Blood-brain Barrier Choline Transporter Research Articles

Comparative in silico analysis of CNS-active molecules targeting the blood-brain barrier choline transporter for Alzheimer's disease therapy.

The online version contains supplementary material available at 10.1007/s40203-024-00245-w.

In silico predictive model to determine vector-mediated transport properties for the blood-brain barrier choline transporter.

The blood–brain barrier choline transporter (BBB-ChT) may have utility as a drug delivery vector to the central nervous system (CNS). We therefore initiated molecular docking studies with the AutoDock and AutoDock Vina (ADVina) algorithms to develop predictive models for compound screening and to identify structural features important for binding to this transporter. The binding energy predictions were highly correlated with r2=0.88, F=692.4, standard error of estimate =0.775, and P-value<0.0001 for selected BBB-ChT-active/inactive compounds (n=93). Both programs were able to cluster active (Gibbs free energy of binding <−6.0 kcal*mol−1) and inactive (Gibbs free energy of binding >−6.0 kcal*mol−1) molecules and dock them significantly better than at random with an area under the curve value of 0.86 and 0.84, respectively. In ranking smaller molecules with few torsional bonds, a size-related bias in scoring producing false-negative outcomes was detected. Finally, important blood–brain barrier parameters, such as the logBBpassive and logBBactive values, were assessed to predict compound transport to the CNS accurately. Knowledge gained from this study is useful to better understand the binding requirements in BBB-ChT, and until such time as its crystal structure becomes available, it may have significant utility in developing a highly predictive model for the rational design of drug-like compounds targeted to the brain.

473 Mesenchymal stromal cells enhance the malignant potential of human colorectal cancer cells by inducing epithelial–mesenchymal transition (EMT)-related phenomena

N-n-Alkylation of nicotine converts it from an agonist into an antagonist at neuronal nicotinic acetylcholine receptor subtypes mediating nicotine-evoked dopamine release. Conformationally restricted analogues exhibit both high affinity and selectivity at this site, and are able to access the brain due to their ability to act as substrates for the blood–brain barrier choline transporter.

3-D-QSAR and docking studies on the neuronal choline transporter

The high affinity neuronal choline transporter (CHT1) is responsible for the uptake of choline into the pre-synaptic terminal of cholinergic neurons. Considering our past experience with modeling the blood–brain barrier choline transporter (BBBCHT) as drug delivery vector to the CNS, we investigated the 3-D-quantitative structure–activity relationship of the neuronal choline transporter. Comparative molecular field analysis (CoMFA) and comparative similarity index analysis (CoMSIA) yielded cross-validated models with a q 2 of 0.5, and a non-cross validated r 2 of 0.8. The electrostatic results of the 3-D-QSAR models are corroborated with a docking study into the bacterial choline transporter. Using this electrostatic map, we propose a putative binding site in a homology model of the CHT1. Knowledge gained from this study is useful to better understand the CHT1 as well as can be used in medicinal chemistry programs targeting this transporter.

Bis-azaaromatic quaternary ammonium salts as ligands for the blood–brain barrier choline transporter

A series of bis-azaaromatic quaternary ammonium compounds containing flexible polymethylenic linkers as well as conformationally restricted linkers were evaluated for their affinity for the blood–brain barrier choline transporter (BBB-ChT). The preliminary structure–activity relationships obtained from this study suggest that incorporating a linear, conformationally restricted linker into the molecule improves affinity for the BBB-ChT.

The Inhibitory Effect of Rivastigmine and Galantamine on Choline Transport in Brain Capillary Endothelial Cells

【The blood-brain barrier (BBB) transport of acetylcholinesterase (AChE) inhibitors, donepezil and tacrine suggested to be mediated by choline transport system in our previous study. Therefore, in the present study, we investigated the interaction of other AChE inhibitors, rivastigmine and galantamine with choline transporter at the BBB. The effects of rivastigmine and galantamine on the transport of choline by conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB cells) were characterized by cellular uptake study using radiolabeled choline. The uptake of [ $^3H$ ]choline was inhibited by rivastigmine and galantamine, with $IC_{50}$ values (i.e. concentration necessary for 50% inhibition) for 1.13 and 1.15 mM, respectively. Rivastigmine inhibited the uptake of [ $^3H$ ]choline competitively with $K_i$ of 1.01 mM, but galantamine inhibited noncompetitively. In addition, the efflux of [ $^3H$ ]choline was significantly inhibited by rivastigmine and galantamine. Our results indicated that the BBB choline transporter may be involved in a part of the influx and efflux transport of rivastigmine across the BBB. These findings should be therapeutically relevant to the treatment of Alzheimer's disease (AD) with AChE inhibitors, and, more generally, to the BBB transport of CNS-acting cationic drugs via choline transporter.】

Predictive screening model for potential vector-mediated transport of cationic substrates at the blood–brain barrier choline transporter

A set of semi-rigid cyclic and acyclic bis-quaternary ammonium analogs, which were part of a drug discovery program aimed at identifying antagonists at neuronal nicotinic acetylcholine receptors, were investigated to determine structural requirements for affinity at the blood–brain barrier choline transporter (BBB CHT). This transporter may have utility as a drug delivery vector for cationic molecules to access the central nervous system. In the current study, a virtual screening model was developed to aid in rational drug design/ADME of cationic nicotinic antagonists as BBB CHT ligands. Four 3D-QSAR comparative molecular field analysis (CoMFA) models were built which could predict the BBB CHT affinity for a test set with an r 2 <0.5 and cross-validated q 2 of 0.60, suggesting good predictive capability for these models. These models will allow the rapid in silico screening of binding affinity at the BBB CHT of both known nicotinic receptor antagonists and virtual compound libraries with the goal of informing the design of brain bioavailable quaternary ammonium analogs that are high affinity selective nicotinic receptor antagonists.

Bis-Pyridinium cyclophanes: Novel ligands with high affinity for the blood–brain barrier choline transporter

A series of bis-pyridinium cyclophane analogs designed as conformationally restricted bis-quaternary ammonium compounds were evaluated for their affinity for the blood–brain barrier (BBB) choline transporter. All the cyclophanes investigated exhibited high affinity compared to choline. Of these compounds, N, N′-(1,10-decanediyl)3,3′-(1,9-decadiyn-1,10-diyl)- bis-pyridinium diiodide ( 5c) and N, N′-(1,9-nonanediyl)3,3′-(1,9-decadiyn-1,10-diyl)- bis-pyridinium dibromide ( 5b) exhibited highest affinity with K i values of 0.8 μM and 1.4 μM, respectively, and constitute some of the most potent BBB choline transporter ligands reported.

Pharmacokinetics of the Novel Nicotinic Receptor Antagonist N,N′-Dodecane-1,12-diyl-bis-3-picolinium Dibromide in the Rat

Plasma and brain concentrations of the nicotinic acetylcholine receptor antagonist and blood-brain barrier choline transporter substrate, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), were analyzed by liquid beta-scintillation spectrometry after administration of [14CH3]bPiDDB to male Sprague-Dawley rats. Plasma concentrations of [14CH3]bPiDDB were determined at 10 time points over 3 h. Absolute plasma bioavailabilities (1, 3, and 5.6 mg/kg s.c.) were 80.3, 68.2, and 103.7%, respectively. bPiDDB (1, 3, and 5.6 mg/kg) gave Cmax values of 0.13, 0.33, and 0.43 microg/ml, respectively, Tmax values of 5.0, 6.7, and 8.8 min, respectively, and t1/2 values of 76.0, 54.6, and 41.7 min, respectively. Mean area under the plasma concentration versus time curve from time zero to infinity (micrograms per minute per milliliter) and mean Cmax (microg/ml) values were dose-dependent (r2=0.9361 and 0.7968, respectively) over the dose range studied. No metabolism of [14CH3]bPiDDB was detected with any dose of bPiDDB administered. Only moderate protein binding (63-65% in plasma and 59-62% in brain supernatant) was observed, which was reversible. Brain concentrations and brain/plasma ratios of bPiDDB after a single 5.6 mg/kg s.c. dose over 5 to 60 min ranged from 0.09 to 0.33 microg/g brain tissue and were maximal at 10 min after injection, representing approximately 0.6% of the administered dose. Brain/blood ratio (0.18 at 5 min to 0.51 at 60 min after injection) was observed, indicating that clearance from brain is slower than clearance from plasma. The results show that bPiDDB is distributed rapidly from the site of injection into plasma, affords good plasma concentrations, and appears to reach brain tissues via facilitated transport by the blood-brain barrier choline transporter to afford therapeutically relevant concentrations in rat brain.

N,N′-Alkane-diyl-bis-3-picoliniums as Nicotinic Receptor Antagonists: Inhibition of Nicotine-Evoked Dopamine Release and Hyperactivity

The current study evaluated a new series of N,N'-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6-C12 methylene linkers as nicotinic acetylcholine receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity, and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity for alpha4beta2* (* indicates putative nAChR subtype assignment) and alpha7* high-affinity ligand binding sites and exhibited no inhibition of DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50 = 2 nM-6 microM; Imax = 54-64%), with N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; C12) being most potent. bPiDDB did not inhibit electrically evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine whether bPiDDB interacts with alpha-conotoxin MII-sensitive alpha6beta2-containing nAChRs, slices were exposed concomitantly to maximally effective concentrations of bPiDDB (10 nM) and alpha-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with alpha6beta2-containing nAChRs. C7, C8, C10, and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10, and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.

Carrier-Mediated Transport of the Quaternary Ammonium Neuronal Nicotinic Receptor Antagonist N,N′-Dodecylbispicolinium Dibromide at the Blood-Brain Barrier

The quaternary ammonium compound N,N'-dodecyl-bispicolinium dibromide (bPiDDB) potently and selectively inhibits nicotinic receptors (nAChRs) mediating nicotine-evoked [(3)H]dopamine release and decreases nicotine self-administration, suggesting that this polar, charged molecule penetrates the blood-brain barrier (BBB). This report focuses on 1) BBB penetration of bPiDDB; 2) the mechanism of permeation; and 3) comparison of bPiDDB to the cations choline and N-octylnicotinium iodide (NONI), both of which are polar, charged molecules that undergo facilitated BBB transport. The BBB permeation of [(3)H]choline, [(3)H]NONI, and [(14)C]bPiDDB was evaluated using in situ rat brain perfusion methods. Cerebrovascular permeability surface-area product (PS) values for [(3)H]choline, [(3)H]NONI, and [(14)C]bPiDDB were comparable (1.33 +/- 0.1, 1.64 +/- 0.15, and 1.3 +/- 0.3 ml/s/g, respectively). To ascertain whether penetration was saturable, unlabeled substrate was added to the perfusion fluid. Unlabeled choline (500 microM) reduced the PS of [(3)H]choline to 0.15 +/- 0.06 microl/s/g (p < 0.01). Likewise, unlabeled bPiDDB (500 microM) reduced the PS of [(14)C]bPiDDB to 0.046 +/- 0.005 microl/s/g (p < 0.01), whereas unlabeled NONI reduced the PS for [(3)H]NONI by approximately 50% to 0.73 +/- 0.31 microl/s/g. The PS of [(14)C]bPiDDB was reduced (p < 0.05) in the presence of 500 microM choline, indicating that the BBB choline transporter may be responsible for the transport of bPiDDB into brain. Saturable kinetic parameters for [(14)C]bPiDDB were similar to those for [(3)H]choline. The current results suggest that bPiDDB uses the BBB choline transporter for approximately 90% of its permeation into brain, and they demonstrate the carrier-mediated BBB penetration of a novel bisquaternary ammonium nAChR antagonist.

Discovery of a novel nicotinic receptor antagonist for the treatment of nicotine addiction: 1-(3-Picolinium)-12-triethylammonium-dodecane dibromide (TMPD)

Limitations in efficacy and high relapse rates of currently available smoking cessation agents reveal the need for more efficacious pharmacotherapies. One strategy is to develop subtype-selective nicotinic receptor (nAChR) antagonists that inhibit nicotine-evoked dopamine (DA) release, the primary neurotransmitter involved in nicotine reward. Simple alkylation of the pyridino N-atom converts nicotine from a potent agonist into a potent antagonist. The classical antagonists, hexamethonium and decamethonium, differentiate between peripheral nAChR subtypes. Using a similar approach, we interconnected varying quaternary ammonium moieties with a lipophilic linker to provide N, N′-bis-nicotinium analogs, affording a lead compound, N, N′-dodecyl-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), which inhibited nicotine-evoked DA release and decreased nicotine self-administration. The current work describes a novel compound, 1-(3-picolinium)-12-triethylammonium-dodecane dibromide (TMPD), a hybrid of bPiDDB and decamethonium. TMPD completely inhibited (IC 50 = 500 nM) nicotine-evoked DA release from superfused rat striatal slices, suggesting that TMPD acts as a nAChR antagonist at more than one subtype. TMPD (1 μM) inhibited the response to acetylcholine at α3β4, α4β4, α4β2, and α1β1ɛδ receptors expressed in Xenopus oocytes. TMPD had a 2-fold higher affinity than choline for the blood–brain barrier choline transporter, suggesting brain bioavailability. TMPD did not inhibit hyperactivity in nicotine sensitized rats, but significantly and specifically decreased nicotine self-administration. Together, the results suggest that TMPD may have the ability to reduce the rewarding effect of nicotine with minimal side effects, a pharmacological profile indicative of potential clinical utility for the treatment of tobacco dependence.

Receptor Surface Models in the Classroom: Introducing Molecular Modeling to Students in a 3-D World

In this study we propose a novel method of teaching interactions of drugs with a receptor, enzyme, or any other macromolecule or protein using plastic molecular models and aluminum foil. In the university setting, computer modeling software or textbooks are often used in this regard, but many students have difficulty interpreting the three dimensionality of drug�macromolecule interaction from these sources. By introducing a receptor surface model (RSM) for a small congeneric series of blood�brain barrier choline transporter (BBB-ChT) substrates as an example, students are able to investigate concepts such as steric interaction and hydrogen-bond interactions that are important concepts in medicinal chemistry and drug design.

Nicotine Exposure Does not Alter Plasma to Brain Choline Transfer

Acute and chronic nicotine exposure in rats is associated with an increase in brain acetylcholine (ACh) transmission. The acquisition of choline for neuronal ACh synthesis occurs primarily via two pathways; first, free choline is transported from the blood across the blood-brain barrier (BBB) and/or second, from synaptic choline generated by either hydrolysis of non-bound ACh or membrane phosphatidylcholine catabolism. To determine if nicotine-induced cholinergic demand is associated with increased choline transport rates into brain, we measured BBB choline transport in naïve and S-(-) nicotine exposed rats (acute and chronic, 4.5 mg/kg/d for 1, 14, 21 and 28 d; osmotic minipumps) using the in situ rat brain perfusion technique. No significant changes in choline uptake after acute or chronic nicotine exposure were observed in whole brain or cortex. Of considerable interest was a significant decrease in regional brain choline uptake measured in the hippocampus after chronic nicotine exposure (28 d). Our data suggest that the increased ACh transmission observed after nicotine exposure does not correlate with increased blood-to-brain transfer of choline. Considering these data and previous literature reports, we propose that the additional free choline required under conditions of nicotine exposure (for ACh synthesis) is primarily recruited from membrane phospholipid metabolism.

3D-QSAR study of bis-azaaromatic quaternary ammonium analogs at the blood–brain barrier choline transporter

Previously, we have developed 3D-QSAR models of the blood–brain barrier (BBB) choline transporter, a transport system that may have utility as a vector for central nervous system drug delivery. In this study, we extended the model by evaluating five bis-azaaromatic quaternary ammonium compounds for their affinity for the choline binding site on the BBB-choline transporter. The compounds, and their affinities for the transporter, were then incorporated into our existing molecular model, in order to update our knowledge on the molecular recognition factors associated with interaction of ligands at the choline binding site. The current compounds are structurally related to previous substrates that we have evaluated, but offer additional three dimensional aspects compared to the series of compounds previously utilized to define the original models. The compounds showed good affinity for the BBB-choline transporter, exhibiting inhibition constants ranging from 10 to 68 μM, as determined by the in situ rat brain perfusion method. Comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods were used to build the new 3D QSAR models. When the new bis-azaaromatic quaternary ammonium compounds were included in the model, the best cross-validated CoMFA q 2 was found to be 0.536 and the non-cross-validated r 2 was 0.818. CoMSIA hydrophobic cross-validated q 2 was 0.506 and the non-cross-validated r 2 was 0.804. This new model was able to better predict BBB-choline transporter affinity of hemicholinium-3 (predicted 65 μM, actual 54 μM), when compared to an earlier model (predicted 316 μM).

Cation Transport Specificity at the Blood?Brain Barrier

The molecular identification, expression and cloning of membrane-bound organic cation transporters are being completed in isolated in vitro membranes. In vivo studies, where cation specificity overlaps, need to complement this work. Cross-inhibition of [3H]choline and [3H]thiamine brain uptake by in situ rat brain perfusion. [3H]Choline brain uptake was not inhibited by thiamine at physiologic concentrations (100 nM). However, choline ranging from 100 nM to 250 microM inhibited [3H]thiamine brain uptake, though not below levels observed at thiamine concentrations of 100 nM. (1) The molecular family of the blood-brain barrier (BBB) choline transporter may be elucidated in vitro by its interaction with physiologic thiamine levels, and (2) two cationic transporters at the BBB may be responsible for thiamine brain uptake.

Molecular modeling studies on the active binding site of the blood?brain barrier choline transporter

The blood–brain barrier choline transporter may have utility as a drug delivery vector to the central nervous system. Surprisingly, this transporter has as yet not been cloned and expressed. We therefore initiated a 3D-QSAR study to develop predictive models for compound binding and identify structural features important for binding to this transporter. In vivo experimental data were obtained from in situ rat brain perfusion studies. Comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods were used to build the models. The best cross-validated CoMFA q2 was found to be 0.47 and the non-cross-validated r2 was 0.95. CoMSIA hydrophobic cross-validated q2 was 0.37 and the non-cross-validated r2 was 0.85. These models rendered a useful approximation for binding requirements in the BBB-choline transporter and, until such time as the cloned transporter becomes available, may have significant utility in developing a predictive model for the rational design of drugs targeted to the brain.

Molecular modeling studies on the active binding site of the blood–brain barrier choline transporter

The blood–brain barrier choline transporter may have utility as a drug delivery vector to the central nervous system. Surprisingly, this transporter has as yet not been cloned and expressed. We therefore initiated a 3D-QSAR study to develop predictive models for compound binding and identify structural features important for binding to this transporter. In vivo experimental data were obtained from in situ rat brain perfusion studies. Comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods were used to build the models. The best cross-validated CoMFA q 2 was found to be 0.47 and the non-cross-validated r 2 was 0.95. CoMSIA hydrophobic cross-validated q 2 was 0.37 and the non-cross-validated r 2 was 0.85. These models rendered a useful approximation for binding requirements in the BBB-choline transporter and, until such time as the cloned transporter becomes available, may have significant utility in developing a predictive model for the rational design of drugs targeted to the brain.

Development of subtype-selective ligands as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release

N-n-Alkylation of nicotine converts it from an agonist into an antagonist at neuronal nicotinic acetylcholine receptor subtypes mediating nicotine-evoked dopamine release. Conformationally restricted analogues exhibit both high affinity and selectivity at this site, and are able to access the brain due to their ability to act as substrates for the blood–brain barrier choline transporter.

Active transport of high-affinity choline and nicotine analogs into the central nervous system by the blood-brain barrier choline transporter.

Cigarette smoking is strongly implicated in the development of cardiovascular disorders. Recently identified nicotinium analogs may have therapeutic benefit as smoking cessation therapies but may have restricted entry into the central nervous system by the blood-brain barrier (BBB) due to their physicochemical properties. Using the in situ perfusion technique, lobeline, choline, and nicotinium analogs were evaluated for binding to the BBB choline transporter. Calculated apparent K(i) values for the choline transporter were 1.7 microM N-n-octyl choline, 2.2 microM N-n-hexyl choline, 27 microM N-n-decylnicotinium iodide, 31.9 microM N-n-octylpyridinium iodide, 49 microM N-n-octylnicotinium iodide (NONI), 393 microM lobeline, and >/=1000 microM N-methylnicotinium iodide. Nicotine and N-methylpyridinium iodide, however, do not apparently interact with the BBB choline transporter. Given NONI's apparent K(i) value determined in this study and its ability to inhibit nicotine-evoked dopamine release from superfused rat brain slices, potential brain entry of NONI via the BBB choline transporter was evaluated. [(3)H]NONI exhibited a BBB transfer coefficient value of approximately 1.6 x 10(-3) ml/s/g and a K(m) of approximately 250 microM. Unlabeled choline addition to the perfusion fluid reduced [(3)H]NONI brain uptake. We hypothesize the N-n-octyl group on the pyridinium nitrogen of NONI facilitates brain entry via the BBB choline transporter. Thus, NONI may have utility as a smoking cessation agent, given its ability to inhibit nAChRs mediating nicotine-evoked dopamine release centrally, and to be distributed to brain via the BBB choline transporter.