Study of the Interaction of Chlorisondamine and Chlorisondamine

Study of the Interaction of Chlorisondamine and Chlorisondamine Analogues with an Epitope of the r-2 Neuronal Acetylcholine Nicotinic Receptor Subunit Hay-Yan J. Wang,† Andrew E. Taggi,‡,§ Jerrold Meinwald,‡ Roy A. Wise,† and Amina S. Woods*,† Behavioral Neuroscience Branch, NIDA-IRP, National Institutes of Health, DHHS, 5500 Nathan Shock Drive, Baltimore Maryland 21224 and Department of Chemistry and Chemical Biology, Cornell University, Ithaca New York 14853 Received November 22, 2004

Chlorisondamine (CHL), a neuronal nicotinic ganglionic blocker, when injected in the cerebral ventricle of rats chronically blocks the increase in locomotion and rearing by subcutaneous nicotine injection. The blocking of the ion channel(s) prevents nicotine from exerting its rewarding effects on the CNS. When administered intraperitoneally, a dose 400-500 times the intracerebroventricular one is needed to cross the blood-brain barrier and to generate the same level of nicotine antagonism, resulting in severe side-effects, thus making it unlikely to be used as a therapeutical compound. Three CHL analogues, 2-(indolin-1-yl)-N,N,N-trimethylethanaminium iodide, 2-(1,3-dioxoisoindolin-2-yl)- N,N,Ntrimethylethanaminium iodide, and 2-(1H-indole-3-carboxamido)- N,N,N-trimethylethanaminium iodide, were synthesized in the hope of circumventing the parent compound’s shortcomings. They all share a modified indole ring, lack the four chlorines CHL carries, and have one tertiary amine and one quaternary amine. The CHL analogues form noncovalent complexes with an epitope of the R-2 nicotinic receptor subunit, GEREE(p)TEEEEEEEDEN, previously proposed as the possible site of CHL interaction. Complexes were analyzed using matrix-assisted laser desorption/ionization mass spectrometry for comparison with CHL. Overall, all three analogues showed better affinity than CHL for complex formation with both the nonphosphorylated and phosphorylated epitopes. Keywords: ganglionic blockers • chlorisondamine analogues • molecular modeling

Introduction The nicotinic acetylcholine receptor (AChR) is the most widely studied and best characterized neuroreceptor. Acetylcholine combines with AChRs to initiate excitatory postsynaptic potential in nerves, or to initiate end plate potentials in muscles.1 In addition to its physiological importance, the neuronal nicotinic acetylcholine receptor (nAChR) is involved in the development of tobacco addiction.2-5 It was shown in animal studies that self-administration of nicotine can be blocked by (bis-) quaternary ammonium compounds such as chlorisondamine (CHL).6 Clarke and colleagues also demonstrated that a single intracerebroventricular (ICV) injection of CHL (5-10 µg free base /rat) could block the nicotine-induced behavioral effects for months.7-10 Although CHL chronically blocks nicotine-induced behavioral effects without blocking the attachment of nicotine at the * To whom correspondence should be addressed. E-mail: awoods@ intra.nida.nih.gov. † Behavioral Neuroscience Branch, NIDA-IRP, National Institutes of Health. ‡ Department of Chemistry and Chemical Biology, Cornell University. § Current address: Andrew E Taggi, Ph.D. DuPont Crop Protection, StineHaskell Research Center, P.O. Box 30, Newark, DE 19714-0030. E-mail: [email protected].

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active site,11 it does not readily cross the blood-brain barrier (BBB). Clarke et al. used 500 times the effective ICV dose for intraperitoneal (i.p.) or subcutaneous (s.c.) injection in rats to obtain a similar level of nicotinic blockade,9,10 resulting in hemodynamic instabilities, diarrhea, and transient paralysis of the animals that lasted up to 72 h due to nonspecific blockage at the neuromuscular junctions, thus negating the therapeutical potentials of CHL as a tool in combating tobacco addiction. To take advantage of the long-lasting effect of CHL, it is paramount to find analogues that could be administered orally, and could readily cross the blood-brain barrier, hence causing minimal side effects. We previously proposed an interaction between CHL and an epitope of the R-2 subunit (GEREE(p)TEEEEEEEDEN) of nAChR.12 Three CHL analogues, 2-(indolin-1-yl)-N,N,N-trimethylethanaminium iodide (AT12), 2-(1,3-dioxoisoindolin-2yl)- N,N,N-trimethylethanaminium iodide (AT26), and 2-(1Hindole-3-carboxamido)- N,N,N-trimethylethanaminium iodide (AT47) (Figure 1), were synthesized in the hope of circumventing the shortcoming of CHL in crossing BBB, while retaining its ability to form a noncovalent complex with the nicotinic receptor epitope. The present study qualitatively, and quantitatively compares the complex forming capabilities of CHL and 10.1021/pr049786g CCC: $30.25

 2005 American Chemical Society

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Chlorisondamine and Chlorisondamine Analogues

Table 1. MH+ List and Relative Abundance (RA) of Noncovalent Complexes between Receptor Epitope and CHL (pH buffered)

Figure 1. (A) The structure of CHL and the three analogue ligands: (B) The surface electrostatic potential of CHL (upper left), AT12 (upper right), AT26 (lower left), and AT47 (lower right). Color spectrum: red: -21.24 kcal/mol; blue: 169.21 kcal/mol.

its three analogues with both the nonphosphorylated, and phosphorylated receptor epitopes. The formation of noncovalent complexes is an indicator of the affinity of CHL and its analogues for the nicotinic receptor epitope. Under identical experimental conditions, the number of molecules complexing with nicotinic epitope determines the affinity.

Material and Methods 1. Receptor Peptides. R-2 nicotinic acetylcholine receptor peptides GEREETEEEEEEEDEN (epitope), and GEREEpTEEEEEEEDEN (p-epitope, where ‘p’ denotes phosphorylation of threonine [T]) were synthesized and purified by the Johns Hopkins School of Medicine Synthesis and Sequencing Laboratory. Epitope stock solutions were made at 1 mM concentration in doubly distilled water. In the study of pH effects on noncovalent complex formation, both peptide solutions were unbuffered; otherwise, they were buffered to a pH of 5-6 by Tris-HCl (T-3038, Sigma Chemical Co., MO). 2. Ligands. Chlorisondamine diiodide (CHL) was acquired from Tocris Cookson Inc. (Ellisville, MO), and made into 10mM stock solution in doubly distilled water. AT12, AT26, and AT47 were custom-synthesized and purified by Andrew Taggi in Prof. Jerrold Meinwald laboratory in the Department of Chemistry and Chemical Biology, Cornell University, and made into 50, 15, and 25mM stock solutions, respectively, in doubly distilled water. The pH of the stock ligand solutions was buffered to 6. 3. Formation of Non-Covalent Complexes. Each ligand was mixed with the nicotinic receptor epitope, and p-epitope (phosphorylated) solutions at the final molar ratios of 2:1, 5:1, and 10:1 (ligand: peptide) where the peptide concentration was

no. of CHL complexed

epitope (m/z)

RA (%)

p-epitope (m/z)

RA (%)

0 1 2 3 4

1982.8 2340.0 2698.1 3056.3 3414.4

100 56 18 3.6 0

2062.8 2420.0 2778.1 3136.3 3494.4

100 53.9 18.9 4.3 0.3

0.5mM and the mixture pH 6. When studying pH effect, the CHL-peptide mixtures were unbuffered, and had a pH of 2. 4. Sample Preparation. 0.7 µL of ligand- peptide mixture is deposited on the target plate followed by an equal volume of a saturated matrix solution of 6-aza-2-thiothymine (ATT) in 50% ethanol-water (Aldrich, Milwaukee, WI,), and air-dried. 5. Instrument. Mass spectra were the average of 50 shots, acquired in linear positive ion mode on a DE-PRO MALDI (PEBiosystems, Framingham, MA), equipped with a nitrogen laser (337 nm) and an extraction voltage of 20 kV. 6. Molecular Modeling and Binding Calculations. Geometry optimizations and surface electrostatic potential of CHL and the three analogues were calculated using Semi-Empirical method at AM1 level using Spartan ‘02 for Windows.13 Surface electrostatic potential profiles are shown in Figure 1B (red: -21.24 kcal/mol; blue: +169.21 kcal/mol). The epitope and p-epitope structures were modeled using DS Modeling 1.1 Suite (Accelrys Inc. San Diego, CA). Their conformations were minimized by CHARMm force field at pH ) 6 using approximate solvent effects (dieletcric constant ) 4). The results are shown in Figure 6. To further compare the number of ligands bound to each peptide, thus the ligand binding efficacies, we weighted the binding through the following conversions. The relative abundance (RA; see first paragraph of “Results”) of a noncovalent complex species was normalized against that of the most abundant noncovalent complex species (set as 1) from a single spectrum. The result is designated as Normalized Complex Abundance (NCA). NCA also represents the normalized abundance of peptide involved in noncovalent complex formation, since in most instances multiple ligand molecules complex with one peptide molecule. The Weighted Bound Ligand Abundance (WBLA) of each complex is acquired by multiplying NCA with its ligand number. The sum of WBLA in each ligand-peptide combination is then divided by the sum of NCA to get Weighted Bound Average (WBA), which is used as the indicator for the number of ligand molecule bound to each peptide. The WBA calculation is listed in Table 5.

Results The molecular ion (MH+) of the peptide in every mixture was the base peak, and was set at 100% relative abundance (RA) in each spectrum after centroiding. The RA of the noncovalent complexes in each spectrum were normalized against the RA of the peptide MH+, and listed alongside the m/z value in Table 1 to Table 4. In each ligand-peptide mixture combination, the number of the ligand complexed with the peptide was quite consistent across the tested molar ratios, except for [AT12+epitope] mixture at 2:1 molar ratio, where no complex was observed. To compare the difference between the ligands involved in complex formation, all the spectra Journal of Proteome Research • Vol. 4, No. 2, 2005 533

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Figure 2. (A) Spectrum of the receptor epitope and chlorisondamine (CHL). The legend “n” preceeding the ligand (CHL) represents the number of ligand molecules involved in the formation of non-covalent complexes. (B) Spectrum of the phosphorylated receptor epitope and CHL.

shown were acquired from the mixtures of 5:1 (ligand: peptide). To enhance the clarity, the complex formed by n Ligand(s) and one peptide will be presented as nLigand-peptide complex, where the identity of the peptide (epitope, or p-epitope) may be further specified if necessary. For example, the complex formed by 2 CHL and one p-epitope is specifically presented as 2CHL-p-epitope complex. A. Effects of pH on the Complex Formation between CHL and the Receptor Epitope. The unbuffered mixture environment allowed the peptides to complex with as many as two CHL molecules, regardless of their phosphorylation status. The RA of 1CHL-peptide complexes were between 20 and 25% of the base peak, while the RA of 2CHL-peptide complexes were between 4 and 6% of the base peak (See Figure 1 in ref 12). Once the mixture environment was adjusted to a pH of 6, each epitope could complex with as many as three CHL, and each p-epitope could complex with as many as four CHL molecules. The RA of 1CHL- and 2CHL-peptide complexes also increased 534

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by 2-3-fold (see Table 1 and Figure 2). The results suggest that a pH closer to physiological pH promotes noncovalent complex formation. The subsequent studies were conducted at a pH of 6. B. Noncovalent Complexes Formation between the Nicotinic Receptor Epitope and the Analogues. CHL: Table 1 lists the MH+ and RA of the noncovalent complexes from the mixtures of CHL and the receptor peptides. The epitope could complex with three CHL molecules (Figure 2A), whereas the p-epitope could complex with as many as four CHL molecules (Figure 2B). However, the RA of 4CHL-p-epitope complex was quite low (0.3%; Figure 2B and Table 1). This could be due to the conformational constraints. AT12: Table 2 lists the MH+ and RA of the noncovalent complexes from the mixtures of AT12 and the receptor epitopes. One epitope could complex with as many as three AT12. However, the RA of the complexes were quite low (Table 2; Figure 3A). Also, as was mentioned earlier, noncovalent com-

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Figure 3. (A) Spectrum of the receptor epitope and AT12. (B) Spectrum of the phosphorylated receptor epitope and AT12. Table 2. MH+ List and RA of Non-covalent Complexes between Receptor Epitope and AT12

All together, the results indicate that phosphorylation of Thr (T) residue promotes the formation of noncovalent complexes.

no. of AT12 complexed

epitope (m/z)

RA (%)

p-epitope (m/z)

RA

0 1 2 3 4 5 6

1982.8 2187.1 2392.4 2597.8 2803.1 3008.4 3213.7

100 8.8 2.6 1.3 0 0 0

2062.8 2267.1 2472.4 2677.8 2883. 3088.4 3293.7

100 48 40.5 23.5 9 3.2 2.3

plexes were not detected in [AT12+epitope] mixture at 2:1 molar ratio. On the other hand, phosphorylation of the epitope drastically enhanced its complex formation with AT12: as many as six AT12 molecules could complex with p-epitope. In addition, the RA of the noncovalent complexes in [AT12+pepitope] mixture increased significantly (Table 2, Figure 3B).

For both CHL and AT12, the highest RA of the noncovalent complexes came from the 1Ligand-peptide complexes. The RA of 2Ligand-, 3Ligand-, or even 4Ligand- peptide complexes showed a linear decrease paralleling the increase in the number of ligand molecule complexing with the epitopes. AT26: Table 3 lists the MH+ and RA of the noncovalent complexes from the mixture of AT26 and the peptides. Although one epitope could complex with as many as six AT26, the RA of these complexes were moderately low (Table 3, Figure 4A). Phosphorylation of the epitope did not further increase the number of AT26 complexed to it. However, such epitope modification did increase the RA of 1AT26- to 5AT26- epitope complexes by roughly 2-3-folds (Table 3; Figure 4B). We also observed a quasi-bell shape distribution in the RA of the complexes, with the 3Ligand- or 4Ligand-epitope complexes as the most abundant species. This observation is in contrast Journal of Proteome Research • Vol. 4, No. 2, 2005 535

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Figure 4. (A) Spectrum of the receptor epitope and AT26. (B) Spectrum of the phosphorylated receptor epitope and AT26. Table 3. MH+ List and RA of Non-covalent Complexes between Receptor Epitope and AT26

Table 4. MH+ List and RA of Non-covalent Complexes between Receptor Epitope and AT47


epitope (m/z)

RA (%)

p-epitope (m/z)

RA

0 1 2 3 4 5 6

1982.8 2215.1 2448.4 2681.7 2915.0 3148.3 3381.5

100 2.4 8.7 15.1 16.4 5.4 3

2062.8 2295.1 2528.4 2761.7 2995.0 3228.3 3461.5

100 8.2 26.5 42.9 32.1 14.1 3.5

to the earlier observations of CHL and AT12 where the highest RA came from the 1Ligand- peptide complexes. AT47: Table 4 lists the MH+ and RA of the noncovalent complexes from the mixture of AT47 and the peptides. One epitope could complex with as many as eight AT47, but the RA of these noncovalent complexes were, again, moderately low. Phosphorylation of the epitope, again, increased the RA 536



epitope (m/z)

RA (%)

p-epitope (m/z)

RA

0 1 2 3 4 5 6 7 8

1982.8 2228.1 2474.5 2720.8 2967.1 3213.5 3459.8 3706.1 3952.4

100 5.2 9.8 11.5 13.1 10.5 6 6.1 0.4

2062.8 2308.1 2554.5 2800.8 3047.1 3293.5 3539.8 3786.1 4032.4

100 5 20 38.5 37.7 26.9 15.1 8.2 2.9

of 2AT47- to 8AT47- peptide complexes by approximately 2-3fold. Nonetheless, phosphorylation of the epitope did not further increase the number of AT47 complexed to it. The RA of the complexes, again, showed a quasi-bell shape pattern.


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Figure 5. (A) Spectrum of the receptor epitope and AT47. (B) Spectrum of the phosphorylated receptor epitope and AT47.

For both peptides, the most abundant complexes were from the 3Ligand- and 4Ligand- peptide complexes (Table 4; Figure 5A and 5B). Weighted Bound Average (WBA): Table 5 lists the calculation of WBA of each ligand to epitope and p-epitope. The higher the number of the WBA, the more ligand bound to the epitope. The WBA of the ligand to p-epitope is generally higher than that to the nonphosphorylated epitope, except for AT26 where the WBA to the p-epitope is slightly lower than that to the epitope.

Discussions The data from Figure 2 and our previous study12 clearly shows that pH has a significant influence on ligand (CHL) binding to the receptor epitopes. It is well-known that, ambient pH can change the folding of peptides and proteins, and subsequently change their functions. In our study, changing the pH from 2 to 6 has dramatically enhanced noncovalent

complex formation between CHL and the receptor peptides. The closer the pH is to 7.4 the more likely the binding of ligand - peptide is to take place. Ab Initio calculations showed that, the tetrachloro- portion of the isoindolium ring of CHL and the isoindole portions of the three analogues carry negative surface electrostatic potentials. The distal quaternary ammonium arms carry moderate to high positive electrostatic potentials. Such arrangement generated dipole moments across CHL and the analogues. The arrangement of the acidic (Asp, Glu, and/or phosphate group) and the basic amino acid residues (Arg) across the receptor epitope also generates a dipole moment. The electrostatic charges on the ligands and the receptor epitopes allow the formation of noncovalent complexes through the delocalized positive charge on the guanidinium group of Arg interacting with the π cloud over the ring system of the ligand molecule via cation-π interaction;12,14-21 whereas the delocalized negative charge of the carboxyl group of acidic residues, or the two Journal of Proteome Research • Vol. 4, No. 2, 2005 537

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Table 5. Calculation of Weighted Bound Average from Complexesa no. of bound ligand

1 2 3 4 5 6 sum WBA

no. of bound ligand

1 2 3 4 5 6 7 8 Sum WBA

CHL-Epitope

CHL-p-Epitope

RA (%)

NCA

WBLA

56 18 3.6

1 0.321 0.064

1 0.642 0.192

1.385 1.325

1.834

NCA

WBLA

53.9 18.9 4.3 0.3

1 0.351 0.080 0.006

1 0.702 0.240 0.024

1.437 1.368

1.966

AT26-Epitope RA (%)

NCA

2.4 8.7 15.1 16.4 5.4 3

AT12-Epitope

RA (%)

NCA

WBLA

8.8 2.6 1.3

1 0.295 0.148

1 0.590 0.444

1.443 1.410

2.034

AT26-p-Epitope WBLA

RA (%)

NCA

WBLA

0.146 0.531 0.921 1 0.329 0.183

0.146 1.062 2.763 4 1.645 1.098

8.2 26.5 42.9 32.1 14.1 3.5

0.191 0.618 1 0.748 0.329 0.082

0.191 1.236 3 2.992 1.645 0.492

3.110 3.454

10.741

2.968 3.220

9.556

AT12-p-Epitope

RA (%)

RA (%)

48 40.5 23.5 9 3.2 2.3

AT47-Epitope RA (%)

5.2 9.8 11.5 13.1 10. 5 6.0 6.1 0.4

NCA

WBLA

1 0.844 0.490 0.188 0.067 0.048 2.637 2.098

1 1.688 1.470 0.752 0.335 0.288 5.533

AT47-p-Epitope

NCA

WBLA

0.397 0.748 0.878 1 0.802 0.458 0.466 0.031 4.780 3.932

0.397 1.496 2.634 4 4.010 2.748 3.262 0.248 18.795

RA (%)

5 20 38.5 37.7 26.9 15.1 8.2 2.9

NCA

WBLA

0.130 0.520 1 0.979 0.699 0.392 0.213 0.075 4.008 3.998

0.130 1.040 3 3.916 3.495 2.352 1.491 0.600 16.024

a RA: Relative Abundance; NCA: Normalized Complex Abundance; WBLA (Weighted Bound Ligand Abondance) ) NCA × No. of Bound Ligand. WBA: Weighted Bound Average. See Material and Methods for detail.

delocalized pairs of electrons on the phosphate group form salt bridges with the positively charged tertiary and/or quaternary amines on the ligands. The combination of such paired interactions is thought to further stabilize the complexes. The receptor epitope is a cytoplasmic domain, having a R-helical,12 or coiled conformation,12,22,23 with a high likelihood (99%) of phosphorylation at the Thr residue. Phosphorylation appears to alter the conformation of the epitope (Figure 6), increasing the exposure of the residue side chain to the external environment, rendering them more accessible to the exogenous ligands for interaction. This view is supported by the increase in number of CHL and AT12 molecules bound to the phosphorylated epitope. In addition, at pH of 6, phosphorylation of the epitope increases the overall negative charge of the peptide from -11 to -12.5, enhancing its overall electronegativity. The combined conformational and electrostatic effects of phosphorylation are thought to be largely responsible for the 2-3-fold increase in the RA of the complexes seen in the Ligand-p-epitope mixture. We previously proposed that CHL binds to the R-helix of the receptor epitope like a V-shaped vice via cation-π and electrostatic interactions.12 The optimized ligand geometries showed that AT47 bare a more clearly defined V-shape structure between the isoindolium ring and the quaternary ammonium than the other ligands (Figure 1B). Although such optimized geometries may not represent the actual conformation of each ligand when binding to the epitope, these modeling calculations do provide a means to predict the binding efficacy between the four ligands. Not surprisingly, the well-defined V-shape conformation of AT47 parallels its superior complex formation with the receptor epitope. The RA of the noncovalent complexes from AT26-epitopes and AT47-epitopes combinations showed a quasi-bell shaped distribution, where the 3Ligand- and 4Ligand-peptides complexes are the predominant species, suggesting favorable 538


Figure 6. Conformations of Epitope (top) and p-Epitope (bottom) at pH ) 6. The guanidinium groups of Arg in both peptides are facing outward. Only the helical backbone, termini, and the residue side-chains are shown in the structures. Green ribbons represent the helical backbone over the peptides.

stoichiometries of 3 or 4 lignads to one receptor epitope. A feasible explanation for this phenomenon is that the additional interactions between the bound ligands thermodynamically enhance such ligand-epitope relationship. Unlike CHL, The π clouds of AT12, AT26, and AT47, are relatively undisturbed,12,14 and are likely to be the platform for aromatic stacking24-26

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between the bound ligands. Puchkaev et al.26 altered one of the three stacked aromatic amino acids residues in cytochrome CYP119 by point-mutation, and caused the melting temperature of this protein to decrease by as much as 10 °C. The result of their study exemplified the reduction of intramolecular stability by disrupting the stacked aromatic rings. In the case of AT26 and AT47, stacking of two or three rings from the bound ligands is thought to further reduce the global minima of the complexes, thus enhancing the observed stoichiometries. Further studies in X-ray crystallography or in molecular mechanic modeling will reveal additional details of these interactions. To allow ring stacking to occur between bound ligands, the flexibility of their quaternary ammonium arms will play a substantial role. The higher degree of rotational freedom relative to the ring’s quaternary ammonium arm, the more conformations the ligand could have. The higher number of conformations would allow a bound ligand to point its ring toward the ring(s) of other bound ligand(s) in a thermodynamically favorable configurations, thus increasing the probability of aromatic stacking. Structurally, AT47 has the highest number of possible conformations, due to the high rotational freedom of its quaternary ammonium arm relative to the indole ring. AT47 also has the highest WBA for the epitope and the p-epitope. Although the individual configuration of the stacked ligands may not be at its geometrical minimum, the energy required to overcome the conformational barriers is predicted to be insignificant. The energy released by aromatic stacking is predicted to exceed that needed for the conformational changes in ligands, and brings the complex to systemic minima. A contrasting example supporting this proposal is seen in AT12. The possible number of conformations in AT12 is restricted by the relative position of its quaternary arm to the ring, and by the planar sp2 bond linking these two structures. Although its π electron system is similar to that in AT47, due to its conformational constraint, the bound AT12 are less likely to reach conformations suitable for stacking. Such a shortcoming is only partially ameliorated by the conformational change in p-epitope. Nonetheless, the WBA of AT12 unveils its underlining inadequacy. Surprisingly, the WBA of AT26 for p-epitope is slightly less than that for the epitope. Although the underlying causes have to be elucidated, the two oxygen atoms on the indole ring are thought to contribute to the discordance by the following two mechanisms. First, despite being less electrophilic than the chlorines in CHL, the oxygen atoms can nonetheless weaken the π cloud by withdrawing the π electron away from the ring (Figure 1B). Second, the negative charge of the phosphate group on p-epitope may repel the approaching rings of AT26 through these two negatively charged oxygen atoms, thus reducing the likelihood of the noncovalent interactions. The distances between the carbon atoms of the two spatially adjacent carboxyl groups (-COO-), and the distance between the carboxyl carbon and the phosphate atom on the p-epitope (measured from the geometrically optimized epitopes) rarely exceed 9 Å (Figure 6). In CHL and its analogues, the distance between the distal quaternary ammonium and the center of the isoindole ring does not exceed 9 Å in most configurations. The overall arrangement of a multi-ligand-peptide complex may be similar to a chemical octopus, the head being the stacked rings, and the extending quaternary ammonium arms its tentacles, grabbing the pray (the acidic residues on the receptor epitope). A ligand’s aromatic ring could bind to the

epitope via cation-π interaction. It is believed that the global energy minima would favor such tertiary structures between the receptor peptides and AT26, or between the receptor peptides and AT47. When such bindings occur in vivo, it will contribute greatly to the effectiveness and the persistence of ligand binding to its recognition sites. The surface electrostatic potential of CHL appears to be the most negative among the four ligands tested. The electronegativity of the tetrachloro- portion in the isoindolium of CHL is thought to repulse the overall negatively charged luminal surface of the tightly connected cerebral endothelial cells that form the BBB (e.g., refs 27,28). The absence of chlorine molecules in the analogues reduced the overall electronegativity, and is predicted to penetrate the BBB better. Together with a design for improved BBB permeability and superior complex forming capability, these three analogues could potentially be better therapeutical agents than CHL for counteracting the rewarding effects of nicotine.

Acknowledgment. The authors gratefully acknowledge Dr. Susanne Moyer (Boston University) for thoughtful discussion in ab Initio calculations; and ONDCP for instruments funding, without which this and other projects could not have been done. References (1) Galzi, J.-L.; Changeux, J.-P. Neuropharmacology 1995, 34(6), 563582. (2) Buisson, B.; Bertrand, D. Trends Pharmacol. Sci. 2002, 23(3), 130136. (3) Dani, J. A.; De Biasi, M. Pharmacol Biochem Behav. 2001, 70(4), 439-446. (4) Lindstrom, J. M. Ann. N Y Acad. Sci. 2003, 998, 41-52. (5) Pidoplichko, V. I.; Noguchi, J.; Areola, O. O.; Liang, Y.; Peterson, J.; Zhang, T.; Dani, J. A. Learn Mem. 2004, 11(1), 60-69. (6) Corrigall, W. A.; Franklin, K. B.; Coen K. M.; Clarke P. B. Psycopharmacol(Berl.). 1992, 107(2-3), 285-289. (7) Clarke, P. B. Br. J. Pharmacol. 1984, 83(2), 527-535. (8) Clarke, P. B.; Kumar, R. Br. J. Pharmacol. 1983, 80(3), 587-594. (9) Clarke, P. B.; Chaudieu, I.; el-Bizri, H.; Boksa, P.; Quik, M.; Esplin, B. A.; C ˇ apek, R. Br. J. Pharmacol. 1994, 111(2), 397-405. (10) El-Bizri, H.; Rigdon, M. G.; Clarke, P. B. Br. J. Pharmacol. 1995, 116(5), 2503-2509. (11) El-Bizri, H.; Clarke, P. B. Br. J. Pharmacol. 1994, 113(3), 917925. (12) Woods, A. S.; Moyer, S. C.; Wang, H. Y.; Wise, R. A. J Proteome Res. 2003, 2(2), 207-212. (13) Wave function Inc., Irvine, CA. (14) Beene, D. L.; Brandt, G. S.; Zhong, W.; Zacharias, N. M.; Lester, H. A.; Dougherty, D. A. Biochemistry 2002, 41(32), 10262-10269. (15) Dougherty, D. A. Science 1996, 271(5246), 163-168. (16) Dougherty, D. A.; Lester, H. A. Nature 2001, 411(6835), 252-255. (17) Gallivan, J. P.; Dougherty, D. A. PNAS 1999, 96(17), 9459-9464. (18) Ma, J. C.; Dougherty, D. A. Chem. Rev. 1997, 97, 1303-1324. (19) Mecozzi, S.; West, A. P., Jr.; Dougherty, D. A. PNAS 1996, 93(20), 10566-10571. (20) Woods, A. S. J. Proteome Res. 2004, 3(3), 478-484. (21) Zhu, W.-L.; Tan, X.-J.; Puah, C. M.; Gu, J.-D.; Jiang, H.-L.; Chen, K.-X.; Felder, C. E.; Silman, I.; Sussman, J. L. J. Phys. Chem. A 2001, 104, 9573-9580. (22) Lacroix, E.; Viguera, A. R.; Serrano, L. J. Mol. Biol. 1998, 284, 173191. (23) Munoz, V.; Serrano, L. Nat. Struct. Biol. 1994, 1(6), 399-409. (24) Maves, S. A.; Sligar, S. G. Protein Sci. 2001, 10(1), 161-168. (25) McGaughey, G. B.; Gagne´, M.; Rappe´, K. J Biol. Chem. 1998, 273(25), 15458-15463. (26) Puchkaev, A. V.; Koo, L. S.; Ortiz de Montellano, P. R. Arch. Biochem. Biophys. 2003, 409(1), 52-58. (27) Hart, M. N.; VanDyk, L. F.; Moore, S. A.; Shasby, D. M.; Cancilla, P. A. J. Neuropathol. Exp. Neurol. 1987, 46(2), 141-153. (28) Sahagun, G.; Moore, S. A.; Hart, M. N. Am. J. Physiol. 1990, 259 (1 Pt 2), H162-166.

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Study of the Interaction of Chlorisondamine and Chlorisondamine

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