Identification of binding sites of prazosin, tamsulosin and

KMD-3213 with a1-adrenergic receptor subtypes

by molecular modeling

Masaji Ishiguro a,*, Yukiyo Futabayashi b, Toshio Ohnuki b, Maruf Ahmed b,Ikunobu Muramatsu c, Takafumi Nagatomo b

a Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimahon-cho, Mishima-gun, Osaka 618-8503, Japanb Department of Pharmacology, Niigata College of Pharmacy, 5-13-2 Kamishinei-cho, Niigata 950-2081, Japan

c Department of Pharmacology, School of Medicine, Fukui Medical University, 23 Shimoaizuki, Matsuoka,

Fukui 910-1193, Japan

Received 15 April 2002; accepted 7 June 2002

Abstract

This investigation was performed to assess the importance of interaction in the bindings of selective and

nonselective a1-antagonists to a1-adrenergic receptor (a1-AR) subtypes using molecular modeling. The a1-

antagonists used in this study were prazosin, tamsulosin and KMD-3213. Molecular modeling was performed on

Octane 2 workstation (Silicon Graphics) using Discover/Insight II software (Molecular Simulations Inc.). Through

molecular modeling, possible binding sites for these drugs were suggested to lie between transmembrane domains

(TM) 3, 4, 5 and 6 of the a1-AR subtypes. In prazosin, the 4-amino group, 1-nitrogen atom and two methoxy

groups of quinazoline ring possibly interact with the amino acids in TM3, TM5 and TM6 of a1-ARs. In

tamsulosin, amine group of ethanyl amine chain, methoxy group of benzene ring and sulfonamide nitrogen of

benzene ring interacts in TM3, TM4 and TM5 of a1-ARs. In KMD-3213, amine of ethyl amine chain and indoline

nitrogen of this compound possibly interact within TM3 and TM5 of a1-ARs. Amide nitrogen of KMD-3213 also

interacts within TM4 of a1A-AR. The results of the present study suggested that prazosin has similar binding sites

in all the a1-AR subtypes while tamsulosin interacts at higher number of sites with a1D-subtype than other a1-AR

subtypes. KMD-3213 being an a1A-AR selective ligand, binds to higher number of sites of a1A subtype than to

other subtypes. All these amino acids are located near the extracellular loop. These findings are consistent with the

previous studies that antagonists bind higher in the pocket closer to the extracellular surface unlike agonist binding.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: a1-antagonists; Molecular modeling; Human cloned CHO cells; Radioligand binding

0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0024 -3205 (02 )02077 -5

* Corresponding author. Tel.: +81-75-962-3742; fax: +81-75-962-2115.

E-mail address: ishiguro@sunbor.or.jp (M. Ishiguro).

www.elsevier.com/locate/lifescie

Life Sciences 71 (2002) 2531–2541

Introduction

The human a1-adrenergic receptors (a1-ARs) are members of the G-protein coupled receptor

superfamily of membrane proteins that mediate the actions of the endogenous catecholamines, the

neurotransmitter norepinephrine, and the hormone epinephrine. Three a1-AR subtypes have been cloned

and pharmacologically characterized: a1A [1,2], a1B [3], and a1D-AR [4,5]. Each a1-AR subtype has a

distinct pharmacology that is recognized through the use of selective ligands. Selective agonists for a1A,

a1B and a1D-AR include methoxamine, AH11110A, noradrenaline respectively and selective antagonists

of these subtypes are WB4101, cyclazosin and BMY 7378 respectively.

To rationally design selective drugs, an understanding of subtype differences in the ligand binding

pockets would be invaluable. Our laboratory has reported the sites of interaction of the nonselective h-antagonists and amino acids of human h1- and h2-AR using molecular modeling [6,7]. In this report, we

identified the amino acid residues of a1-AR subtypes involved in antagonist binding. Although agonist

binding in a1-ARs is fairly well understood and involves residues located in transmembrane domains 3

through 6, there are few reports about antagonist binding. Waugh et al. recently reported [8] two

phenylalanine residues in the transmembrane domain 7 of the a1A-adrenergic receptor that are major

sites of antagonist interaction. Earlier studies showed that, two a1A-AR antagonists, phentolamine and

WB4101, were conferred by interactions with three consecutive amino acid residues in the extracellular

loop 2 of a1A-AR [9] and a phenylalanine residue at the surface of transmembrane domain 2 in the a1A-

AR accounted for a1A- versus a1D- niguldipine selectivity [10]. Studies suggested that in contrast to

agonist binding, which is localized to the interior core of the receptor, antagonists interact with residues

closer to the extracellular surface of adrenergic receptors, above the plane of agonist binding pocket

[9,11,12].

In this investigation, eight a1-antagonists were tested for their affinities. Three a1-antagonists viz.,

prazosin, tamsulosin and KMD-3213 having comparatively higher affinities for a1-AR subtypes have

been chosen for molecular modeling with a view to large number of binding sites in a1-ARs which was

apparent from their high binding affinities for the different subtypes of a1-AR. It would be of interest to

assess the interactions between chemical structures of these compounds and amino acids of a1-ARs at

the molecular level using molecular modeling.

Thus, the purpose of this study was to determine, through molecular modeling, whether differences in

ligand (prazosin, tamsulosin and KMD-3213) binding affinity to a1-ARs result from the different

interactions of the functional groups of the ligands with specific amino acid residues of the human a1-

ARs.

Materials and methods

Membrane preparation

For the cloned a1-ARs, CHO cells were transfected with the cDNA clones of human a1A-, a1B-, and

a1D-AR. The harvested cells were suspended in ice-cold assay buffer (Tris–HCL 50 mM, EDTA 1 mM,

pH 7.4), sonicated and centrifuged at 3000 � g for 10 min. The supernatant was then centrifuged at

80,000 � g for 30 min, and the resulting pellet was resuspended in binding assay buffer and used for

binding experiments.

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–25412532

Protein assay

Protein contents of the membrane concentration were measured by the method of Bradford using

bovine serum albumin as the standard [13].

Radioligand binding experiments

In saturation binding experiments, the membranes were incubated with various concentrations of

[3H]-Prazosin for 45 min at 30 jC. Total incubation volume for [3H]-Prazosin was 500 Al (a1A- 5f 10

Ag, a1B- 5f 10 Ag, a1D- 50f 100 Ag protein/tube). Nonspecific binding was defined as binding in the

presence of 1 AM tamsulosin for [3H]-Prazosin.

In competition binding experiments, membranes were incubated with 200 pM [3H]-Prazosin and

unlabelled drugs for 45 min at 30 jC. Specific binding of [3H]-Prazosin was approximately 91%, 96%

and 94% of the total binding for cloned a1A-, a1B-, and a1D-AR cells. Reactions were terminated by

rapid filtration under vacuum onto Whatman GF/C filters presoaked in 0.3% polyethyleneimine for over

30 mins. The filters were then washed three times with 6 ml of ice-cold 50 mM Tris–HCl (pH 7.4) and

kept in a vial with 2 ml scintillation fluid overnight. The filter bound radioactivity was determined by

liquid scintillation counting. Experiments were conducted in duplicate. Binding affinities of [3H]-

Prazosin and unlabelled drugs were expressed as Kd and negative logarithm of the equilibrium

dissociation constant, pKi.

Table 1

Binding affinity of 3H-Prazosin for cloned human a1-AR subtypes

Receptors Kd (pM) Bmax (fmol/mg protein)

a1A (4) 177.8 F 38.9 5118.8 F 438.8

a1B (4) 28.9 F 13.3 4164.4 F 1271.8

a1D (3) 71.7 F 14.3 151.54 F 6.0

The data shown indicate mean F SEM and numbers in parentheses indicate number of experiments.

Table 2

Competition Binding parameters in cloned human a1-AR subtypes

a1A a1B a1D

Tamsulosin 9.73 F 0.69 9.2 F 0.26 10.29 F 0.26

Prazosin 9.72 F 0.36 9.75 F 0.35 10.10 F 0.29

KMD-3213 9.36 F 0.54 7.99 F 0.16 8.06 F 0.06

Terazosin 8.06 F 0.38 9.34 F 0.06 8.87 F 0.07

HV-723 8.6 F 0.76 8.38 F 0.09 8.96 F 0.09

Amosulalol 8.19 F 0.04 7.91 F 0.16 8.65 F 0.11

Ketanserin 7.69 F 0.7 8.28 F 0.28 8.04 F 0.07

Clonidine 5.67 F 0.1 6.19 F 0.34 6.56 F 0.11

The data represent the pKi values and are means F SEM of three to four different experiments, each performed in duplicate.

pKi = � log (Ki), where Ki is in nM.

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–2541 2533

Fig. 1. Chemical structures of a1-AR antagonists.

Fig. 2. Amino acid sequences in TM3 to TM6 of a1-AR subtypes. Bold letters represent interactive amino acids with different

functional groups of prazosin, tamsulosin and KMD-3213.

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–25412534

Fig. 3. Lateral view of A) Complex of prazosin at the ligand binding site of a1A-, a1B- and a1D-ARs. B) Complex of tamsulosin

at the ligand binding site of a1-ARs. C) Complex of KMD-3213 at the ligand binding site of a1-ARs. Numbers with TM and EL

denote those of transmembrane helices and extracellular loops.

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–2541 2535

Data analysis

Nonlinear regression analysis of saturation and competition binding assay was performed using Prism

(GraphPad Software, San Diego, CA, USA). Data are presented as the mean F standard error of mean

(sem). Differences between groups were analysed using a one-way ANOVA, to obtain a p value.

Drugs

The drugs used and their sources were as follows: (-)-(R)-1-(3-hydroxypropyl)-5-[2- [[2-[2-(2,2,2-

trifluoroethoxy) phenoxy] ethyl] amino] propyl] indoline-7-carboxamide (KMD-3213) from Kissei

Pharmaceutical Co. Ltd. (Matsumoto, Japan); HV-723 from Hokuriku Pharmaceutical Co. Ltd.;

amosulalol and tamsulosin HCl from Yamanouchi Pharmaceutical Co. Ltd.; prazosin HCl from Sigma;

terazosin from Mitsubishi Chemical Industries (Tokyo, Japan); ketanserin from Research Biochemicals

International (Maryland, USA); clonidine HCl from Boehringer Ingelheim Japan (Kawanishi, Japan);

and [3H]-Prazosin (2856.4 GBq/mM) from NEN (Boston, USA).

KMD-3213 was dissolved in dimethylsulphoxide and diluted in binding assay buffer for binding

studies. Prazosin was dissolved in 50% ethanol and diluted in binding assay buffer. Other drugs were

dissolved in milli Q water and diluted with binding assay buffer.

Molecular modeling system

An Octane 2-workstation (Silicon Graphics) and Discover/Insight II (Molecular Simulations Inc., San

Diego, CA) software were used for the molecular modeling of human a1-ARs.

Fig. 3 (continued).

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–25412536

Methods of molecular modeling

The coordinates of the a-carbon positions were determined by an overlay of the putative a1-AR TM

residues with the TM coordinates derived from rhodopsin [14] using the Insight II molecular modeling

software. The boundaries of the putative TM domains were determined with an algorithm based on the

weighted pair-wise comparisons of adjacent residues [15]. In addition, the conformations of complicated

and three-dimensional amino acid groups were revised by the rotational isomer library, and this three-

dimensional structure was finally optimized by Discover 3. Each ligand was then docked into the

binding site manually and the initial complex model was energy-optimized by the molecular dynamics

calculation and the simulated annealing using Discover 3.

Results

Radioligand binding experiments for cloned a1-AR subtypes

In saturation experiments, specific binding of [3H]-prazosin was determined using all the cloned

membranes of the a1-AR subtypes. The affinity constant and the receptor density for all the membranes are

Fig. 4. Scheme of bindings of prazosin with a1-ARs. Interaction between the functional groups and amino acids is shown in

dotted lines indicating electrostatic interactions.

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–2541 2537

shown in the Table 1. Most of the tested drugs monophasically inhibited this binding. Table 2 shows the

binding affinities (pKi) for a1-AR antagonists at the three subtypes. In general, most of the antagonists

showed high affinity to the three subtypes. Among them, we choose prazosin, tamsulosin and KMD-3213

for molecular modeling studies. The structures of these three compounds are given in Fig. 1. For better

understanding of the a1-AR subtypes, the sequences of these receptors are given in Fig. 2.

Molecular modeling

Since the receptor structures built by use of the coordinates of rhodopsin showed a maneuver binding

sites for ligands, TMs3 and 4 were moved to secure an enough space for ligands. Fig. 3 (A) depicts the

binding sites between amino acids of seven TMs of a1-ARs and the functional groups of prazosin. Binding

profiles between ligands and a1-ARs taken from the side are shown. The docking studies of prazosin into

the all a1-AR subtypes suggested that the 4-amino group and 1-nitrogen atom on quinazoline ring possibly

interact with the carboxyl group of Asp106(a1A),125(a1B),176(a1D) in TM3 and the hydroxyl group of

Ser188(a1A),207(a1B),258(a1D) in TM5 by hydrogen bonds. Two methoxy groups of quinazoline ring

may also interact with hydroxyl group of Thr111(a1A), 130(a1B),181(a1D) in TM3 and the hydroxyl group

of Ser192(a1A),211(a1B),262(a1D) in TM5 and the carbonyl group between the piperazine and furan rings

Fig. 5. Scheme of bindings of tamsulosin with a1-ARs. Interaction between the functional groups and amino acids is shown in

dotted lines indicating electrostatic interactions.

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–25412538

are presumed to form a hydrogen bond with Ser296 (a1A),318(a1B),372(a1D) in TM6. The results show

that these interaction sites between prazosin and the amino acids in interacting helices are almost same in

case of all the a1-AR subtypes as shown schematically in Fig. 4.

Fig. 3 (B) depicts the binding sites between amino acids of seven TMs of a1-ARs and the functional

groups of tamsulosin. Binding profiles between ligands and a1-ARs taken from the side are shown.

Important and possible binding sites in a1A are (1) the amino group of phenethyl amine moiety with the

carboxylic acid of Asp106 in TM3 and (2) the methoxy group of benzene ring with the hydroxyl group

of Ser188 in TM5. The two binding sites with a1B are (1) the amino group of phenethyl amine moiety

with carboxylic acid of Asp125 in TM3 and (2) the methoxy group of the benzene ring with the hydroxyl

group of Ser207 in TM5. In case of a1D, tamsulosin binds with three sites (1) the amino group of the

phenethyl amine moiety with carboxylic acid of Asp176 in TM3, (2) the methoxy group of the benzene

ring with the hydroxyl group of Ser258 in TM5 and (3) sulfonamide nitrogen of the benzene ring with

Glu237 of TM4. Fig. 5 schematically shows the binding sites with tamsulosin in a1-ARs.

On the other hand, KMD-3213 showed almost similar binding sites of a1 subtypes to those of

tamsulosin as evident fromFigs. 3 (C) and 6, suggesting that the amino group of the ethyl aminemoiety and

indoline nitrogen of this compound possibly interact with the carboxyl group of Asp106 (a1A) Asp125

(a1B) Asp176 (a1D) in TM3 by a hydrogen bond andwith the hydroxyl group of Ser188 (a1A) Ser207 (a1B)

Ser 258 (a1D) in TM5 respectively. Amide nitrogen of KMD-3213 also forms an ionic bond with the

carboxyl group of Gln167 in TM4 of a1A-AR.

Fig. 6. Scheme of bindings of KMD-3213 with a1-ARs. Interaction between the functional groups and amino acids is shown in

dotted lines indicating electrostatic interactions.

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–2541 2539

Discussion

The mechanism how antagonists bind to the a1-AR is still not well understood. However, some

reports have been published showing the important sites of interaction with antagonists in a1-AR [8–

10]. In the present study, we found that a1-AR antagonists prazosin, tamsulosin and the a1A-AR

selective KMD-3213 showed consistent binding profiles with a1-AR as was hypothesized from their

binding affinity for all the a1-AR subtypes. This study suggests that the different binding affinity is due

to different binding interactions of these compounds with the a1-AR subtypes.

The present study showed that the main functional groups of prazosin, i.e., a range of the quinazoline-

ring substituents and the carbonyl groups possibly interact with Asp106 (TM3), Thr111 (TM3), Ser192

(TM5), Ser188 (TM5) and Ser296 (TM6) of a1A-AR, Asp125 (TM3), Thr130 (TM3), Ser211 (TM5),

Ser207 (TM5) and Ser318 (TM6) of a1B-AR and Asp176 (TM3), Thr181 (TM3), Ser262 (TM5), Ser258

(TM5) and Ser372 (TM6) of a1D-AR. As evident from the model of complex between prazosin and a1-

AR subtypes (Fig. 3 (A)), the number of binding sites of prazosin in all the subtypes is the same.

Prazosin interacts with amino acids in the same positions and in the identical helices of all the a1-AR

subtypes. This may account for the nonselectivity of prazosin to all the a1-AR subtypes.

The functional groups of tamsulosin, i.e., ethylamine nitrogen, sulphonamide nitrogen and methoxy

oxygen of the benzene ring may interact with Asp106 (TM3) and Ser188 (TM5) of a1A-AR, Asp125

(TM3) and Ser207 (TM5) of a1B-AR and Asp176 (TM3), Glu237 (TM4) and Ser258 (TM5) of a1D-AR.

Sulphonamide nitrogen of tamsulosin would interact with Glu237 in TM4 of a1D-AR. This is probably

due to the more acidic nature of sulphonamide and the longer bond lengths of the C–S and S–N bonds,

which would enable the sulphonamide to bind with Glu237 of a1D-AR.

Ethylamine nitrogen, amide nitrogen and indoline nitrogen of KMD-3213 possibly form hydrogen

bonds with Asp106 (TM3), Gln167 (TM4) and Ser188 (TM5) of a1A-AR, Asp125 (TM3) and Ser207

(TM5) of a1B-AR and Asp176 (TM3) and Ser258 (TM5) of a1D-AR. Glu186 (a1B) being negatively

charged would form a salt bridge with positively charged Lys185 (a1B) in EL2 and Glu237 (a1D) with

Lys236 (a1D) in TM4. The bonding energy of the amide group in KMD-3213 is not sufficient to break

the salt bridges in a1B and a1D. Thus, glutamates (Glu) at positions 186 and 237 in a1B and a1D-ARs

respectively, may not contribute to the interaction with KMD-3213. On the other hand, Arg166 (a1A)

at the same position of lysine in a1B may not interact with the neutral residue Gln167 (a1A). Therefore,

Gln167 may be able to orient to the ligand-binding site for binding with the amide group of KMD-

3213.

Our investigation showed that glutamate at position 237 of transmembrane 4 in a1D-AR and

glutamine at position 167 of transmembrane 4 in a1A-AR clearly justifies the selective binding profile

of tamsulosin and KMD-3213 for a1D- and a1A-AR respectively. Moreover, the interacting amino acids

of the a1-AR subtypes with the antagonists tested are located closer to the extracellular loop, which is

consistent with the earlier findings [9,11,12] and the binding affinity of prazosin, tamsulosin and KMD-

3213 is related to the occupancy of the binding pockets of the a1-AR subtypes.

Acknowledgements

This research was in part supported by a grant from the Promotion and Mutual Aid Corporation for

Private Schools of Japan.

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–25412540

References

[1] Perez DM, Piascik MT, Malik N, Gaivin R, Graham RM. Cloning, expression and tissue distribution of the rat homologue

of the bovine a1c-adrenergic receptor provide evidence for its classification as the a1a subtype. Molecular Pharmacology

1994;46:823–31.

[2] Schwinn DA, Lomasney JN, Loreng W, Szklut PJ, Fremau RT, Yang-Feng TL, Caron MG, Lefkowitz RJ, Cotecchia S.

Molecular cloning and expression of the cDNA for a novel a1-adrenergic receptor subtype. Journal of Biological Chem-

istry 1990;265:8183–9.

[3] Cotecchia S, Schwinn RR, Randell RR, Lefkowitz RJ, Caron MG, Kobilka BK. Molecular cloning and expression of the

cDNA for the hamster a1-adrenergic receptor. Proceedings of the National Academy of Sciences of the United States of

America 1988;85:7159–63.

[4] Perez DM, Piascik MT, Graham RM. Solution-phase library screening for the identification of rare clones: isolation of an

a1d-adrenergic receptor cDNA. Molecular Pharmacology 1991;40:876–83.

[5] Lomasney JW, Cotecchia S, Lorenz W, Leung WY, Schwinn DA, Yang-Feng TL, Braunstein M, Lefkowitz RJ, Caron

MG. Molecular cloning and expression of the cDNA for the a1-adrenergic receptor. Journal of Biological Chemistry

1991;266:6365–9.

[6] Hattori K, Ishiguro M, Ohnuki T, Nakamura T, Muramatsu I, Nagatomo T. Identification of Binding Sites of Bopindolol

and its two metabolites with h1-adrenoceptors by Molecular Modeling: Comparison with h2-adrenoceptors. Pharmacology

1999;58:183–9.

[7] Nagatomo T, Ishiguro M, Ohnuki T, Hattori K, Hosohata Y, Takatsu N, Katayama H, Watanabe K. Studies on relation-

ships between chemical structure and h-blocking potency of Bopindolol and its two metabolites. Life Sciences 1998;62:

1597–601.

[8] Waugh DJJ, Gaivin RJ, Zuscik MJ, Gonzalez-Cabrera P, Ross SA, June Y, Perez DM. Phe-308 and Phe-312 in trans-

membrane domain 7 are major sites of a1-adrenergic receptor antagonist binding: Imidazoline agonists bind like antag-

onists. Journal of Biological Chemistry 2001;276(27):25366–71.

[9] Zhao MM, Hwa J, Perez DM. Identification of critical extracellular loop residues involved in a1-adrenergic receptor

subtype-selective antagonist binding. Molecular Pharmacology 1996;50:1118–26.

[10] Hamaguchi N, True TA, Saussy Jr DL, Jeffs PW. Phenylalanine in the second membrane spanning domain of

a1a-adrenergic receptor determines subtype selectivity of dihydropyridine antagonists. Biochemistry 1996;35:14312–7.

[11] Wurch T, Colpaert FC, Pauwels PJ. Chimeric receptor analysis of the ketanserin binding site in the human 5-Hydroxy-

tryptamine1D receptor: importance of the second extracellular loop and fifth transmembrane domain in antagonist binding.

Molecular Pharmacology 1998;54(6):1088–96.

[12] Li X, Varga EV, Stropova D, Zalewska T, Malatynska E, Knapp RJ, Roeske WR, Yamamura HI. y-Opioid receptor: the

third extracellular loop determines naltrindole selectivity. European Journal of Pharmacology 1996;300(1–2):R1–2.

[13] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle

of protein-dye binding. Analytical Biochemistry 1976;72:248–54.

[14] Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Trong IL, Teller DC, Okada T, Stenkamp RE,

Yamamoto M, Miyano M. Crystal structure of Rhodopsin: A G protein-coupled receptor. Science 2000;289:739–45.

[15] Reik RP, Handshumacher ND, Sung SS, Tan M, Glynias MJ, Schluchter MD, Novotny J, Graham RM. Evolutionary

conservation of both the hydrophilic and hydrophobic nature of transmembrane residues. Journal of Theoretical Biology

1995;172:245–58.

M. Ishiguro et al. / Life Sciences 71 (2002) 2531–2541 2541

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