Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT 2A , 5-HT 2C , and 5-HT 2B , an inverse agonist at 5-HT 7 , a partial agonist at D 2, D 5 and 5-HT 6 , an agonist at 5-HT 1A and D 4 receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT 2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.

Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.

The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.

Funding: This work was funded by National Institutes of Health Grants #1F31MH091921 to MSF and RO1MH61887, U19MH82441, the National Institutes of Mental Health Psychoactive Drug Screening Program and the Michael Hooker Chair in Pharmacology to BLR; P30 ES 019776 and T32 ES 012870 to GWM and AIB, and and NIH GM71630 and GM71896 to BKS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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.

Most antipsychotics share common G protein-coupled receptor (GPCR) targets including the D 2 dopamine and 5-HT 2A serotonin receptors. [ 5 – 7 ] Beyond these shared targets, antipsychotic compounds exhibit diverse receptor affinity profiles. For example, the atypical antipsychotic clozapine binds with nanomolar affinities to nearly 50 targets ( Fig 2 ). [ 8 ] This polypharmacological profile has been suggested as a path forward in therapeutic drug development.[ 6 , 9 ] Patterns of pharmacological activity responsible for antipsychotic efficacy remain the subject of ongoing investigations. This polypharmacology approach presents a conundrum for drug discovery efforts as it is impossible to design a compound for a polypharmacological “target” (a pattern of molecular activity that engenders therapeutic efficacy) that has not yet been elucidated. We therefore used ethnobotanical records of Nelumbo nucifera and Nymphaea caerulea medicinal properties as suggestive evidence for potential therapeutic efficacy of a novel polypharmacological profile. Our in silico predictions of all phytochemicals identified in Nelumbo nucifera suggest that nuciferine (and its metabolites) cross the blood-brain barrier and have multiple protein targets. Furthermore, nuciferine has been shown to cross the blood brain barrier in rats.[ 10 ] These predictions and previously reported data suggest that nuciferine has a rich polypharmacology that is responsible for its psychotropic effects. We therefore investigated the in vitro and in vivo properties of nuciferine using cell-based pharmacology assays and animal behavioral models of antipsychotic drug action.

The lotus plants, Nelumbo nucifera and Nymphaue caerulea, have been used by cultures, both past and present, for their medicinal properties.[ 1 ] In eastern medicine, one of the cited potential medical effects of the lotus is “calming emotional disturbance”.[ 2 ] The alkaloid nuciferine ( Fig 1 ) is thought to be responsible for the psychotropic effects of Nelumbo nucifera and Nymphaea caerulea, though its pharmacological properties are not entirely clear. Macko and colleagues [ 3 ] observed that nuciferine produces effects similar to those of the antipsychotic chlorpromazine in rodents. Bhattacharya and colleagues [ 4 ] observed that nuciferine produces antipsychotic-like behavior in rats, including inhibition of the conditioned avoidance response and amphetamine-induced behaviors. The behavioral effects previously observed in rodents, in addition to the cited potential medical effects of the lotus in eastern medicine, led the authors to hypothesize that nuciferine has a pharmacological profile similar to that of antipsychotic medications.

2. Materials and Methods

2.1 Drugs Nuciferine was purchased from Sequoia Research Products (Pangbourne, United Kingdom) and Angene (Hong Kong, China) and was dissolved in DMSO at 10 mM for in vitro studies or in 0.9% saline with 1 drop 85% lactic acid per 50 ml for animal studies. D-amphetamine (AMPH), phencyclidine (PCP), and 2,5-dimethoxy-4-iodoamphetamine (DOI) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were dissolved in 0.9% saline. Clozapine (Sigma-Aldrich) was dissolved in 0.2% acetic acid—2% cyclodextran solution. For animal studies, all drugs were administered intraperitoneally unless otherwise noted.

2.2 Bioinformatics The profile of phytochemicals in Nelumbo nucifera was obtained from Mukherjee et al.[11] The similarity ensemble approach (SEA) was utilized to predict molecular targets for each phytochemical, using Scitegic ECFP4 fingerprints on a target panel extracted from a “binding” subset of ChEMBL-12 [12] and standardized as previously described. [13, 14] Briefly, The SEA [13–15] uses the chemical similarity of a bait molecule, against that of a set of ligands annotated to a target, to predict whether the bait molecule will modulate that target. Briefly, SEA calculates the similarity of the bait molecule to every annotated ligand, typically using topological fingerprints such as ECFP4. Similarity is calculated as the Tanimoto coefficient (Tc), the number of feature (bits) in common between the bait molecule and any given ligand, divided by the total number of features (bits) in the two molecules; identical molecules will have Tc values of 1.0. The Tc values above a threshold value against all the ligands for the target are averaged, and that Tc is compared to that expected for a set of ligands of similar size that would be expected at random. An E-value is calculated by calculating a Z-score for the observed average Tc vs. the ligand set, then plotting this value against an extreme value distribution and using the BLAST sequence comparison machinery. This E-value represents the likelihood of seeing the similarity one does, between the bait molecule and the known ligands for any given target, compared to what one would expect at random. This calculation is repeated against all of the > 2500 targets in ChEMBL [16] (https://www.ebi.ac.uk/chembl/). The blood-brain barrier penetrability of each compound was predicted using the online blood-brain barrier prediction (BBB) server [17] (http://www.cbligand.org/BBB/).

2.3 Psychoactive drug screening program affinity and functional profiling The NIMH Psychoactive Drug Screening Program (PDSP) has published standardized methods for radioligand binding assays and functional assays. [9, 18–20] Full details of the methods used in the radioligand receptor assays and the functional assays are described in the PDSP Assay Protocol book (http://pdsp.med.unc.edu/pdspw/binding.php). For affinity determination, nuciferine was subjected to primary radioligand binding assays tested at a single 10 μM concentration to displace 50% of the radioligand at a given receptor target. If a more than 50% of the radioligand was displaced, nuciferine was selected for a secondary binding assay tested at 11 concentrations in triplicate in competition with the radioligand to generate an IC50 and Ki. Binding assays were performed in 96-well plates with 125 μL per well in appropriate binding buffer using radioligand at or near the Kd. Plates are incubated at room temperature in the dark for 90 min. Reactions are stopped by vacuum filtrations onto 0.3% polyethyleneimine soaked 96-well filter mats using a 96-well Filtermate harvester, followed by at least three washes of cold wash buffer. Scintillation (MeltiLex) cocktail is melted onto dried filters and radioactivity is counted using a Wallac Trilux Microbeta (Perkin Elmer). For receptor functional assays, Gs or Gi-coupled receptor activation was measured using a split-luciferase cAMP biosensor, GloSensor (Promega), and Gq-coupled receptor activation was measured as calcium flux using Fluo-4 Direct Dye (Invitrogen). HEKT cells (ATCC) transiently transfected or cells stably expressing the receptor were plated into 384-white (GloSensor) or black plates (Calcium flux) in DMEM containing 1% dialyzed FBS at least 6 hours to 24 hours before the assay. For GloSensor, the media was decanted and replaced with 20 μL drug buffer per well (HBSS, 20 mM HEPES, pH 7.4) containing GloSensor substrate. Cells were challenged with 10 μL of nuciferine or positive control (3X) to generate 16 point concentration curves and incubated for 15 minutes. For Gs-mediated cAMP accumulation, plates were read immediately. For Gi-mediated cAMP inhibition, 10 μL of isoproterenol (200 nM final concentration) was added to stimulate cAMP via endogenous β-adrenergic receptors and plates were read 15 minutes later. Luminescence was measured using Wallac TriLux Microbeta (Perkin Elmer) and luminescent counts per second (LCPS) was plotted. For Gq-mediated calcium flux, media was decanted and replaced with 20 μL drug buffer containing 2.5 mM probenecid and Fluo-4 dye and allowed to incubate for at least one hour at 37°C and 5% CO 2 in a humidified incubator. Afterwards, 10 μL of nuciferine or positive control (3X) was added per well for 16 point concentrations and fluorescence was measured using FLIPRTETRA (Molecular Devices). Maximum-fold increase over basal fluorescence was plotted. Results were analyzed using nonlinear regression to obtain EC 50 using Graphpad Prism 5.0.

2.4 Dynamic light scattering to test for colloidal aggregation Nuciferine was diluted into filtered water from a 10 mM stock in DMSO with 50 mM potassium phosphate, pH 7.0. Measurements were made at room temperature using a DynaPro MS/X (Wyatt Technology) with a 55 mW laser at 826.6 nm. The laser power was 100%, and the detector angle was 90° with samples run in duplicate.

2.5 AmpC β-lactamase assay counterscreen to test for colloidal aggregation AmpC β -lactamase inhibition was measured in 50 mM potassium phosphate (pH 7.0) at room temperature as described. [21] Nuciferine was diluted from a 10 mM stock in DMSO and incubated with 1 nM AmpC for 5 minutes before the reaction was initiated by adding 92 μM CENTA substrate (Tydock Pharma; Modena, Italy). The final reaction volume was 1 mL. Change in absorbance was monitored at 405 nm for 2 minutes using an HP 8453 UV-Vis spectrophotometer. The assay was performed in duplicate in methacrylate cuvettes.

2.6 Vesicular monoamine transporter (VMAT2) study Cell culture: HEK293 cells (ATCC) lines stably expressing hDAT or hDAT and hVMAT2 constructs were cultured at 37°C and 5% CO 2 in DMEM with 10% FBS. All constructs were made in pcDNA3.1 (Life Technologies). hDAT and hVMAT2 expressing constructs contained a neomycin or zeocin resistance gene, respectively. Plasmids were transfected into HEK293 cells with Lipofectamine 2000. Stable cell lines were generated by repetitive rounds of limiting dilutions in selective media. Double stable cell lines were created by transfecting HEK-hDAT stable cells with the hVMAT2 construct and selecting for both plasmids with both neomycin and zeocin. Whole cell 3H-dopamine (DA) uptake: Cells were plated into 48-well plates one day before uptake was performed. Cells were washed with 0.5 ml uptake buffer (4 mM Tris, 6.25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl 2 , 1.2 mM MgSO 4 , 5.6 mM D-glucose, 1.7 mM ascorbic acid, and 1 μM pargyline, pH 7.4). Cells were incubated with 225 μl uptake buffer with or without the indicated concentration of nuciferine for 15 minutes. After incubation, 25 μl uptake buffer containing 3H-DA and DA was added for a final concentration of 20 nM 3H-DA and 1 μM DA. Cells were incubated at 37°C for 20 minutes or for the time indicated. Nonspecific uptake was determined in the presence of 10 μM nomifensine. Uptake was terminated by aspirating uptake buffer and washing each well twice with 0.5 ml ice-cold uptake buffer. Cells were lysed in 0.1 N NaOH and transferred to vials containing 3 ml scintillation cocktail. Radioactivity was quantitated using a Beckman LS6500 counter. Data were analyzed in Graph Pad Prism 5.0. Vesicular 3H-DA Uptake: Cells were plated in 10 cm dishes and grown to 100% confluency. Cells were washed with warm PBS without Ca2+ or Mg2+and resuspended in uptake buffer (25 mM HEPES, 100 mM potassium tartrate, 100 μM EDTA, 50 μM EGTA, pH 7.4). Cells were homogenized with a glass/Teflon homogenizer 30 times on ice. The homogenate was centrifuged at 8000xg for 8 minutes at 4°C. Protein content of the resulting supernatant was determined by BCA assay. Uptake assays utilized 100 μg of protein in complete uptake buffer (uptake buffer with 1.7 mM ascorbate, 2 mM Mg2+-ATP salt, pH 7.4) and 20 μM tetrabenazine (TBZ) to define specific uptake. Samples were incubated in uptake buffer with or without the indicated concentration of nuciferine for 10 minutes at 30°C followed by addition of 1 μM dopamine with a 2% tracer of 3H-DA. Samples were incubated for 5 minutes at 30°C with gentle shaking. The assay was terminated by addition of 5 ml ice-cold assay buffer before filtration through 0.5% PEI-soaked Whatman GF/F filters (Brandel Inc., Gaithersburg, MD). Filters were then placed in vials containing 3 ml scintillation fluid and counted using a Beckman LS6500. Data were analyzed in Graph Pad Prism 5.0.

2.7 Animals University of North Carolina at Chapel Hill: The head-twitch response, locomotor activity, and catalepsy studies were run with male C57BL/6J mice bred at UNC. University of Arkansas for Medical Sciences: For DOI discrimination studies, adult male NIH Swiss mice weighing approximately 25 g were obtained from Harlan Laboratories (Indianapolis, IN, USA) and housed 3 mice per cage upon arrival. For PCP discrimination studies, adult male Sprague-Dawley rats weighing approximately 250 g were obtained from Harlan Laboratories (Indianapolis, IN, USA) and housed 3 rats per cage upon arrival. Virginia Commonwealth University: For clozapine substitution studies, adult male 129S2/SvHsd inbred mice were bred in-house. Duke University: adult male and female C57BL/6J mice, wild type (WT) mice, and dopamine transporter knockout (DAT-KO) mice were used in PPI experiments. The DAT mice were generated by heterozygous matings. DAT mice were group-housed in an environmentally-controlled room on a 14:10-h light/dark cycle (lights on 0800 h). The C57BL/6J, NIH Swiss, and 129S2/SvHsd mice and the Sprague-Dawley rats were housed in environmentally-controlled rooms on a 12:12-h light/dark cycle (lights on at 0700 hr). All experiments were conducted with approved protocols from the Institutional Animal Care and Use Committees of the university associated with each principal investigator: The University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee, the Duke University Institutional Animal Care and Use Committee, the Virginia Commonwealth University Institutional Animal Care and Use Committee, and the University of Arkansas for Medical Sciences Animal Care and Use Committee. It should be noted that the range of species and breeds used in these studies was due to the “convenience and availability” nature of the collaboration, in which nuciferine was tested in the established experimental protocols of the collaborating laboratories dependent upon the availability of animals and openings in the experimental schedule.

2.8 Head-twitch responses The head-twitch response procedure has been described elsewhere.[22–24] In brief, mice were injected with either nuciferine (1.0, 3.0, or 10.0 mg/kg, i.p.) or vehicle, n = 4 mice/condition. Fifteen minutes later, mice were injected with 1.0 mg/kg DOI (i.p.) and immediately placed in an observation chamber (new cage without bedding). Head-twitches (operationally defined as a rapid rotational jerk of the head that can be distinguished from species-appropriate grooming or scratching behaviors) were counted for 20 minutes in 5 minute bins. For the time-course study, mice were pretreated with 3.0 mg/kg nuciferine (i.p.) at 60, 45, 30, 15, or 0 minutes (co-injection) prior to the 1.0 mg/kg DOI (i.p.) injection, and head-twitches were counted as described above. In one experiment, mice (n = 4 per condition) were pretreated with an injection (s.c.) of 3.0 mg/kg nuciferine or vehicle 15 minutes prior to 1.0 mg/kg DOI injection (i.p.) and head-twitches were counted as described above. All experiments were performed by 3 observers, with 2 observers blinded to the experimental conditions which were evenly distributed. Power analyses were performed with the resulting data. The two highest doses of nuciferine tested (10.0 and 3.0 mg/kg), had 0.96 and 0.88 power to detect significance (α = 0.05). As these experiments were performed blinded and in distinct mice, further replication was not performed.

2.9 Open field activity Locomotor activity was assessed in AccuScan activity monitors (41X41X30 cm; AccuScan Instruments, Columbus, OH) with photocells spaced at 1.52 cm as described. [22] In these monitors, the photocells create a grid of light beams, and breaks in the light beams (caused by the mouse) are recorded. The Accuscan software then calculates the total distance travelled (amongst other measurements) by analyzing the sequential order of beam breaks in the grid. Horizontal activity was measured as the total distance traveled in centimeters and was recorded in 5-minute bins. PCP-induced hyperlocomotor activity: mice (n = 16) previously acclimated to activity chambers were placed into the chambers for 15 minutes. Mice were then injected (i.p.) with either vehicle or 3 or 10 mg/kg nuciferine and returned to the chamber for 15 min. Subsequently, mice were injected with 6.0 mg/kg PCP (i.p.) and returned to the chamber for 90 minutes. Induction of AMPH-induced hyperlocomotor activity followed an identical protocol except locomotor activity was recorded for a total of 75 minutes and 3 mg/kg AMPH was used (i.p.).

2.10 Catalepsy procedure Mice (n = 3) were initially injected (i.p.) with vehicle (0.9% saline/0.2% lactic acid), 10.0 mg/kg nuciferine, or 1.0 mg/kg haloperidol. Mice were placed upright on a 45° angled screen. The time required for the animal to move all four paws was scored in seconds (maximum of 5 min) and is reported as the latency to movement. An extended delay to move on the inclined screen test is indicative of drug-induced catalepsy. Power analyses were performed with the resulting data. The experiment had 100% power to detect a significant difference (α = 0.05) between nuciferine and haloperidol at the 60 minute timepoint.

2.11 DOI drug discrimination Adult male NIH Swiss mice (n = 6) were trained to respond under an FR5 reinforcement schedule by presentation of evaporated milk in daily sessions using procedures similar to those previously described. [25] Mice were trained in drug discrimination via injection of saline (VEH) or 0.3 mg/kg R(-)-DOI presented in a pseudo-random order, with the constraint that no animal could receive the same injection for more than 3 consecutive sessions (1 session / day). Response assignments were counterbalanced across trials. Drugs were administered i.p. and pre-treatment time was 10 minutes. During each training session the overall response rate, overall distribution of responses on the drug-injection lever, and the distribution of responses on this same lever prior to delivery of the first reinforcer were analyzed. When animals reliably achieved a level of >85% correct responding prior to delivery of the first reinforcer over 3 consecutive sessions, a substitution test occurred the following day. During test sessions, a multiple component cumulative dosing procedure was used, and no responses were reinforced. Each component was terminated after the emission of five responses on either lever. Mice were then removed from the chamber, administered the next cumulative dose, and returned to the chamber. Ten minutes later, levers were re-extended into the experimental chamber. In this manner, four doses of drug could be tested over ~40 min in a single session. The distribution of responses between the two levers was expressed as a percentage of total responses emitted on the drug-appropriate lever. Response rate was calculated for each session by dividing the total number of responses emitted on both levers by the elapsed time prior to 5 responses on either lever. Nuciferine was administered 15 minutes prior to the first injection of DOI.

2.12 PCP drug discrimination Adult male Sprague-Dawley rats (n = 5) were trained to respond under an FR20 schedule and were reinforced by presentation of food pellets in daily sessions using procedures similar to those previously described. [26] Rats were trained in drug discrimination via a pre-session injection of saline (VEH) or 3 mg/kg PCP chosen in a pseudo-random order (coin flip), with the same constraints, criteria, and dosing procedures as described above except that during test sessions, a given component of the cumulative dosing procedure was terminated after the emission of 20 responses on either lever. Drugs were administered i.p. and pretreatment time was 10 minutes. As described above for DOI, four doses of drug could be tested in a single ~40 min test session. Nuciferine was administered 15 minutes prior to the first injection of PCP.

2.13 Clozapine drug discrimination study Adult male B6129 hybrid mice (n = 12) were trained to respond under a FR10 schedule and were reinforced by presentation of sweetened milk as described previously. [27] The drug and vehicle lever positions were counterbalanced between groups to control for olfactory cues. [28] All injections were given subcutaneously with a pre-session injection time of 30 minutes. Training occurred on a double alternation injection schedule with two days of VEH followed by two days of CLZ and repeated (VEH, VEH, CLZ, CLZ, VEH, VEH etc.). In order for a mouse to pass a training day it had to meet three criteria: (1) complete the first fixed ratio (FR) on the condition-appropriate lever, (2) at least 80% of the total responses were made on the condition-appropriate lever, and (3) at least 10 responses per minute were made during the session. Drug testing was conducted approximately two times per week with at least two training days in between. To be eligible for testing, mice were required to pass both a drug and vehicle training day consecutively. During drug substitution tests, animals were injected subcutaneously with nuciferine (0.1, 0.3, 1.0, 3.0, 10.0 mg/kg) and placed in the operant chamber after 30 minutes. Responses on both levers were reinforced.

2.14 Prepulse inhibition (PPI) PPI of the acoustic startle response was conducted as described elsewhere [29] using SR-LAB chambers (San Diego Instruments, San Diego, CA). To determine whether nuciferine could ameliorate or normalize PPI, two separate experiments were conducted. In the first, C57BL/6J mice were administered VEH, 5, or 10 mg/kg nuciferine (i.p.) and returned to their home-cages for 15 min. Subsequently mice were treated with either VEH or 6 mg/kg PCP (i.p.) and placed into the PPI apparatus for a 5 min habituation prior to the onset of testing. In the second study, WT and DAT-KO mice were given VEH or 2.5, 5, or 10 mg/kg nuciferine or 2 mg/kg clozapine (i.p.) and returned to their home-cages. Fifteen min later DAT mice were habituated to the PPI apparatus for 5 min and testing began. The startle trials consisted of a 40 msec burst of 120dB white-noise; prepulse trials consisted of 20 msec prepulse stimuli that were 4, 8, or 12 dB above the white-noise background (64dB) and were followed 100 msec later by the 120dB acoustic startle stimulus. Non-stimulus or null trials consisted of the 64dB white-noise background. PPI responses were calculated as a percentage score for each prepulse intensity, where %PPI = [1–(prepulse trials/startle-only trials)]*100.