Mechanism of Off-Target Interactions and Toxicity ... - ACS Publications

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Mechanism of Off-Target Interactions and Toxicity of Tamoxifen and Its Metabolites Maria Flynn,† Kali Amelia Heale,† and Laleh Alisaraie*,†,‡ †

School of Pharmacy, Memorial University of Newfoundland, A1B 3V6 St. John’s, Newfoundland, Canada Department of Chemistry, Memorial University of Newfoundland, A1B 3X7 St. John’s, Newfoundland, Canada

‡

S Supporting Information *

ABSTRACT: Tamoxifen is an estrogen modulator that acts to competitively inhibit the binding of endogenous estrogens. It is widely used for treatment of breast cancer; however, analogous with many antineoplastic agents, tamoxifen is associated with numerous adverse effects, most prominently nausea. We have identified several off-target receptors of tamoxifen and 22 of its metabolites that include histamine H1 and H3, and muscarinic M1, M4, and M5 subtypes, and dopamine D2 receptor. We have shown how they are associated with tamoxifen and its metabolites’ toxicity through a comprehensive computational analysis of their interaction modes, which were also compared to that of the related endogenous substrates of each receptor. The results were further evaluated using available in vivo and in vitro data. The presented work provides foundational knowledge toward the determination of the precise mechanism of nausea induction, and in particular, interactions of tamoxifen and its metabolites with the receptors involved in that biomolecular pathway. This study can assist in predicting the potential undesired effects of the chemicals with common pharmacophores or similar fragments to that of tamoxifen and its metabolites and serve drug discovery research in developing more effective and tolerable tamoxifen analogues or chemotherapeutic agents.

1. INTRODUCTION Breast cancer is estimated to be the most common type of cancer diagnosis in females and one of the leading causes of cancer-related death in women.1,2 Breast cancer is highly heterogeneous. The molecular expression profiles allow for classification into subgroups, based upon the expression of hormone receptors (estrogen and progesterone) and human epidermal growth factor, which aids in the determination of disease prognosis.1 Tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]ethyldimethylamine citrate) is a selective estrogen receptor modulator (SERM) utilized in the treatment of early and advanced stages of estrogen receptor-positive breast cancer, in both pre- and postmenopausal females, as well as in males.3 Tamoxifen is used as adjuvant therapy in patients receiving primary treatment of surgery or radiation. Also, it may be used for breast cancer prevention in individuals deemed high-risk.4,5 SERMs exert their actions in a tissue-specific manner. This class of drugs may act to agonize or antagonize the estrogen receptors, depending on the presence of estrogen in the tissue. Tamoxifen exerts its main antineoplastic effects through its antiestrogenic effects.6 The drug acts to competitively inhibit the binding of endogenous estrogens to the estrogen receptors, thus reducing estrogen-mediated cell proliferation.6 Transcription activation of the estrogen receptor is mediated by two separate activation functions, AF-1 and AF-2. AF-1, located in the N-terminus, is activated via growth factors of the MAP kinase signaling pathway. AF-2, within the C-terminus, is activated by agonist binding. © 2017 American Chemical Society

Tamoxifen binds within the C-terminal ligand binding domain of the nuclear estrogen receptors, thus binding within the same site of the endogenous ligand, estradiol.7 Tamoxifen binding causes a conformational change in the receptor protein, which varies from that of the endogenous ligand via its α12 helix (residue 536−544 in ER α). α12 binds to hydrophobic residues of helices α3 (residues 342−361) and α5 (residues 372−394). This conformational change results in a partial blockade of the AF-1 binding site, prohibiting coactivator binding.7 Thus, the receptor is inactivated, preventing gene activation and mRNA transcription.8 Toxicity calculations carried out using the ADMET Predictor determined tamoxifen and the vast majority of its metabolites to be toxic at the estrogen receptor in rats, ranging from a 49−98% chance of toxicity. This calculation further verifies the mechanism of action of this chemical compound. The agonistic properties of tamoxifen at the estrogen receptors are responsible for some favorable physiological effects, such as slowing bone loss and reducing serum lipid levels.6 Analogous with many antineoplastic agents, tamoxifen is associated with numerous adverse effects. Increased risk of cardiovascular events, such as stroke, pulmonary embolism, and deep vein thrombosis,5,9 as well as endometrial cancer, have been reported.9 Symptomatic, non-life-threatening adverse effects such as vasomotor symptoms (hot flashes), increased vaginal discharge, and gastrointestinal irritation (nausea, Received: May 1, 2017 Published: May 31, 2017 1492

DOI: 10.1021/acs.chemrestox.7b00112 Chem. Res. Toxicol. 2017, 30, 1492−1507

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Chemical Research in Toxicology Table 1. Possible Metabolites of Tamoxifen Formed through Enzymatic Biotransformation3

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Chemical Research in Toxicology Table 1. continued

diarrhea) have also been observed.9 In general, nausea is the predominant adverse effect associated with this drug and antineoplastic agents in general. The numerous metabolites of tamoxifen have the potential to interact with a variety of offtarget receptors within the body, indicating a possible mechanism of the mediation of drug toxicity which will be further discussed within this work.

gradient of 0.001 kJ/mol, the quality of the structures was assessed using PROCHECK18 to evaluate the stereochemical quality and analyze the residue geometry and protein tertiary structure.18 The PROCHECK assessments confirmed the quality of the minimized structures based on, for instance, the high percentage of the residues being in the core regions of the Ramachandran plot. Accordingly, D2 structure has 89.5% of its residues in the core and 9.3% in the allowed region, H3 has 92.9% in the core and 5.7% in the allowed, M1 has 93.3% in the core and 4.7% in the allowed, M4 has 94.4% in the core and 4.6% in the allowed and as well, M5 has 93.5% of the residues in the core and 4.5% in the allowed region. It is noteworthy that the crystal structures of the M1 and M4 receptors were recently solved. Thus, they also were acquired form the Protein Data Bank, M1 (5DSG PDB code)19 and M4 (5CXV PDB code),19 and compared with the folding of the minimized homology model of the respective receptors. We observed a very low root-mean-square deviation (RMSD < ∼1.0 Å) and a high percentage of identity (above 98%) in their folding. Also, the layout of the residues particularly in the binding sites were almost identical, which further confirmed the high quality of the modeled M1 and M4 structures and thus the accuracy of the modeling protocol employed for the simulation of D2 and H3. Proteins retrieved from the Protein Data Bank contained water molecules. Thus, for the preparation of the proteins structures for ligand docking, any water molecules present were removed; polar hydrogens were added to the proteins while nonpolar hydrogens were excluded. The ligand structures were built up as all-atom entities and were energy minimized using the Tripos force field. The library of tamoxifen and its metabolites, as well as the natural substrates, were docked into the protein binding site in separate docking experiments using FlexX 3.1 embedded in the LeadIT software package (v.2.1.3).20−22 The protein−ligand interactions were predicted by FlexX through the use of its incremental construction strategy. There are three phases to the FlexX docking algorithm including selection of a set of base fragments, placing the base fragments into the active site, and constructing the complex incrementally and calculating the interaction

2. MATERIALS AND METHODS A library of tamoxifen and 22 possible metabolites3 were built up using Sybyl-X 2.1.1 (www.certara.com) (Table 1). Each metabolite was minimized using the Tripos force field with a 0.001 kJ/mol energy gradient through 1000 iterations. The structure of histamine, dopamine, acetylcholine, and 17β-estradiol (E2), the endogenous substrate of each receptor tested, were also built up and minimized using the Tripos force field embedded in the Sybyl-X software package. The structure of each protein was acquired through either the Protein Data Bank (PDB)10 or homology modeling. The crystal structure of estrogen receptor α (3ERT), with a resolution of 1.90 Å,7 was retrieved from the PDB,10 along with that of estrogen receptor β (2FSZ) with a resolution of 2.20 Å,11 and the histamine H1 receptor (3RZE) with a 3.10 Å resolution.12 The structures of the histamine H3 receptor with Uniprot13 sequence codes of (Q9Y5N1), dopamine D2 receptor (P14416), and muscarinic receptors M1 (P11229), M4 (P08173), and M5 (P08912) were determined via homology modeling, which was performed using the Phyre Protein Homology Recognition Engine.14 D2 was simulated based on the β-2-adrenergic receptor as the template (2RH1)15 and muscarinic receptors based on the structure of human M2 muscarinic acetylcholine receptor (3UON)16 as the template, all of which was done with 100% prediction confidence as rated by the Phyre. The templates were used to model the target proteins using the SWISS-MODEL workspace.17 Following the removal of geometric strain of the modeled structures by energy minimization using the Tripos force field with an energy 1494


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Chemical Research in Toxicology energies via a Böhm scoring function that results in ranking the docking solutions.20−22 The docking solution with highest binding energy was further studied to analyze each ligand binding profile against its targeted protein. The residues in a spherical region surrounding each ligand’s center-of-mass were set up as the binding region. To determine the best conformation of each ligand with the lowest binding energy and the highest interactions with the binding site, the radius around the center was optimized. Thus, for each ligand the optimized binding site radius varied depending on the ligand chemical structure and size as well as the target proteins’ structure and the potential involving residues effective on the interactions with the ligand. The binding site of agonists and antagonists of estrogen receptor α is located within the C-terminal ligand binding domain. The hydrophobic binding pocket is formed by helices α3 (residues 342− 361), α8 (residues 424−438), and α11 (residues 497−526).7 (Z)-4Hydroxy-tamoxifen is part of the PDB structure 3ERT; an area of 6.5 Å radius around this reference ligand was used for the determination of the binding site for the docking ligands in the library. The second estrogen receptor, estrogen receptor β, contains two possible binding sites for tamoxifen and its metabolites: the C-terminal ligand binding domain and an allosteric binding site.11 The second binding site overlaps with the coactivator binding site. The PDB structure (2FSZ) contains 4-hydroxy-tamoxifen in both possible binding sites; a 6.5 Å radius around this reference ligand was used as the target area for docking. In the dopamine D2 receptor, the agonist binding site is defined by helices α3 (residues 104−137), α4 (residues 151−171), α5 (residues 187−220), and α6 (residues 336−369)23,24 (Figure 1). A 10 Å radius around Asp114 of the D2 receptor was used to define the binding site, as this amino acid is known to form a salt bridge with the amino group of dopamine.23,24The G-protein coupled histamine H1 receptor contains a hydrophobic pocket formed by the α3 (residues 96−130), α5 (residues 188−216), and α6 helices (residues 408−441)12 (Figure 1). The PDB structure contains the antagonist doxepin ((3E)-3(dibenzo[b,e]oxepin-11(6H)-ylidene)-N,N-dimethylpropan-1-amine) bound to the ligand binding site. The structure of doxepin is similar to that of tamoxifen and the metabolites as it contains a ring system with two aromatic rings, a double bond between the rings and a short carbon chain with a terminal tertiary amine, similar to tamoxifen and some of its metabolites. As tamoxifen acts to antagonize this receptor as well, a radius of 10 Å around this reference ligand was used as the target area for the docking of histamine, tamoxifen, and its metabolites. The histamine H3 receptor contains a hydrophobic binding pocket for both agonists and antagonists, formed by transmembrane helices α3 (residues 104−137), α4 (residues 141−178), and α5 (residues 196−225).25 A 10 Å radius around the Tyr115 aromatic residue was used as the target site for docking (Figure 1). The antagonist binding site of the muscarinic M1 receptor is defined by helices α3 (residues 95−128), α4 (residues 139−168), α5 (residues 181−214), α6 (residues 362−390), and α7 (residues 397−432). Acetylcholine binds to a site between transmembrane helices α3, α6, and α7.26 To define the binding site of the M1 receptor, Asp105 was used as the center of the 10 Å radius sphere in the binding pocket. This amino acid was chosen as it is critical for both agonist and antagonist binding, and is involved in electrostatic interactions with amino groups found in agonists and antagonists.26 The M4 receptor binding site was defined by a 10 Å radius around Asp78. The antagonist binding site of the M5 receptor is located between transmembrane α2 (residues 62- 93), α3 (residues 99−133), α4 (residues 142−166), α5 (residues 189−215), α6 (residues 435− 467), and α7 (residues 472−495). Asp110 was chosen as the center of a 10 Å radius spherical region in the pocket and is involved in both electrostatic interactions and hydrogen bonds with agonists and antagonists.27 ADMET Predictor version 7.2.0001, developed by Simulation Plus (www.simulations-plus.com), was utilized to preform calculations to aid in the determination of various pharmacokinetic and pharmacodynamic properties of tamoxifen and its known metabolites under

Figure 1. Binding sites and their importance involving helices for accommodation of tamoxifen and its metabolites in (A) dopamine D2 receptor, (B) histamine H1 receptor, (C) histamine H3 receptor, (D) muscarinic M1 receptor, (E) muscarinic M4 receptor, and (F) muscarinic M5 receptor. physiological conditions with pH 7.4. The Metabolism Module in ADMET Predictor contains human P450 enzyme kinetic models for five important recombinant CYP isozymes: 1A2, 2C9, 2C19, 2D6, and 3A4. 1495


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Chemical Research in Toxicology Table 2. Calculated Values of Selected ADME Properties of Tamoxifen and Its Metabolites

tamoxifen (E)/(Z)endoxifen (E)/(Z)4hydroxytamoxifen N-desmethyltamoxifen (E)/(Z)-4′hydroxytamoxifen 3-hydroxytamoxifen α-hydroxytamoxifen (E)/(Z)-4′-hydroxy-Ndesmethyltamoxifen 3-hydroxy-Ndesmethyltamoxifen α-hydroxy-Ndesmethyltamoxifen tamoxifen-N-oxide N,Ndidesmethyltamoxifen N-desmethyltamoxifen3-O-glucuronide (E)/(Z)-Ndesmethyltamoxifen4-O-glucuronide (E)/(Z)-tamoxifen-4O-glucuronide tamoxifen-3-Oglucuronide tamoxifen-Nglucuronide

human jejunal effective permeability (cm/s × 10−4)

Vd (L/kg)

% unbound to plasma proteins

6.64 5.50 5.97

5.62 2.43 4.10

5.06 3.54 4.35

1.57 2.00 1.35

5.53 3.64 4.86

high high low

1.02 0.87 0.91

0.15 0.53

6.12 5.88

4.27 4.14

5.44 4.38

1.97 1.42

4.15 4.75

high low

1.00 0.87

0.0379 0.0575 0.104

0.541 0.544 0.153

6.01 5.12 5.44

3.93 3.84 2.57

4.31 4.26 3.67

1.35 2.20 2.06

4.93 4.02 3.55

low high high

0.92 0.80 0.83

0.0192

0.0898

0.152

5.51

2.27

3.50

2.04

3.69

high

0.88

0.0411

0.0575

0.164

4.90

2.82

4.12

2.91

2.96

high

0.80

0.116 0.0189

0.151 0.103

0.164 0.177

3.21 5.36

0.62 3.41

1.56 4.50

15.49 3.01

1.07 3.61

low high

0.34 0.65

1.56

0.122

0.122

1.76

0.26

0.73

7.75

1.75

low

−1.10

1.51

0.109

0.0727

1.82

0.26

0.72

7.49

1.81

low

−1.16

3.32

0.0839

0.149

2.17

0.29

0.73

5.53

2.14

low

−1.19

3.35

0.0941

0.18

2.12

0.29

0.73

5.68

2.08

low

−1.14

2.62

2.09

0.11

−0.54

0.25

0.35

37.76

−0.54

low

−1.49

solubility in simulated fasted state gastric fluid (mg/mL)

solubility in simulated fasted state intestinal fluid (mg/mL)

solubility in simulated fed state intestinal fluid (mg/mL)

0.121 0.0195 0.313

0.0152 0.0947 0.0407

0.561 0.151 0.534

0.005 0.354

0.0343 0.0459

0.299 0.788 0.021

log P

3. RESULTS AND DISCUSSION 3.1. ADMET Properties of Tamoxifen. As the required ADMET properties of tamoxifen and its metabolites have not been experimentally determined, we have assessed them using ADMET predictor. The results demonstrate that tamoxifen absorption after oral administration occurs slowly, with peak serum concentrations present 3−6 h post single-dose administration.28 Solubility of the drug molecule in the gastrointestinal tract is crucial in promoting absorption; we calculated the solubility of tamoxifen in both gastric and intestinal fluid, as well as in both fed and fasted states (Table 2). The solubility in simulated fasted state gastric fluid is 1.21 × 10−1 mg/mL, while simulated fasted intestinal fluid is 1.52 × 10−2 mg/mL, and simulated fed intestinal fluid is 5.61 × 10−1 mg/mL. These results indicate a better dissolution, thus better absorption, of the drug in the fed state. The lipophilicity of the molecule is also an important factor in determining drug absorption, as it is necessary for passage through cellular membranes. log P (the octanol−water partition coefficient) for tamoxifen was calculated to be 6.64, illustrating the degree of its lipophilicity and supporting the experimental evidence of slow oral absorption, as discussed previously. The human jejunal effective permeability value calculated for tamoxifen was 5.62 × 10−4 cm/s; this value illustrates the absorption of this drug through this imperative area of the small intestine in terms of surface area permeability for this specific molecule. Tamoxifen displayed the highest level of human jejunal permeability compared to that of other molecules tested (Table 2). The volume of distribution (Vd) of tamoxifen was calculated to be 5.06 L/kg, illustrating that this drug is well distributed

log D

qualitative likelihood of crossing BBB

log brain/ blood partition coefficient

over the whole organism. The Vd values of tamoxifen metabolites ranged from 0.35 L/kg, for tamoxifen-N-glucoronide, to 5.44 L/kg, for N-desmethyltamoxifen, showing each individual metabolite is distributed within body tissues to a varying degree, dependent upon the path of metabolism (Table 2). The Vd and clinical action of the drug is related to the percentage of drug that is unbound to plasma proteins; for tamoxifen, 1.57% was calculated to be unbound, thus displaying a high affinity for the plasma proteins. Metabolites of tamoxifen displayed similar binding patterns, with the majority of calculated values between 1.35 and 3.01% unbound. Metabolites of higher polarity displayed reduced binding to plasma proteins (Table 2). The log D value (octanol−water distribution coefficient) of tamoxifen was calculated to be 5.53. Metabolite values ranged from −0.54, tamoxifen-N-glucuronide, to 4.93, 3-hydroxytamoxifen, showing different distributions of metabolites based upon polarity. Calculations of the brain/blood partition coefficient and the qualitative likelihood of crossing the blood−brain barrier (BBB) allow for predictions of the probability of tamoxifen and the metabolites entering the brain and interacting with off-target receptors located within. The qualitative likelihood of crossing the BBB calculation classified tamoxifen and 7 of its metabolites to have high probability (including 3-hydroxy-N-desmethyltamoxifen, αhydroxy-N-desmethyltamoxifen, α-hydroxytamoxifen, N,N-didesmethyl-tamoxifen, (E)/(Z)-4′-hydroxy-N-desmethyltamoxifen, (E)/(Z)-endoxifen, and N-desmethyltamoxifen). The log brain/blood partition coefficient for tamoxifen was 1.02, supporting the hypothesis of the high probability of BBB 1496


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Chemical Research in Toxicology Table 3. Calculated Values of Selected Metabolic Properties of Tamoxifen and Its Metabolites

tamoxifen (E)/(Z)endoxifen (E)/(Z)4hydroxytamoxifen N-desmethyltamoxifen (E)/(Z)-4′hydroxytamoxifen 3-hydroxytamoxifen α-hydroxytamoxifen (E)/(Z)-4′-hydroxy-Ndesmethyltamoxifen 3-hydroxy-Ndesmethyltamoxifen α-hydroxy-Ndesmethyltamoxifen tamoxifen-N-oxide N,Ndidesmethyltamoxifen N-desmethyltamoxifen3-O-glucuronide (E)/(Z)-Ndesmethyltamoxifen4-O-glucuronide (E)/(Z)-tamoxifen-4O-glucuronide tamoxifen-3-Oglucuronide tamoxifen-Nglucuronide

CYP 3A4 substrate

CYP 3A4 Vmax (nmol/min/nmol enzyme)

CYP 3A4 Km (μM)

CYP 3A4 CLint (μL/ min/mg HLM protein)

CYP 2D6 substrate

CYP2D6 Vmax (nmol/min/nmol enzyme)

CYP 2D6 Km (μM)

CYP 2D6 CLint (μL/ min/mg HLM protein)

yes yes yes

4.94 1.52 2.38

23.2 20.7 22.7

64.5 8.15 11.7

yes yes yes

5.36 3.25 10.4

0.664 0.465 2.38

64.5 55.9 35.0

yes yes

3.81 1.71

21.0 18.6

20.2 10.2

yes yes

2.34 13.9

0.138 2.91

136 38.3

yes yes yes

2.80 10.4 1.15

22.8 24.0 16.5

13.6 47.9 7.71

yes yes yes

10.8 12.4 4.20

2.20 3.85 0.585

39.3 25.7 57.4

yes

1.61

20.8

8.59

yes

3.03

0.402

60.3

yes

6.85

20.5

37.1

yes

7.25

0.801

72.4

yes yes

1.35 1.72

42.2 29.4

3.54 6.50

yes yes

5.95 1.47

1.04 0.264

45.6 44.4

no

no

no

no

yes?

4.35

3.05

159

no

yes?

5.13

3.45

165

no

no

no

distribution from the previous calculation. Values of the metabolites with a high probability of crossing the BBB were 0.88, 0.80, 0.80, 0.65, 0.83, 0.87, and 1.00, demonstrating varying levels of distribution (Table 2). The average serum concentrations of tamoxifen, 4-hydroxytamoxifen and N-desmethyltamoxifen, were 127 Ag/L, 4.7 Ag/ L, and 227 Ag/L, respectively, for patients with a 24-h interval dosing regimen of 20 mg of tamoxifen.29 Steady-state serum concentrations of tamoxifen are attained within 3−8 weeks of continual use. Activity levels of the metabolites in human tissues have been described by Mürdter et al.3 Distribution of tamoxifen into the breast tumor tissue appears to correlate with serum concentrations; however, there are interindividual variations.3,30 Tamoxifen undergoes extensive cytochrome P450 (CYP450) mediated biotransformation, resulting in numerous possible phase I and II metabolites (Table 3). This occurs predominantly through CYP3A4, CYP3A5, and CYP2D63,31 (Figure 2). Flavin-containing monooxygenase (FMO) also contributes to the metabolism, to a lesser extent. Tamoxifen is administered as a pure Z-enantiomer; hence, metabolites are most commonly present in Z isomeric formation. However, E-enantiomers are formed to a lesser extent and have been shown to have decreased activity31 (Table 3 and Table 4). The main metabolic pathway of tamoxifen involves demethylation of the tertiary amine of tamoxifen (N26, Table 3), mediated predominantly by CYP3A4 and CYP3A5, forming the metabolite, N-desmethyltamoxifen. 4-Hydroxylation of tamoxifen also occurs, although to a lesser extent. This metabolic pathway is mediated via CYP2D6 and produces the metabolite 4-hydroxytamoxifen. Endoxifen, the third most

Figure 2. Biotransformation pathway of tamoxifen and the selected products, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen.

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Chemical Research in Toxicology Table 4. Interactions of Tamoxifen and Metabolites with Identified Potential Off-target Proteinsa total binding score (kJ/mol) docked ligand

D2 receptor

H1 receptor

H3 receptor

M1 receptor

M4 receptor

M5 receptor

dopamine histamine acetylcholine tamoxifen endoxifen 4-hydroxytamoxifen N-desmethyltamoxifen (E)-4-hydroxytamoxifen (Z)-4′-hydroxytamoxifen (E)-4′-hydroxytamoxifen 3-hydroxytamoxifen α-hydroxytamoxifen (E)-endoxifen (Z)-4′-hydroxy-N-desmethyltamoxifen (E)-4′-hydroxy-N-desmethyltamoxifen 3-hydroxy-N-desmethyltamoxifen α-hydroxy-N-desmethyltamoxifen tamoxifen-N-oxide N,N-didesmethyltamoxifen N-desmethyltamoxifen-3-O-glucuronide (Z)-N-desmethyltamoxifen-4-O-glucuronide (E)-N-desmethyltamoxifen-4-O-glucuronide (Z)-tamoxifen-4-O-glucuronide (E)-tamoxifen-4-O-glucuronide tamoxifen-3-O-glucuronide tamoxifen-N-glucuronide

−14.76 NB NB −14.86 −17.06 −7.21 −16.12 −16.83 −11.05 −15.90 −16.66 −14.06 −15.00 −15.18 −15.29 −18.58 −18.30 −10.10 −17.41 −11.47 −3.94 −9.53 −12.51 −13.23 −13.48 −10.28

NB −19.33 NB −24.84 −31.26 −26.42 −32.91 −29.82 −23.03 −22.44 −30.98 −24.45 −31.26 −29.30 −28.11 −34.74 −30.17 −24.01 −29.52 −7.60 −27.50 −24.40 NB NB NB −22.51

NB −14.74 NB NB −13.13 NB −12.78 NB −20.00 NB −21.08 −7.61 NB −24.22 NB −19.84 −13.01 NB −11.64 NB NB NB NB NB NB NB

NB NB −7.59 −17.25 −13.36 −9.57 −19.79 −12.62 −16.63 −14.68 −17.97 NB −16.61 −19.13 −19.90 −19.55 −16.04 NB −10.09 NB NB NB NB NB NB NB

NB NB −5.72 −18.89 −23.52 −23.65 −17.80 −18.30 −23.65 −18.09 −17.72 −23.32 −19.91 −22.06 −23.20 −22.93 −23.81 −15.49 −17.53 −27.32 −24.77 −20.73 −22.76 −21.04 −27.21 −16.13

NB NB −6.47 −15.38 −12.05 −13.12 −18.06 −17.19 −20.37 −12.93 −16.16 −11.16 −22.81 −17.53 −22.19 −15.43 −9.91 −2.49 −12.50 −22.36 NB −9.59 0.25 −2.99 NB NB

Metabolites found in high concentrations are in italic font, and endogenous ligands are underlined. “NB” indicates no appropriate binding to the binding site. a

A number of metabolites had higher Km values, illustrating the reduction of the respective concentration for the half-rate enzymatic reaction of these derivatives. These metabolites include 3-hydroxytamoxifen, α-hydroxy-N-desmethyltamoxifen, α-hydroxytamoxifen, (E)/(Z)-4′-hydroxytamoxifen, (E)/(Z)-4hydroxytamoxifen, and tamoxifen-N-oxide. Metabolites with higher Km values were calculated to have higher Vmax values in comparison to those of tamoxifen as well. The CLint values of metabolites were similar to that of tamoxifen. N-Desmethyltamoxifen, however, had a much larger value of 136 μL/min/mg HLM protein. This information confirms that CYP2D6 is the major enzyme involved in the transformation of Ndesmethyltamoxifen to endoxifen. The Km value of Ndesmethyltamoxifen for CYP2D6 is significantly lower in comparison to that for CYP3A4, further supporting this hypothesis (0.138 μM and 21 μM, respectively). The CLint values for CYP2D6 are significantly higher than those of CYP3A4; thus, this enzyme plays a larger role in the clearance of these drug molecules. The higher Vmax values for the majority of compounds interacting with CYP2D6 compared to CYP3A4 demonstrate a higher rate of catalysis, with less concentration dependent saturation of this enzyme. Tamoxifen and a number of metabolites were also determined to interact with CYP1A2 and CYP2C8, to a lesser degree. The ADMET program did not afford any information regarding CYP3A5, a major enzyme in this metabolic pathway, which would have provided further insight into the enzyme kinetics of tamoxifen metabolism. Although N-desmethyltamoxifen is the most predominant metabolite, contributing to approximately 92% of tamoxifen’s metabolism,32,33 its affinity for the estrogen receptors is

prominent metabolite of tamoxifen, is formed from the metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen. This occurs via 4-hydroxylation of N-desmethyltamoxifen, mediated by CYP2D6, in addition to N-demethylation of 4hydroxytamoxifen by CYP3A4 and CYP3A531 (Figure 2). The metabolism of tamoxifen and its metabolites was examined by qualitative assessment of the molecule being a substrate to various CYP and UGT enzymes, via ADMET predictor software. Tamoxifen and all phase I metabolites were observed to be potential substrates of CYP3A4, and the Michaelis−Menten Vmax and Km constants of CYP3A4 for tamoxifen are 4.94 nmol/min/nmol enzyme and 23.2 μM, respectively. The Vmax values of the metabolites were below that of tamoxifen for the majority, aside from 5.13 nmol/min/nmol enzymes for tamoxifen-3-O-glucoronide, 6.85 nmol/min/nmol enzymes for α-hydroxy-N-desmethyltamoxifen, and 10.4 nmol/ min/nmol enzymes α-hydroxytamoxifen. The metabolites that interact with CYP3A4 displayed close Km values to that of tamoxifen, showing similar concentration for their corresponding half-rate enzymatic reaction. The intrinsic clearance (CLint) of tamoxifen is 64.5 μL/min/mg HLMprotein. The intrinsic clearance for all metabolites was determined to be less than that of tamoxifen (Table 3). Calculations involving CYP2D6 were also performed, and as with CYP3A4, tamoxifen and all phase I metabolites were found to be substrates of CYP2D6. The Vmax and Km values for tamoxifen with this enzyme are 5.36 nmol/min/nmol enzyme and 0.664 μM, respectively, with a CLint value of 64.5 μL/min/ mg HLM protein (Table 3). 1498


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Chemical Research in Toxicology considerably low.29 Both 4-hydroxytamoxifen and endoxifen are shown to be 30 to 100 times more potent than tamoxifen itself.29,33 Endoxifen is present in up to 10-fold higher serum concentrations, in comparison to 4-hydroxytamoxifen;32 thus, it is considered to be the active metabolite.3,31 Therefore, tamoxifen may be classified as a prodrug.34 These results are in line with the ADMET predictor calculations of the quantitative measure of estrogen receptor toxicity in rats (100% × IC50 (estrogen)/IC50 (compound)), and N-desmethyltamoxifen was found to have a predicted toxicity level of 0.7239, compared to 32.0409 and 25.4874 of 4-hydroxytamoxifen and endoxifen, respectively. Tamoxifen was determined to have a toxicity level of 0.8914, and this result verifies the classification of this compound as a prodrug. The predicted toxicity values represent the relative binding affinity (RBA) of a molecule determined by a competitive binding assay, which is a dimensionless value demonstrating the percent of IC50 ratios. As the RBA increases, it shows higher binding affinity and likelihood for endocrine related toxicity. Kennel et al. have reported a rat-based study that looks into various factors which potentially affect tamoxifen endocrine effects.35 The metabolites of tamoxifen are inactivated via glucuronidation and sulfation by a number of uridine diphosphate glucuronosyltransferases (UGTs) and cytosolic sulfotransferases (SULTs).34 Glucuronidation is the most predominant pathway, accounting for approximately 75% of biliary excretion products.36 Tamoxifen and a number of metabolites were determined, via ADMET predictor, to interact primarily with UGT 1A1 and 1A3 in the mediation of glucuronidation. However, the sulfate conjugates may also interact with these receptors. The main excretion of tamoxifen and its metabolites occurs through bile,29 in which enterohepatic recirculation may occur.37 Therefore, predominant excretion occurs via feces; however, urinary excretion may also occur to a lesser degree.38 The elimination half-life of tamoxifen can range from 5 to 7 days.28 Differences in systemic concentrations of endoxifen have been seen in patients possessing polymorphic CYP2D6 enzymes.34 3.2. Evaluation of Tamoxifen Binding to the Desired Target. The estrogen receptors are the desired target of tamoxifen and its metabolites. These receptors are present in high concentrations in the breast tissue of those diagnosed with estrogen receptor positive breast cancer. Competitive inhibition of these receptors prevents the natural ligand, estradiol, from binding, thus reducing estrogen-mediated cell proliferation and inhibiting tumor growth.6 This is the main mechanism by which tamoxifen exerts its antineoplastic effects. 3.2.1. Estrogen Receptor α. 17β-Estradiol (E2) is the most potent endogenous substrate of the estrogen receptors (ER).39 It binds to the C-terminal ligand binding domain of ER α with a binding energy of −14.43 kJ/mol. The hydrogen of the phenol group acts as a hydrogen bond donor forming a bond with the carbonyl oxygen of Glu353, the oxygen atom of the E2 phenol group acts as a hydrogen bond acceptor, interacting with a hydrogen atom of Arg394 primary amine. Phe404 of ER α forms a π−π interaction with the phenol ring of E2. van der Waals interactions are formed between the hydrophobic steroidal backbone of the substrate and Leu349, Leu391, Leu525, Gly521, Ile424, Ala350, Leu346, Met343, Leu428, Met388, Met421, Leu384, and Leu387 (Figure 3). Tamoxifen, as well as its three prominent metabolites, all bind within the same receptor binding site as does the natural

Figure 3. Interaction of tamoxifen, estradiol, 4-hydroxytamoxifen, endoxifen, and N-desmethyltamoxifen with the C-terminal ligand binding domain of estrogen receptor α.

substrate (E2) (Figure 3). The binding energies of these drugs are considerably lower in comparison to that of the endogenous ligand, thus displaying the superior binding affinity of tamoxifen and its metabolites. Tamoxifen binds with an energy of −27.14 kJ/mol, endoxifen with −35.39 kJ/mol, 4-hydroxytamoxifen with −34.58 kJ/mol, and N-desmethyltamoxifen with −25.99 kJ/mol. All four exogenous ligands form a hydrogen bond between the secondary amine hydrogen and the carbonyl oxygen of Asp351. This interaction appears vital in anchoring the substrates into the binding site. Tamoxifen and its metabolites contain a hydrophobic ring system consisting of an ethyl group, as well as three aromatic rings which are connected by a double bond. The ring system along with the carbon chain of the dimethylaminomethyl side chain may participate in van der Waals interactions with surrounding hydrophobic residues. Both endoxifen and 4-hydroxytamoxifen form two additional hydrogen bonds, with Arg 394 and Glu353, illustrating the reason behind their stronger binding affinity (Figure 3). 3.2.2. Estrogen Receptor β. There are two possible binding sites of tamoxifen and its metabolites in the second estrogen receptor (ER β). First, the C-terminal ligand binding domain, and second, an allosteric binding site within the coactivator binding region11 (Figure 4). The ligands bind to the ligand binding domain with a much higher affinity compared to that of the allosteric site. This is shown by the differences in binding energies. In the C-terminal ligand binding domain, the natural substrate E2 binds with a binding strength of −19.50 kJ/mol. Its hydroxyl group is able to act as a hydrogen bond donor to the carbonyl oxygen of Glu305 and the sulfur of Met295. The steroidal backbone of the natural substrate forms hydrophobic interactions with a number of residues located in the first binding site. Tamoxifen, 1499


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endoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen bind with scores of −21.52, −29.51, −28.33, and −23.95 kJ/ mol, respectively. Tamoxifen and its three main metabolites all form an ionic interaction and hydrogen bonds between the ligand amino group and Asp303. Endoxifen and 4-hydroxytamoxifen form additional hydrogen bonds with Glu305, as well as Arg346 with 4-hydroxytamoxifen. The highly hydrophobic ring system of tamoxifen and the metabolites are able to form hydrophobic interactions with a number of residues (Figure 5). The allosteric binding site, located in the coactivator binding domain, composes the second ER β binding site, in which

tamoxifen and its metabolites may interact. Evidence of this binding site was shown through electron density mapping of ER β with the ligand 4-hydroxytamoxifen.11 This binding site has a lower affinity for the ligands compared to that of the ligand binding domain. Estradiol binds with a score of −7.35 kJ/mol, tamoxifen binds with −7.85 kJ/mol, endoxifen with −7.42 kJ/mol, 4-hydroxytamoxifen with −5.94 kJ/mol, and Ndesmethyltamoxifen with −8.60 kJ/mol. In agreement with Wang et al.,11 binding of ligands to this secondary site appears to be mediated mainly via hydrophobic and van der Waals interactions. E2 interacts with Trp335, Glu332, and Asp303 to form three hydrogen bonds, whereas hydrophobic residues, Leu306, Trp335, and Asp303, also interact with the steroidal backbone of estradiol. Tamoxifen and N-desmethyltamoxifen interact with the allosteric binding site in a similar manner. Both form two hydrogen bonds with the residues Glu332 and Trp335. Also, an ionic bond is formed between the negatively charged oxygen atom of Glu332 and the positively charged amino group, and there are van der Waals interactions between a number of lipophilic residues and the three aromatic rings and ethyl group. Endoxifen and 4-hydroxytamoxifen display a similar interaction profile to one another. Hydrogen bonds are formed with the carbonyl oxygen of Leu306 and Asp303 with the hydrogen atom of the hydroxyl group of the phenol ring and the hydrogen of the amino group, respectively. An ionic interaction is formed between the cationic amino group and the anionic oxygen of Asp303. Hydrophobic and van der Waals interactions also occur between the hydrophobic portions of the ligands and nearby hydrophobic residues (Figure 6). 3.3. Off-target Receptors Associated with Tamoxifen Induced Nausea. Nausea is a subjective adverse effect often

Figure 5. Interaction of tamoxifen, estradiol, 4-hydroxytamoxifen, endoxifen, and N-desmethyltamoxifen with the C-terminal ligand binding domain of estrogen receptor β.

Figure 6. Interaction of tamoxifen, estradiol, 4-hydroxytamoxifen, endoxifen, and N-desmethyltamoxifen with the allosteric binding site of estrogen receptor β.

Figure 4. Binding of tamoxifen to (A) the ligand binding domain. (B) The allosteric binding domain of estrogen receptor β.

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Figure 7. Structures of tamoxifen, dopamine, histamine, and ACh.

An extensive list of off-target receptors of tamoxifen is available (Table S2). 3.3.1. Dopamine D2 Receptor. Dopamine D2 receptors are located along the pathways of nausea and vomiting, both peripherally and centrally. Tamoxifen and its metabolites act as agonists of this receptor, leading to its activation. Agonists of the D2 receptor have the ability to directly trigger the peripheral pathway, in the gastrointestinal tract, as well as the chemoreceptor trigger zone. This will, in turn, trigger a cascade of events leading to the activation of the vomiting center and thus the sensation of nausea.40 Binding of tamoxifen to the dopamine D2 receptor is a probable mechanism of inducing this common adverse effect. Tamoxifen may interact with both central and peripheral dopamine D2 receptors to produce nausea.42 While tamoxifen binds to the dopamine D2 receptor with a similar affinity to the natural substrate, dopamine (−14.86 and −14.76 kJ/mol, respectively), both endoxifen and N-desmethyltamoxifen bind to the agonist binding site with stronger binding affinities (−17.06 and −16.12 kJ/mol), along with a number of other metabolites, which are formed to a lesser extent (Table 4). Dopamine shares its major pharmacophores with tamoxifen and its metabolites, which makes the agonistic properties of this drug on the D2 receptors plausible. Each contains an aromatic ring with an amide side chain; the structures also contain highly lipophilic regions which contribute to binding (Figure 7). Dopamine binds to the D2 receptor by forming hydrophobic interactions between its aromatic ring and carbon chain and the residues Ile183 and Phe360. Two of the primary amine hydrogens act as hydrogen bond donors, forming bonds with Asp114 and Tyr387. The oxygen atoms of both the meta- and para-hydroxyl groups act as hydrogen bond acceptors forming two more hydrogen bonds with the side chains of Ile184 and Tyr379. An electrostatic interaction occurs between the positively charged amine and the negatively charged oxygen atom of Asp114 (Figure 8). Tamoxifen, endoxifen, and N-desmethyltamoxifen form numerous hydrophobic and van der Waals interactions between the lipophilic residues in the binding site and highly hydrophobic portions of the structures. Tamoxifen acts as a hydrogen bond donor, forming a bond with the residue Tyr379. Hydrogen bonds are formed among endoxifen, Ser194, and

described as the urge to vomit, associated with autonomic responses such as salivation, sweating, pallor, and tachycardia.40 There are four main emetogenic pathways that, when stimulated, result in the activation of the brain’s vomiting center, thus prompting the sensation of nausea and the vomiting reflex. This includes (i) the cerebral cortex, (ii) the peripheral pathways, (iii) the chemoreceptor trigger zone, and (iv) vestibular input.40 The main neurotransmitters involved in the activation of the nausea pathways are 5-hydroxytryptamine (5HT), dopamine, and substance P.41 Various receptors, such as the 5-hydroxytryptamine 3 (5HT3), 5-hydroxytryptamine 4 (5HT4), dopamine D2 (D2), neurokinin-1 (NK1), μ-opioid (μ), acetylcholine muscarinic (AChM), and histamine H1 (H1) receptors, are located throughout the proposed pathways of nausea, and activation of these receptors will result in initiation of the respective nausea pathway.40 Mechanoreceptors and chemoreceptors located in the gastrointestinal tract34 can also participate in inducing the sensation of nausea.40 The primary mechanism of chemotherapy-induced nausea and vomiting is mediated by the abdominal vagal afferent nerves of the peripheral pathway. Enteroendocrine cells, located within the GIT mucosa, contain a number of local mediators, including 5HT, substance P, and cholecystokinin. Corresponding receptors located on the terminal ends of the vagal afferent nerves are also present. Antineoplastic agents, through either direct mucosal or blood-borne mechanisms, stimulate enteroendocrine cells to release said mediators, thus resulting in activation of the receptors and stimulation of the vagal afferents.41 Antineoplastic agents may also affect the chemoreceptor trigger zone. The chemoreceptor trigger zone is located on the base of the fourth ventricle of the brain; this area contains a relatively permeable region of the BBB.40,41 This area of the brain contains receptors for serotonin, dopamine, cholecystokinin, and opioids.40 The chemoreceptor trigger zone is activated through blood and cerebrospinal fluid-borne detection of chemicals and hormones. Activation of either pathway in turn leads to activation of the vomiting center, which contains acetylcholine, histamine, opioid, and cholecystokinin receptors.40 This center, located within the medulla, consists of numerous dissociated neuronal areas.41 Activation of this area, via afferent inputs, prompts the sensation of nausea.40 1501


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Figure 8. Interaction of dopamine, endoxifen, and N-desmethyltamoxifen with the agonist binding site of the dopamine D2 receptor agonist binding site.

Figure 9. Interaction of tamoxifen, histamine, 4-hydroxytamoxifen, endoxifen, and N-desmethyltamoxifen with the binding site of this histamine H1 receptor.

Cys182. N-Desmethyltamoxifen interacts with Tyr379 forming a hydrogen bond between the secondary amine hydrogen and the oxygen atom of the tyrosine phenol side chain (Figure 8). 3.3.2. Histamine Receptor. Histamine shares similar pharmacophores with tamoxifen and many of its metabolites, containing an amine moiety attached to an ethane chain. The structure of tamoxifen is much bulkier than that of histamine, as it contains a total of three aromatic rings, compared to the single imidazole ring of histamine; hence, it can participate in additional van der Waals and other hydrophobic interactions (Figure 7). 3.3.2.1. Histamine H1 Receptor. Histamine H1 receptors can be found within the vomiting center of the medulla. Tamoxifen is known to act as an antagonist to the histamine H1 receptor.43 Many of the metabolites of tamoxifen bind to the H1 receptor with high affinity, greater than that of the endogenous ligand, histamine (Table 4). Histamine, tamoxifen, and the metabolites bind to the same site of the histamine H1 receptor, as both agonists and antagonists are known to interact within the same region.12 Histamine binds to the H1 receptor with an affinity of −19.33 kJ/mol (Table 4). It is able to form noncovalent interactions within the site, such as hydrogen bonds with Asp107, Try431, and Asp178, as well as an ionic interaction between the molecule’s positively charged primary amine and Asp107. This interaction anchors the substrate into the binding pocket and is essential for binding to the H1 receptor.12 Hydrophobic interactions occur with nonpolar amino acids within the binding pocket, such as Lys179 and Ile454 (Figure 9). Tamoxifen and the primary metabolites all are shown to bind to this receptor with a higher affinity than histamine, the

endogenous substrate. Tamoxifen binds with a binding score of −24.84 kJ/mol, endoxifen with −31.26 kJ/mol, 4-hydroxytamoxifen with −26.42 kJ/mol, and N-desmethyltamoxifen with32.91 kJ/mol (Table 4). The increased affinity of these molecules for the H1 receptor depicts competition at the receptor binding site, in which tamoxifen and the metabolites are preferentially bound. This will result in receptor antagonism and reduction of activation mediated by histamine. Tamoxifen, endoxifen, and Ndesmethytamoxifen have similar interaction profiles with the H1 receptor. Each forms a hydrogen bond between the molecule’s amino hydrogen and Lys179, and between the ether oxygen and Asp178. Endoxifen also interacts with Thr194, forming an additional hydrogen bond. A salt bridge is also formed among the positively charged amine of tamoxifen, endoxifen, and N-desmethyltamoxifen and the negatively charged side chain of the Asp178 residue. 4-Hydroxytamoxifen forms a single hydrogen bond with the binding site, between the hydroxyl oxygen of Ser111 and the hydroxyl hydrogen of phenol ring of this metabolite. van der Waals and hydrophobic interactions occur between the lipophilic side chains of residues and the hydrophobic portions of the metabolites (Figure 9). 3.3.2.2. Histamine H3 Receptor. The interaction of histamine with the histamine H3 receptors located on the enterocromaffin cells of the gastrointestinal tract has been shown to inhibit the release of serotonin.44 Serotonin receptors, within the gastrointestinal tract and the central nervous system, play a vital role in both of the main pathways in which chemotherapy induced nausea and vomiting are initiated.40 H3 receptor antagonists, such as tamoxifen and its metabolites, are able to antagonize this inhibitory effect, thus resulting in an 1502


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suggests that the SERMs may act as an agonist or partial agonist instead of a pure antagonist of the muscarinic receptor. Acetylcholine, the endogenous substrate of the muscarinic receptors, contains an ester attached to a quaternary amine by an ethane chain, quite similar to the dimethylaminoethyl side chain found in tamoxifen and its derivatives (Figure 7). The shared pharmacophores allow tamoxifen to occupy the acetylcholine muscarinic binding site in a manner that is homologous to the endogenous substrate. 3.3.3.1. Muscarinic M1 Receptor. Agonists of the muscarinic M1 receptor bind to the hydrophobic pocket among transmembranes α2, α6, and α7. Antagonists, however, bind among helicies α3−α7.26 Acetlycholine interacts with the M1 receptor with a binding score of −7.59 kJ/mol, a low affinity when compared to that of tamoxifen and the metabolites (Table 4). Acetylcoline interacts with the receptor by forming two hydrogen bonds between the carbonyl oxygen of the ester moiety and the amino acid residues of Tyr404 and Tyr106. Hydrophobic interactions occur among Tyr404, Try381, Tyr106, and the methyl groups of the qutaernary ammonium, the ethyl chain, and the carbonyl moiety (Figure 11).

increased concentration of serotonin release, causing the activation of the peripheral pathway via the abdominal vagal afferent nerves. The binding of both agonists and antagonists occurs within the same site of the H3 receptor. Histamine binds to the receptor with an affinity of −14.74 kJ/mol, and each of the main metabolites binds to this receptor with a slightly lower affinity, −13.13 kJ/mol for endoxifen and −12.78 kJ/mol for Ndesmethyltamoxifen. No docking solution was found for tamoxifen and 4-hydroxytamoxifen binding to the substrate binding site of this receptor. The amino acid, Asp114, forms an ionic bond with all ligands examined, and this interaction appears to be necessary for binding both agonists and antagonists to the receptor binding site. Histamine forms a hydrogen bond with Ser405, while antagonists endoxifen and N-desmethyltamoxifen bind with the thiol hydrogen of Cys118. van der Waals and hydrophobic interactions occur between residues within the hydrophobic binding pocket and histamine as well as the antagonists (Figure 10).

Figure 10. Interaction of endoxifen, N-desmethyltamoxifen, and histamine with the binding site of the histamine H3 receptor.

Metabolites of tamoxifen, formed to a lesser extent, appear to have stronger interactions with this receptor, such as (Z)-4′hydroxy-N-desmethyltamoxifen with −24.22 kJ/mol, 3-hydroxytamoxifen with −21.08 kJ/mol, and (Z)-4′-hydroxytamoxifen with −20.00 kJ/mol (Table 4). These results indicate that although the primary metabolites bind to the histamine H3 receptor with a lesser affinity than the endogenous substrate, competitive inhibition and antagonism of this receptor is still possible when the metabolites are present in high enough concentrations or in the absence of histamine. 3.3.3. Muscarinic Receptor. Muscarinic acetylcholine receptors, M1, M4, and M5, are expressed within the diffuse group of neurons in the medulla, otherwise known as the vomiting center. Competition experiments of the selective estrogen modulator clomiphene with the muscarinic receptor were conducted by Ben-Bauch et al.45 Clomiphene has the same mechanism of action and a very similar structure to that of tamoxifen. The results of these experiments showed that the SERM did in fact bind to the muscarinic receptor and had the ability to displace muscarinic antagonists. The Kd value obtained for clomiphene was lower than those of known antagonists and similar to those of agonists.45 This information

Figure 11. Interaction of tamoxifen, acetylcholine, 4-hydroxytamoxifen, endoxifen, and N-desmethyltamoxifen with the binding site of the muscarinic M1 receptor.

Tamoxifen interacts with this receptor with an affinity of −17.25 kJ/mol, endoxifen with −13.36 kJ/mol, 4-hydroxytamoxifen with −9.57 kJ/mol, and N-desmethyltamoxifen with −19.79 kJ/mol. Tamoxifen binds in a similar manner to the M1 receptor, by forming two hydrogen bonds to the ether oxygen with the same residues, Tyr106 and Tyr404. More hydrophobic interactions can be formed as tamoxifen contains multiple aromatic rings and hydrophobic moieties. This is likely the reason why tamoxifen binds the M1 receptor with a much 1503


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Tamoxifen, endoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen bind the M4 receptor with higher affinities of −18.89, −23.52, −23.65, and −17.80 kJ/mol, respectively. Tamoxifen and 4-hydroxytamoxifen interact with the M4 receptor in a similar way by forming electrostatic interactions between the cationic amino groups and the anionic oxygen of Asp432, hydrogen bonds with the ether oxygen of tamoxifen and the phenol oxygen of 4-hydroxytamoxifen and Tyr89, and hydrophobic interactions with amino acids such as Asp432, Trp435, Phe186, and Ile187. 4-Hydroxytamoxifen forms an additional hydrogen bond with the molecule’s ether oxygen and Asn432. The binding of endoxifen and N-desmethyltamoxifen is also comparable. Both ligands form two hydrogen bonds with Tyr113, the first with the drug molecule’s amino hydrogen and the second the ether oxygen. A third hydrogen bond is formed with the other amino hydrogen and Tyr416. Endoxifen forms an additional hydrogen bond with the phenol hydrogen and Ser436. All ligands also interact with a number of lipophilic residues forming hydrophobic interactions (Figure 12). These results indicate the preferential binding of tamoxifen and the metabolites compared to that of the endogenous substrate, resulting in competition at the receptor binding site favoring the interaction with tamoxifen and its derivatives. Other metabolites of this medication, which are formed to a lesser extent, also interact with the M4 receptor at a high affinity (Table 4). This interaction results in the activation or partial activation of this receptor, resulting in the cholinergic response, as also occurred in the M1 receptor. 3.3.3.3. Muscarinic M5 Receptor. Acetylcholine binds the M5 subtype of muscarinic receptor with an affinity of −6.47 kJ/ mol, which, similar to both M1 and M4 receptors, is a much lower affinity than that of tamoxifen and its examined metabolites. Acetylcholine interacts with Tyr111 and Tyr481 to form two hydrogen bonds, both with the carbonyl oxygen of the ester moiety. This endogenous substrate is also involved in hydrophobic interactions with the nonpolar residues within the binding site, such as Tyr111, Tyr481, and Tyr458. Tamoxifen binds to the M5 receptor with a binding score of −15.38 kJ/ mol, endoxifen with a score of −12.05 kJ/mol, 4-hydroxytamoxifen with −13.12 kJ/mol, and N-desmethyltamoxifen with −18.06 kJ/mol (Table 4). Tamoxifen and 4-hydroxytamoxifen both form a hydrogen bond with the carbonyl oxygen of Asn115, which occurs between the amino hydrogen in tamoxifen and the phenol hydrogen in 4-hydroxytamoxifen. 4-Hydroxytamoxifen forms an additional hydrogen bond with the thiol moiety Cys484 and its ether oxygen, and as well undergoes an electrostatic interaction with Asp110 and the positively charged amino group. Both endoxifen and N-desmethyltamoxifen form two hydrogen bonds with the side chain of Ser114, the first with an amino hydrogen and the second with the ether oxygen. Numerous hydrophobic and van der Waals interactions occur between the hydrophobic residues of the binding site and the highly hydrophobic structure of tamoxifen and its metabolites (Figure 13). (E)-Endoxifen, N-desmethyltamoxifen-3-O-glucuronide, and (E)-4′-hydroxy-N-desmethyltamoxifen bind the M5 receptor with the highest affinities, −22.81, −22.36, and −22.19 kJ/mol, respectively (Table 4). As stated previously, the interaction of tamoxifen and the metabolites with this receptor will result in activation or partial activation. This activation results in a signal cascade causing

stronger binding affinity in comparison to that of the natural ligand. Endoxifen and 4-hydroxy tamoxifen interact in a similar manner. Each forms an ionic bond with Asp105 and the protonated amino group and as well forms hydrogen bonds between the ether oxygen and the thiol hydrogen of Cys407, and the amine hydrogen with the carbonyl oxygen of Asp105. Endoxifen also forms a third hydrogen bond between the Asn110 carbonyl oxygen and the hydroxy portion of the phenol ring. N-Desmethyltamoxifen interacts by forming two hydrogen bonds with Ser109, one with the secondary amine hydrogen, and the second with the ether moiety. The hydrophobic portions of this ligand interact with lipophilic side cains of the residues (Figure 11). The metabolites found to interact with the highest affinity are N-desmethyltamoxifen (−19.79 kJ/mol), (E)-4′-hydroxy-Ndesmethyltamoxifen (−19.90 kJ/mol), and (Z)-4′-hydroxy-Ndesmethyltamoxifen (−19.13 kJ/mol) (Table 4). Each metabolite tested, that was discovered to interact with this receptor, was determined to have a higher affinity compared to that of acetylcholine, thus resulting in agonism of the M1 receptor, giving rise to cholinergic effects which aid in the mediation of nausea and vomiting. 3.3.3.2. Muscarinic M4 Receptor. Acetlycholine interacts with the muscarinic M4 receptor with a binding score of −5.72 kJ/mol; this result is significantly lower than that of tamoxifen and its derviatives (Table 4). This interaction is mediated by the formation of a hydrogen bond between the carbonyl oxygen of the ester moiety and the amino hydrogen of Ile187, as well as hydrophobic interactions with Trp435 and Phe186 residues (Figure 12).

Figure 12. Interaction of tamoxifen, acetylcholine, 4-hydroxytamoxifen, and N-desmethyltamoxifen with the binding site of the muscarinic M4 receptor. 1504


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desmethyltamoxifen display hydrogen bonding involving the amino hydrogen and Tyr379. If the hydrogen binding of this compound was reduced, the affinity for the dopamine D2 receptor would also decrease, thus resulting in potentially diminished levels of nausea for patients requiring this drug. Modification to tamoxifen to prevent the formation of these causative hydrogen bonds is possible, and a bulky protecting group (such as tert-butyl) may be added to either the hydroxyl or amino end of the molecule to provide steric hindrance and prevent the formation of such noncovalent interactions. If the added group contained atoms which increase the polarity of the overall molecule, it may also aid in decreasing the partition of the molecule through the BBB, thus reducing the overall drug concentration available to interact with these receptors centrally. Further studies must be conducted to determine if such structural changes will have an influence on the ligand’s overall effectiveness and binding to the site of action. This study can serve drug discovery research in developing more effective and tolerable tamoxifen analogues or other novel chemotherapeutic agents.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00112. rate of occurrence of 490 potential side effects; codes of adverse effects; and tamoxifen potential off-target protein interactions (PDF)

Figure 13. Interaction of tamoxifen, acetylcholine, 4-hydroxytamoxifen, endoxifen, and N-desmethyltamoxifen with the binding site of the muscarinic M5 receptor.

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stimulation of the vomiting center, mediating the nausea and vomiting response, a common cholinergic effect.

AUTHOR INFORMATION

Corresponding Author

*Memorial University, School of Pharmacy, Health Sciences Centre, 300 Prince Philip Dr., St. John’s, N.L., Canada, A1B 3V6. Phone: 709-777-8430. E-mail: [email protected].

4. CONCLUSIONS Nausea is a prominent adverse effect experienced by the vast majority of patients ingesting antineoplastic drugs. Structural modifications of these drugs may allow for reduction in the number of off-target interactions, thus resulting in a decrease in the severity of adverse effects. The novel information provided here is valuable for the modification and creation of new therapeutics. Tamoxifen and a vast majority of metabolites are found to interact with the dopamine D2 receptor with favorable binding and high affinity. These receptors are present in both the periphery (in the gastrointestinal tract) and centrally (in the chemo receptor trigger zone). Therefore, they may be activated by drug molecules that are able to cross the BBB, as well as those that are present in the gastrointestinal tract, either through initial intake of the pure drug molecule tamoxifen or through biliary secretion or enterohepatic recirculation of metabolites. This receptor is very prominent in the nausea and vomiting pathway and is currently a target for a number of antiemetic drugs. By preventing the interaction with this receptor, we may be able to significantly reduce the nausea experienced by patients taking this drug. Tamoxifen and its metabolites interact with this receptor predominantly through noncovalent hydrogen bonding. The majority of metabolites were observed to form hydrogen bonds between either the hydroxyl oxygen of each compound and Cys182 of the D2 receptor or between the amine hydrogen of each compound and Asp114, or both. Tamoxifen and N-

ORCID

Laleh Alisaraie: 0000-0002-8874-5909 Funding

This work was supported by grants from Memorial University of Newfoundland (# 208892) awarded to L.A., APOTEX pharmaceutical company (# 211261), and ACENET summer research fellowship awarded to M.F. (# 210535) for this project. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Compute Canada for free access to their computer facilities and technical support.

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ABBREVIATIONS H, histamine; M, muscarinic; D, dopamine; SERM, selective estrogen receptor modulator; AF, activation function; ADMET, absorption, distribution, metabolism, elimination, and toxicity; PDB, Protein Data Bank; CYP 450, cytochrome P450; 5HT, 5hydroxytryptamine; AChM, acetylcholine muscarinic; NK, neurokinin; UGT, uridine diphosphate glucuronosyltransferase; SULT, cytosolic sulfotransferase 1505


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(19) Thal, D. M., Sun, B., Feng, D., Nawaratne, V., Leach, K., Felder, C. C., Bures, M. G., Evans, D. A., Weis, W. I., Bachhawat, P., Kobilka, T. S., Sexton, P. M., Kobilka, B. K., and Christopoulos, A. (2016) Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature 531, 335−340. (20) Rarey, M., Kramer, B., and Lengauer, T. (1997) Multiple automatic base selection: protein-ligand docking based on incremental construction without manual intervention. J. Comput.-Aided Mol. Des. 11, 369−384. (21) Rarey, M., Kramer, B., and Lengauer, T. (1999) Docking of hydrophobic ligands with interaction-based matching algorithms. Bioinformatics 15, 243−250. (22) Rarey, M., Kramer, B., Lengauer, T., and Klebe, G. (1996) A Fast Flexible Docking Method using an Incremental Construction Algorithm. J. Mol. Biol. 261, 470−489. (23) Kalani, M. Y. S., Vaidehi, N., Hall, S. E., Trabanino, R. J., Freddolino, P. L., Kalani, M. A., Floriano, W. B., Kam, V. W. T., and Goddard, W. A. (2004) The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists. Proc. Natl. Acad. Sci. U. S. A. 101, 3815− 3820. (24) Teeter, M. M., Froimowitz, M., Stec, B., and DuRand, C. J. (1994) Homology Modeling of the Dopamine D2 Receptor and Its Testing by Docking of Agonists and Tricyclic Antagonists. J. Med. Chem. 37, 2874−2888. (25) Dastmalchi, S., Hamzeh-Mivehroud, M., Ghafourian, T., and Hamzeiy, H. (2008) Molecular modeling of histamine H3 receptor and QSAR studies on arylbenzofuran derived H3 antagonists. J. Mol. Graphics Modell. 26, 834−844. (26) Peng, J. Y.-c., Vaidehi, N., Hall, S. E., and Goddard, W. A. (2006) The Predicted 3D Structures of the Human M1 Muscarinic Acetylcholine Receptor with Agonist or Antagonist Bound. ChemMedChem 1, 878−890. (27) Huang, X., Zheng, G., and Zhan, C.-G. (2012) Microscopic Binding of M5 Muscarinic Acetylcholine Receptor with Antagonists by Homology Modeling, Molecular Docking and Molecular Dynamics Simulation. J. Phys. Chem. B 116, 532−541. (28) Fabian, C., Sternson, L., and Barnett, M. (1980) Clinical pharmacology of tamoxifen in patients with breast cancer: comparison of traditional and loading dose schedules. Cancer Treat. Rep. 64, 765− 773. (29) Hutson, P. R., Love, R. R., Havighurst, T. C., Rogers, E., and Cleary, J. F. (2005) Effect of Exemestane on Tamoxifen Pharmacokinetics in Postmenopausal Women Treated for Breast Cancer. Clin. Cancer Res. 11, 8722−8727. (30) Daniel, P., Gaskell, S. J., Bishop, H., Campbell, C., and Nicholson, R. I. (1981) Determination of tamoxifen and biologically active metabolites in human breast tumours and plasma. Eur. J. Cancer Clin. Oncol. 17, 1183−1189. (31) Jager, N. G. L., Rosing, H., Schellens, J. H. M., Linn, S. C., and Beijnen, J. H. (2014) Tamoxifen dose and serum concentrations of tamoxifen and six of its metabolites in routine clinical outpatient care. Breast Cancer Res. Treat. 143, 477−483. (32) Stearns, V., Johnson, M. D., Rae, J. M., Morocho, A., Novielli, A., Bhargava, P., Hayes, D. F., Desta, Z., and Flockhart, D. A. (2003) Active Tamoxifen Metabolite Plasma Concentrations After Coadministration of Tamoxifen and the Selective Serotonin Reuptake Inhibitor Paroxetine. J. Natl. Cancer Inst. 95, 1758−1764. (33) Desta, Z., Ward, B. A., Soukhova, N. V., and Flockhart, D. A. (2004) Comprehensive Evaluation of Tamoxifen Sequential Biotransformation by the Human Cytochrome P450 System in Vitro: Prominent Roles for CYP3A and CYP2D6. J. Pharmacol. Exp. Ther. 310, 1062−1075. (34) Binkhorst, L., Kloth, J. S. L., Wit, A. S., Bruijn, P., Lam, M. H., Chaves, I., Burger, H., Alphen, R. J., Hamberg, P., Schaik, R. H. N., Jager, A., Koch, B. C. P., Wiemer, E. A. C., Gelder, T., Horst, G. T. J., and Mathijssen, R. H. J. (2015) Circadian variation in tamoxifen pharmacokinetics in mice and breast cancer patients. Breast Cancer Res. Treat. 152, 119−128.

REFERENCES

(1) Yang, G., Nowsheen, S., Aziz, K., and Georgakilas, A. G. (2013) Toxicity and adverse effects of Tamoxifen and other anti-estrogen drugs. Pharmacol. Ther. 139, 392−404. (2) Siegel, R., Naishadham, D., and Jemal, A. (2012) Cancer statistics, 2012. Ca-Cancer J. Clin. 62, 10−29. (3) Mürdter, T. E., Schroth, W., Bacchus-Gerybadze, L., Winter, S., Heinkele, G., Simon, W., Fasching, P. A., Fehm, T., The German, T., Group, A. I. C., Eichelbaum, M., Schwab, M., and Brauch, H. (2011) Activity Levels of Tamoxifen Metabolites at the Estrogen Receptor and the Impact of Genetic Polymorphisms of Phase I and II Enzymes on Their Concentration Levels in Plasma. Clin. Pharmacol. Ther. 89, 708− 717. (4) Fisher, B., Costantino, J. P., Wickerham, D. L., Cecchini, R. S., Cronin, W. M., Robidoux, A., Bevers, T. B., Kavanah, M. T., Atkins, J. N., Margolese, R. G., Runowicz, C. D., James, J. M., Ford, L. G., and Wolmark, N. (2005) Tamoxifen for the Prevention of Breast Cancer: Current Status of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl. Cancer Inst. 97, 1652−1662. (5) Smigel, K. (1998) Breast Cancer Prevention Trial shows major benefit, some risk. J. Natl. Cancer Inst. 90, 647−648. (6) Osborne, C. K. (1998) Tamoxifen in the Treatment of Breast Cancer. N. Engl. J. Med. 339, 1609−1618. (7) Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen. Cell 95, 927−937. (8) Beato, M., and Sanchez-Pacheco, A. (1996) Interaction of Steroid Hormone Receptors with the Transcription Initiation Complex. Endocr. Rev. 17, 587−609. (9) Fisher, B., Costantino, J. P., Wickerham, D. L., Redmond, C. K., Kavanah, M., Cronin, W. M., Vogel, V., Robidoux, A., Dimitrov, N., Atkins, J., Daly, M., Wieand, S., Tan-Chiu, E., Ford, L., Wolmark, N., Breast, o. N. S. A., and Investigators, B. P. (1998) Tamoxifen for Prevention of Breast Cancer: Report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl. Cancer Inst. 90, 1371− 1388. (10) Berman, H. M., W, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) The Protein Data Bank. Nucleic Acids Res. 28, 235−242. (11) Wang, Y., Chirgadze, N. Y., Briggs, S. L., Khan, S., Jensen, E. V., and Burris, T. P. (2006) A second binding site for hydroxytamoxifen within the coactivator-binding groove of estrogen receptor β. Proc. Natl. Acad. Sci. U. S. A. 103, 9908−9911. (12) Shimamura, T., Shiroishi, M., Weyand, S., Tsujimoto, H., Winter, G., Katritch, V., Abagyan, R., Cherezov, V., Liu, W., Han, G. W., Kobayashi, T., Stevens, R. C., and Iwata, S. (2011) Structure of the human histamine H1 receptor complex with doxepin. Nature 475, 65− 70. (13) Consortium, T. U. (2015) UniProt: a hub for protein information. Nucleic Acids Res. 43, D204−D212. (14) Kelley, L. A., and Sternberg, M. J. E. (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363−371. (15) Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G. F., Thian, F. S., Kobilka, T. S., Choi, H.-J., Kuhn, P., Weis, W. I., Kobilka, B. K., and Stevens, R. C. (2007) High-Resolution Crystal Structure of an Engineered Human β-2 Adrenergic G Protein− Coupled Receptor. Science 318, 1258−1265. (16) Haga, K., Kruse, A. C., Asada, H., Yurugi-Kobayashi, T., Shiroishi, M., Zhang, C., Weis, W. I., Okada, T., Kobilka, B. K., Haga, T., and Kobayashi, T. (2012) Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature 482, 547−551. (17) Bordoli, L., Kiefer, F., Arnold, K., Benkert, P., Battey, J., and Schwede, T. (2008) Protein structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 4, 1−13. (18) Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283−291. 1506


Article

Chemical Research in Toxicology (35) Kennel, P., Pallen, C., Barale-Thomas, E., Espuña, G., and Bars, R. (2003) Tamoxifen: 28-day oral toxicity study in the rat based on the Enhanced OECD Test Guideline 407 to detect endocrine effects. Arch. Toxicol. 77, 487−499. (36) Kiyotani, K., Mushiroda, T., Imamura, C. K., Hosono, N., Tsunoda, T., Kubo, M., Tanigawara, Y., Flockhart, D. A., Desta, Z., Skaar, T. C., Aki, F., Hirata, K., Takatsuka, Y., Okazaki, M., Ohsumi, S., Yamakawa, T., Sasa, M., Nakamura, Y., and Zembutsu, H. (2010) Significant Effect of Polymorphisms in CYP2D6 and ABCC2 on Clinical Outcomes of Adjuvant Tamoxifen Therapy for Breast Cancer Patients. J. Clin. Oncol. 28, 1287−1293. (37) Fromson, J. M., Pearson, S., and Bramah, S. (1973) The Metabolism of Tamoxifen (I.C.I. 46,474) Part I: In Laboratory Animals. Xenobiotica 3, 693−709. (38) Soininen, K., Kleimola, T., Elomaa, I., Salmo, M., and Rissanen, P. (1986) The Steady-State Pharmacokinetics of Tamoxifen and its Metabolites in Breast Cancer Patients. J. Int. Med. Res. 14, 162−165. (39) Kuiper, G. G. J. M., Carlsson, B., Grandien, K., Enmark, E., Häggblad, J., Nilsson, S., and Gustafsson, J.-Å. (1997) Comparison of the Ligand Binding Specificity and Transcript Tissue Distribution of Estrogen Receptors α and β. Endocrinology 138, 863−870. (40) Harris, D. G. (2010) Nausea and vomiting in advanced cancer. Br. Med. Bull. 96, 175−185. (41) Hesketh, P. J. (2008) Chemotherapy-Induced Nausea and Vomiting. N. Engl. J. Med. 358, 2482−2494. (42) Hiemke, C., and Ghraf, R. (1984) Interaction of non-steroidal antiestrogens with dopamine receptor binding. J. Steroid Biochem. 21, 663. (43) Watts, C. K., and Sutherland, R. L. (1987) Studies on the ligand specificity and potential identity of microsomal antiestrogen-binding sites. Mol. Pharmacol. 31, 541−551. (44) Racké, K., Reimann, A., Schwörer, H., and Kilbinger, H. (1996) Regulation of 5-HT release from enterochromaffin cells. Behav. Brain Res. 73, 83−87. (45) Ben-Baruch, G., Schreiber, G., and Sokolovsky, M. (1982) Cooperativity pattern in the interaction of the antiestrogen drug clomiphene with the Muscarinic receptors. Mol. Pharmacol. 21, 287− 293.

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