may enhance antagonist activity. These results demonstrate that CoMFA is capable of generating a statistically crossvalidated 3-D QSAR model that can successfully distinguish between agonist and antagonist 5-HT_1A ligands. To the best of our knowledge, this is the first time this or any other QSAR method has been successfully applied to the correlation of structure with IA rather than potency or affinity. The analysis has suggested various structural features associated with agonist and antagonist behaviors of 5-HT_1A ligands and thus should assist in the future design of drugs that act via 5-HT_1A receptors. © 1993 John Wiley & Sons, Inc.     

Partial agonists and subunit selectivity at NMDA receptors

Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N-methyl-D-aspartic acid (NMDA) receptor class. Development of these ligands seems to be a difficult task because of the conserved region in the binding site of the NMDA receptor subunits. A few scaffolds have been developed showing potential to differentiate between the NMDA receptors.     

Controlled on-chip stimulation of quantal catecholamine release from chromaffin cells using photolysis of caged Ca2+ on transparent indium-tin-oxide microchip electrodes

Photorelease of caged Ca(2+) is a uniquely powerful tool to study the dynamics of Ca(2+)-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca(2+), together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 100 nm thick transparent indium-tin-oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (approximately 20 microm by 20 microm). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca(2+) cage NP-EGTA and Ca(2+) indicator dye fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca(2+), a uniform rise of [Ca(2+)](i) within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We observed a burst of amperometric spikes upon rapid elevation of [Ca(2+)](i) and a "priming" effect of sub-stimulatory [Ca(2+)](i) on the response of cells to subsequent [Ca(2+)](i) elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca(2+) is a suitable stimulation technique for higher-throughput studies of Ca(2+)-dependent exocytosis on transparent electrochemical microelectrode arrays.     

3‐Fluoro‐N‐methyl‐D‐aspartic acid (3F‐NMDA) Stereoisomers as Conformational Probes for Exploring Agonist Binding at NMDA Receptors

N‐Methyl‐D ‐aspartate (NMDA) is the prototypical agonist of the NMDA receptor subtype of ionotropic glutamate receptors. Stereogenic placement of a C? F bond at the 3‐position of (S)‐NMDA generates either the (2S,3S)‐ or (2S,3R)‐ diastereoisomers of 3F‐NMDA. The individual diastereoisomers were prepared by synthesis in enantiomerically pure forms and it was found that (2S,3S)‐3F‐NMDA is an agonist with a comparable potency to NMDA itself, whereas the (2S,3R)‐diastereoisomer has negligible potency. The difference in potency of these stereoisomers is attributed to a preference of the C? F bond (2S,3S)‐3F‐NMDA to adopt a gauche conformation to the C? N⁺ bond in the binding conformation, whereas the (2S,3R)‐3F‐NMDA forces these bonds anti, losing electrostatic stabilisation, to achieve the required binding conformation. These observations illustrate the utility of stereoselective fluorination in influencing the molecular conformation of β‐fluorinated amino acids and thus probing the active conformations of bioactive compounds at receptors.     

Virtual screening of organic molecule databases. Design of focused libraries of potential ligands of NMDA and AMPA receptors

A system of virtual screening of organic molecule databases is designed, which permits preprocessing of databases, molecular docking to a three-dimensional model of receptor, and post-processing of the results obtained. Using this screening system, it is possible to reproduce positions of the known ligands in the glutamate sites of the NMDA and AMPA receptors and in the glycine site of the NMDA receptor, to substantially enrich the database with potentially active compounds, and to distinguish between the agonistic and antagonistic character of the action of these compounds in the case of docking to the open and closed forms of the binding sites. Based on the results of screening of a database of low-molecular-weight organic compounds (total of 135,000 structures) using models of the open and closed forms of the glutamate and glycine sites of the NMDA receptor and of the glutamate site of the AMPA receptor, focused libraries of potential agonists and antagonists of these sites were designed.     

Global physicochemical properties as activity discriminants for the mGluR1 subtype of metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are important as candidate therapeutic targets for many neurological disorders. In the present work, the focus has been on the mGluR1 subtype, where agonists have a proconvulsant profile while antagonists exert anticonvulsant activity. Identification of molecular determinants for the inhibition of mGluR1 provides a new avenue for the discovery and development of novel anticonvulsant drugs. Spatial configuration of key groups alone cannot explain activation selectivity at this specific receptor subtype. In fact, all known agonists and antagonists acting at mGluR1 can accommodate the same critical moieties in a similar geometric arrangement that corresponds to the extended conformation of glutamate. Therefore, other factors must account for the differences in activation. This study presents the results of an analysis of a large suite of steric, topological, electrostatic, and thermodynamic molecular properties calculated for a representative set of potent mGluR1 agonists and antagonists. Global steric parameters and the total nonpolar area provide discrimination between the mGluR1 agonists and antagonists considered in the present work. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 2018–2027, 2001     

Modelling of ORL1 receptor-ligand interactions

An opioid receptor like (ORL1) receptor is one of a family of G-protein-coupled receptors (GPCR); it represents a new pharmaceutical target with extensive therapeutic potential for the regulation of important biological functions such as nociception, mood disorders, drug abuse, learning or cardiovascular control. Although the crystal structure of the inactive form of the ORL1 receptor has been determined, little is known about its activation. By using X-ray structures of the β2-adrenegic receptor in its inactive (2RH1) and active (3P0G) states as templates, inactive and active homology models of the ORL1 receptor were constructed. Structurally diverse sets of strongly binding antagonists and agonists were docked with both ORL1 receptor forms. The major receptor-ligand interactions responsible for antagonist and agonist binding were identified. Although both sets of ligands, agonists and antagonists, bind to the same region of the receptor, they occupy partially different binding pockets. Agonists bind to the inactive receptor in a slightly different manner than antagonists. This difference is more pronounced in binding to the active ORL1 receptor model and points to the amino acids at the extracellular end of TM6, suggesting that this region is important for receptor-activation.