Selectivity of 2-dimethylamino-5 or 6-phenyl-1,2,3,4-tetrahydronapthylene and their Derivatives for Serotonin 2 vs. Histamine 1 G-protein Coupled Receptors

Material Information

Selectivity of 2-dimethylamino-5 or 6-phenyl-1,2,3,4-tetrahydronapthylene and their Derivatives for Serotonin 2 vs. Histamine 1 G-protein Coupled Receptors
Felsing, Daniel
Publication Date:


Subjects / Keywords:
Agonists ( jstor )
Automatic picture transmission ( jstor )
Complementary DNA ( jstor )
Ligands ( jstor )
Pendants ( jstor )
Phenyls ( jstor )
Receptors ( jstor )
Serotonin receptors ( jstor )
Side effects ( jstor )
Transfection ( jstor )
Drug abuse
Undergraduate Honors Thesis


Medications for drug abuse and obesity are still lacking, and of importance based on their widespread effects among our population. Activation of serotonin 2C receptors may modulate an anti-addictive behavioral response to both food and psychostimulant. Activation of Serotonin 2A and 2B receptors must be avoided due to harmful side effects. Histamine receptors are closely related to Serotonin receptors and activation triggers negative side affects associated with many antipsychotics. (±)-2-dimethylamino- 5 or 6-phenyl-1,2,3,4-tetrahydronapthylene (APT) and their derivatives showed 4-10 fold selectivity for 5-HT2 receptors over H1. 5 APT derivatives show increased selectivity over their 6 APT counter parts. 2-diethylamino-5-phenyl-mCF3-1,2,3,4-tetrahydronapthylene and 2-diethylamino-5-phenyl-mCl-1,2,3,4-tetrahydronapthylene may potentiate into therapeutic drugs. APT compounds tend to show selectivity for Serotonin 2B receptors over 2A, 2C, H1. ( en )
General Note:
Awarded Bachelor of Science; Graduated May 3, 2011 summa cum laude. Major: Chemistry, Emphasis/Concentration: Biochemistry
General Note:
College/School: College of Liberal Arts and Sciences
General Note:
Advisor: Raymond Booth

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Daniel Felsing. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.


This item is only available as the following downloads:

Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EYD619PSE_I2H8AA INGEST_TIME 2017-06-01T19:08:33Z PACKAGE AA00057483_00001


Felsing D 1 Selectivity of 2di methylamino-5 or 6-phenyl-1,2,3,4tetrahydronapthylene and their Derivatives for Serotonin 2 vs. Histamine 1 G-protein Coupled Receptors. By: Daniel Felsing PI: Dr. Raymond Booth, Department of Medicinal Chemistry Undergraduate Thesis April 6 2011


Felsing D 2 Abstract Medications for drug abuse and obesity are still lacking, and of importance based on their widespread effects among our population. Activation of serotonin 2C receptors may modulate an anti-addictive behavioral response to both food and psychostimulant. Activation of Serotonin 2A and 2B receptors must be avoided due to harmful side effects. Histamine receptors are closely related to Serotonin receptors and activation triggers negative side affects associated with many antipsychotics. ( )-2-dimethylamino5 or 6-phenyl-1,2,3,4-tetrahydronapthylene (APT) and their derivatives showed 4-10 fold selectivity for 5HT 2 receptors over H 1. 5 APT derivatives show increased selectivity over their 6 APT counter parts. 2-diethylamino-5-phenyl-mCF 3 1,2,3,4-tetrahydronapthylene and 2-diethylamino-5-phenylmC l-1,2,3,4tetrahydronapthylene ma y potentiate into therapeutic drugs. APT compounds tend to show selectivity for Serotonin 2B receptors over 2A, 2C, H1. Keywords: Serotonin 2A, 2B, 2C, receptors (5HT 2 ), Histamine 1 receptors ( H1 ), Binding affinity ( Ki ), (1R, 3S)-( )-trans-1-phenyl-3-dimethylamino1,2,3,4-tetrahydronapthalene (PAT), 2-diethylamino-5-phenyl-1,2,3,4-tetrahydronapthylene (5-APT)


Felsing D 3 Introduction Obesity and drug addiction are growing problems in the United States, and throughout the world. In 2007, the Center for Disease Control reported that 68% of Americans were said to be overweight, (with a body mass index (BMI) >25), and 34% obese (with a BMI >30). i The incidence of obesity continues to rise is predicted to be the leading cause of preventable death in America by 2015. ii Although the epidemic of obesit y shows no signs of regressing, the FDA has not approved any new diet medications in the last decade. Similarly, psychostimulant addiction is a growing problem ,especially methamphetamine addiction. According to the National Survey on Drug Abuse in 2005, 512,000 people had used methamphetamine within the previous month, and according to a separate study in 2008, 1.4 million people admitted using cocaine within the last month. iii Serotonin receptors in the brain are known to modulate both dietary control and the reinforcing effects of addictive psychostimulants. For this reason, drug development targeting these receptors has great potential and will be the focus of this study. Serotonin: There are at least 14 distinctive serotonin (5-Hydroxytryptamine or 5-HT) receptors classified into 7 different families, 5HT 1-7 With the exception of the 5HT 3 receptor, a ligand gated Na and K channel, all are G-Protein couple receptors (GPCRs) The 5HT 2 family consists of three separate receptors subtypes: 5HT 2A 5HT 2B and 5HT 2C iv The human 5HT 2C receptor is located almost exclusive ly in the brain and is expressed in many different regions such as the basal ganglia, choroid plexus, cortex, and limbic system. v 5-HT 2A and 5HT 2B receptors are located throughout the body, including in the brain, GI tract, cardiovascular system and smooth muscle tissue. 5HT 2 receptors


Felsing D 4 activate G q proteins which leads to stimulation of phospholipase C (PLC), and formation of inositol phosphates (IP 3 ) and diacylglycerol (DAG) as second messengers. vi 5HT 2A and 5HT 2B receptors are known to be involved in regulating blood pressure and cognitive processes such as emotion and memory. iv 5-HT receptors modulate multiple neurotransmitters, both excitatory and inhibitory, such as: glutamate, dopamine nor-epinephrine, GABA and others. iv For example, the 5HT 2C receptor attenuates dopamine release in multiple areas of the rat brain such as the striatum vii hippocampus viii nucleus acumbens, and vental tegmental area (VTA). ix x Dopamine release in these brain regions, known as the mesolimbic pathway; is associated with subjective feelings of reward. It is widely recognized that addictive drugs and other reinforcers, including food, activate this pathway. Therefore, agonists of the 5HT 2C receptor ma y decrease dopamine release in this pathway are potential therapeutic tools in breakin the positive reinforcing effects of food and psychostimulant patholog y in addiction. 5HT 2 effects: Compelling evidence suggests that activation of the 5HT 2C receptor causes anorexigenic effects and reduc es cocaine-seeking behavior and cocaine relapse in rodents. xi 5HT 2C agonists also reduce amphetamine-induced locomotor activity xii A similar reduction in locomotor activity occurred after blockade of the 5HT 2A receptor xiii This evidence suggests blockade of 5HT 2A and activation of 5HT 2C ma y produce similar attenuation of amphetamine locomotor effects. Bolstering these findings, 5HT 2C knockout mice show enhanced locomotor and reinforcing effects of ps ychostimulants. 11 Hallucinogenic drugs like lysergic acid diethylamide (LSD) and psilocybin activate the 5HT 2A receptors in the brain which lead to alterations in cognition and mood and


Felsing D 5 ultimately producing hallucinations. xiv Peripheral effects of 5-HT 2A agonism include attenuation of intraocular pressure. The 5HT 2A agonist AL3466 2 produced this effect, and because it is unable to cross the bl o od -brain barrier AL 3466 2 does not produce hallucinogenic effects. xv Both 5HT 2A and 5HT 2B receptors contribute to the regulation of blood pressure. The selective 5HT 2A antagonist, ketanserin, is classified by the World Health Organization (WHO) and the National Institute of Health (NIH) as an antihypertensive and is available for use in Europe. xvi Activation of 5HT 2B is known to produce deleterious side effects. Patients who were prescribed the diet drug fenfluramine showed increase prevalence of pulmonary hypertension xvii and cardiac valvulopathy associated with 5HT 2B agonism. xviii It was later shown that this effect was mediated by the activation of 5HT 2B receptors. anorexigenic effect however, was likely mediated by 5HT 2C agonism, which subsequently activated melanocortin M 3 and M 4 receptors, which are known to affect eating habits. xix It is apparent that agonists of 5HT 2A and 5HT 2B receptors produce deleterious side effects and should be discarded as therapeutic drugs. However, they can still be beneficial in delineating molecular determinants for affinity and function of such receptors. Locaserin was a 5HT 2C mediated antiobesity drug candidate in Stage 3 clinical trials, but it was deemed unsafe due to carcinogenic effects observed in rats. xx Lorcaserin is an agonist at all three receptors with 15-fold selectivity of 5HT 2C over 5HT 2A and 100-fold affinity of 5HT 2C over 5HT 2B xxi xxii Although no 5HT 2A -mediated psychotomimetic effects or 5HT 2B cardiac effects were reported, in a clinical trial merely a third of the individuals receiving the highest dose of lorcaserin lost 5% of their pretreatment body weight showing less than ideal efficacy. Other diet drugs have also targeted this 5HT 2C mediated


Felsing D 6 anti-obesity effect, but the FDA, due to harmful side effects, has not approved any within the last decade. Histamine: Another homologue, the histamine 1(H 1 ) receptor is more closely related genetically to the 5HT 2 family than any other histamine receptor. For this reason many ligands targeting the 5HT 2 receptors also bind H 1 H 1 receptor agonism can lead to diarrhea, respiratory distress, and hypotension typically connected with a peripheral allergic response. xxiii Most pharmacotherapeutic H 1 ligands that have been developed are H 1 antagonists that block peripheral allergic responses. xxiv On the other hand, antagonism or inverse agonism of H 1 ma y lead to short-term weight gain. xxv This is a common side effect of many atypical antipsychotics like clozapine, olanzapine, and ziprasidone, which have H 1 inverse-agonist properties PAT: As noted earlier, t he re are multiple potential therapeutic uses of a specific 5HT 2C receptor agonist that either has poor affinity for or is an antagonist at 5HT 2B and 5HT 2A receptors However recently a small (molecular weight<300), lipophilic molecule, (1R, 3S)( )-trans-1-phenyl-3-dimethylamino1,2,3,4-tetrahydronapthalene (PAT) was shown to be a full agonist for the 5HT 2C and a competitive antagonist at the 5HT 2A 5HT 2B and H 1 receptors. xxvi Recent in vivo studies show ed that PAT has brain penetrating properties, being able to block a central 5HT 2A mediated behavioral response the 2,5-Dimethoxy-4iodoamphetamine (DOI) induced head twitch response (unpublished observations). These unique characteristics of PAT and its derivatives could translate in vivo to a new


Felsing D 7 pharmacotherapy for obesity, psychosis, cocaine addiction, and other neuropsychiatric disorders. Figure 1. Lead compound PAT, (1R, 3S)-()-trans-1-phenyl-3-dimethylamino1,2,3,4tetrahydronapthalene There is a shared 80% sequence homology between the 7 trans-membrane domains (TMD) where ligands can bind, of 5HT 2C and 5HT 2A receptors, 70% between 5HT 2 C and 5HT 2B receptors, and a mere 44% homology between 5HT 2 C and H 1 receptors. xxvii The shared homology creates a serious challenge for developing ligands that selective ly bind to 5HT 2A, 2B, 2C and H 1 receptors. Differences in ligand binding to these receptors can be explored to gain knowledge of the molecular determinants that govern selective ligand binding. PAT shows specificity towards 5HT 2C and H 1 over 5-HT 2A and 5HT 2B having K i values of 37.6 3.02, 1.95 0.51, 410 38, 1200 6.8nM, respectively. xxviii The specificity of PAT for 5HT 2C over 5HT 2A and 5HT 2B is 10 and 30 -fold, respectively (low K i translates into high affinity). This specificity should translate in vivo to less drug necessary to produce the desired effect, along with there being a smaller chance of triggering 5HT 2A and 5HT 2B


Felsing D 8 mediated detrimental side affects. However, since PAT is an effective antagonist with high specificity for H 1 one should expect PAT to cause similar effects, such as drowsiness, as other first generation antihistamines like diphenhydramine whose K i is 11.5 nM xxviii APT: With the goal of further developing a selective 5HT 2C agonist, a series of compounds, structurally similar to the 5-HT2 C agonist PAT, were synthesized. ( )-2dimethylamino5 or 6-phenyl-1,2,3,4-tetrahydronapthylene (APT) contains the same 2dimethylamino-1,2,3,4-tetrahydronapthelene structure. However, the pendant phenyl ring bound to carbon 1 of the cyclohexyl ring for PAT, and has been moved to either the 5 or 6 position on the phenyl ring of tetrahydronapthelene structure. The pendant phenyl ring in the APT molecules is bound to an sp 2 carbon, which severely limits rotation, and forces the pendant phenyl ring to be planar to the tetrahydronapthelene structure. This steric hindrance is thought to be the main cause for a >1000 loss of affinity for the H 1 receptor of APTs compared to PAT. xxix The pendant phenyl ring of PAT is attached to a flexible sp 3 carbon, allowing it to rotate freely and accommodate multiple conformations in binding t o the receptor.


Felsing D 9 Figure 2. Structure of test compound, 5-APT, 2-diethylamino-5-phenyl-1,2,3,4tetrahydronapthylene Molecular interactions, Modeling: The specific molecular determinants that at the 5HT 2A, 5HT 2B 5HT 2C and H 1 are not fully understood, and much less is understood of the 5HT 2B and H 1 binding pockets compared to 5HT 2A and 5HT 2C The aspartate residue D3.32 is known through mutagenesis studies to be crucial for ligand binding due to its ability to form an ionic bond with protonated amine moieties in ligands. xxx This residue is conserved in 5HT 2A, 2B, 2C and H 1 receptors. The APT test compounds, with their dimethylamide moiety, should form this interaction and have the capability to bind. Other amino acid residues thought to be relevant to ligand binding in this family of GPCRs are F5.47, F5.48 xxxi W6.48, F6.51, and F6.52 xxxii These aromatic residues are located in the binding pocket and can possibly interact with the two phenyl rings in PAT mediated through interactions. Differences in binding affinities between PAT and APT derivatives could be due to loss or gain of these interactions caused by the movement of the


Felsing D 10 pendant phenyl ring. Another difference between the receptors that may be exploited is the residue at position 5.46. This residue is different in three of the four receptors and is known to affect binding of indoleamines as well as ergolines. xxxiii In 5-HT2 A this residue is a serine, in 5HT 2C it is an alanine, and in H 1 it is an asparagine. This difference could help create specificity for ligands between the individual receptors. In the present experiments, HEK cells expressing 5HT 2A 5HT 2B 5HT 2C and H 1 receptors were used to isolate potential drug targets and determine affinities of 8 APT analogues. These studies will help elucidate molecular determinants of 5HT 2 and H 1 receptor ligands and will help guide future mutagenesis studies and 3D molecular models of human WT 5HT 2A 5HT 2B 5HT 2C as well as H 1 Results In order to carry out saturation and competition binding isotherms, a Bradford assay was performed to determine the concentration of protei n in each membrane preparation. The samples were compared to a standard curve of BSA, with concentrations ranging from 0-2000 ug/ul of protein.


Felsing D 11 Figure 3. Standard Curve of Absorbance vs. Protein Concentration of BSA, used to obtain approximate protein concentrations of membrane preparations. Shown below in Table 1 are the concentrations of protein in each membrane preparation from the Bradford assay and standard curve of BSA. 5HT 2C and H 1 receptors were highly expressed, consistently having protein concentrations over 300 ug/ul. The 5HT 2A and 5HT 2B receptors were expressing at lower levels in the range of 200-300 ug/ul, however binding was still evident and transfection confirmed. 0.000 0.500 1.000 1.500 2.000 2.500 0 500 1000 1500 2000 2500 Absorbance Protein Concentration (ug/ul) BSA standard curve


Felsing D 12 Receptor P rotein Concentration (ug/ ul) 5 HT 2A 219 5 HT 2B 293 5 HT 2C 530 H 1 322 Table 1. Protein concentration of WT 5HT 2A 5HT 2B 5HT 2C H 1 via Bradford assay. Saturation binding isotherms were performed to validate transfection procedure, and to obtain K D estimates for analysis in competition binding experiments. The standard errors are in parenthesizes Figure 4. Saturation binding of [ 3 H]-ketanserin to 5HT 2A confirming transfection and to obtain K D values of radioligand.


Felsing D 13 Table 2 represents experimentally determined K d values and compares them to values accepted by the PDSP, PDSP values were used in analysis of competition assays. E xperimental K D PDSP xxviii K I [ 3 H] ketanserin @ 5 HT 2A 0.58 (0.04) 2.25 [ 3 H] mesulergine @ 5 HT 2B 1.27 (0.33) 1.95 [ 3 H] mesulergine @ 5 HT 2C 1.85 (1.17) 1.4 0 [ 3 H] mepyramine @ H1 0.77 (0.39) 0.8 0 Table 2. Experimentally determined K D i HT 2A 5HT 2B 5HT 2C H 1. The experimental K d values of the radioligands were all below 2 nM, and within the optimal range of radioligands to accurately discriminate other test ligand s binding affinities. The experimental K d values of 0.58, 1.27, 1.85 and 0.77 nM for 5HT 2A 5HT 2B 5HT 2C H 1 respectively, were in range of the corresponding K i values published by PDSP which confirms successful cDNA transfection. The largest variance of 1.67 nM, was found for [ 3 H] -ketanserin binding to the 5HT 2A receptor. The other experimental K D had minimal differences of 0.67, 0.40, and 0.03 nM for the radioligands of 5HT 2B 5-HT 2C and H 1 receptors, respectivel y. Competition binding isotherms were performed to deduce K i s, in nM, for each APT compound at 5HT 2A 5HT 2B 5HT 2C as well as H 1 with the standard error in parenthesizes.


Felsing D 14 Figure 5. Competition binding of 5-APT of WT 5HT 2A 5HT 2B 5HT 2C H 1 GPCRs. The competition binding curves of 5AP T at the four receptors show that it is selective for 5HT 2B > 5HT 2C > 5HT 2A > H 1 receptors. 5 HT2a 5 HT2b 5 HT2c H1 n 5 APT 289 (97) 26 (7) 132 (47) 1068 (118) 2,2,2,2 5 APT mCF3 113 (10) 40 (10) 63 (9) 1448 (8) 3,3,3,3 5 APT mOMe 566 (12) 14 (3) 66 (8) 1160 (30) 2,2,2,2 5 APT mCl 89 16 42 570 1,1,1,1 6 APT 493 (93) 173 (33) 344 (14) 1487 (56) 3,2,3,3 6 APT mCF3 351 (34) 38 (2) 321 (37) 920 (28) 2,2,2,2 6 APT mOMe 410 (67) 315 (5) 945 (51) 875 (165) 2,2,2,2 6 APT mCl 156 (0.5) 153 (54.5) 334 (17.5) 872 (288.5) 2,2,2,2 219 2313 (103) 342 1168 (2) 1378 (256) 2,1,2,2 PAT 410 (38) 1200 (7) 38 (3) 2 (0.5) published Table 3. K i (nM) values of test compounds at WT 5HT 2A 5HT 2B 5HT 2C H 1 GPCRs. Shown in table 3 are the experimentally determined K i values of 5 and 6-APT along with their derivatives at 5HT 2A 5HT 2B 5HT 2C H 1 receptors, the standard error in parenthesizes, and the replication number of each experiment for each compound.


Felsing D 15 These data show that both the 5 and 6-APT compounds are selective for 5HT 2 receptors over H 1 The 5-APT compounds in general, have around 10-fold increased affinities for the 5HT 2C over H 1 receptor. The 6-APT compounds only show a modest 23 fold increase in affinities for 5HT 2B and 5HT 2C receptors over H 1 the only exception being the 6-APT mCF 3 compound. This compound showed a 10-fold increase in affinity over the other 6-APT compounds for the 5HT 2B receptor. This compound along with 5-APT compounds showed a 30-fold increase in affinity for 5HT 2B over H 1 The only APT compounds to show a statistical difference in affinity for 5HT 2A receptor were the 5 and 6APT mCl compounds. These showed a 4-fold higher affinity for 5HT 2A over the other APT compounds. The 219 compound showed a decrease in affinity from the APT compounds of 6-fold for 5HT 2A and 4 fold for 5HT 2C However the 219 compound, without a pendant phenyl ring, showed no statistical difference in affinities for the H 1 receptor. Ligand H 1 affinit y 5 APT 3090 (410) 5 APT mCF 3 6270 (210) 5 APT mOMe 3200 (90) 5 APT mCl 2650 (50) 6 APT 9570 (730) 6 APT mCF 3 2740 (270) 6 APT mOMe 1220 (270) 6 APT mCl 1310 (140) Table 4. Reported K i Data for test compounds at H 1 GPCR. 1 In table 4 are the published K i values 31 of the APT derivatives at the H 1 receptor, along with their associated standard errors. These published data show the lack of binding


Felsing D 16 affinity of the 5-APT compounds for the H 1 receptor. Also, that electro negative meta substituents of the 6-APT compounds increase affinity 37 -fold for the H 1 receptor. Discussion The main finding of these studies was that the APT compounds had high affinity for the 5HT 2 receptors, whereas there affinity for the H 1 receptor was relatively poor most 1 M. The 5-APT compound and its derivatives showed a slightly increased affinity for both 5HT 2C and 5HT 2B over their 6-APT counterparts Substituents at the meta-position enhanced the affinity of the 6-APT compounds for the H 1 receptor. The data for 5HT 2A seem to show no difference between that of either the 5-APT 6-APT, PAT or their derivatives. However, the 219 compound shows that a pendant phenyl ring increases binding for 5HT 2A 5 -fold. The APT compounds showed an increased affinity for 5-HT 2B over the other receptors studied. Substituents at the meta-position seem t o enhance affinity of the 6-APT compounds for the H 1 receptor. For example, 6-APT generated the worst binding of 1487 nM. It s derivatives maintained a consistently 2-fold higher affinity, also reaffirmed by published data Substituents on the 5-APT structures however, did not significantly affect H 1 binding as some were both slightly attenuated and slightly increased Even though subtle differences in affinities between the APT compounds exist, none of the APT compounds for H 1 receptor were near that of PAT, but were reduced by three orders of magnitude. This difference suggests major steric implications dependant on the position of the pendant phenyl ring for H 1 affini ty this difference could help enable exclusion of H 1 mediated side effects of new drugs. A more rigid structure locked into position, limits


Felsing D 17 binding to H 1 to a greater extent than binding to the 5HT 2 receptors. This aspect gives clues into differences of the binding pockets between 5HT 2 and H 1 receptors, as binding of APT compounds was not hindered for 5HT 2 receptors nearly as much as the binding to the H 1 receptors. Th e 5-APT compounds show a greater specificity for the 5HT 2 receptors compared to the 6-APT compounds; and therefore produce a greater chance of being a therapeutic tool This makes sense, in a 3D view, 5-APT derivatives closely resembles that of PAT, the position in space of the metasubstituents of the pendant phenyl ring is almost identical The movement of the phenyl ring to a locked position (i.e. APTs vs. PATs) does not affect the affinity of the 5HT 2 receptors nearly as much as the H 1 receptors. Specificity for the 5HT 2C and 5HT 2B receptors seems to be lost as the pendant phenyl moves further away from its position in PAT (i.e. 5-APT vs. 6-APT). Polar substituents in the meta position of the 5-APT compounds increase affinity for the 5HT 2C receptor by 2 to 3 -fold, y et that does not seem to be the case for the 6-APT compounds, as no statistical difference was observed. The 5-APT compounds show a much higher selectivity in binding for 5HT 2B receptor than does PAT, with differences in affinities around 20 -fold. This difference provides insight to the model of the 5HT 2B receptor as to which not much is known. A mere position change of the pendant phenyl must remove a sizable steric clash and or form a new interaction with undetermined amino acids. Most likely, a steric clash between the pendant phenyl and amino acid was relieved and allowed the compounds to gain access and bind easier to 5HT 2B Even though these compounds are not that different in position of atoms in a space filling model, the affinities drastically differ. This removal of a steric clash is validated further ity affinity


Felsing D 18 for 5HT 2B over the other receptors is 4 to 10 -fold, and shows that the pendant phenyl is not as necessary for binding to 5HT 2B but may impede it depending on position. Although the halogenated APT compounds did improve affinity at 5HT 2B some up to 10 -fold. This suggests that the phenyl ring, which changed position in the APT compounds, may also positively contribute to affinity. The pendant phenyl ring was proven vital for the binding of APTs and PATs to 5HT 2A and 5HT 2C receptors. Binding was reduced around 10-fold for these receptors when the pendant phenyl ring was removed, i.e. for compound 219. This supports the claim that ce rtain aromatic amino acids, like the ones previously mentioned in the fifth and sixth transmembrane domains, could likely be important in forming interactions to stabilize the ligand-receptor complex and increase binding with PAT and APT compound derivatives. The experimental data and published data of the ligands for the H 1 receptor agree in trend of K i values. The ligands with the worst affinity were 6-APT 5-APT mCF3, and 5-APT Those with the best affinity were 5 and 6-APT mCl and 6-APT mOMe. Previously published data values were obtained using [ 3 H] mepyramine and stable producing Chinese hamster ovarian (CHO) cells to clone H 1 receptors of guinea pig brain. Slight variants in binding and function are known to occur between similar receptors of different species, such as human H 1 vs rat H 1 or guinea pig H 1 xxxiv Although K i values are not identical, which could be due to a number of variants in methods, trends still agree. One reason that may explain the discrepancy is the difference in dilutions, H 1 binding may have been over-estimate d due to lack of dilute solution, only up to 10nM concentrations of compound were used.


Felsing D 19 The 5-APT m CF 3 and 5-APT mCl show enough selectivity for 5HT 2 receptors that they may present a viable candidate depending on function analysis. These must be analyzed for function to show if the deleterious side effects of 5HT 2A and 5HT 2B activation occur as it binds with high affinity to these receptors. Although little variance in structure would suggest similar function to that of PAT for the 5HT 2 receptors. Future studies: Finish the 3n, possibly more for publication. Determine function of viable drug targets at wild type receptors using Phospholipase C or Inositiol Phosphate assay. Determine useful mutant strains of receptors to guide molecular modeling. Methods Test Ligands: The ligands tested were racemic mixtures of both ( +/ -) isomers. The nine ligands tested were 2-diethylamino-5 or 6-phenyl-1,2,3,4-tetrahdronapthylene derivatives synthesized by Dr. Ray 2-diethylamino-5-phenyl-1,2,3,4-tetrahydronapthylene 2-diethylamino-5-phenyl-mCF 3 -1,2,3,4-tetrahydronapthylene 2-diethylamino-5-phenyl-mOCH 3 -1,2,3,4-tetrahydronapthylene 2-diethylamino-5-phenyl-mCl-1,2,3,4-tetrahydronapthylene 2-diethylamino-6-phenyl-1,2,3,4-tetrahydronapthylene 2-diethylamino-6-phenyl-mCF 3 -1,2,3,4-tetrahydronapthylene 2-diethylamino-6-phenyl-mOCH 3 -1,2,3,4-tetrahydronapthylene 2-diethylamino-6-phenyl-mCl-1,2,3,4-tetrahydronapthylene


Felsing D 20 2-diethylamino-1,2,3,4-tetrahydronapthylene (219) cDNA isolation: A population of E. Coli bacteria containing a plasmid for one of each of the four wild HT 2A 5HT 2B 5HT 2C and H 1 ) was grown in LB broth for 18 hours. A Promega PureYield TM Plasmid Midiprep kit was used according to manufacturers protocol to isolate complementary DNA (cDNA) for future transfection. The quality and concentration of the cDNA was determined by obtaining spectrophotometric readings at 280 and 260 nm. Only cDNA with excellent purity (i.e. 1.8-2.0 of A 260 A 280 ) and yield (>0.4 g L was used for transfections. Cell Line: Human Embryonic Kidney (HEK) cells were grown in 10cm plates in sterile Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% antibiotic solution at 37 C in an incubator, and were transfected with 5HT 2A 5HT 2B 5HT 2C or H 1 DNA when they reached 75% confluenc y. Transient transfections: HEK cells were transfect this experiment. For each plate of cells transfected, two 14ml Falcon tubes were filled with 2.5mL of filtered optiMEM, in one tube 20 g of cDNA of the desired GPCR and in the other 40 L of lipofectamine 2000 reagent bought from Invitrogen, in a sterile fume hood. The two tubes were allowed to incubate at room temperature for 5 minutes then combined and mixed. The mixture was allowed to incubate for another 20 minutes when 5ml of DMEM containing 5% dialyzed FBS was added. HEK cells were carefully washed, by slowly adding as to not dislodge the cells from binding to the plate, with


Felsing D 21 Buffered Saline (DPBS). The DPBS was aspirated and the combined 10mL mixture of cDNA, lipofectamine, optiMEM, and DMEM containing 5% dialyzed FBS was again carefully added as to not disrupt the HEK cells. The cells were then placed back in the incubator for 48 hours to allow high expression levels of the receptor. Membrane Harvesting: The membranes of the transfected HEK cells were harvested approximately 48 hours after transfection. The cells were dislodged from the plate by vigorously pipetting assay buffer (50mM Tris HCl, 10mM MgCl 2 6H 2 O, 0.1mM EDTA at pH 7.3) onto the cells repeatedly until all the cells have been dislodged from the plate. The solution containing the transfected cells were placed in a centrifuge tube and centrifuged. The centrifuge tube was drained and 10mL of new ice-cold assay buffer was added to the cell pellet and homogenized by a Tekmar Tissumizer. The homogenized solution was centrifuged. The tube was drained and 2.5mL of new assay buffer was added to the cell pellet and homogenized. The membrane sample was then labeled and stored in an 80C freezer until use in a binding assay. Bradford Assay: Bradford reagent was obtained through a Pierce BCA Protein assay kit bought from Thermo Scientific, and manufacturers protocol was followed. A 50:1 ratio of reagents was made and added to 25 L of homogenized membrane preparation to dilute 8 times. The mixture was incubated at 37 C for 30 minutes and measurements were made spectrophotometrically at 562nm, and compared to a standard curve made from BSA In -vitro binding assays: Compounds:


Felsing D 22 Mianserin hydrochloride and tripolidine hydrochloride were both purchased from Sigma Aldrich. The radioligands [ 3 H] -mesulergine (specific activity 85.4 Ci mmol, Lot #6 442 68) were used for 5HT 2B and 5HT 2C assays, [ 3 H] -ketanserin (specific activity 53.4 Ci mmol, Lot #621931) was used for 5HT 2A as says, the radioligand [ 3 H] -mepyramine (specific activity 37 Ci mmol, Lot #632150) was used for H 1 assays. The nine compounds for testing were previously synthesized by established methods. 29 Figure 6 Mesulergine, radioligand used for 5HT 2B and 5HT 2C receptors. Figure 7. Ketanserin, radioligand used for 5HT 2A receptor.


Felsing D 23 Figure 8. Mepyramine, radioligand used for H 1 receptor. Saturation binding assays: 5HT 2A 5HT 2B 5HT 2C and H 1 receptor binding site density (B max ) and affinity (K d ) were determined with [ 3 H] -ketanserin, [ 3 H] -mesulergine, [ 3 H] -mesulergine, and [ 3 H] mepyramine, respectively. Membrane preparations were analyzed for protein concentration using a Bradford assay while comparing that to a standard curve of Bovine Serum Albumin (BSA). The membranes were incubated with 7 varying concentrations of their associated radioligand (0.1-10nM) for 60 minutes on a rotator at room temperature to determine their total binding. Nonspecific binding was determined by using 40 M concentrations of mianserin for the 5HT 2 receptors and tripolidine for the H 1 receptor. After incubation, free radioligand was separated from bound radioligand by rapid vacuum filtration using Mach 2 cell harvester, with GF/B grade filters presoaked with 0.3% polyethyleneimine. Filters with membrane bound radioligand were placed in vials with 2mL of scintillation cocktail (ScintiVerse), and placed in a Beckman LS6500 liquid


Felsing D 24 scintillation counter 24 hours later for 3 H liquid scintillation. All measurements were done in triplicate. Competition binding assays: The inhibitory affinity (K i ) of the previously mentioned nine compounds was measured for 5HT 2A 5HT 2B 5HT 2C and H 1 receptors using [ 3 H] -ketanserin, [ 3 H] mesulergine, [ 3 H] -mesulergine, and [ 3 H] -pyrilamine as the radioligands, respectively. The four associated membranes were incubated with their K d concentrations of radioligand 2.25, 1.9, 1.4, and 0.8nM respectively, and a series of 7 dilutions, (from 0.1-10,000 nM) for each test compound,. The membranes incubated for 60 minutes on a rotator at room temperature to determine their total binding. Nonspecific binding was determined by using 40 M concentrations of mianserin and tripolidine for 5HT 2 receptors and H 1 receptor respectively. After incubation, free radioligand was separated from bound radioligand by rapid vacuum filtration using Mach 2 cell harvester, with GF/B grade filters presoaked with 0.3% polyethyleneimine. Filters with membrane bound radioligand were placed in vials with 2mL of scintillation cocktail and counted in a Beckman LS6500 liquid scintillation counter 24 hours later for 3 H liquid scintillation. All measurements were done in triplicate. Data Analysis: Binding data was analyzed by fitting a non-linear regression curve in Graphpad 5.2 to obtain K d and B max values for saturation binding and IC 50 values for competition binding of each ligand at 5HT 2A 5HT 2B 5HT 2C H 1 receptors O ne-site K i model was used for the nonlinear regression test. Ligand affinity for the receptors in competition assays was transformed from IC 50 into K i values by the equation:


Felsing D 25 Figure 9. Where L is the concentration of radioligand used, and K d affinity for an individual receptor. Radioligand K d values were obtained individually, but the values used in analysis of competition binding data, found on PDSP, were 2.25, 1.95, 1.40, 0.80 nM respectively. 2 8 Actual concentrations of radioligand delivered were determined for analysis by counting 25 L of the radioligand stock solution in 2mL of scintillation cocktail and converted from cpm to concentration via Graphpad 5.2.


Felsing D 26 Referenc es i Center for Disease Control and Prevention. ii Fox M. Study predicts 75 percent overweight in US by 2015. Reuters 2007 Jul 19. iii National Institite on Drug Abuse. DrugPages Cocaine.html, DrugPages Methamphetamine.html. iv Ho yer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saexena PR, Humphrey PA. International Union of Pharmacology Classification of Receptors for 5Hydroxytryptamine (Serotonin). The American Society for Pharmacology and Experimental Therapeutics. 1994, 46 (2):158-193. v Saltzman AG, Mores B, Withman MM, Ivanshchenko Y, Jaye M, Felder S. Cloning of the human serotonin 5-HT2 and 5-HT1C receptor subtypes. Biochem Biophys Res Commun. 1991, 181:1469-7148. vi Ra ymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, Grewal JS, Garnovskaya MN. Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther. 200, 92:179-212. vii Eberle-Wang K, Mikeladze Z, Uryu K, Chesselet MF, Pattern of expression of the serotonin 2C receptor messenger RNA in the basal ganglia of adult rats. J Comp Nuerology. 1997, 384(2):233-247 viii Pasqualetti M, Ori M, Nardi I, Castagna M, Cassano GB, Marazziti D, Distribution and cellular localization of the serotonin type 2C receptor messenger RNA in human brain. Neuroscience. 1999, 92: 60111. ix De Deurwaerdre P Navailles S Berg KA, Clarke WP Spampinato U Constitutive activity of the serotonin2C receptor inhibits in vivo dopamine release in the rat striatum an d nucleus accumbens. J Neurosci. 2004, 24(13):323541. x Di Matteo V, Di Giovanni G, Di Mascio M, Esposito E. Biochemical and electrophysiological evidence that RO 60-0175 inhibits mesolimbic dopaminergic function through serotonin(2C) receptors. Brain Res. 2000, 865:8590. xi Enhanced locomotor, reinforcing, and neurochemical effects of cocaine in serotonin 5hydroxytryptamine 2C receptor mutant mice. J Neurosci 2002, 22:10039 10045. xii Fletcher PJ, Sinyard J, Higgins GA. The effects of the 5HT 2C receptor antagonist SB242084 on locomotor activity induced by selective, or mixed, indirect serotonergic and dopaminergic agonists. Psychopharmacology 2006, 187:51525. xiii Weiner DM, Burstein ES, Nash N, Croston GE, Currier EA, Vanover KE, Harves, Donahue E, Hansen HC, Anderson SM, et al. 5-Hydroxytryptamine 2A receptor inverse agonists as anti ps ychotics. J pharmacol Ecp Ther 2001 299: 26876. xiv Gonzalez-Maeso J, Weisstaub NV, Zhou M, Chan P, Ivic L, Ang R, Lira A, Bradley-Moore M. Ge Y, Zhou Q, SEalfon SC, Gingrach JA, Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior. Neuron. 2007 53:439-52. xv Sharif NA, McLaughlin MA, Kelly CR. AL-34662: a potent, selective, and efficacious ocular hypotensive serotonin-2 receptor agonist J of ocular pharmacology and therapeutics 2007, 23: 1 13.


Felsing D 27 xvi Van der Starre PJ, Solinas C. Ketanserin in the treatment of protamine-induced pulmonary hypertension. Texas Heart Institute journal 1996, 23: 301 304. xvii Launay HUmbert M, Simonneau G, et al. Function of the serotonin 5-hydroxy tr yptamine 2B receptor in pulmonary hypertension. Nat Med 2002, 8: 1129-1135. xviii Fitzgerald LW, Burn TC, Brown BS, PATterson JP, Corjay MH, Valentine PA, Sun JH, Link JR, Abbaszade I, Hollis JM, et al. Possible role of valvular serotonin 5-HT(2B) receptor in the cardiopathy associated with fenfluramine. Mol Pharmacol. 2000, 57: 7581. xix Xu Y, Jones JE, Lauzon DA, Anderson JG, Balthasar N, Heisler LK, Zinn AR, Lowell BB, Elmquist JK. A serotonin and melanocortin circuit mediates D-fenfluramine anorexia J Neurosci. 2010, 30(44):146304. xx Larken C. FDA staff says Arena diet pill linked to cancer. Sept 15 2010. xxi Smith SR, Prosser W, Donahue D, Anderson C, Shanahan W. Lorcaserin Phase 2b clinical Stud. American Diabetes Association, 200 6. xxii Jensen MD. Potential role of new therapies in modifying cardiovascular risk in overweight patients with metabolic risk factors. Obesity. 2006, 14: 143S-149S. xxiii Hill SJ, Ganellin CR, Timmerman H, et al. International Union of Pharmacology. XIII. Classification of histamine receptors. Pharmacol. Rev. 1997, 49 (3): 253 78. xxiv Zhang, M. Q., Leurs, R., Timmerman, H. HistamineH1receptor antagonists. In Burgers Medicinal Chemistry and Drug Discovery, 5thed; Wol M. E., Ed.; John Wiley: New York, 1997, 5:495 559. xxv Kroese WK, Hufeisen SJ, Popadak BA, Renock SM, Steinber S, Ernsberger P, Jathilake K, Melzer H, Roth BL. H1 -histamine receptor affinity predicts short-term weight gain for typical and atypical antipsychotic drugs. 2003 28(3):519-26 xxvi : xxvii Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin, G.R., Mylecharane, E.J., Saxena, P.R., Humphrey, P.P. International Union of Pharmacology classification of receptors for 5hydroxytryptamine (Serotonin). Pharmacol Rev. 1994, 46:157-2033 xxviii PDSP, Psychoactive Drug Screening Program; 2005, 2010. BL Roth, Director. NIMH Contract NO2MH80002, University of North Caroline, Chapel Hill, NC. http: xxix Ghoneim OM, Jacqueline AL, Golbraikh A, Tropsha A, and Booth R.G. Novel ligands for the human histamine H 1 receptor: Synthesis, pharmacology, of 2-dimethylamino-5-(6)-phenyl-1,2,3,4tetrahydronaphthalenes xxx Kristiansen, K., Dahl, S.G. Molecular modeling of serotonin, ketanserin, ritanserin and their 5-HT2C receptor interactions. Eur J Pharmacol. 1996, 306: 195210. xxxi Roth, B.L., Shoham, M., Choudhary, M.S., Khan, N. Identification of conserved aromatic residues essential for agonist binding and second messenger production at 5hydroxytryptamine2A receptors. Mol Pharmacol. 1997, 52: 259-66.


Felsing D 28 xxxii Choudhary MS, Craigo S, Roth BL. Identification of receptor domains that modify ligand binding to 5-hydroxytryptamine2 and 5-hydroxytryptamine1c receptors. Mol Pharmacol. 1992, 42: 627-633. xxxiii Almaula N, Ebersole BJ, Ballesteros JA, Weinstein H, and Sealfon SC. Contribution of a Helix 5 locus to selectivity of Hacinogenic and Nonallucinogenic Ligands for the Human 5Hydroxytraptamine 2A and 5-Hydroxytraptamine 2C Receptors: Direct and Indirect effects on Ligand Affinity Mediated b the Same Locus. Molecular Pharmacolgy. 1996, 50:3442. xxxiv Straer A, Wittmann HJ Kunze M, Elz S, Seifert R Molecular Basis for the Selective Interaction of Synthetic Agonists with the Human Histamine H1-Receptor Compared with the Guinea Pig H1-Receptor. Mol Pharmacolog. 2009, 75:454 465.