Design and Discovery of Functionally Selective Serotonin 2C (5-HT_2C) Receptor Agonists

On the basis of the structural similarity of our previous 5-HT_2C agonists with the melatonin receptor agonist tasimelteon and the putative biological cross-talk between serotonergic and melatonergic systems, a series of new (2,3-dihydro)­benzofuran-based compounds were designed and synthesized. The compounds were evaluated for their selectivity toward 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors in the calcium flux assay with the ultimate goal to generate selective 5-HT_2C agonists. Selected compounds were studied for their functional selectivity by comparing their transduction efficiency at the G protein signaling pathway versus β-arrestin recruitment. The most functionally selective compound (+)-<b>7e</b> produced weak β-arrestin recruitment and also demonstrated less receptor desensitization compared to serotonin in both calcium flux and phosphoinositide (PI) hydrolysis assays. We report for the first time that selective 5-HT_2C agonists possessing weak β-arrestin recruitment can produce distinct receptor desensitization properties

DAT modulation by nuciferine.

<p>(a) Concentration-response curves of nuciferine on vesicular uptake in isolated vesicles, (b) concentration-response curves of nuciferine on vesicular uptake in HEK cells transfected with DAT and VMAT2, (c) concentration response curves of nuciferine on vesicular uptake in HEK cells transfected with DAT. Data are presented as a percentage of the response to vehicle control. The data represent concentration-response curves of normalized data with respect to vehicle performed in triplicate (mean +/- s.e.m.; n = 3).</p

In silico and in vitro characterization of nuciferine.

<p>In silico and in vitro characterization of nuciferine.</p

PPI responses to nuciferine in mouse models of hypoglutamatergia and hyperdopaminergia.

<p>Nuciferine rescued PPI in the former, but not in the latter model. (a-c) Null activity (a), startle activity (b), and PPI (c) for C57BL/6J mice treated with vehicle (Veh), 5 or 10 mg/kg nuciferine (Nuc), and/or phencyclidine (PCP). (d-g) Null activity (d), startle activity (e), and PPI for wild-type (WT) (f) and dopamine transporter knockout (DAT-KO) mice (g) given Veh, 2 mg/kg clozapine (CLZ) or 2.5–10 mg/kg Nuc. N = 8–17 mice/group in the C57BL/6J experiment; *<i>p</i><0.05, compared to Veh/Nuc groups; +<i>p</i><0.05, compared to the Veh and PCP groups; †<i>p</i><0.05, compared to all other groups. N = 9–17 mice/genotype/treatment in the DAT experiment; ^<i>p</i><0.05, WT versus KO within dose; #<i>p</i><0.05, dose effect within genotype; &<i>p</i><0.05, overall drug effect regardless of genotype.</p

Nuciferine functional activity at the dopamine D₂ receptor as measured via D₂-mediated G_i signaling in HEKT cells.

<p>(a) Concentration-response curve of nuciferine (red) compared to dopamine (black) and aripiprazole (blue). (b) Concentration-response curve of dopamine (DA) in the presence of 1 μM nuciferine (red) or clozapine (green) showing rightward-shift of DA indicative of competitive antagonist activity. The data represent concentration-response curves of normalized data with respect to dopamine performed in triplicate (mean +/- s.e.m.; n = 3).</p

Comparison of the polypharmacology of nuciferine with atypical and typical antipsychotics.

<p>The empirical affinity values of three antipsychotics are shown (clozapine, haloperidol, aripiprazole). The empirical nuciferine affinity profile from this study is shown in comparison. Values for clozapine, haloperidol, and aripiprazole compiled from data available on the PDSP website accessed 20150428. Only “PDSP verified” data was used for the figure. Data entries of “>10,000” were entered as 10,000 μM.</p

Bioinformatic predictions of lotus phytochemicals.

<p>X axis = number of unique predicted targets, as predicted by SEA. Each unique target included all affinity classes. Y axis = prediction of blood brain barrier penetration using the online blood-brain barrier prediction (BBB) server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150602#pone.0150602.ref017" target="_blank">17</a>] (<a href="http://www.cbligand.org/BBB/" target="_blank">http://www.cbligand.org/BBB/</a>). Higher values are predicted to pass the blood brain barrier. Size of circles = mean of -log10(e-value) for SEA-predicted targets. Larger circles indicate stronger confidence of predicted targets. Color of circles = Mean of max T values. Warmer colors (red) indicate better molecule-molecule matching, a second measure of prediction confidence. Y axis reference line (0.0815) and X axis reference line (17) are the average value for the world’s most widely prescribed psychiatric medications (aripiprazole and quetiapine).[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150602#pone.0150602.ref074" target="_blank">74</a>] These predictions suggest that nuciferine and its metabolites (O-nornuciferine, lirinidine) may be responsible for the psychotropic effects reported in humans. Chemical structures of nuciferine, O-nornuciferine, and lirinidine provided.</p

Locomotor studies of nuciferine.

<p>(a) Nuciferine suppresses the PCP-induced hyperlocomotor response. Data are presented as total distance travelled in 5-minute bins (left) and as cumulative distance travelled between minute 45 and minute 60 (right). N = 18 mice; ^p<0.05, compared to PCP group. (b) Nuciferine (3.0 mg/kg, 15 minute pretreatment) enhances the hyperlocomotor effect of amphetamine (3.0 mg/kg) administration. N = 14 mice; * < 0.001 compared to Amphetamine group. Data are presented as above.</p

Drug Discrimination studies of nuciferine.

<p>(a,d) Nuciferine blocks the discriminative stimulus of DOI at doses that do not affect the rate of responding. (b,e) Nuciferine does not block the discriminative stimulus of PCP at any dose tested. (e,f) Nuciferine substitutes for clozapine (solid circles) but only at a dose that suppresses response rate (empty circles). Data are presented as % training-drug appropriate responding and response rate per second.</p

Cataleptic properties of nuciferine.

<p>Cataleptic properties of nuciferine.</p