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1.  Attenuation of Compulsive-Like Behavior Through Positive Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors in Non-Induced Compulsive-Like Mice 
Nicotinic α4β2 receptors are the most abundant subtypes of nicotinic acetylcholine receptors (nAChRs) expressed in brain regions implicated in obsessive compulsive disorder (OCD). These receptors are known to modify normal and addictive behaviors by modulating neuronal excitability. Desformylflustrabromine (dFBr) is a novel, positive allosteric modulator (PAM) of high acetylcholine sensitivity (HS) and low acetylcholine sensitivity (LS) α4β2 nAChRs. The present study tested the hypothesis that positive allosteric modulation of α4β2 receptors by dFBr will attenuate compulsive-like behavior in a non-induced compulsive-like mouse model. Male mice (Mus musculus) selected for compulsive-like nesting behavior (NB; 48 animals; 12 per group) received acute (once) and chronic (every day for 32 days) subcutaneous injection of dFBr at 2, 4 and 6 mg/kg doses. Saline was used as a control (0 mg/kg). Compulsive-like NB was assessed after 1, 2, 3, 4, 5 and 24 h, while compulsive-like marble burying (MB) and anxiety-like open field (OF) behaviors were performed 2 h after dFBr administration. In the acute administration protocol, dFBr dose dependently attenuated NB and MB. Rapid effects (1–2 h after drug administration) of dFBr on MB and NB were observed for the chronic administration which was in congruence with the acute study. Chronic administration also revealed sustained suppression of NB by dFBr following 5 weeks of treatment. In both the acute and chronic regimen dFBr did not modulate OF behaviors. This research demonstrates the novel role of positive allosteric modulation of α4β2 nicotinic receptors by dFBr as a translational potential for OCD.
PMCID: PMC5214813
desformylflustrabromine (dFBr); α4β2 nicotinic receptors; positive allosteric modulator; obsessive compulsive disorder; non-induced compulsive-like mice
2.  Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES✩ 
A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5–10 mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2− interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors.
PMCID: PMC3752001  PMID: 22732654
Alpha4beta2; Alpha7; Nicotinic acetylcholine receptor; Ligand gated ion channels; Positive allosteric modulation; HEPES; desformylflustrabromine; dFbr; Two-electrode voltage clamp; Mutagenesis
3.  Improved Surface Modification Approach for Micromechanical Biosensors 
We have investigated the sensing performance of protein-based microcantilever biosensors prepared from multiple surface conjugation chemistries. The 11-mercaptoundecanoic acid monolayers were prepared according to both traditional and modified processes. In three protein-based biosensors, the modified process improved microcantilever sensing performance by increasing the bending amplitude, a critical step toward developing a cost-effective microcantilever-based sensor platform for medical diagnostics and environmental and drug screening applications. Scanning electron microscopy (SEM) images demonstrated that proteins immobilized on the microcantilever surface using the modified chemistry approach formed a compact layer.
PMCID: PMC3633073  PMID: 18154314
4.  Synthesis of desformylflustrabromine and its evaluation as an α4β2 and α7 nACh receptor modulator 
Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of α4β2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.
PMCID: PMC3633077  PMID: 17604168
Nicotinic cholinergic receptors; Allosteric modulators
5.  Deconstruction of the α4β2 Nicotinic Acetylchloine (nACh) Receptor Positive Allosteric Modulator des-Formylflustrabromine (dFBr) 
Journal of medicinal chemistry  2011;54(20):7259-7267.
des -Formylflustrabromine (dFBr; 1), perhaps the first selective positive allosteric modulator of α4β2 neuronal nicotinic acetylcholine (nACh) receptors, was deconstructed to determine which structural features contribute to its actions on receptors expressed in Xenopus ooycytes using 2-electrode voltage clamp techniques. Although the intact structure of 1 was found optimal, several deconstructed analogs retained activity. Neither the 6-bromo substituent nor the entire 2-position chain is required for activity. In particular, reduction of the olefinic side chain of 1, as seen with 6, not only resulted in retention of activity/potency but in enhanced selectivity for α4β2 versus α7 nACh receptors. Pharmacophoric features for the allosteric modulation of α4β2 nACh receptors by 1 were identified.
PMCID: PMC3200116  PMID: 21905680
6.  Identification of critical residues in loop E in the 5-HT3ASR binding site 
BMC Biochemistry  2002;3:15.
The serotonin type 3 receptor (5-HT3R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family.
Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147) to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR.
Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, mCPBG, d-tc and lerisetron. 5-HT and mCPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.
PMCID: PMC117120  PMID: 12079500
7.  Functional group interactions of a 5-HT3R antagonist 
BMC Biochemistry  2002;3:16.
Lerisetron, a competitive serotonin type 3 receptor (5-HT3R) antagonist, contains five functional groups capable of interacting with amino acids in the 5-HT3R binding site. Site directed mutagenesis studies of the 5-HT3AR have revealed several amino acids that are thought to form part of the binding domain of this receptor. The specific functional groups on the ligand that interact with these amino acids are, however, unknown. Using synthetic analogs of lerisetron as molecular probes in combination with site directed mutagenesis, we have identified some of these interactions and have proposed a model of the lerisetron binding site.
Two analogs of lerisetron were synthesized to probe 5-HT3R functional group interactions with this compound. Analog 1 lacks the N1 benzyl group of lerisetron and analog 2 contains oxygen in place of the distal piperazine nitrogen. Both analogs show significantly decreased binding affinity to wildtype 5-HT3ASRs. Mutations at W89, R91, Y142 and Y152 produced significant decreases in binding compared to wildtype receptors. Binding affinities of analogs 1 and 2 were altered only by mutations at W89, and Y152.
Based on the data obtained for lerisetron and analogs 1 and 2, we have proposed a tentative model of the lerisetron binding pocket of the 5-HT3ASR. According to this model, The N-benzyl group interacts in a weak interaction with R91 while the benzimidazole group interacts with W89. Our data support an interaction of the distal amino nitrogen with Y142 and Y152.
PMCID: PMC116678  PMID: 12079499

Results 1-7 (7)