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Structural and Functional Exploration of 5-HT2A and its Response to Lysergic Acid Diethylamide (LSD) Laura Glastra Chemistry Department, Pacific Lutheran University, Tacoma WA 98477, USA INTRODUCTION

Serotonin receptors are a class of G-protein coupled receptors (GPCRs). These serotonin receptors are located in regions of the brain related to the visual cortex, limbic system, basal ganglia, and olfactory nuclei (Frazer et al., 1999). The actions of different GPCRs vary based on amino acid sequences and protein structures, which interact differently based on the specific ligand. Ligands such as hallucinogens effect a wide range of receptors including the majority of the serotonin family. The most potent known hallucinogen, lysergic acid diethylamide (LSD), has a high affinity for the 2A serotonin receptor (5-HT2A) (Nichols, 2012). LSD’s hallucinogenic effects would not be possible without the 5-HT2A receptor, but the exact mechanism of action has not yet been discovered. The following review is the product of extensive research into the various known mechanisms that are involved in the binding and action of LSD with the 5-HT2A

receptor.

BACKGROUND Serotonin, or 5-hydroxytryptamine (5-HT), is a neurotransmitter derived from the amino acid tryptophan (Nichols et al., 2001) (Figure 1). It is found in the brains of various organisms from nematodes such as C. elegans to vertebrates (Nichols et al., 2001). Its function varies between organisms, and in C. elegans it is responsible for egg

laying and other relatively simple behaviors (Nichols et al., 2001). This evolution history suggest that the receptor is highly conserved. The focus of this review will be on the action of the human receptor. In humans, 5-HT is involved with more complex behaviors like sleep cycle, mood, and memory (Nichols et al., 2001). Serotonin neurons within vertebrates are all found

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throughout the raphe nuclei (RN) (Nichols et al., 2001). 5-HT is an inhibitory transmitter that is produced by neurons within the RN of the midbrain (Nichols et al., 2001). The RN branch from the brainstem and can be found throughout the majority of the brain (Nichols et al., 2001). It is thought that the neurons located here may be involved in inhibitory processes that prevent the brain from overstimulation (Nichols et al., 2001). When the activity or production of 5-HT decreases it is no longer effective at inhibiting other neurons in the reaction chain (Nichols et al., 2001). This results in the brain becoming increasingly more active (Nichols et al., 2001). There are seven families, or groups, of serotonin receptors ranging from 5-HT1

to 5-HT7 (Frazer et al., 1999). Of these families of serotonin receptors, the 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT2 belong to the G-protein receptor superfamily (Frazer et al., 1999). Each of these 5-HT families is a single-subunit protein receptor (Frazer et al., 1999). The 5-HT2A receptor is influenced by a variety of hallucinogenic drugs (Nichols et al., 2001). The effect of hallucinogenic

drugs would not be possible without the 5-HT2A receptor (Nichols et al., 2001). The potency of hallucinogens can therefore be determined by their affinity for the 5-HT2A receptor (Nichols et al., 2001). The 5-HT2A receptor is highly concentrated in the prefrontal cortex and other areas of the brain’s cortical regions (Frazer et al., 1999). Cortical regions of the brain include the claustrum which is connected to the visual cortex, the limbic system involved in the endocrine and autonomic nervous system, which are involved in emotion, learning, and memory function; the basal ganglia that is responsible for habit based learning and motor behaviors, and the olfactory nuclei, which are highly evolved in vertebrates and are involved with odor information processing (Frazer et al., 1999). There are primarily three types of chemicals that act as agonists for the 5-HT2A receptor. These include tryptamines, ergolines, and phenethylamines (Figure 1). Tryptamines are the agonists most closely related to serotonin, which is the natural neurotransmitter. Ergolines are

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tetracyclic molecules derived from alkaloids in ergot fungus. An example of such a molecule, the most potent of the known psychedelics, is lysergic acid diethylamide (LSD) (Nichols, 2012). Some studies have proposed the following to explain the general mechanism of LSD’s effects on serotonin:

In view of the localization of the raphe nuclei close to the brain stem’s consciousness-alerting system, called the ascending reticular activating system (ARAS), it is suggested that LSD could influence the gating function of this system for afferent sensory information. Thus a reduction in serotonin release mediated by inhibitory presynaptic serotonin receptors would be likely to reduce the tonic inhibition in the ARAS due to serotonin and thus allow abnormal stimulation of the visual and other relevant areas of the brain, causing hallucination (Bradford et al., 1986).

Structure of LSD.

LSD is a relatively planar molecule with a molecular mass of 323 g/mol and chirality at two positions in the molecule (Nichols, 2012). These chiral centers are at carbons 5 and 8, which have strong influence over the psychoactive properties of the molecule (Figure 2). The carbons must be in a 5R, 8R - configuration for LSD to be biologically active. This relationship of configuration and biological activity is seen throughout all ergolines (Nichols, 2012). There are many non-psychoactive forms of LSD, however little research has been done in clinical settings with the exceptions of those that occurred in the 1950s and 1960s prior to the legal changes that occurred in that 1970s. These studies found that the psychoactive and biologically active properties of LSD change in response to reduction of double bonds, halogenation, and alkylation (Figure 2). The (+)-LSD or d-LSD form is the psychoactive form that had previously been used for therapy. Epimerization of LSD readily occurs at the 8-position, producing (+)-isolysergic acid diethylamide (Figure 2). This isomer is not an active hallucinogen, further supporting the importance of the

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specific configuration required at the 5 and 8 chiral carbons (Nichols, 2012). Halogenating the 2-position of LSD resulted in molecules that acted as antagonists of the 5-HT2A receptor, rather than agonists such as (+)-LSD (Figure 2). Antagonists such as these molecules act to inhibit binding of serotonin (Nichols, 2012). Reduction of the 9,10-double bond was another alteration made to LSD in early research (Figure 2). This reduction also removed the hallucinogenic activity of the molecule. In tryptamines, similar reductions of the highest pi-bond orbitals did not have the same effect on hallucinogenic activity. This signifies that the 9,10-double bond is an important characteristic of the molecule’s psychoactive properties, though it is unclear why (Nichols, D. E). A reduction of the 2,3-double bond was also conducted, and the molecule remained biologically active afterwards (Figure 2). The onset of the psychoactive effects were slower, however, suggesting that metabolic changes may alter the conformation of the molecule in the body. Hallucinogenic effects were still

reported, though it was found to be approximately eight times less intense than LSD itself (Nichols, 2012). This reduction occurs on the indole group of the LSD molecule. Some studies have suggested that this functional group is a distinguishing factor on how various psychedelics function. LSD and other hallucinogens with indole ring systems preferentially inhibit cells from releasing serotonin, rather than affecting the serotonin receptors post synaptic regulation (Passie, et. al.). Alkylation of the N(6)-methyl group on LSD has been found to produce much more potent psychoactives in vitro rodent behaviors. It is anticipated that these effects on rodents are likely to be comparable to those in humans (Nichols, 2012). Serotonin Receptor (5-HT2A) Structure. The serotonin receptor has a length of 471 amino acids and a molecular mass of 52,603 g/mol (Figure 3) (UniProt). It is a G-protein coupled receptor (GPCR), which stands for guanine nucleotide triphosphate binding proteins. Of the GPCR families, serotonin is in the

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largest, which is the Rhodopsin, or A Family (Isberg et al., 2011). Previously it was suggested that receptors produced intracellular metabolites as a result of ligand binding, but it is now believed that GPCRs have the effect they do because of interactions that occur between the receptor with the G-protein (Byrne et al., 2004). Interaction of the G-protein with its coupled receptor allows for modulation of different effector systems such as ion channels, adenylyl cyclase, and phospholipase C, which the 5-HT2A receptors activate (Frazer et al., 1999). Characteristics of G-proteins include the presence of seven transmembrane alpha helices (TM1-TM7), an intracellular carboxy terminus, and an extracellular amino terminus. The most conserved feature of GPCRs is the seven transmembrane structure (Byrne et al., 2004, J. H.). Each G-protein complex has a specific receptor protein that weaves through the seven transmembrane segments. It is this protein’s amino terminus that extends out of the cell membrane, and its carboxy terminus that is found inside the cytoplasm of the cell. Intracellular loops, or segments, of the protein are

found between TM1 and TM2, TM3 and TM4, and TM5 and TM6. These segments are denoted i1, i2, and i3 respectively. The extracellular loops are connected between TM2 and TM3, TM4 and TM5, and TM6 and TM7. Similarly, these are denoted as e1, e2, and e3 respectively (Byrne et al., 2004, J. H.) (Figure 4). The seven domains that the protein wraps through are composed of about 24 hydrophobic amino acids. It is in the center of these proteins where the binding site is located. Amino acids of the transmembrane domains point in toward the binding site. These inward pointing amino acids influence the binding affinity of ligands (Byrne et al., 2004, J. H.). These generalized structural characteristics describe the basic structure of the 5-HT2A receptor. CURRENT RESEARCH Current research on the 5-HT2A receptor is limited because the exact structure has not yet been identified. It has not been identified exactly because X-ray crystallography techniques are especially challenging for membrane proteins (Rodriguez et al., 2014).

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Without definitive knowledge of the protein’s structure, little research has been able to make conclusions on the specific molecular function behind the receptor’s mechanisms. The research that will be explored in this review focus on the methodology behind identifying the structure and why LSD may have such a high affinity for this receptor. One approach made in identifying the structure of 5-HT2A is using 5-HT2A homology modeling. Homology modeling begins with use of another molecule sharing similar characteristics to the receptor of interest. An inactive

β2-adrenoceptor (β2AR) was used and

then altered to fit characteristics of active GPCR structures (Isberg et al.,

2011). Alterations to the β2AR were

made to adjust angle positioning of TM5 and TM6, alter the i3 segment sequence from LQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLK to AQQQESATTQKA, insert a Gαs peptide backbone, and move R1313.50 to a rotamer with interaction of the G-protein (Figure 5) (Isberg et al., 2011).

Models of 5-HT2A have been produced in silico through steered molecular dynamics simulations (Isberg et al., 2011). Since structure of the receptor is unknown, the model was steered towards a structure that fit the characteristics of known GPCRs. Specific distance constraints were set for bond lengths based on X-ray crystallography and mutagenesis data.

These were included in the simulations as pairwise (donor - acceptor) atom distance harmonic constraints (default between hydrogen and acceptor: 1.8 Ǟ; G-protein and helix 8: 2.2 Ǟ; protonated nitrogen in ligand and - D1553.32 gamma carbon: 3.0 Ǟ (Isberg et al., 2011).

These binding characteristics lead to movement of helices and side-chains energy changes. These changes are thought to continue until the active form of the molecule is achieved (Isberg et al., 2011). In silico studies demonstrate the importance of side-chain rotameric actions. Change in rotameric position is referred to as a “toggle switch” (Tautermann et al., 2011). Toggling can occur strongly in response to activation

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by agonists while the binding site holds a similar conformation in the active and inactive state (Tautermann et al., 2011). Constraints imposed in the silico modeling method include the aforementioned binding lengths, ligand-receptor binding, G-protein-receptor binding, interhelical receptor binding, and helical backbone binding of the receptor and G-protein to prevent local distortions and unfolding at truncated sites (Isberg et al., 2011). Hydrogen bonding was a key restriction emplaced to stabilize hydrophobic networks throughout the helices. These constraints were imposed to activate the receptor models. Extracellular Face and Ligand Binding Site. Helical shifts induce tightening of the overall binding site inside of the helices. Tightening is required for proper binding of the ligand to occur because of the proximity and arrangement of the ligand and receptor complex. (Isberg et al., 2011; Shapiro et al., 2002). Changes in proximity of the ligand and binding site interactions occur because of side chain rotations, helical tilting, and helical rotating. The main observed

cause leading to activation of the receptor occurs due to the rotations in the side chains (Isberg et al., 2011). The largest movements observed through the molecular dynamics modeling occurred when constraints were first implemented and when the restraints of the helical backbone were released (Isberg et al., 2011). The largest known movement of the extracellular surface is an inward tilt of TM6. Another movement is the shift of TM3 moving closer to TM5 (Isberg et al., 2011). Additionally, TM7 shifts to the side toward TM1, likely occurring as a response to make room for the repositioning of TM6. As a result of the TM7 shift, TM 1 and TM2 both shift sideways as well. The shift of TM7 is away from the ligand binding site, but the helix maintains good hydrogen bonding with N-benzyl groups of the ligand. It is thought to be a tyrosine molecule that induces hydrogen bonding with the N-benzyl moiety (Isberg et al., 2011). The TM6 helix also has significant interactions with the TM3 helix through strong ionic forces (Shapiro et al., 2002). Changes in movement of TM6

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are proposed to be a result of the interaction the helix has during the binding of the agonist (Shapiro et al., 2002). This hypothesis has been tested through site directed mutagenesis and molecular modeling. This interaction may proceed via aromatic residues of TM6. The interactions and movement of TM6 lead to disruptions in ionic bonding between it and the TM3 helix. This is hypothesized to be a wide ranging interaction of agonist bound 5-HT receptors that are coupled to G-proteins given the similarities in amino acid sequences of 5-HT receptors (Shapiro et al., 2002). It is possible these interactions are through the side chains of the TM6 helix’s hydrophobic residues (Shapiro et al., 2002). The TM6 helix has three hydrophobic regions composed of different combinations of cysteine, arginine, isoleucine, leucine, phenylalanine, tyrosine, valine, and asparagine. Alanine substitution in these three regions induces strong Van der Waals forces with adjacent helices through their residues (Shapiro et al., 2002).

The ionic and hydrophobic interactions that occur through residues within the TM6 helix have key stabilizing effects for the inactive 5-HT2A receptor (Shapiro et al., 2002). When agonist binding occurs, TM6 does not maintain its stabilizing properties, and activation of the receptor begins. Side chain interactions are important because of changes in hydrogen bonding that can have drastic effects on the molecular functions and interactions with other side chains and helices (Shapiro et al., 2002). Intracellular Face and G-Protein Binding Site. TM6 is also involved in the intracellular, or cytoplasmic, movements of the receptor (Figure 6). To make room for the G-protein the TM6 shifts outward, which is an example of the “global toggle-switch method.” In order for the TM6 movement to occur, the TM5 moves sideward to provide space. This mechanism may not be universal to GPCRs since different interprotein interactions occur through differences in receptor sequences (Isberg et al., 2011).

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The intracellular loop i2 may be involved in specific binding reactions of hallucinogenic and non-hallucinogenic drugs (Figure 4). Implications of this involve different pharmacological actions of the receptor based on the specific hallucinogen (Perez-Aguilar et al., 2014). The global toggle-switch model is a proposed method for the different functional mechanisms that occur with the i2 loop and different ligands. CONCLUSION In summary, 5-HT2A is G-protein coupled receptor. It is characterized by seven transmembrane helices (TM1-TM7) as well as extracellular and intracellular loops. Each helix and loop interact with the binding ligand, but some do more than others. The TM6 helix in particular has many effects that result in changes of other helices as well. This includes TM5 and TM3. Various methods have been used to attempt construction of the protein’s structure, but this has proved extremely difficult. It is a membrane-bound protein, which are challenging to

crystallize. Modern techniques have shifted towards molecular dynamics modeling. These have provided relatively accurate models based off of ligand binding tests conducted in the studies. One challenge remaining is determining individual interactions of internal amino acids with the ligand when binding in the active site. The knowledge behind this mechanism for LSD would provide insight as to why a molecule with relatively similar traits to serotonin has as high of an affinity as it does for the 5-HT2A receptor. ACKKNOWLEDGMENTS The author thanks Dr. Tonn for her support and guidance in this research as well as providing the CHEM403 student body with the opportunity to conduct their own choices of research. Additional thanks are attributed to Pacific Lutheran University for access to journal articles and other resources. Finally, it is of great appreciation to the authors cited in this review for their dedication and efforts to furthering the knowledge of the scientific community and general population.

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FIGURES Figure 1. Structures of various types of agonists on the 5-HT2A receptor. There are similarities between tryptamines and ergolines, though tryptamines tend to have more similar binding characteristics as serotonin given similarities of the indole ring structure and amine groups (Nichols, 2012).

Figure 2. Structures of lysergic acid diethylamide (LSD). The key characteristic is the indole ring, which is similar to serotonin’s structure and an important part of the molecule involved in the binding to the active site (Wikimedia Commons).

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Figure 3. Represented is the “canonical” sequence of the 5-HT2A receptor, with a length of 471 amino acids and a molecular mass of 52, 603 g/mol (UniProt).

Figure 4. General structure for a G-protein coupled receptor, where purple circles indicate conserved amino acids throughout the entire G-protein family. The protein displayed is mAChR in its M3 isoform. The diagram provides the general structural characteristics of G-proteins, where there are seven intermembrane helices, an extracellular amino terminus, and an intracellular carboxy terminus. The second diagram consisting of parts A, B, and C shows the seven transmembrane domains with the center as the binding site. These are not the structure for the 5-HT2A receptor, but it displays a generalized formation of how the ligand binds in the active site. Displayed here is a catecholamine binding in βAR. This model also demonstrates the stabilizing hydroxyl groups within the active site (Byrne et al., 2004).

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Figure 5. Extracellular view of the transmembrane helices involved in the 5-HT2A

receptor. The magenta shows the structure of β2AR while the brown depicts the 5-HT2A

receptor modeled through molecular dynamics. Tightening of the helices is the most important function involved in binding of the active site. This involves transmembrane helices 3, 5 and 6. Changes to the TM5 and TM6 present in this figure were made during the molecular dynamics modeling (Isberg et al., 2011).

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FIGURE 11.15 Amino acid sequence and predicted domain topology of the M3 isoform of mAChR. The transmembranedomains are TM1–TM7. The NH2 terminus of the protein is at the left and extends into the extracellular space. The COOHterminus is intracellular and is at the right; i1 to i4 are the four intracellular domains. The conserved disulfide bond (–S–S–)connects extracellular loop 2 to loop 3. The dashes in the amino acid sequence represent inserts of various lengths that are not shown.Conserved amino acids for all members of the G protein-coupled receptor family of receptors are marked in purple. The amino acidstaking part in ACh binding to the receptor are highlighted in yellow. Note that all amino acids associated with ligand binding lie inapproximately the same horizontal plane across the receptor. Adapted from Trends Pharmacol. Sci., Vol. 14, 1993.

© 2004 Elsevier Inc. All rights reserved. FROM MOLECULES TO NETWORKS John H. Byrne, James L. Roberts

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FIGURE 11.14 (A) Diagram showing the approximate position of the catecholamine binding site in βAR. The transmitter binding site isformed by amino acids whose side chains extend into the center of the ring produced by the seven transmembrane domains (TM1–TM7).Note that the binding site exists at a position that places it within the plane of the lipid bilayer. (B) A view looking down on a model of βARidentifying residues important for ligand binding. The seven transmembrane domains are represented as gray circles labeled TM1 though TM7.Amino acids composing the extracellular domains are represented as green bars labeled e1 through e4. The disulfide bond (–S–S–) that links e2 to e3also is shown. Each of the specific residues indicated makes stabilizing contact with the transmitter. (C) A view looking down on a model of mAChRidentifying residues important for ligand binding. Stabilizing contacts, mainly through hydroxyl groups (–OH), are made with the transmitter on fourof the seven transmembrane domains. The chemical nature of the transmitter (i.e., epinephrine versus ACh) determines the type of amino acids necessaryto produce stable interactions in the receptor binding site (compare B and C). Adapted from Strosberg (1990).

© 2004 Elsevier Inc. All rights reserved. FROM MOLECULES TO NETWORKS John H. Byrne, James L. Roberts

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Figure 6. Intracellular depiction for the above model of 5-HT2A and β2AR protein superimposition. (Isberg et al., 2011)

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CITATIONS Passie, T.; Halpern, J. H.; Stichtenoth, D. O.; Emrich, H. M.; Hintzen, A. The pharmacology of lysergic acid diethylamide: a review. CNS Neuroscience & Therapeutics 2008, 14, 295-314. Frazer, A.; Hensler, J. G. Serotonin receptors. In Basic Neurochemistry: Molecular, Cellular, and Medical Aspects, 6th edition [Online]; Siegel, G. J.; Agranoff, B. W.; Albers, R. W., et al., Eds; Lippincott-Raven: Philadelphia, 1999. https://www.ncbi.nlm.nih.gov/books/NBK28234/ (Accessed October 20, 2016). Nichols, C. D.; Sanders-Bush, E. Serotonin receptor signaling and hallucinogenic drug action. The Heffter Review of Psychedelic Research 2001, 2, 73-79. Nichols, D. E. Structure-activity relationships of serotonin 5-HT2A agonists. WIREs Membrane Transport & Signaling 2012, 1, 559-579. doi: 10.1002/wmts.42 Isberg, V.; Balle, T.; Sander, T.; Jørgensen, F. S.; Gloriam, D. E. G-protein and agonist-bound serotonin 5-HT2A receptor model activated by steered molecular dynamics simulations. J. Chem. Inf. Model. 2011, 51, 315-325. Rodriguez, D.; Ranganathan, A.; Carlsson, J. Strategies for improved modeling of GPCR-drug complexes: blind predictions of serotonin receptors bound to ergotamine. J. Chem. Inf. Model. 2014, 54, 2004-2021. Tautermann C. S.; Pautsch, A. The implication of the first agonist bound activated GPCR X-ray structure on GPCR in silico modeling. ACS Med. Chem. Lett. 2011, 2, 414-418. Bradford, H. F. Chemical Neurobiology: An Introduction to Neurochemistry; W. H. Freeman and Company: New York, 1986; pp 424-427. Byrne, J. H.; Roberts, J. L., Eds. From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience; Elsevier Academic Press: San Diego, 2004; pp 319-331. Shapiro, D. A.; Kristiansen, K.; Weiner, D. M.; Kroeze, W. K.; Roth, B. L. Evidence for a model of agonist-induced activation of 5-hydroxytryptamine 2A serotonin receptors that involves the interruption of a strong ionic interaction between helices 3 and 6. J. Biol. Chem. 2002, 277, pp 11441-11449. Wikimedia Commons. https://commons.wikimedia.org/wiki/File:LSD-2D-skeletal-formula-and-3D-models.png (accessed November 31, 2016). Uniprot. http://www.uniprot.org/uniprot/P28223 (accessed November 31, 2016).

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Perez-Aguilar, J. M.; Shan, J.; LeVine, M. V.; Khelashvili, G.; Weinstein, H. A functional selectivity mechanism at the serotonin-2a GPCR involves ligand-dependent conformations of intracellular loop 2. J. Am. Chem. Soc. 2014, 136, 16044-16054.