Glycine Substitution Effects on the Supramolecular Morphology and Rigidity of Cell-Adhesive Amphiphilic Peptides.

Self-assembling peptides that are capable of adopting ß-sheet structures can generate nanofibers that lead to hydrogel formation. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a ß-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, glycine substitution in an amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing and led to twisted ß-sheet structures and to nanofiber bundling, which likely led to the stiffened hydrogel. Importantly, the supramolecular structures were accurately predicted by molecular dynamics simulations, demonstrating the helpfulness of these techniques for the identification of self-assembling peptides. The hydrogel formed by the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective in modulating self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.

Comparative evaluation of two glycine transporter 1 radiotracers [11C]GSK931145 and [18F]MK-6577 in baboons.

Glycine transporter type-1 (GlyT1) has been proposed as a target for drug development for schizophrenia. PET imaging with a GlyT1 specific radiotracer will allow for the measurement of target occupancy of GlyT1 inhibitors, and for in vivo investigation of GlyT1 alterations in schizophrenia. We conducted a comparative evaluation of two GlyT1 radiotracers, [(11) C]GSK931145, and [(18) F]MK-6577, in baboons. Two baboons were imaged with [(11) C]GSK931145 and [(18) F]MK-6577. Blocking studies with GSK931145 (0.3 or 0.2 mg/kg) were conducted to determine the level of tracer specific binding. [(11) C]GSK931145 and [(18) F]MK-6577 were synthesized in good yield and high specific activity. Moderately fast metabolism was observed for both tracers, with ∼ 30% of parent at 30 min post-injection. In the brain, both radiotracers showed good uptake and distribution profiles consistent with regional GlyT1 densities. [(18) F]MK-6577 displayed higher uptake and faster kinetics than [(11) C]GSK931145. Time activity curves were well described by the two-tissue compartment model. Regional volume of distribution (VT ) values were higher for [(18) F]MK-6577 than [(11) C]GSK931145. Pretreatment with GSK931145 reduced tracer uptake to a homogeneous level throughout the brain, indicating in vivo binding specificity and lack of a reference region for both radiotracers. Linear regression analysis of VT estimates between tracers indicated higher specific binding for [(18) F]MK-6577 than [(11) C]GSK931145, consistent with higher regional binding potential (BPND ) values of [(18) F]MK-6577 calculated using VT from the baseline scans and non-displaceable distribution volume (VND ) derived from blocking studies. [(18) F]MK-6577 appears to be a superior radiotracer with higher brain uptake, faster kinetics, and higher specific binding signals than [(11) C]GSK931145.

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT(3)R.

In silico re-engineering of a neurotransmitter to activate KCNQ potassium channels in an isoform-specific manner.

Voltage-gated potassium (Kv) channel dysfunction causes a variety of inherited disorders, but developing small molecules that activate Kv channels has proven challenging. We recently discovered that the inhibitory neurotransmitter γ-aminobutyric acid (GABA) directly activates Kv channels KCNQ3 and KCNQ5. Here, finding that inhibitory neurotransmitter glycine does not activate KCNQs, we re-engineered it in silico to introduce predicted KCNQ-opening properties, screened by in silico docking, then validated the hits in vitro. Attaching a fluorophenyl ring to glycine optimized its electrostatic potential, converting it to a low-nM affinity KCNQ channel activator. Repositioning the phenyl ring fluorine and/or adding a methylsulfonyl group increased the efficacy of the re-engineered glycines and switched their target KCNQs. Combining KCNQ2- and KCNQ3-specific glycine derivatives synergistically potentiated KCNQ2/3 activation by exploiting heteromeric channel composition. Thus, in silico optimization and docking, combined with functional screening of only three compounds, facilitated re-engineering of glycine to develop several potent KCNQ activators.

Regulation of neurosteroid allopregnanolone biosynthesis in the rat spinal cord by glycine and the alkaloidal analogs strychnine and gelsemine.

The neurosteroid allopregnanolone (3alpha,5alpha-THP) is well characterized as a potentially therapeutic molecule which exerts important neurobiological actions including neuroprotective, antidepressant, anxiolytic, anesthetic and analgesic effects. We have recently observed that neurons and glial cells of the rat spinal cord (SC) contain various key steroidogenic enzymes such as 5alpha-reductase and 3alpha-hydroxysteroid oxido-reductase which are crucial for 3alpha,5alpha-THP biosynthesis. Furthermore, we demonstrated that the rat SC actively produces 3alpha,5alpha-THP. As the key factors regulating neurosteroid production by nerve cells are unknown and because glycine is one of the pivotal inhibitory neurotransmitters in the SC, we investigated glycine effects on 3alpha,5alpha-THP biosynthesis in the rat SC. Glycine markedly stimulated [(3)H]-progesterone conversion into [(3)H]3alpha,5alpha-THP by SC slices. The alkaloid strychnine, well-known as a glycine receptor (Gly-R) antagonist, blocked glycine stimulatory effect on 3alpha,5alpha-THP formation. Gelsemine, another alkaloid containing the same functional groups as strychnine, increased 3alpha,5alpha-THP synthesis. The stimulatory effects of glycine and gelsemine on 3alpha,5alpha-THP production were additive when the two drugs were combined. These results demonstrate that glycine and gelsemine, acting via Gly-R, upregulate 3alpha,5alpha-THP biosynthesis in the SC. The data also revealed a structure-activity relationship of the analogs strychnine and gelsemine on neurosteroidogenesis. Possibilities are opened for glycinergic agents and gelsemine utilization to stimulate selectively 3alpha,5alpha-THP biosynthetic pathways in diseases evoked by a decreased neurosteroidogenic activity of nerve cells.

Activity of novel lipid glycine transporter inhibitors on synaptic signalling in the dorsal horn of the spinal cord.

BACKGROUND AND PURPOSE: Inhibitory neurotransmission plays an important role in controlling excitability within nociceptive circuits of the spinal cord dorsal horn. Loss of inhibitory signalling is thought to contribute to the development of pathological pain. Preclinical studies suggest that increasing inhibitory glycinergic signalling is a good therapeutic strategy for treating pain. One approach to increase synaptic glycine is to inhibit the activity of the glycine transporter 2 (GlyT2) on inhibitory nerve terminals. These transporters are involved in regulating glycine concentrations and recycling glycine into presynaptic terminals. Inhibiting activity of GlyT2 increases synaptic glycine, which decreases excitability in nociceptive circuits and provides analgesia in neuropathic and inflammatory pain models. EXPERIMENTAL APPROACH: We investigated the effects of reversible and irreversible GlyT2 inhibitors on inhibitory glycinergic and NMDA receptor-mediated excitatory neurotransmission in the rat dorsal horn. The effect of these drugs on synaptic signalling was determined using patch-clamp electrophysiology techniques to measure glycine- and NMDA-mediated postsynaptic currents in spinal cord slices in vitro. KEY RESULTS: We compared activity of four compounds that increase glycinergic tone with a corresponding increase in evoked glycinergic postsynaptic currents. These compounds did not deplete synaptic glycine release over time. Interestingly, none of these compounds increased glycine-mediated excitatory signalling through NMDA receptors. The results suggest that these compounds preferentially inhibit GlyT2 over G1yT1 with no potentiation of the glycine receptor and without inducing spillover from inhibitory to excitatory synapses. CONCLUSIONS AND IMPLICATIONS: GlyT2 inhibitors increase inhibitory neurotransmission in the dorsal horn and have potential as pain therapeutics. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Antibacterial activity of remifentanil and mixtures of remifentanil and propofol.

STUDY OBJECTIVE: To investigate the antibacterial activity of glycine, which is contained in remifentanil, when combined with propofol. DESIGN: Prospective study. SETTING: Departments of anesthesiology and microbiology of a university hospital. MEASUREMENTS: Growth of the microorganisms Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans in propofol 1%; saline dilutions of remifentanil at one-, 10-, and 100-microg/mL concentrations; and 1:1 mixtures of propofol with remifentanil solutions was determined. MAIN RESULTS: Remifentanil inhibits bacterial growth in a concentration-dependent manner. The antibacterial effects were more pronounced with Staphylococcus aureus and Pseudomonas aeruginosa at cultures obtained at the fifth hour. The inhibition of bacterial growth was less influenced with Escherichia coli and Candida albicans. CONCLUSIONS: Propofol and remifentanil mixtures decreased bacterial growth, and combinations may reduce the infectious complications from accidentally contaminated propofol.

Novel indole-2-carboxamide and cycloalkeno[1,2-b]indole derivatives. Structure-activity relationships for high inhibition of human LDL peroxidation.

Series of indole-2-carboxamide and cycloalkeno[1,2-b]indole derivatives were synthesized and evaluated in order to determine the necessary structural requirements for a high inhibition of human LDL copper-induced peroxidation. Various modulations were systematically performed on the indole and cycloalkeno[1,2-b]indole nuclei as well as on the carboxamide moiety. The best compounds (3c, 3e, 7c, 7f, 7h, 7g, and 7o) are between 5 and 30 times more active than probucol itself. Two of these compounds (3c and 7o) were selected for complementary in vitro and in vivo investigations, which have shown additional properties of interest for the treatment and the prevention of atherosclerosis injuries. Compound 3c was found to have some antiinflammatory properties while compound 7o was proved to protect endothelial cells from the direct cytotoxicity of oxidized LDL with some additional calcium channel blocking properties.

Substituted indole-2-carboxylates as in vivo potent antagonists acting as the strychnine-insensitive glycine binding site.

A series of indole-2-carboxylates bearing suitable chains at the C-3 position of the indole nucleus was synthesized and evaluated in terms of in vitro affinity using [3H]glycine binding assay and in vivo potency by inhibition of convulsions induced by N-methyl-D-aspartate (NMDA) in mice. 3-[2-[(Phenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-carboxyl ic acid (8) was an antagonist at the strychnine-insensitive glycine binding site (noncompetitive inhibition of the binding of [3H]TCP, pA2 = 8.1) displaying nanomolar affinity for the glycine binding site (pKi = 8.5), coupled with high glutamate receptor selectivity (> 1000-fold relative to the affinity at the NMDA, AMPA, and kainate binding sites). This indole derivative inhibited convulsions induced by NMDA in mice, when administered by both iv and po routes (ED50 = 0.06 and 6 mg/kg, respectively). The effect of the substituents on the terminal phenyl ring of the C-3 side chain was investigated. QSAR analysis suggested that the pKi value decreases with lipophilicity and steric bulk of substituents and increases with the electron donor resonance effect of the groups present in the para position of the terminal phenyl ring. According to these results the terminal phenyl ring of the C-3 side chain should lie in a nonhydrophobic pocket of limited size, refining the proposed pharmacophore model of the glycine binding site associated with the NMDA receptor.

(E)-3-(2-(N-phenylcarbamoyl)vinyl)pyrrole-2-carboxylic acid derivatives. A novel class of glycine site antagonists.

The synthesis and preliminary biological evaluation of novel (E)-3-(2-(N-phenylcarbamoyl)-vinyl)pyrrole-2-carboxylic acids bearing alkyl, acyl, alkoxy, phenyl, and halo substituents at the 4- and 5-positions of the pyrrole ring are reported. These compounds were studied for their in vitro affinity at the strychnine-insensitive glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor complex. In the [3H]glycine binding assay (E)-4,5-dibromo-3-(2-(N-phenylcarbamoyl)vinyl)pyrrole-2-carboxylic acid 6w (pKi = 7.95 +/- 0.01) and the 4-bromo-5-methyl 6j (pKi = 7.24 +/- 0.01) and 4,5-dimethyl 6g (pKi = 6.70 +/- 0.03) analogues were the most active compounds of the series. Qualitative structure-activity analysis points to a negative correlation between bulk of the C-4 and C-5 substituents and affinity which is enhanced by halo-substituents. QSAR analysis by the Hansch descriptors F, R, pi, and MR, on a subset of compounds with pKi > or = 4, indicates that electron-withdrawing groups at C-4 and C-5 enhance the affinity. Bulk and lipophilicity are also relevant for the substituents at these positions. 6g was found to be a full antagonist (alpha = 0; enhancement of the [3H]TCP binding). The in vivo potency of 6g, 6j, and 6w was evaluated by the inhibition of NMDA-induced convulsions in mice by both the i.v. and po routes; 6w was the most active compound (ED50 = 3 x 10(-3) (0.8-10) g/kg, i.v. and 30 x 10(-3) (4.5-61) g/kg, p.o.). The results of this study indicate that the 3,4-disubstitutedpyrrole-2-carboxylate represents a novel template for the design of new glycine antagonists.

Comparison of the actions of glycine and related amino acids on isolated third order neurons from the tiger salamander retina.

Whole cell voltage and current clamp recordings were obtained from third order neurons isolated from the salamander retina. Using cross desensitization, the structure-function relationship of short chain amino acids on the glycine receptor were examined. L-Serine, L-alanine, beta-alanine and taurine all cross desensitized with glycine, but did not show significant cross desensitization with GABA. This indicates that these amino acids act at the glycine receptor. The order of potency was glycine >> beta-alanine > taurine >> L-alanine > L-serine. TAG, a reputed selective taurine antagonist, was equally effective in blocking taurine and glycine currents. There is no evidence for distinct receptors for taurine. Amino acids with larger moieties at the alpha carbon, such as threonine and valine, produced inactive ligands. Placing a methyl group on the amine of glycine or esterification of the carboxyl group also greatly reduced activity. Based on these modifications of the glycine molecule, it appears that selectivity at the glycine receptor results in part from steric restrictions at all three sites in the glycine chain. The steric interference is most critical at the carboxyl and amino ends, and less limiting at the alpha carbon. Doses of L-serine that had only slight effects in voltage clamp experiments, nevertheless produced large effects in current clamp experiments. This indicates that several endogenous amino acids can have significant effects on membrane voltage, even when their shunting activity may be small. High concentrations of agonists produced desensitization in the voltage clamp records, but there was little evidence of desensitization in the current clamp experiments. These results indicate that several endogenous amino acids can activate the glycine receptor, but there is no evidence for a discrete receptor for taurine, beta-alanine, L-alanine or L-serine. Since all these endogenous amino acids have similar amino and acid terminals, reduction in potency results from steric interference around the alpha carbon. This graded potency may have functional significance in mediating inhibition.

Characterization of the binding of two novel glycine site antagonists to cloned NMDA receptors: evidence for two pharmacological classes of antagonists.

The potency of two novel glycine site antagonists, GV150,526A and GV196,771A, was assessed by their ability to inhibit the binding of [(3)H]-MDL105,519 to cell homogenates prepared from mammalian cells transfected with either NR1-1a, NR1-2a, NR1-1a/NR2A, NR1-1a/NR2B, NR1-1a/NR2C or NR1-1a/NR2D NMDA receptor clones. The inhibition constants (K(i)s) for GV150,526A displacement of [(3)H]-MDL105,519 binding to either NR1-1a or NR1-2a expressed alone were not significantly different and were best fit by a one-site binding model. GV150,526A inhibition to NR1-1a/NR2 combinations was best fit by a two-site model with the NR1-1a/NR2C having an approximate 2 - 4 fold lower affinity compared to other NR1-1a/NR2 receptors. The K(i)s for GV196,771A displacement of [(3)H]-MDL105,519 binding to NR1-1a, NR1-2a and all NR1-1a/NR2 combinations was best fit by a two-site binding model. There was no significant difference between the K(i)s for the binding to NR1-1a and NR1-2a; NR1-1a/NR2A receptors had an approximate 4 fold lower affinity for GV196,771A compared to other NR1-1a/NR2 combinations. The K(i)s for both GV150, 526A and GV196,771A for the inhibition of [(3)H]-MDL105,519 binding to membranes prepared from adult rat forebrain were determined and compared to the values obtained for binding to cloned NMDA receptors. The K(i)s for a series of glycine site ligands with diverse chemical structures were also determined for the inhibition of [(3)H]-MDL105,519 binding to NR1-1a/NR2A receptors. L689,560 displayed similar binding characteristics to GV150,526A. It is suggested that glycine site antagonists may be divided into two classes based on their ability to distinguish between NR1 and NR1/NR2 receptors with respect to binding curve characteristics.

Allosteric modulation of glycine receptors is more efficacious for partial rather than full agonists.

Allosteric modulation of [3H]strychnine binding to glycine receptors (GlyRs) was examined in synaptosomal membranes of rat spinal cord. An allosteric model enabled us to determine the cooperativity factors of the allosteric agents with [3H]strychnine and glycine bindings (alpha and beta, respectively). We modified the allosteric model with a slope factor because the slope values of the displacement curves of partial agonists (beta-alanine, taurine and gamma-aminobutyric acid) were beyond unity. The slope factor was reduced only by 100 microM propofol. Further, propofol showed positive cooperativity (beta < 1) stronger with taurine than with glycine. The extent of the positive cooperativity of propofol was nearly independent from the potencies and structures of partial agonists. The steroidal alphaxalone and minaxolone also potentiated taurine better than glycine. Alphaxalone exerted weak negative cooperativity with [3H]strychnine binding. Displacement by taurine is attenuated by granisetron and m-chlorophenylbiguanide representing negative cooperativity (beta >> 1) greater than with glycine. The results suggest a developmental role of elevated perinatal levels of taurine and neurosteroids as well as a better allosteric modulation of decreased agonist efficacies for impaired glycine receptor-ionophores.

[3H]1-aminocyclopropanecarboxylic acid, a novel probe for strychnine- insensitive glycine receptors.

[3H]1-Aminocyclopropanecarboxylic acid (ACPC) exhibits high affinity, specific binding to strychnine-insensitive glycine receptors. In extensively washed rat forebrain membranes, the specific binding of [3H]ACPC was optimal at 25 degrees C in the presence of 10 mM MgCl2. Comparable levels of specific [3H]ACPC binding were obtained using centrifugation and filtration for separation of bound from free radioligand. [3H]ACPC labels two sites with Kdl and Bmax1 values of 129 +/- 34 nM and 2.30 +/- 0.37 pmol/mg protein and Kd2 and Bmax2 values of 7.26 +/- 1.69 microM and 20.6 +/- 2.2 pmol/mg protein for the high and low affinity sites, respectively. The Kd of [3H]ACPC (66 nM) estimated under non-equilibrium conditions (koff = 8.91 +/- 0.78 x 10(-3) s-1; kon = 1.35 x 10(-4) nM-1 s-1) was similar to the value obtained for the high affinity site obtained by equilibrium binding. The Kd1 of[3H]ACPC is in good agreement with the previously reported Ki values of ACPC to inhibit the binding of other glycinergic ligands including [3H]glycine, [3H]5,7-dichlorokynurenic acid (5,7-DCKA) and [3H]L-689,560 ((+/-)-4-(trans)-2-carboxy-5,7-dichloro-4- phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline). Moreover, the potencies of a series of glycine site ligands, including glycine. ACPC, 1-aminocyclobutanecarboxylic acid (ACBC), 5,7-DCKA, 7-chlorokynurenic acid (7-CKA), R(+)-3-amino-1-hydroxy-2- pyrrolidine (HA-966) and D-serine, to inhibit [3H]ACPC binding were highly correlated with their potencies to inhibit [3H]glycine and [3H]5,7-DCKA binding (r2 = 0.98-0.51). These results demonstrate that [3H]ACPC is a useful tool for examining the neurochemical and pharmacological properties of strychnine-insensitive glycine receptors.

3-(2-Carbamoylvinyl)-4,5-dimethylpyrrole-2-carboxylic acids as ligands at the NMDA glycine-binding site: a study on the 2-carbamoylvinyl chain modification.

Twenty 4,5-dimethylpyrrole-2-carboxylic acids (5a-t) with different 2-carbamoylvinyl chains in position 3 were prepared to further investigate the relationships between structure and in vitro affinity for the strychnine-insensitive glycine-binding site. None of these compounds was superior to (E)-3-(N-phenyl-2-carbamoylvinyl)-4,5-dimethylpyrrole-2-carb oxylic acid III (pKi = 6.70), which was taken as a reference standard, but overall the results obtained indicate that the N-phenyl-2-carbamoylvinyl substituent of III may be replaced with the N-(1-adamantyl)-2-carbamoylvinyl group as in 5h (pKi = 6.20) without considerable loss of affinity. This finding adds to previous knowledge.

An all-in-one adhesive does not etch beyond hybrid layers.

Continuous etching of aggressive all-in-one adhesives occurs in wet dentin tubules after polymerization of the adhesives. This study challenged the hypothesis that unpolymerized acidic monomers from an aggressive all-in-one self-etching adhesive continue to etch beyond dentin hybrid layers. Dentin surfaces bonded with Adper Prompt L-Pop were sectioned into 0.3-mm-thick slabs. Some of the slabs were stored in water (pH 6.8) or glycine buffer (pH 11.1) for six weeks and then examined by CLSM, SEM, and TEM. The rest were immersed in a biomimetic remineralizing medium for up to 4 months. Morphologic analysis indicated no difference in demineralization thickness between the two 6-week storage groups. However, increased permeability and loss of integrity occurred along the base of the hybrid layers in the glycine buffer group, but not in the water storage group. These findings were also confirmed by the results of biomimetic remineralization along the bases of those hybrid layers.

Synthesis and pharmacological properties of novel glycine antagonists.

The NMDA receptor is an ionotropic receptor complex widely distributed in the central nervous system and its activation, particularly in hypoxic conditions such as stroke, traumatic head injury and hypoglycemia, results in a massive influx of calcium ions into the post-synaptic neurones, leading to cell death through the activation of several neurotoxic cascades. The NMDA receptor is a unique ionotropic receptor complex because its activation requires the simultaneous binding of glutamate and glycine and selective antagonists at the glycine binding site are endowed with a better side-effect profile than competitive NMDA antagonists. Then, considerable efforts have been devoted to find potent and selective ligands, resulting in the identification of several classes of glycine antagonists. The research at Glaxo Wellcome has been aimed at the identification of novel in vivo active glycine antagonists, and led to the synthesis and pharmacological characterization of a number of novel, potent and systemically active compounds belonging to different chemical classes.

Identifying agonistic and antagonistic mechanisms operative at the GABA receptor.

Based on our molecular modeling investigations of the glycinergic receptor, we expanded our studies to similarly investigate the GABAergic receptor. New data suggest there may exist a slightly different agonistic mechanism for the molecules described herein as compared to glycine. The origin of this is undoubtedly the fact that, while glycine has a positive and two negative binding sites, it is significantly shorter than GABA and the other GABA agonists. Clearly, discovery of more glycine agonists is needed to further clarify this point. Moreover, we find a remarkedly different antagonistic mechanism exists for this phylogenetically newer inhibitory system in the central nervous system (CNS) than recently reported for strychnine and eight weaker glycine antagonists. We used GABA and six agonists (muscimol, dihydromuscimol, THIP, isoguvacine, trans-3-aminocyclopentane-1-carboxylic acid, piperidine-4-sulfonic acid) and five antagonists (bicuculline-N15-methobromide, R5135, pitrazepin, iso-THAZ and securinine) to derive our conclusions. We found that each of the agonists have three clearly defined atoms that can serve as attachment points at the GABAA receptor site. One of the three attachment atoms includes a carbonyl or carboxylate oxygen. The role of the carbonyl or carboxylate atom is very important. First, we theorize that a rapid two-point attachment occurs (one from the positive end and one from one of the other two negative atoms on the ligand) at the recognition site in the receptor where GABA or a GABAergic agonist binds. The positive end of the agonist perhaps associates through hydrogen bonding to a beta-carboxyl group in one of the aspartate molecules in the polypeptide. The negative attachment points perhaps bind through hydrogen bonding to arginine molecules in this polypeptide. The second negative site in the agonist immediately triggers a conformational change by pulling together the aforementioned groups by electrostatic attraction, and hence opening the chloride channel. We propose the carbonyl oxygen is partly responsible for triggering the opening by formation of a double hydrogen bond to arginine. We postulate that this attraction is the first step inducing the conformational change. In the case of the GABA antagonists investigated, a fourth attachment site was not found. In fact only two sites have been identified similar to the group II glycine antagonists. Our data support a hypothesis for GABAergic antagonist activity which suggests that the antagonist simply binds to the recognition site and blocks the neurotransmitter, GABA, from entering this site thereby preventing the opening of the chloride channel; it just stays closed. This mechanism is different from the mechanism proposed for the large number of Group I glycine antagonists (Aprison et al.: J Neurosci Res 41: 259-269, 1995).

Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection.

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine.