Serotonin as a Drug Receptor for Li+: A Computational Study · Draft 2 Abstract. Although Li+ is widely used to treat bipolar disorder, its antidepressant pharmacological mechanism

Draft

Serotonin as a Drug Receptor for Li+: A Computational

Study

Journal: Canadian Journal of Chemistry

Manuscript ID cjc-2017-0665.R1

Manuscript Type: Article

Date Submitted by the Author: 24-Jan-2018

Complete List of Authors: Meek, Autumn; Krembil Research Institute, Fundamental Neurobiology Morzycki, Alexander; Krembil Research Institute, Fundamental Neurobiology Carter, Michael; Dalhousie University Faculty of Medicine Barden, Christopher; Krembil Research Institute, Fundamental Neurobiology

Weaver, Donald; Krembil Research Institute, Fundamental Neurobiology

Keyword: Serotonin, Lithium, Bipolar Disorder, Molecular Modelling, Depression

Is the invited manuscript for consideration in a Special

Issue?: Dalhousie

https://mc06.manuscriptcentral.com/cjc-pubs

Canadian Journal of Chemistry

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Serotonin as a Drug Receptor for Li+:

A Computational Study

Autumn R. Meek, Alexander Morzycki, Michael D. Carter, Christopher Barden, Donald F.

Weaver*

Krembil Research Institute, University Health Network, University of Toronto, 60 Leonard Ave.,

Toronto, Ontario, M5T 2S8, Canada

Corresponding Author.

*E-mail: [email protected]

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Abstract. Although Li+ is widely used to treat bipolar disorder, its antidepressant

pharmacological mechanism of action remains unelucidated. Herein, based on molecular

modelling studies, we present the novel hypothesis that one possible receptor for Li+ is serotonin

(5-hydroxytryptamine, 5-HT), a small molecule neurotransmitter rather than a large

macromolecular protein; the resulting Li+/5-HT drug-receptor complex subsequently interacts

with “upstream” macromolecular receptors, such as the 5-HT1A receptor, differently than 5-HT

alone, producing an enhanced antidepressant effect. The notion that a neurotransmitter could

itself be a receptor for a therapeutic is an interesting receptor cascade concept. Using molecular

mechanics, semi-empirical and ab initio levels of theory, the potential interactions between Li+

and 5-HT and between Li+ and 5-HIAA (5-hydroxyindoleacetic acid, a 5-HT metabolite) were

examined. Molecular dynamics simulations were then used to examine how Li+ affects the

binding of 5-HT to its target 5-HT1A antidepressant receptor. The results of these calculations

suggest that Li+ can interact with 5-HT, and in such a way that it modifies the ligand-protein

interactions.

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Keywords: Serotonin. Lithium. Molecular modelling. Molecular dynamics. Bipolar disorder.

Depression.

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Introduction

For more than a century, exogenously administered Li+ (usually as lithium carbonate) has

been used to treat unipolar and bipolar depressive disorders.1-5 Despite this widespread use,

Li+’s pharmacological mechanism of action remains unknown.6 Past research has suggested

Li+’s ability to influence dopamine, glutamate, γ-aminobutyric acid (GABA), and serotonin (5-

hydroxytryptamine; 5-HT) receptor systems, both pre and post-synaptically;7-13 Li+ has also been

shown to modulate adenylyl cyclase,14 the phosphoinositol signaling pathway,15 arachidonic acid

turnover,16 levels of PLA2,17 and COX-2 expression.1 Amongst these many proposed

mechanisms, the interactions between Li+ and serotonergic biochemistry have been particularly

well-studied.8-13 This is not surprising given the extensive study of 5-HT’s role in depression,

mediated by a variety of receptors, particularly the 5-HT1A receptor.

During a screening campaign of compounds present in normal human brain, we identified

Li+ as a potential endogenous antidepressant compound. Although most antidepressant drugs

have well characterized receptors, Li+ does not; there is no clearly identified Li+ receptor (LiR).

Classically, a drug receptor is a macromolecule such as an enzyme or membrane-associated

protein whose biological function is altered or inhibited by the binding of a drug. Herein, based

upon a comprehensive series of molecular modelling experiments, we present the possibility that

serotonin is a candidate LiR. The notion that a neurotransmitter could itself be a receptor is a

novel concept. The Li+/5-HT drug-receptor complex subsequently binds to the 5-HT1A receptor

differently than 5-HT alone, producing an enhanced antidepressant effect and exemplifying how

Li+ alters the biological effects of its receptor. The notion that a neurotransmitter (which

subsequently interacts with a downstream macromolecular receptor) could itself be a receptor for

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a therapeutic is an interesting receptor cascade concept, opening the door to the design of Li+-

like new chemical entities capable of binding to 5-HT to elicit a therapeutic response.

Methods

To determine if Li+ exerts its mechanism of action by binding to 5-HT and/or 5-HIAA (5-

hydoxyindoleacetic acid, a 5-HT metabolite; Fig. 1), a multi-theory approach was taken. Initial

analysis was performed using molecular mechanics (MM) studies to determine project viability,

followed by semi-empirical and density functional theory (DFT) calculations. Molecular

dynamics (MD) simulations were also used to further elucidate the potential of 5-HT acting as a

receptor for Li+. At the MM level, a variety of solvent conditions were used to mimic the

environment in which Li+ and 5-HT/5-HIAA may be found in vivo; temperatures of 295 K and

310K were selected for MD simulations to mimic conditions available for potential in vitro

experiments.

Molecular Mechanics Studies: Geometry optimizations of potential interactions between Li+

and 5-HT or 5-HIAA were performed in the molecular operating environment (MOE)18 software

suite employing the CHARMM22 force field.19 Energy minimizations were completed for four

charge states (neutral, anionic, cationic, zwitterionic) to match an obtained crystal structure

(unpublished data, Weaver group); all charge states were examined to determine if interactions

would form regardless of the local environment in which the compound might be found.

Conformational searches of 5-HIAA in neutral and anionic forms were performed (as no crystal

structure is available), and these identified structures were used for further study.

In vacuo MM studies: For each charge state of 5-HT and 5-HIAA, a systematic series of

potential interactions with a Li+ cation was examined. Li+ was oriented 3.0 Å away (a distance

from which attraction or repulsion can occur without significant ligand deformation) from each

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of seven identified regions within 5-HT and 5-HIAA, designated as OH, A, B, N, CHA, CHB,

NH2 (for 5-HT), and OH, A, B, N, CHA, CB, CO, and COH (for 5-HIAA) (See Fig. 1).

For each result, the total system energy (Etot), van der Waals energy (Evdw), and the electrostatic

energy (Eele) was recorded, along with the final location of Li+ in regards to each region of 5-HT

or 5-HIAA. The binding energies were computed by subtracting the individual energies of Li+

and 5-HT or 5-HIAA (in their energy minimized form) using the following general formula:

∆E = E (Li+-LiR) – E (Li+) – E(LiR) Eq. 1

Solution phase MM studies: Tetrahydrofuran (THF, dielectric constant = 7.6) was used

as a model solvent to mimic the dielectric constant of the interior of a protein20. A THF solvent

box was constructed to provide adequate surroundings for Li+ and 5-HT or 5-HIAA, and was

energy minimized; the system was set up such that 5-HT or 5-HIAA and Li+ were placed

centrally in the solvent box, and overlapping solvent were either removed or relocated to a less

hindered location. Simulations in water were performed by placing Li+ and 5-HT/5-HIAA in an

initial orientation, followed by solvation with a periodic box of water having a 6.0 Å boundary.

Semi-Empirical Methods: Semi-empirical optimizations were performed using the AM121

Hamiltonian as implemented in Gaussian09.22 For each charge state a model of 5-HT or 5-HIAA

was constructed and subjected to a conformational scan to find the lowest energy structure. The

identified dihedral angles were set to rotate in increments of 60° (H-N-C-C, N-C-C-C), 45° (C-

C-C-C), or 180° (H-O-C-C) for 5-HT and in increments of 60° (C-C-C-O), 45° (C-C-C-C) or

180° (H-O-C-C) for 5-HIAA . Relaxed redundant coordinates were selected for the grid scan

using closed-shell AM1, a charge state of 0 (neutral and zwitterion), +1 (cation) or -1 (anion),

and a singlet spin state. To produce a zwitterionic charge state for 5-HT, the optimization was

performed with implicit THF present. The lowest energy structure was identified using closed-

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shell AM1, a singlet spin state, and a charge set to the appropriate value of the system. Li+ was

positioned 3.0 Å away from the various regions of 5-HT or 5-HIAA as identified in Fig. 1.

Binding energies were calculated using Eq. 1.

Density Functional Theory Methods: DFT calculations were performed with the B3LYP

functional and 6-31+G(d,p) basis set using Gaussian09.22,23 An initial conformational scan was

set up for each charged state of 5-HT/5-HIAA using relaxed redundant coordinates. Systems

were set up as closed shell B3LYP, in the singlet state, and with a charge reflecting the state of

system (-1, 0, or +1). Dihedral angles were scanned in increments of 180° (H-O-C-C), 60° (C-C-

C-N, C-C-N-H), and 45° (C-C-C-C) for 5-HT, and 180° (H-O-C-C), 60° (C-C-C-O, C-C-O-H),

and 45° (C-C-C-C) for 5-HIAA. Li+ was located 3.0 Å away from the various regions of 5-HT

or 5-HIAA, as identified in Fig. 1.

Molecular Dynamics Studies: MD simulations were performed in MOE24 using the PFROSST

force field to initially optimize the system.25-27 Systems were set up such that three molecules of

5-HT or 5-HIAA, six Li+ cations, and 42 Na+ cations were present – to represent a 2:1 ratio of

ion to small molecule, with an excess of sodium to mimic biological conditions. Explicit

solvation was applied using a 4.0 Å margin water cube and Cl- counter ions were present;

systems were equilibrated for a pH of 7.4, and a NaCl concentration of 120 mM. Simulations of

the interactions between Li+ and 5-HT/5-HIAA were performed at two temperatures: 295 K and

310 K, with periodic boundary conditions in place.

Each of the two optimized systems (5-HT or 5-HIAA) was used as the starting point for

the MD simulations. The sample time was set to 0.1 ps, with a time step of 0.001 ps, no

constraints were applied, and non-rigid water molecules were selected. An equilibration phase

was set for 100 ps, with a pressure of 101 kPa, a temperature of either 295 K or 310 K, and a

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heavy atom tether of r=(0.5, 100). The production phase was set for 500 ps, with a pressure of

101 kPa, and a temperature of either 295 K or 310 K. The Nosé-Poincaré-Andersen (NPA)

algorithm28,29 was used. Upon completion of the simulation, distances were measured between

each of the heavy atoms of 5-HT/5-HIAA and each of the Li+ ions present in the system for the

500 ps production phase. An interaction was assumed if the distance between Li+ and the

selected atom of 5-HT/5-HIAA was less than 4.0 Å. If interactions occurred between Li+ and one

of the heavy atoms, the distance between Li+ and any associated hydrogen atoms was also

measured.

Homology Modelling: A homology model of the 5-HT1A serotonin receptor protein was

constructed in MOE using the crystal structure (4IAQ)30 of 5-HT1B as a template. 5-HT1A has

43.9% sequence identity with 5-HT1B. Sequences were aligned, gap modeling was permitted, and

fine refinement was selected for both the intermediate and final models. Hydrogen atoms and

charges were adjusted using the PFROSST force field. The final generated model was subjected

to a site finder search to identify the likely binding pocket for 5-HT. The first identified binding

pocket is composed of 38 amino acid residues (see Supplementary Data); a second pocket was

identified based on the location of the ligand in the crystal structure used to generate the

homology model and is composed of 33 amino acid residues31,32. After initial docking, a third

smaller pocket was also identified, composed of the 21 residues.

Docking and Molecular Dynamics: A structure of cationic 5-HT was generated using the

LowModeMD conformational search function in MOE. This cationic 5-HT was docked into each

of the three identified pockets of the homology generated model of 5-HT1A using induced fit

docking (allowing for free side chain movement): pocket 1 generated 24 poses, pocket 2

generated 28 poses, and pocket 3 generated 23 poses. Of the retained poses, several were

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identified through careful screening to identify potential, unique locations of serotonin. For these

identified poses, a Li+ was introduced into the system in the vicinity of serotonin, followed by

energy minimization. Each system had an additional 2 Li+ ions and 12 Na+ placed around the

protein, and a 5 Å margin water box. Energy minimization was performed using the PFROSST

force field, and R-field electrostatics. To compare the ion effect, two identical systems were

generated where the three Li+ ions were converted to either Na+ or H+ ions. Each system was

subjected to a 5 ns MD simulation using the NPA algorithm at 310 K – 1000 ps of equilibration

were followed by 4000 ps of the production phase. The final 1000 ps were examined at 50 ps

intervals to identify interactions occurring between 5-HT, the receptor, and the cation.

Results and Discussion

Molecular Mechanics Results: Table 1 summarizes the observed binding locations of Li+ as

well as the average binding energy for cationic 5-HT in vacuo, THF and H2O (Results of all

charge states are found in the Supplementary Table S1). In the gas phase, 5-HT interacts with Li+

in the region of the OH group, introducing THF alters these interactions to include some contact

between Li+ and the indole ring as well. The interactions between Li+ and 5-HT in an aqueous

environment are more varied; however, the indole ring features prominently in the interactions.

Table 2 summarizes the results of anionic 5-HIAA interacting with Li+. In the gas phase, anionic

5-HIAA interacts with Li+ solely at the carboxylate group, whereas in THF, some interactions

with the ring system also occur. When H2O is present, 5-HIAA interactions are not significantly

altered from those observed when THF is present. Optimized systems favour binding interactions

between Li+ and the indole ring of both charged species, as observed in Figures 2 and 3.

The overall results of the molecular mechanics studies suggest that the interactions

between Li+ and 5-HT/5-HIAA vary depending on the solvent environment; this suggests that

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the interactions occurring within a protein environment will not be the same as those occurring in

the aqueous extracellular environment. The water molecules appear to exert a charge shielding

effect for those molecules with anionic or cationic functional groups present: Cationic 5-HT

demonstrated few interactions in vacuo and in THF, whereas the presence of H2O allowed for

some interactions both with the indole ring and for positioning near the charged amino group.

The results of the 5-HIAA optimizations with Li+ shifted slightly from interacting with Li+

mainly near the carboxylic acid in vacuo and THF, to interacting with the indole ring as well as

the acid functional group in water. The MM results suggested that 5-HT and its metabolite of 5-

HIAA could interact with Li+ in a variety of environments and molecular charge states. The

results also suggest that the type of interaction/location of interactions between Li+ and 5-HT/5-

HIAA is dependent on the environment within which they are located. Potentially, Li+ may be

transported to the protein in a pincer-type complex of 5-HT-Li+, with a shift in Li+ location

towards the hydroxyl region of 5-HT upon entry into the protein receptor.

Although the force field used for modelling these interactions does not fully account for

the potential electrostatic interactions that can occur between a cation and indole ring, the results

were used primarily as a screening tool to determine if the proposed concept was plausible. As

the interactions appeared to be possible (regardless of the charge state of 5-HT/5-HIAA),

research continued using more in depth computational approaches.

Semi-Empirical Results: The results of the semi-empirical optimization of Li+ and 5-HT (Table

1) suggest that Li+ interacts with the indole ring system, with a preference towards the phenyl

ring component. There are some minor conformational changes observed in 5-HT when Li+ is

present; the amino chain can be more, or less flexed towards the indole ring. When optimized at

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the AM1 level of theory, Li+ interacts with 5-HIAA near the indole ring (Table 2): Li+ fits into a

pincer type pocket, interacting with both the carboxylate group and the indole ring.

Density Functional Theory Results: Li+ and 5-HT interacted more strongly than observed at

the semi-empirical QM level: Figure 4 demonstrates one of the similar poses resulting from

optimization using AM1 and B3LYP/6-31+G(d,p). The results in Table 1 show that Li+ interacts

primarily with the phenyl region of the indole ring, with some interaction with the hydroxyl

group occurring also. These results suggest that Li+ can interact with cationic 5-HT in an

environment similar to the interior of a protein receptor such as 5-HT1A. Similar to the results

observed at the semi-empirical level, there is flexibility observed in the positioning of the amino

chain of 5-HT (see Supplementary Figure 1).

MD Simulation Results: The results of the MD simulations of 5-HT/5-HIAA and Li+ in water

are summarized according to the interactions between Li+ and the heavy atoms of each molecule.

If interactions occurred between Li+ and a heavy atom, any connected hydrogen atoms were also

included in the results. Detailed results can be found in the supplementary data section. MD

simulations were performed at 2 temperatures, 295K and 310K as these temperatures represent

those commonly used for in vitro testing as well as normal human body temperature.

The results of the MD simulation of 5-HT and Li+ at 295 K indicate that they interact in a

measurable quantity of time for all three 5-HT molecules present in the simulation; furthermore,

Li+ can interact with more than one atom of 5-HT at a time. Figure 5 represents the regions of 5-

HT with which Li+ is interacting, summarized for each of the three molecules present in the

system. 5-HT (1) interacts mainly near the nitrogen atom of the pyrrole ring of the indole, while

5-HT (2) interacts mainly with the hydroxyl group. 5-HT (3) interacts with Li+ in three different

regions: near the pyrrole ring, near the alkyl chain, and with the phenyl ring near the hydroxyl

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group. Five of the six Li+ ions interact with 5-HT at 295 K (Table 3): Those ions that do interact

show a range in time length of interaction from very brief to almost one fifth of the total time

range studied.

Increasing the temperature of the system to 310 K results in different interactions

between Li+ and 5-HT. Figure 8 summarizes the locations where Li+ interacts with 5-HT at 310

K: 5-HT (1) interacts mainly with the hydroxyl group and the phenyl ring of the indole, while 5-

HT (2) interacts only with the hydroxyl group, and 5-HT (3) interacts with Li+ over the entire

molecule. Li+ (Table 4) demonstrates the capacity to interact with two 5-HT molecules

simultaneously at 310 K; however, only five of the six ions interact with 5-HT.

The results of the MD simulations of 5-HIAA and Li+ ions in water at 295 K are

summarized in Figure 7 (detailed results are found in the supplementary data). 5-HIAA (3)

interacts with Li+ at the carboxylic acid group and some interactions with the hydroxyl group and

the top half of the indole were also observed; Li+ interacts with 5-HIAA (1) mainly near the

pyrrole ring of the indole and with the carboxylic acid functional group; there are also some

interactions with hydroxyl group and the remaining area of the indole ring. 5-HIAA (2) interacts

with Li+ over the entire surface of the molecule, with an emphasis on the regions near the

hydroxyl group and phenyl ring, as well as the carboxylic acid.

The summary of Li+ interactions in Table 5 suggests that all ions present can interact with the

metabolite of 5-HT and that one Li+ ion can interact with two 5-HIAA species simultaneously.

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When the temperature is increased to 310 K there is a shift in the interactions observed

between Li+ and 5-HIAA. Figure 11 represents a summary of the regions of the metabolite where

each Li+ ion interacts. 5-HIAA (1) interacts with Li+ primarily with the carboxylic acid and

pyrrole ring, while 5-HIAA (2) interacts mainly near the amine of the pyrrole ring of the indole.

Li+ interacts with 5-HIAA (3) for the entire length of the production phase at the carboxylic acid

site; however, there are measurable interactions with every atom present in the molecule.

Similar to the results observed at 295K, all Li+ ions showed measurable interactions with 5-

HIAA at 310 K. Simultaneous interactions with a two-to-one ratio of metabolite to Li+ were also

observed.

Table 7 summarizes the interactions occurring between each 5-HT/5-HIAA molecule and Li+,

where each unique time was accounted for. At 295K, 5-HIAA spends an average of 70.7% of the

time interacting with Li+ compared to 11.0% for 5-HT. With a temperature increase to 310 K, 5-

HT spends an average of 24.7% of the time interacting with Li+, while 5-HIAA spends 47.9% of

the time forming interactions.

The results of MD simulations of a two-to-one ratio of Li+ to 5-HT or 5-HIAA suggest

that even when an excess of Na+ is present in the system, Li+ can still bind to/interact with the

target molecules. At 295 K, all three molecules of 5-HT interact with Li+ for a measurable

amount of time, with one of the molecules interacting for a much longer time. On average, the

side of the indole ring containing the nitrogen atom and the hydroxyl group forms the most

interactions with Li+. When the temperature of the system is increased to 310 K, the length of

time Li+ interacts with 5-HT increases. While interactions occur with the hydroxyl region for all

three molecules, every atom of one molecule interacts with a Li+ ion over the entire duration of

the dynamics run; the region near the terminal amine spends the most time interacting with Li+.

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The simulations provide evidence that temperature does play a significant role in how the

molecules interact with Li+.

At 295 K, 5-HIAA also demonstrates the capacity to bind to Li+; given the presence of a

carboxylic acid functional group, that Li+ would spend the length of the simulation interacting

with this region is not unexpected. 5-HIAA also demonstrates to interact with Li+ at any atomic

position; however, there is a preference for the upper half of the indole and the carboxylic acid

region, followed by the region of the hydroxyl group. When the temperature is increased to 310

K, there is still one molecule in the system that spends the entire time interacting with Li+ at the

carboxylic acid site. The nitrogen of the indole ring is also a common binding site for Li+.

Averaging the total times of each MD simulation (counting only unique time points) shows that

as the temperature increases, so too does the amount of time Li+ interacts with 5-HT, doubling

both the average time and total length of time for interactions. The opposite trend is observed for

5-HIAA, where an increase in temperature results in a decrease in binding. At each temperature,

5-HIAA spends more total time interacting with Li+ than does 5-HT, likely due to the presence

of the negatively charged carboxylic functional group, which is a natural attractant for the

positively charged Li+ ion.

Looking at the Li+ ions present in the system, all ions present in the metabolite systems

interact with the target molecules, whilst only five of the six ions interact with 5-HT. At 295 K

Li+ spends more time interacting with 5-HIAA than 5-HT; while this also remains true at 310 K,

there is a drop in the amount of time Li+ interacts with 5-HIAA and an increase in the time Li+

interacts with 5-HT. The favourability of Li+ interactions with 5-HT at 310K are a good

indication that increasing Li+ levels in the brain will be beneficial and result in increased

interactions with 5-HT.

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Receptor Docking and Molecular Dynamics: The generated homology model of 5-

HT1A was deemed acceptable for docking studies – loop modelling did not produce significantly

different results as there were very few unorganized regions of the protein. Having identified

several unique docked poses of 5-HT in the receptor, and having optimized the full protein with

Li+ present in the vicinity, all atom molecular dynamics simulations were performed for 5

different systems. Of the five systems, one retained all three cations within the vicinity of 5-HT;

this provides the system for which the most conclusions can be drawn and is thus identified as

System 1 in the results. Graph 1 summarizes the number of interactions occurring for System 1

between 5-HT and its receptor when each cation is present in the pocket with it, over 21 separate

time points. The most differences are observed when Na+ is present in the pocket with 5-HT –

the location of 5-HT in the pocket is shifted significantly when compared to its location with Li+

or H+ present in the pocket. For clarity, Graph 1 only indicates amino acid residues where

interactions occurred for at least 25 % of the observed time points. For detailed results of the

observed interactions at each time point and a summary of the measurable binding interactions,

see Graph S1 and Table S15 in the supplementary data. Data for the remaining four systems is

given in the supplementary data section.

Figure 9 provides a snapshot of the location of serotonin in the presence of each cation after 5 ns

of MD simulations. While 5-HT in the presence of Li+ and H+ is located in a similar region of the

pocket, 5-HT in the presence of Na+ has shifted more significantly. Measurable bonding

interactions, in the form of hydrogen bonds, π-H, and ionic interactions, are observed for all

three cationic systems. Figure 10 shows the preferred binding sites for serotonin in the presence

of a cation, where measurable bonds are identified.

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When Li+ is present, the amino group prefers hydrogen bond interactions with Asn386,

while the CH2 immediately connected to the amino group forms hydrogen bonds with Asp116;

the hydroxyl group forms hydrogen bonds to the backbone atoms of Ala383. When Li+ is

exchanged for Na+, the interactions shift such that the amino group forms hydrogen bonds (and

ionic interactions) with the Asp116 residue, while the hydroxyl group forms hydrogen bonds

with Tyr390. When Li+ is exchanged for H+, the amino group forms hydrogen bonds with

Asn386, and both hydrogen bond and ionic interactions with Asp116; hydrogen bonds form

between the indole ring and Thr188, and between the hydroxyl group and Gln97.

An examination of the location of the cations over time is summarized in Graph 2, and

shows that the cation present will also interact in different regions within the pocket. All three

ions also are located close enough to serotonin for potential electrostatic interactions to occur:

measurable bonds were not observed for this particular system; however, some of the other

systems did show metal-type interactions occurring between the various cations and 5-HT.

The effect of the ion on 5-HT and its receptor is observable in the final shape of the

receptor. Figure 11 shows the opening of the 5-HT1A receptor after 5 ns MD simulations with

Li+, Na+, and H+. When Li+ is present, the receptor pore is closed to a greater extent, suggesting

that Li+ helps 5-HT bound in the receptor to remain in the receptor. It is clear that the presence of

Li+ has a more significant impact than either of the other ions; while having a simple H+ present

suggests there may be some closure of the pore, having only Na+ present results in a very open

pore, suggesting that 5-HT may be able to leave the receptor more easily. Similar results are

observed in a total of 4 of the 5 MD simulations that were run: one system was rather conclusive

in identifying that is not the correct pose for 5-HT to dock to 5-HT1A as none of the ions stayed

in the vicinity of 5-HT, and the receptor pore remained open.

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These combined methodologies may further be verified through in vitro assays to provide

more evidence to support 5-HT as a receptor for lithium.

Conclusions

The physiological role of Li+ is widespread and linked to many neurochemical processes

and exhibits neuroprotective effects34,35. The therapeutic window for Li+ salts has a narrow range

between effectiveness and toxicity; however, it is still a preferred treatment for bipolar

disorder34,35. The results of the computational models presented suggest that a viable mechanism

of action for Li+ salts is modulation of the 5-HT receptor protein. This may only be one of the

mechanistic roles that Li+ plays within the brain, and it may exert its effects through multiple

pathways: for instance, Li+ is also a competitive inhibitor of magnesium and thus impacts the

role of GSK3β, an enzyme linked to inflammation that is dysfunctional in many diseases33.

Little is understood about the mechanistic action of Li+ as either an endogenous or

exogenous antidepressant; in particular, where it binds in the brain to exert its effects,

particularly within the serotonergic system. Through the use of multiple levels of theory and

computational methods, we show that the receptor for Li+ may be the 5-HT molecule itself. MM

calculations determined that both 5-HT and its 5-HIAA metabolite can form interactions with Li+

in gas phase, protein-like, and aqueous environments. QM calculations using AM1 and B3LYP

also verified that 5-HT and 5-HIAA could interact with Li+ in a “pincer-type” configuration, and

that these interactions are energetically favourable. MD simulations of Li+ in a 2:1 ratio with 5-

HT/5-HIAA in an aqueous environment containing a biologically relevant concentration of Na+

also resulted in binding interactions between the two. A shift was also noted in that the number

of interactions between 5-HT and Li+ increased as the temperature increased.

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Using homology modeling, a binding pocket was identified in the 5-HT1A receptor at

which 5-HT binds. Examination of the results of various docked poses following dynamics

simulations indicates that Li+ impacts how 5-HT binds to the 5-HT1A receptor, as evidenced by a

change in the opening of the receptor pore. These computational methods all support the

hypothesis that 5-HT acts as a receptor for Li+; furthermore, they also suggest that

mechanistically, Li+ exerts its impact by altering how 5-HT binds to the receptor, versus altering

the receptor itself.

Funding Sources. We acknowledge funding from the Krembil Foundation.

References

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(3) Judd, L. L. et al. Psychosocial disability in the course of bipolar I and II disorders: a

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Caption List:

Figure 1. Identification of the functional groups/regions of 5-HT (left) and 5-HIAA (right)

Figure 2. Cationic 5-HT and Li+ face on (left) and side view (right) after optimization in H2O.

Figure 3. 5-HIAA (anionic form) and Li+: face on (left) and side view (right) after optimization in

H2O.

Figure 4. Examples of 5-HT interacting with Li+ at the AM1 level of theory in vacuo (left) and

B3LYP/6-31G+(d,p) level of theory in vacuo (right).

Figure 5. Identified regions of 5-HT interacting with Li+ at 295 K. 5-HT (1), 5-HT (2), and 5-HT (3)

are represented by A, B and C.

Figure 6. Identified regions of 5-HT interacting with Li+ at multiple sites at 310 K. 5-HT (1), 5-HT

(2), and 5-HT (3) are represented by A, B and C.

Figure 7. Identified regions of 5-HIAA interacting with Li+ at multiple sites at 295 K. 5-HIAA (1),

5-HIAA (2), and 5-HIAA (3) are represented by A, B and C.

Figure 5. Identified regions of 5-HIAA interacting with Li+ at multiple sites at 310 K. 5-HIAA (1),

5-HIAA (2), and 5-HIAA (3) are represented by A, B and C.

Figure 9: Location of 5-HT in the 5-HT1A receptor after 5 ns of MD simulations. 5-HT is colour

coordinated to its respective cation: green for Li+, purple for H

+, and orange for Na

+.

Figure10: Amino acid residues forming bonds with 5-HT in the presence of a cation: Blue

indicates interactions occurring when Li+ is present, pink indicates interactions when H

+ is

present, and orange indicates interactions when Na+ is present.

Figure 11: Final configuration of the receptor pore of 5-HT1A after 5 ns of MD simulation. Yellow indicates the pocket surrounding 5-HT.

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Table 1. Summary of the optimization of Li+ and cationic 5-HT: Percentage of systems in which

Li+ is interacting with each receptor site.

Table 2. Summary of the MM optimization of Li+ and anionic 5-HIAA: Percentage of systems in

which Li+ is interacting with each receptor site.

Table 3. Summary of the total Li+ interactions with 5-HT at 295 K.


*Interacts with both 5-HT (1) and 5-HT (3), 2.34% of the time.

Table 5: Summary of the total Li+ interactions with 5-HIAA at 295 K.

*Interacts with both 5-HIAA (1) and 5-HIAA (3) 6.62% of the time. **Interacts with both 5-HIAA

(1) and 5-HIAA (3) 1.44% of the time

Table 6. Summary of the total Li+ interactions with 5-HIAA at 310 K.

*Interacts with both 5-HIAA (1) and 5-HIAA (3) 24.88% of the time.

Table 7. Summary of the total % of interaction time between 5-HT/5-HIAA and Li+.

Graph 1: Summary of the interactions occurring between serotonin and its receptor in the

presence of a cation, as observed over 21 time points.

Graph 2: Summary of the interactions occurring between a cation and the 5-HT1A serotonin

receptor, as observed over 21 time points.

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Table 1. Summary of the optimization of Li+ and cationic 5-HT: Percentage of systems in which

Li+ is interacting with each receptor site.

Average ∆E

OH A B N CHA CHB NH3+

(kcal/mol)

22% 0% 0% 0% 0% 0% 0% -2.40

Average ∆E


(kcal/mol)

22% 22% 0% 0% 11% 11% 0% 3.06

Average ∆E


(kcal/mol)

11% 56% 56% 56% 22% 0% 44% 15.43

Average ∆E


(kcal/mol)

0% 71% 14% 43% 0% 0% 0% 35.75

Average ∆E


(kcal/mol)

45% 100% 73% 73% 0% 0% 0% 22.84

Interactions (in vacuo)

Interactions (THF)

Interactions (H2O)

Interactions (AM1)

Interactions (B3LYP/6-31+G(d,p))

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��

��

Average ∆E

OH A B N CHA CB CO CO-

(kcal/mol)

0% 0% 0% 0% 0% 0% 100% 100% -37.07

Average ∆E


(kcal/mol)

18% 45% 45% 27% 55% 64% 64% 45% -14.08

Average ∆E


(kcal/mol)

27% 55% 55% 45% 18% 36% 27% 45% -17.55

Average ∆E


(kcal/mol)

0% 27% 64% 36% 45% 36% 27% 100% -118.02

Average ∆E


(kcal/mol)

0% 57% 57% 0% 57% 100% 43% 100% -160.91

C

Interactions (THF)

Interactions (H2O)

Interactions (AM1)

Interactions (B3LYP/6-31+G(d,p))

��

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��

�

Lithium Ion 5-HT (1) 5-HT (2) 5-HT (3)

1 - - 13.72

2 - 0.24 -

3 - - -

4 - 0.26 -

5 3.46 - -

6 2.24 - 19.60

Total % of Interaction Time

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*Interacts with both 5-HT (1) and 5-HT (3), 2.34% of the time.

Lithium Ion 5-HT (1) 5-HT (2) 5-HT (3)

1 - - 22.96

2 - 1.00 -

3 - - 9.84

4 9.94 - 26.02

5 - - -

6* 4.70 - 9.66


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��

�� !"��#�� $"�%�%�&�� !"��#�

�� $"�!�''&��

�

Lithium Ion 5-HIAA (1) 5-HIAA (2) 5-HIAA (3)

1* 6.62 - 100.00

2** 2.44 - 38.78

3 - 0.58 -

4 42.58 - -

5 - 44.00 -

6 - 39.12 -


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Table 6. Summary of the total Li+ interactions with 5-HIAA at 310 K.

*Interacts with both 5-HIAA (1) and 5-HIAA (3) 24.88% of the time.

Lithium Ion 5-HIAA (1) 5-HIAA (2) 5-HIAA (3)

1* 24.88 - 100.00

2 2.66 - -

3 - 5.12 -

4 6.78 - -

5 - 6.04 -

6 - - 20.16


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Table 7. Summary of the total % of interaction time between 5-HT/5-HIAA and Li+.

295 K

Serotonin 5-HIAA

1 5.70 1 46.52

2 0.50 2 65.48

3 26.92 3 100.00

Average = 11.04 Average = 70.67

Total = 31.36 Total = 100.00

310 K

Serotonin 5-HIAA

1 13.78 1 33.24

2 1.00 2 10.42

3 59.28 3 100.00

Average = 24.69 Average = 47.89

Total = 65.34 Total = 100.00

% Interaction Time

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Serotonin as a Drug Receptor for Li+: A Computational Study · Draft 2 Abstract. Although Li+ is widely used to treat bipolar disorder, its antidepressant pharmacological mechanism