Computational analysis of non-competitive antagonist arylguanidine-α7 nAChR complexes.

meta-Chlorophenylguanidine (1) is a non-competitive α7 nicotinic acetylcholine receptor (nAChR) antagonist. Here we examined the hydrogen bond donor role of the anilinic N1-H on the inhibitory effect of 1 by preparing its N1-CH3 counterpart 2. Analog 2 was found to be at least as potent as 1 as a non-competitive α7 nAChR antagonist in a patch-clamp assay. To establish a structural basis for the mode of interaction of guanidines 1 and 2, we generated 100 homology models of the hα7 nAChR. This was followed by Connolly surface (SYBYL-X2.1) and blind docking (AutoDock 4.1) studies to identify eight possible binding pockets, two of which were supported by empirical data and employed in our docking studies. The optimized model-ligand complexes were analyzed using a Hydropathic INTeractions (HINT) analysis in order to compare and contrast different binding pockets and modes. We identified a potential allosteric binding site and distinct rotameric binding modes for 1 and 2 at α7 nAChRs. These differences in the binding orientations minimized the importance of an anilinic NH function for the antagonist activity at nACh receptors.

Controlled drug-release system based on pH-sensitive chloride-triggerable liposomes.

New pH-sensitive lipids were synthesized and utilized in formulations of liposomes suitable for controlled drug release. These liposomes contain various amounts of NaCl in the internal aqueous compartments. The release of the drug model is triggered by an application of HCl cotransporter and exogenous physiologically relevant NaCl solution. HCl cotransporter allows an uptake of HCl by liposomes to the extent of their being proportional to the transmembrane Cl(-) gradient. Therefore, each set of liposomes undergoes internal acidification, which, ultimately, leads to the hydrolysis of the pH-sensitive lipids and content release at the desired time. The developed system releases the drug model in a stepwise fashion, with the release stages separated by periods of low activity. These liposomes were found to be insensitive to physiological concentrations of human serum albumin and to be nontoxic to cells at concentrations exceeding pharmacological relevance. These results render this new drug-release model potentially suitable for in vivo applications.

Nicotine suppresses hyperexcitability of colonic sensory neurons and visceral hypersensivity in mouse model of colonic inflammation.

Recently, we reported that nicotine in vitro at a low 1-µM concentration suppresses hyperexcitability of colonic dorsal root ganglia (DRG; L(1)-L(2)) neurons in the dextran sodium sulfate (DSS)-induced mouse model of acute colonic inflammation (1). Here we show that multiple action potential firing in colonic DRG neurons persisted at least for 3 wk post-DSS administration while the inflammatory signs were diminished. Similar to that in DSS-induced acute colitis, bath-applied nicotine (1 µM) gradually reduced regenerative multiple-spike action potentials in colonic DRG neurons to a single action potential in 3 wk post-DSS neurons. Nicotine (1 µM) shifted the activation curve for tetrodotoxin (TTX)-resistant sodium currents in inflamed colonic DRG neurons (voltage of half-activation changed from -37 to -32 mV) but did not affect TTX-sensitive currents in control colonic DRG neurons. Further, subcutaneous nicotine administration (2 mg/kg b.i.d.) in DSS-treated C57Bl/J6 male mice resulted in suppression of hyperexcitability of colonic DRG (L(1)-L(2)) neurons and the number of abdominal constrictions in response to intraperitoneal injection of 0.6% acetic acid. Collectively, the data suggest that neuronal nicotinic acetylcholine receptor-mediated suppression of hyperexcitability of colonic DRG neurons attenuates reduction of visceral hypersensitivity in DSS mouse model of colonic inflammation.

{alpha}7-nAChR-mediated suppression of hyperexcitability of colonic dorsal root ganglia neurons in experimental colitis.

Controlled clinical trials of nicotine transdermal patch for treatment of ulcerative colitis have been shown to improve histological and global clinical scores of colitis. Here we report that nicotine (1 microM) suppresses in vitro hyperexcitability of colonic dorsal root ganglia (DRG) (L(1)-L(2)) neurons in the dextran sodium sulfate (DSS)-induced mouse model of acute colonic inflammation. Nicotine gradually reduced regenerative multiple-spike action potentials in colitis mice to a single action potential. Nicotine's effect on hyperexcitability of inflamed neurons was blocked in the presence of an alpha(7)-nicotinic acetylcholine receptor (nAChR) antagonist, methyllicaconitine, while choline, the alpha(7)-nAChR agonist, induced a similar effect to that of nicotine. Consistent with these findings, nicotine failed to suppress hyperexcitability in colonic DRG neurons from DSS-treated alpha(7) knockout mice. Furthermore, colonic DRG neurons from DSS-treated alpha(7) knockout mice were characterized by lower rheobase (10 +/- 5 vs. 77 +/- 13 pA, respectively) and current threshold (28 +/- 4 vs. 103 +/- 8 pA, respectively) levels than DSS-treated C57BL/J6 mice. An interesting observation of this study is that 8 of 12 colonic DRG (L(1)-L(2)) neurons from control alpha(7) knockout mice exhibited multiple-spike action potential firing while no wild-type neurons did. Overall, our findings suggest that nicotine at low 1 microM concentration suppresses in vitro hyperexcitability of inflamed colonic DRG neurons in a mouse model of acute colonic inflammation via activation of alpha(7)-nAChRs.

In vitro and in vivo characterization of a novel negative allosteric modulator of neuronal nAChRs.

In this study, we compared the in vitro and in vivo neuronal nicotinic acetylcholine receptor (nAChR) properties of 1,2,3,3a,4,8b-hexahydro-2-benzyl-6-N,N-dimethylamino-1-methylindeno[1,2,-b]pyrrole (HDMP, 4) to that of negative allosteric modulator (NAM), PCP. Patch-clamp experiments showed that HDMP exhibited an inhibitory functional activity at α7 nAChRs with an IC(50) of 0.07 µM, and was 357- and 414-fold less potent at α4ß2 and α3ß4 nAChRs, with IC(50)s of 25.1 and 29.0 µM, respectively. Control patch-clamp experiments showed that PCP inhibited α7, α4ß2 and α3ß4 nAChRs with IC(50)s of to 1.3, 29.0 and 6.4 µM, respectively. Further, HDMP did not exhibit any appreciable binding affinity to either α7 or α4ß2 nAChRs, suggesting its action via a non-competitive mechanism at these neuronal nAChR subtypes. The in vivo study showed that HDMP was a potent antagonist of nicotine-induced analgesia in the tail-flick (AD(50)=0.008 mg/kg), but not in the hot-plate test. All together, our in vitro and in vivo data suggest that HDMP is a novel NAM of neuronal nAChRs with potent inhibitory activity at α7 nAChR subtype at concentrations ≤ 1µM that are not effective for α4ß2 and α3ß4 nAChRs.

MD-354 selectively antagonizes the antinociceptive effects of (-)nicotine in the mouse tail-flick assay.

RATIONALE: (-)Nicotine produces antinociceptive effects in rodents. meta-Chlorophenylguanidine (MD-354), an analgesia-enhancing agent, binds at 5-HT(3) and alpha(2)-adrenoceptors and potentiates the antinociceptive effects of an "inactive" dose of clonidine. The present study examined the actions of MD-354 on (-)nicotine-induced antinociception. MATERIALS AND METHODS: Mouse tail-flick and other assays were employed. RESULTS: In the tail-flick assay, (-)nicotine (ED(50) = 1.66 mg/kg) but not MD-354 produced dose-related antinociceptive effects. Administered in combination with (-)nicotine (2.5 mg/kg), MD-354 (AD(50) = 3.4 mg/kg) did not potentiate, but effectively antagonized the antinociceptive actions of (-)nicotine. In a mouse hot-plate assay, MD-354 failed to modify (-)nicotine responses. In combination with a locomotor activity-suppressing dose of (-)nicotine, MD-354 (up to 17 mg/kg) failed to antagonize (-)nicotine-induced hypolocomotion. In a rat drug discrimination paradigm using (-)nicotine as training drug, MD-354 produced saline-appropriate responding; in combination with the training dose of (-)nicotine, MD-354 failed to antagonize the nicotine cue. CONCLUSIONS: MD-354 selectively antagonizes the antinociceptive actions of (-)nicotine in the tail-flick, but not in the hot-plate assay, or either the motor effects, or discriminative stimulus effects of (-)nicotine. The most parsimonious explanation is that MD-354 might act as a negative allosteric modulator of alpha 7 nACh receptors, and radioligand binding and functional data are provided to support this conclusion.

Rat neuronal nicotinic acetylcholine receptors containing alpha7 subunit: pharmacological properties of ligand binding and function.

AIM: To compare pharmacological properties of heterologously expressed homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) with those of native nAChRs containing alpha7 subunit (alpha7* nAChRs) in rat hippocampus and cerebral cortex. METHODS: We established a stably transfected HEK-293 cell line that expresses homomeric rat alpha7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat alpha7* nAChRs in rat hippocampus and cerebral cortex. We used [(125)I]-alpha-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the alpha7 nAChRs expressed in this cell line were studied using whole-cell current recording. RESULTS: The newly established cell line, KXalpha7R1, expresses homomeric alpha7 nAChRs that bind [(125)I]-alpha-bungarotoxin with a K(d) value of 0.38+/-0.06 nmol/L, similar to K(d) values of native rat alpha7* nAChRs from hippocampus (K(d)=0.28+/-0.03 nmol/L) and cerebral cortex (K(d)=0.33+/-0.05 nmol/L). Using whole-cell current recording, the homomeric alpha7 nAChRs expressed in the cells were activated by acetylcholine and (-)-nicotine with EC(50) values of 280+/-19 micromol/L and 180+/-40 micromol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of alpha7 nAChRs, alpha-bungarotoxin (IC(50)=19+/-2 nmol/L) and methyllycaconitine (IC(50)=100+/-10 pmol/L). A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric alpha7 nAChRs and native alpha7* receptors in rat brain, but it also revealed several notable differences. CONCLUSION: This newly established stable cell line should be very useful for studying the properties of homomeric alpha7 nAChRs and comparing these properties to native alpha7* nAChRs.

3'-Fluoro substitution in the pyridine ring of epibatidine improves selectivity and efficacy for alpha4beta2 versus alpha3beta4 nAChRs.

The analog of epibatidine having a fluoro substituent at the 3' position of the pyridine ring has been recently developed and shown to possess binding affinity in the pM range to alpha4beta2 nAChRs and in the nM range to alpha7 nAChRs and to exhibit potent agonist activity in nicotine-induced analgesia tests. Here we used patch-clamp technique in a whole-cell configuration to compare functional activity of 3'-fluoroepibatidine to that of epibatidine by itself on recombinant alpha4beta2, alpha7 and alpha3beta4 neuronal nAChRs. The agonist effect of (+/-)-epibatidine was partial and yielded comparable EC50s of 0.012 microM (72% efficacy) and 0.027 microM (81% efficacy) at alpha4beta2 and alpha3beta4 nAChRs, respectively, but was full at alpha7 nAChRs with an EC50 of 4.8 muM. Testing of the analog at different concentrations revealed that it acts as a full agonist with an EC50 of 0.36 microM at alpha4beta2 nAChRs and induces partial agonist effect (66% efficacy) at alpha7 nAChRs with an EC50 of 9.8 microM and an IC50 corresponding to 225 microM. In contrast, the analog caused only 24% maximal activation at the range of concentrations from 0.1 to 100 microM and, in addition, induced an inhibition of alpha3beta4 nAChR function with an IC50 of 8.3 microM. Our functional data, which are in agreement with previous binding and behavioral findings, demonstrate that 3'-fluoro substitution in the pyridine ring of epibatidine results in an improved pharmacological profile as observed by an increased efficacy and selectivity for alpha4beta2 versus alpha3beta4 nAChRs.

New noninvasive methodology for real-time monitoring of lipid flip.

A new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of the cascade-blue-labeled lipid through complex formation with a membrane-impermeable cyclen-tetranaphthalenethiourea synthetic receptor for this dye. The high affinity of the receptor to cascade-blue label allows the use of micromolar concentrations of this receptor during the experiment. At these low concentrations, the receptor does not interfere with the membrane integrity and, therefore, renders this new methodology less invasive to the model and cell membranes than commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Unlike with the NBD-dithionite assay, where the fluorescence quenching of the NBD group is achieved through its chemical modification, this new assay relies on the noncovalent interactions between cascade-blue label and the receptor. Therefore, the quenching can be reverted by either competitive displacement of the lipid-attached label with a water-soluble substrate or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. We demonstrate that this new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments.

Synthesis and pharmacological characterization of nicotinic acetylcholine receptor properties of (+)- and (-)-pyrido-[3,4-b]homotropanes.

(+/-)-Pyrido[3,4-b]homotropane [(+/-)-1] is a conformationally rigid analogue of nicotine (2) or nornicotine (3) that showed high affinity for nicotinic acetylcholine receptors. Even though the synthesis and potent activity of this highly interesting compound was originally reported in 1986 (Kanne, D. B.; Ashworth, D. J.; Cheng, M. T.; Mutter, L. C.; Abood, L. G. Synthesis of the first highly potent bridged nicotinoid. 9-Azabicylo[4.2.l]nona[2,3-c]pyridine (pyrido[3,4-b]homotropane). J. Am. Chem. Soc. 1986, 108, 7864-7865), the individual optical isomers have not been prepared and studied. In this study, we report the synthesis of (+)- and (-)-1 and show that (+)-1 has Ki = 1.29 nM at the alpha4beta2* nAChR and has over 260 times higher affinity than (-)-1. Single-crystal X-ray analysis of an intermediate used to prepare the isomers established the absolute stereochemistry as (1S,6S)-(+)-1 and (1R,6R)-(-)-1. Surprisingly, both isomers failed to produce antinociception in the mouse tail-flick and hot-plate assays, engender nicotine-like responding in rat drug discrimination, or alter current amplitude in alpha4beta2- and alpha3beta4-containing cells. However, (-)-1 antagonized nicotine-induced antinociception with an ED50 of 0.07 microg/kg in the tail-flick assay. The reason for this unusual pharmacology is unknown, but it is possible that (-)-1 is acting at a non-epibatidine-sensitive receptor subtype to antagonize nicotine's effects in the tail-flick assay.

2-Fluoro-3-(4-nitro-phenyl)deschloroepibatidine is a novel potent competitive antagonist of human neuronal alpha4beta2 nAChRs.

A patch-clamp technique in a whole-cell configuration was used to examine the functional activity of recently developed 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs on two major subtypes of neuronal nicotinic acetylcholine receptors (nAChRs), alpha4beta2 and alpha3beta4, that predominate in the central and peripheral nervous systems, respectively. These epibatidine analogs have been shown previously to possess high binding affinity to alpha4beta2 but not to alpha7 nAChRs and to inhibit nicotine-induced analgesia in behavioral pain tests. The 2-fluoro-3-(4-nitro-phenyl)deschloroepibatidine (4-nitro-PFEB) exhibited the most pronounced antagonist activity among these analogs when tested electrophysiologically on alpha4beta2 nAChRs. It inhibited acetylcholine (ACh)-induced currents in a concentration-dependent manner with an IC(50) value of 0.1 microM and produced complete inhibition at approximately 1 microM concentration. 4-Nitro-PFEB at 0.1 microM concentration produced a 4-fold rightward shift in the ACh concentration-response curve without altering maximum ACh-induced response. This inhibitory effect of 4-nitro-PFEB was voltage- and use-independent and was partially reversible at its 1 microM concentration. The rise and decay kinetics of ACh-induced currents was not altered in the presence of 4-nitro-PFEB. In contrast to alpha4beta2 nAChRs, this compound did not affect alpha3beta4 nAChR-mediated currents at < or =1 microM (IC(50) approximately 63.9 microM). Overall, these functional data agree with previous binding and behavioral findings and suggest collectively that 4-nitro-PFEB is the most effective and selective antagonist of alpha4beta2 versus alpha3beta4 and alpha7 nAChRs among the tested analogs, acting on alpha4beta2 nAChR through a competitive mechanism with a potency 17-fold higher than that of dihydro-beta-erythroidine.

Differential modulation of beta2 and beta4 subunits of human neuronal nicotinic acetylcholine receptors by acidification.

We have shown previously that acidification increases the affinity of agonists to rat alpha3beta4 nicotinic acetylcholine receptors (nAChR) and accelerates both the activation and decay kinetics of agonist-induced currents recorded from human embryonic kidney 293 cells stably expressing the receptor (Abdrakhmanova et al., 2002b). Here, we report on experiments examining the effect of rapid acidification on four different subtypes (alpha3beta4alpha5, alpha4beta2, alpha3beta2, and alpha3beta2alpha5) of human neuronal nAChRs stably expressed in tsA201 cells using a piezoelectric device for rapid (<5 ms) solution application. Application of ACh, at its EC(50) concentration for each nAChR subtype, at pH values 7.4 and 6.0, showed that acidification, similarly to that reported for rat alpha3beta4 acetylcholine receptors (AChRs), increased the amplitude and accelerated the activation and decay kinetics of the currents in human alpha3beta4alpha5 AChRs by increasing their affinity to the agonist. In sharp contrast, acidification reduced the amplitude but accelerated the decay kinetics of the current in all human beta2-containing nAChR subtypes (alpha3beta2, alpha3alpha5beta2, alpha4beta2) examined in this study. Brief application of ACh at saturating concentration (1 mM) on alpha3beta4alpha5 AChRs induced a "rebound current" upon rapid washout of the agonist at pH 7.4, but no "rebound current" was observed in alpha3beta2 AChRs. Surprisingly, acidification, pH 6.0, applied only during the agonist pulse also accelerated the decay kinetics of the "rebound current". Our data provide evidence for the specificity of proton-induced modulation of neuronal nAChRs based on their beta subunit composition. Furthermore, in alpha3beta4alpha5 AChR, we find that protonation effects may persist, after washout of acidic solutions, consistent with proton-induced conformational changes of the receptor.

Ion channel formation from a calix[4]arene amide that binds HCl.

The ion transport activity of calix[4]arene tetrabutylamide 1,3-alt 2 was studied in liposomes, planar lipid bilayers, and HEK-293 cells. These experiments, when considered together with (1)H NMR and X-ray crystallography data, indicate that calix[4]arene tetrabutylamide 2 (1) forms ion channels in bilayer membranes, (2) mediates ion transport across cell membranes at positive holding potential, (3) alters the pH inside liposomes experiencing a Cl(-) gradient, and (4) shows a significant Cl(-)/SO(4)(2)(-) transport selectivity. An analogue, calix[4]arene tetramethylamide 1, self-assembles in the presence of HCl to generate solid-state structures with chloride-filled and water-filled channels. Structureminus signactivity studies indicate that the hydrophobicity, amide substitution, and macrocyclic framework of the calixarene are essential for HCl binding and transport. Calix[4]arene tetrabutylamide 2 is a rare example of an anion-dependent, synthetic ion channel.

Protons enhance the gating kinetics of the alpha3/beta4 neuronal nicotinic acetylcholine receptor by increasing its apparent affinity to agonists.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system. Although there is a vast literature on the molecular, structural and pharmacological properties of neuronal nAChR, little is known of their pH regulation. Here we report that rapid acidification (pH 6.0) enhances the current through the alpha3/beta4 recombinant nAChRs expressed stably in human embryonic kidney 293 cells and accelerates its activation kinetics without altering selectivity. Acidification also strongly accelerates the decay kinetics ("desensitization") of cytisine- and nicotine-evoked currents (pK(a) approximately 6.1), but the effect is somewhat smaller with acetylcholine and carbachol (undetermined pK(a) values), suggesting that protonation of the agonist contributes to the relaxation of the current. Transient increases of [H(+)](o) from pH 7.4 to 6.0, during the time course of decay of the current, enhances the current and accelerates its decay kinetics in a manner similar to reactivation of current by higher concentrations of agonists. We suggest that protons interact with multiple extracellular sites on alpha3/beta4 nAChRs, decreasing the effective EC(50) values of the agonist and accelerating gating kinetics, in part by promoting agonist-induced block. We speculate that corelease of protons with ACh from the secretory vesicles may induce rapid and reversible conformational changes in the slowly "desensitizing" alpha3/beta4 nAChRs, leading to accelerated signaling.