Design, synthesis, and evaluation of a novel prodrug, a S-trityl-l-cysteine derivative targeting kinesin spindle protein.

Kinesin spindle protein (KSP) is expressed only in cells undergoing cell division, and hence represents an attractive target for the treatment of cancer. Several KSP inhibitors have been developed and undergone clinical trial, but their clinical use is limited by their toxicity to rapidly proliferating non-cancerous cells. To create new KSP inhibitors that are highly selective for cancer cells, we optimized the amino acid moiety of S-trityl-l-cysteine (STLC) derivative 1 using in silico modeling. Molecular docking and molecular dynamics simulation were performed to investigate the binding mode of 1 with KSP. Consistent with the structure activity relationship studies, we found that a cysteine amino moiety plays an important role in stabilizing the interaction. Based on these findings and the structure of GSH, a substrate of γ-glutamyltransferase (GGT), we designed and synthesized the prodrug N-γ-glutamylated STLC derivative 9, which could be hydrolyzed by GGT to produce 1. The KSP ATPase inhibitory activity of 9 was lower than that of 1, and LC-MS analysis indicated that 9 was converted to 1 only in the presence of GGT in vitro. In addition, the cytotoxic activity of 9 was significantly attenuated in GGT-knockdown A549 cells. Since GGT is overexpressed on the cell membrane of various cancer cells, these results suggest that compound 9 could be a promising prodrug that selectively inhibits the proliferation of GGT-expressing cancer cells.

Newly Synthesized Oxygenated Xanthones as Potential P-Glycoprotein Activators: In Vitro, Ex Vivo, and In Silico Studies.

P-glycoprotein (P-gp) plays a crucial role in the protection of susceptible organs, by significantly decreasing the absorption/distribution of harmful xenobiotics and, consequently, their toxicity. Therefore, P-gp has been proposed as a potential antidotal pathway, when activated and/or induced. Knowing that xanthones are known to interact with P-gp, the main goal was to study P-gp induction or/and activation by six new oxygenated xanthones (OX 1-6). Furthermore, the potential protection of Caco-2 cells against paraquat cytotoxicity was also assessed. The most promising compound was further tested for its ability to increase P-gp activity ex vivo, using everted intestinal sacs from adult Wistar-Han rats. The oxygenated xanthones interacted with P-gp in vitro, increasing P-gp expression and/or activity 24 h after exposure. Additionally, after a short-incubation period, several xanthones were identified as P-gp activators, as they immediately increased P-gp activity. Moreover, some xanthones decreased PQ cytotoxicity towards Caco-2 cells, an effect prevented under P-gp inhibition. Ex vivo, a significant increase in P-gp activity was observed in the presence of OX6, which was selectively blocked by a model P-gp inhibitor, zosuquidar, confirming the in vitro results. Docking simulations between a validated P-gp model and the tested xanthones predicted these interactions, and these compounds also fitted onto previously described P-gp induction and activation pharmacophores. In conclusion, the in vitro, ex vivo, and in silico results suggest the potential of some of the oxygenated xanthones in the modulation of P-gp, disclosing new perspectives in the therapeutics of intoxications by P-gp substrates.

From UTP to AR-C118925, the discovery of a potent non nucleotide antagonist of the P2Y2 receptor.

The G protein-coupled P2Y2 receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y2R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y2 receptor discovered using the endogenous P2Y2R agonist UTP as the chemical starting point.

Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp.

Naphthol radical couplings determine structural features and enantiomeric excess of dalesconols in Daldinia eschscholzii.

Understanding how simple molecules are pieced together in organisms may aid biotechnological manipulation and synthetic approaches to complex natural products. The mantis-associated fungus Daldinia eschscholzii IFB-TL01 produces the unusually structured immunosuppressants (±)-dalesconols A and B, along with their congener (±)-dalesconol C, with the (-)-enantiomers in excess. Here we report that these structural and stereochemical peculiarities of dalesconols A-C are a result of promiscuous and atropselective couplings of radicals derived from 1,3,6,8-tetrahydroxynaphthalene, 1,3,8-trihydroxynaphthalene and 1,8-dihydroxynaphthalene. The observed (-)-enantiomeric excess is found to depend on the dominance of particular conformers of naphthol dimer intermediates, which are ligands of laccase.

Target-mediated metabolism and target-mediated drug disposition of the DPPIV inhibitor AMG 222.

Pharmacokinetic and metabolism aspects of AMG 222 interaction with target enzyme, dipeptidylpeptidase IV (DPPIV) were investigated. Inhibition of recombinant human DPPIV by AMG 222 was measured. IC(50) decreased as preincubation time increased. k(off), k(on) and K(d) were measured. Dilution assay indicated a long dissociation half-life (730 min) relative to DPPIV inhibitor vildagliptin. AMG 222 is a slow-on, tight-binding, slowly reversible inhibitor of DPPIV. Amide and acid metabolites arising from hydrolysis of AMG 222's cyano group were formed slowly by rhDPPIV, but not by microsomes or S9. The amide metabolite was converted to the acid metabolite by rhDPPIV, but not by an active site mutant. These metabolites of AMG 222 are formed by target-mediated metabolism of the cyano group, similar to vildagliptin. Human plasma protein binding of [(14)C]AMG 222 was saturable and concentration-dependent. After 30 min, [(14)C]AMG 222 was 80.8% bound at 1 nM and binding decreased to 29.4% above 100 nM. The plasma DPPIV concentration (4.1 nM) and human plasma AMG 222 concentrations that inhibit DPPIV, occurred in the range of concentration-dependent binding. Target-mediated drug disposition influences AMG 222 pharmacokinetics, similar to DPPIV inhibitor, linagliptin.

P-glycoprotein-mediated active efflux of the anti-HIV1 nucleoside abacavir limits cellular accumulation and brain distribution.

P-glycoprotein (P-gp)-mediated efflux at the blood-brain barrier has been implicated in limiting the brain distribution of many anti-HIV1 drugs, primarily protease inhibitors, resulting in suboptimal concentrations in this important sanctuary site. The objective of this study was to characterize the interaction of abacavir with P-gp and determine whether P-gp is an important mechanism in limiting abacavir delivery to the central nervous system (CNS). In vitro and in vivo techniques were employed to characterize this interaction. Abacavir stimulated P-gp ATPase activity at high concentrations. The cellular accumulation of abacavir was significantly decreased by approximately 70% in Madin-Darby canine kidney II (MDCKII)-MDR1 monolayers compared with wild-type cells and was completely restored by the P-gp inhibitors ((R)-4-((1aR,6R,10bS)-1,2-difluoro-1,1a,6,10b-tetrahydrodibenzo(a,e)cyclopropa(c)cycloheptan-6-yl)-alpha-((5-quinoloyloxy)methyl)-1-piperazineethanol, trihydrochloride) (LY335979) and N-[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide (GF120918). Directional flux experiments indicated that abacavir had greater permeability in the basolateral-to-apical direction (1.58E-05 cm/s) than in the apical-to-basolateral direction (3.44E-06 cm/s) in MDR1-transfected monolayers. The directionality in net flux was abolished by both LY335979 and GF120918. In vivo brain distribution studies showed that the AUC(plasma) in mdr1a(-/-) CF-1 mutant mice was approximately 2-fold greater than the AUC(plasma) in the wild type, whereas the AUC(brain) in the mutant was 20-fold higher than that in the wild type. Therefore, the CNS drug targeting index, defined as the ratio of AUC brain-to-plasma for mutant over wild type, was greater than 10. These data are the first in vitro and in vivo evidence that a nucleoside reverse transcriptase inhibitor is a P-gp substrate. The remarkable increase in abacavir brain distribution in P-gp-deficient mutant mice over wild-type mice suggests that P-gp may play a significant role in restricting the abacavir distribution to the CNS.

Selectivity of the multidrug resistance modulator, LY335979, for P-glycoprotein and effect on cytochrome P-450 activities.

Overexpression of ATP-dependent drug efflux pumps, P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP), confers multidrug resistance to tumor cells. Modulators of multidrug resistance block the action of these pumps, thereby sensitizing cells to oncolytics. A potent Pgp modulator is LY335979, which fully sensitizes Pgp-expressing cells at 0.1 microM in cytotoxicity assays and for which Pgp has an affinity of 59 nM. The present study examines its effect on MRP1-mediated drug resistance and cytochrome P-450 (CYP) activity and its ability to serve as a Pgp substrate. Drug resistance was examined with HL60/ADR and MRP1-transfected HeLa-T5 cells. Drug cytotoxicity was unaffected by 1 microM LY335979; leukotriene C4 uptake into HeLa-T5 membrane vesicles was unaffected. Because the substrate specificity of Pgp and CYP3A overlap, the effect of LY335979 on the 1'-hydroxylation of midazolam by CYP3A in human liver microsomes was examined. The apparent K(i) was 3.8 microM, approximately 60-fold higher than the affinity of Pgp for LY335979. The modulator's effect on Pgp was evaluated with Pgp-overexpressing CEM/vinblastine (VLB)(100) and parental CCRF-CEM cells. Both cell lines accumulated [(3)H]LY335979 equally well and did not efflux [(3)H]LY335979 during a 3-h incubation, indicating that it is not a substrate of Pgp. Equilibrium-binding studies with CEM/VLB(100) plasma membranes and [(3)H]LY335979 showed that Pgp had a K(d) of 73 nM, which is in good agreement with the previously determined K(i) value. Thus, LY335979 is an extremely potent Pgp, and not MRP1 or MRP2, modulator and has a significantly lower affinity for CYP3A than for Pgp.

Liquid chromatography/nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry identification of novel metabolites of the multidrug resistance modulator LY335979 in rat bile and human liver microsomal incubations.

Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. An N-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2-3 hr with approximately 100 ng of material in the LC/NMR microprobe. With approximately 1.5 microg of material injected onto the column, 1H-1H correlation spectroscopy spectra could be acquired overnight. Along with LC/MS data, the LC/NMR technique facilitated direct identification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursued.

Binding of 5H-dibenzo[a,d]cycloheptene and dibenz[b,f]oxepin analogues of clozapine to dopamine and serotonin receptors.

Series of 5,11-dicarbo- and 11-carbo-5-oxy-10-(1-alkyl-1,2,3,6-tetrahydro-4 pyridinyl) analogues and a 11-carbo-5-oxy-10-(1-methyl-4-piperidinyl) analogue of the atypical antipsychotic agent clozapine were prepared and tested for binding to the dopamine D-2L and D-4 and serotonin S-2A and S-2C receptors. Some of these analogues were found to have dopamine D-2L and D-4 and serotonin S-2A and S-2C receptor binding activities as high as or higher than those of clozapine, indicating that neither the diazepine structure nor the piperazine ring present in clozapine is essential for high antidopamine activity and or for high dopamine D-4 selectivity (Ki for the dopamine D-2L receptor/Ki for the dopamine D-4 receptor). Increasing in the effective size of the alkyl substituent at the tertiary amine nitrogen atom in the 1,2,3,6-tetrahydro-4-pyridinyl moiety in the 5H-dibenzo[a,d]cycloheptene series reduces the affinity for the dopamine D-4 receptor, but in the dibenz[b,f]oxepin series, no significant change in binding affinity to the dopamine D-4 receptor was observed. Equal or slightly higher affinity for the serotonin S-2A and S-2C receptors was observed for the 10-(1-ethyl-1,2,3,6-tetrahydro-4- pyridinyl) analogues in both series, but for the 10-[1,2,3,6-tetrahydro-1-(2-propenyl)-4- pyridinyl] analogues, any favourable steric factor is overshadowed by an unfavorable electronic effect as a result of change in the basicity of the tertiary amino group in the pyridinyl moiety. Replacement of three of the four nitrogen atoms in clozapine with three carbon or two carbon atoms and an oxygen atom and removal of the chlorine atoms gives 10-(1,2,3,6-tetrahydro-1- methyl-4-pyridinyl)dibenzo[a,d]cycloheptene and 10-(1-methyl-4-piperidinyl)dibenz[b,f]oxepin, each having twice the binding activity to the dopamine D-4 receptor as does clozapine and a dopamine D-4 selectivity equal to that of clozapine.

Metabolism of amineptine in rat, dog and man.

After oral administration of amineptine (7-[(10-11)-dihydro-5H-dibenzo(a,d)cycloheptane-5yl] amino heptanoic acid), an original tricyclic antidepressant, seven metabolites were isolated from urine and plasma of rat, dog and man. The metabolic pathways were similar for the three species studied. The two major pathways consisted of the beta-oxidation of the heptanoic side chain leading to pentanoic (first step) and propanoic (second step) side chain metabolites and the hydroxylation of the dibenzocycloheptyl ring on carbon atom 10 (C10) causing the formation of two diastereoisomers. Lactamization by internal dehydration of beta-oxidized metabolites appeared to be a minor route of biotransformation. Conjugation reactions were of minor importance in the rat, in contrast to findings for dog and man. Urinary elimination was the major route of excretion in man while in dog and in rat faecal excretion was predominant.

Farmacologia da amineptina: síntese e atualizaçäo

A amineptina é um antidepressivo tricíclico com a capacidade única de diminuir seletivamente a captaçäo de dopamina (DA) sem afetar a captaçäo de noradrenalina (NA) e serotonina (5HT). O efeito é demonstrado tanto in vitro como in vivo através do uso de metodologia apropriada. A amineptina pode ser diferenciada da anfetamina tanto com base em parâmetros farmacológicos como bioquímicos. In vivo, a amineptina aumenta o ácido homovanílico estriatal sem afetar os níveis de outros metabólicos da DA, nomeadamente, o ácido 3,4, dihidrozoxifenilacético (DOPAC) e 3-metoxitramina (3MT). No entanto, usando doses relativamente altas de amineptina, o nível extracelular de DOPAC - avaliado pelo uso de pulso voltamétrico - diminui preferencialmente altas de amineptina, o nível extracelular de DOPAC - avaliado pelo uso de pulso voltamétrico - diminui preferencialmente no núcleo accumbens, mas näo no estriado. O tratamento crônico com amineptina, tal como com outros antidepressivos, induz uma "down-regulation" dos receptores beta-adrenérgicos. A amineptina penetra no cérebro e seus efeitos farmacológicos säo provavelmente devidos mais à forma inaltereada do que a seus dois metabólitos principais

Structure-activity relationships of philanthotoxin analogs and polyamines on N-methyl-D-aspartate and nicotinic acetylcholine receptors.

The effects of varying the structure of philanthotoxin (PhTX) were investigated on binding of the channel blockers: [3H]perhydrohistrionicotoxin (H12-HTX) to the nicotinic acetylcholine receptor (nACh-R) of Torpedo electric organ and [3H]MK-801 [( 3H]-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine maleate) to the N-methyl-D-aspartate receptor (NMDA-R) of rat brain cortex. The four moieties of PhTX (tyrosine, butyrate, spermine and the terminal amino group) were modified or conjugated resulting in 36 compounds. Although the potencies of the PhTX analogs on both receptors were higher with increasing lipophilicity and the polyamine chain length, there was considerable divergence between the two receptors' channels in the structural activity requirements for blockade by PhTX analogs. A major difference was the more critical role of the amine terminal for inhibition of the nACh-R than the NMDA-R, whereas the reverse might be true for the tyrosine moiety. The potency range of PhTX analogs on [3H]H12-HTX binding was 1070, but only 21 on [3H]MK-801 binding. Adding a lysine or arginine onto the spermine moiety increased the compound's potency on the nACh-R with little effect on the NMDA-R. Because spermine is a component of PhTX, the effects of five polyamines were also studied. Spermine and spermidine potentiated [3H]MK-801 binding, whereas putrescine, cadeverine and agmatine inhibited it. In presence of glutamate, higher concentrations of all polyamines inhibited [3H]MK-801 binding. On the nACh-R, spermine, spermidine and agmatine inhibited [125I]alpha-bungarotoxin and also [3H]H12-HTX binding in presence of carbamylcholine. The complex nature of PhTX interactions with the two receptors suggests that PhTX may bind to two sites: an external polyamine binding site and a channel binding site.

Characterization of polyamines having agonist, antagonist, and inverse agonist effects at the polyamine recognition site of the NMDA receptor.

The endogenous polyamines spermine and spermidine increase the binding of [3H]MK-801 to NMDA receptors. This effect is antagonized by diethylenetriamine (DET). We report here that spermine increases the rates of both association and dissociation of binding of [3H]MK-801, suggesting that it increases the accessibility of the binding site for MK-801 within the ion channel of the receptor complex. 1,10-Diaminodecane (DA10) inhibited the binding of [3H]MK-801. This effect was due to a decrease in the rate of association with no change in the rate of dissociation of [3H]MK-801. The effect of DA10 was not mediated by an action of DA10 at the binding sites for glutamate, glycine, Mg2+, or Zn2+, and was attenuated by DET. This suggests that DA10 acts at the polyamine recognition site. In hippocampal neurons the NMDA-elicited current was decreased by DA10, an effect opposite to that of spermine. The effects of spermine and DA10 were selectively blocked by DET. It is concluded that DA10 acts as a negative allosteric modulator or inverse agonist at the polyamine recognition site of the NMDA receptor.

Interaction of strychnine-insensitive glycine binding with MK-801 binding in brain synaptic membranes.

Strychnine-insensitive [3H]glycine binding was detected in brain synaptic membranes treated with Triton X-100 using a filtration assay method. The binding was a time-dependent, inversely temperature-dependent, and reversible process with a relatively high affinity for the neuroactive amino acid. Scatchard analysis revealed that Triton treatment doubled both the affinity and density of the binding sites, which consisted of a single component. The binding was not only displaced by structurally-related amino acid such as D-serine and D-alanine, but also inhibited by some peptides containing glycine, including glycine methylester and N-methylglycine. These ligands invariably potentiated the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]- cyclohepten-5,10-imine ([3H]MK-801), a noncompetitive antagonist for the N-methyl-D-aspartate-sensitive subclass of the central excitatory amino acid receptors, in a concentration-dependent manner. Among various endogenous tryptophan metabolites, kynurenic acid significantly inhibited the strychnine-insensitive [3H]glycine binding. The Triton treatment did not affect the pharmacological profile of [3H]MK-801 binding sites. These results suggest that brain synaptic membranes treated with Triton X-100 are useful in evaluating the strychnine-insensitive and kynurenate-sensitive binding sites of glycine, which are functionally linked to N-methyl-D-aspartate- sensitive receptor channels.

Distribution of [3H]MK-801 binding in the turtle retina: an autoradiographical study.

Frozen sections (15 microns) of the turtle (Pseudemys scripta elegans) retina were incubated in 1.6 nM [3H]MK-801. Autoradiograms were generated using dry autoradiographical techniques. A band of [3H]MK-801 labelling was observed in the outer plexiform layer and cells were occasionally labelled in the inner nuclear and ganglion cell layers. This pattern of labelling was enhanced by preincubation in 1.0 mM glycine but not affected by pre-incubation in 3.0 mM N-methyl-D-aspartate (NMDA). We did not observe labelling associated with somata of bipolar cells and photoreceptors, or in the inner plexiform layer. The distribution of label suggests that [3H]MK-801 was bound to horizontal cells and occasional ganglion and/or amacrine cells. We have previously reported that exposure of L-type horizontal cells to MK-801 irreversibly altered the intracellular response of the cell to exogenous NMDA. The present finding strongly suggests that these physiological effects of MK-801 were due to specific binding of MK-801 to the NMDA receptor complex on the horizontal cell membrane.

Characterization of D-3,4-cyclopropylglutamates as N-methyl-D-aspartate receptor agonists.

The 4 configurational isomers of D-3,4-cyclopropylglutamate (D-CGA) have been synthesized and analyzed for their interactions as excitatory amino acid recognition sites. Additionally, functional assessment of the action of these compounds at the N-methyl-D-aspartate (NMDA) receptor was performed. All 4 analogs function as agonists at the NMDA receptor as evidenced by their ability to stimulate [3H]MK-801 binding to the coupled PCP recognition site. Furthermore, the rank order of potency of these compounds in stimulating [3H]MK-801 binding corresponds with their Ki values for the displacement of NMDA-selective L-[3H]glutamate and [3H]CGS-19755 binding (D-CGA-C greater than D-CGA-B greater than D-CGA-D greater than D-CGA-A). The D-CGA-C isomer has affinity and potency at the NMDA receptor similar to the endogenous agonist, L-glutamate. This high potency coupled with greater specificity than L-glutamate, makes D-CGA-C a potentially useful pharmacological tool for the study of this receptor.

Rat brain N-methyl-D-aspartate receptors require multiple molecules of agonist for activation.

N-Methyl-D-aspartate (NMDA) receptors mediate important physiological and pathological processes, including long term potentiation and neuronal excitotoxicity. Elucidation of mechanisms underlying NMDA receptor functioning will promote understanding of the molecular bases of NMDA receptor-mediated processes. The localization of the phencyclidine (PCP) receptor within the ionophore of the NMDA receptor-gated ion channel permits the binding of PCP receptor ligands to serve as a functional marker of channel activation. We have previously demonstrated that the highly selective PCP receptor ligand [3H]MK-801 displays multiexponential kinetics of association, indicating that the NMDA receptor functions according to a multistate model. Using the fast component of [3H]MK-801 binding to PCP receptors as a marker for activated NMDA channels, we demonstrate here a Hill coefficient of 2 for activation of NMDA channels by L-glutamate. A multistate model of NMDA receptor functioning analogous to the model known to account for the functioning of nicotinic cholinergic and gamma-aminobutyric acidA receptors fits well to our experimental data, supporting the concept that the NMDA receptor is properly classified in the Class 1 superfamily of ligand-gated channels.

Role of hydrogen bonding in ligand interaction with the N-methyl-D-aspartate receptor ion channel.

Displacement of [3H]MK-801 (dizocilpine, 1) binding to rat brain membranes has been used to evaluate the affinities of novel dibenzocycloalkenimines related to 1 for the ion channel binding site (also known as the phencyclidine or PCP receptor) on the N-methyl-D-aspartate (NMDA) subtype of excitory amino acid receptor. In common with many other agents having actions in the central nervous system, these compounds contain a hydrophobic aromatic moiety and a basic nitrogen atom. The conformational rigidity of these ligands provides a unique opportunity to evaluate the importance of specific geometrical properties that influence active-site recognition, in particular the role of the nitrogen atom in hydrogen-bonding interactions. The relative affinities (IC50s) of hydrocarbon-substituted analogues of 1 and ring homologated cyclooctenimines illustrate the importance of size-limited hydrophobic binding of both aryl rings and of the quaternary C-5 methyl group. Analysis of the binding of a series of the 10 available structurally rigid dibenzoazabicyclo[x.y.z]alkanes, by using molecular modeling techniques, uncovered a highly significant correlation between affinity and a proposed ligand-active site hydrogen bonding vector (r = 0.950, p less than 0.001). These results are used to generate a pharmacophore of the MK-801 recognition site/PCP receptor, which accounts for the binding of all of the known ligands.

(+)-[3H]MK-801 binding sites in postmortem human brain.

The pharmacological specificity and the regional distribution of the N-methyl-D-aspartate receptor-associated 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) binding sites in human postmortem brain tissue were determined by binding studies using (+)-[3H]MK-801. Scatchard analysis revealed a high-affinity (KD = 0.9 +/- 0.2 nM, Bmax = 499 +/- 33 fmol/mg of protein) and a low-affinity (KD = 3.6 +/- 0.9 nM, Bmax = 194 +/- 44 fmol/mg of protein) binding site. The high-affinity site showed a different regional distribution of receptor density (cortex greater than hippocampus greater than striatum) compared to the low-affinity binding site (cerebellum greater than brainstem). The rank order pharmacological specificity and stereoselectivity of the high-(cortex) and low-(cerebellar) affinity binding sites were identical. However, all compounds tested showed greater potency at the high-affinity site in cortex. The results indicate that (+)-[3H]MK-801 binding in human postmortem brain tissue shows pharmacological and regional specificity.