Identification of binding sites of prazosin, tamsulosin and KMD-3213 with α1-adrenergic receptor subtypes by molecular modeling

Ishiguro, Masaji; Futabayashi, Yukiyo; Ohnuki, Toshio; Ahmed, Maruf; Muramatsu, Ikunobu; Nagatomo, Takafumi

doi:10.1016/s0024-3205(02)02077-5

Cited by 35 publications

(28 citation statements)

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“…Molecular modeling of prazosin to a 1b -AR receptors predicted that made strong electrostatic interactions towards Asp125 in transmembrane helix 3 and hydrogen bonding with Thr130 in transmembrane helix 3 of a 1b -AR receptor. 11) We, therefore, constructed and characterized alanine mutations of these amino acid residues by site-directed mutagenesis. Our findings are consistent with the molecular modeling data that these mutations are important for prazosin binding which have been evident from the markedly decrease in affinity of prazosin for the mutant receptors.…”

Section: )mentioning

confidence: 99%

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

Takahashi

Hossain

Ahmed

et al. 2007

Biological & Pharmaceutical Bulletin

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The a 1 -adrenergic receptors (a 1 -ARs) belong to the super family of G-protein coupled receptors (GPCRs) that transmit a variety of signals across cell membrane. The a 1 -ARs are important for many cardiovascular diseases such as atherosclerosis and hypertension, 1,2) restenosis after coronary dilation, 3) myocardial hypertrophy and cardiac arrhythmia. 4,5) There are three subtypes of a 1 -ARs (a 1a -, a 1b -, and a 1c -AR) and a 1b -AR is one of them. Recent experiments suggested that a 1b -ARs are involved in cardiac hypertrophy and neurodegeneration in a 1b transgenic mice. 4,6)The a 1b -ARs are structurally characterized by seven transmembrane helices connected by alternating extracellular and intracellular loops. Whereas ligand binding involves extracellular portion of the receptor, the intracellular regions mediate the interaction of the receptor with G proteins as well as other signaling and regulatory proteins. Stimulation of the a 1b -ARs by catecholamines activates proteins of the Gq family, resulting in the production of inositol phosphates via the activation of phospholipase C (PLC). 7)It is of now-a-days interest to give more emphasis for identification of more selective ligands to fully elucidate the functional role of different subtypes. Molecular modeling and site-directed mutagenesis studies are helpful to determine the activity of currently existing agents and the development of completely novel therapeutic agents. 8)Prazosin, the prototype of a family of agents that contains a piperazinyl quinazoline nucleus, is a very potent and selective a 1 -adrenergic antagonist. Its affinity for a 1 receptor is about 1000-fold greater than that for a 2 receptors. Interestingly, the drug also is a relatively potent inhibitor of cyclic nucleotide phosphodiesterases, and it was originally synthesized for this purpose.9) Prazosin appears to depress baroreflex function in hypertensive patients.10) Based on radio-ligand binding studies, it was shown that prazosin has relatively lower affinity towards a 1a -and a 1b -AR receptor than a 1c -AR receptors. 11)However, the molecular mechanism by which prazosin binds to a 1b -AR receptors remains undetermined. Molecular modeling of prazosin to a 1b -AR receptors predicted that made strong electrostatic interactions towards Asp125 in transmembrane helix 3 and hydrogen bonding with Thr130 in transmembrane helix 3 of a 1b -AR receptor.11) We, therefore, constructed and characterized alanine mutations of these amino acid residues by site-directed mutagenesis. Our findings are consistent with the molecular modeling data that these mutations are important for prazosin binding which have been evident from the markedly decrease in affinity of prazosin for the mutant receptors. MATERIALS AND METHODSDrugs Niguldipine(ϩ), tamsulosin, prazosin, and ketanserin were obtained from RBI (Research Biochemical Incorporated, Natick, MA, U.S.A.).DNA Constructs A cDNA clone encoding human a 1b -AR receptor in the plasmid vector (pCR3) was introduced into the DH5a strain of E. coli by th...

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Section: )mentioning

confidence: 99%

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

Takahashi

Hossain

Ahmed

et al. 2007

Biological & Pharmaceutical Bulletin

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“…Ser258 mutation to alanine increased Effects of Glu237Ala mutant on antagonist binding to α 1d -AR Similar to the mutation of Ser258 in TMD V or Glu237 in TMD IV also resulted in increased binding affinity to prazosin and tamsulosin in [ 3 H]prazosin displacement studies. As proposed in the literature by Ishiguro et al (17), Glu237 in TMD IV of α 1d -AR being negatively charged might form a salt bridge with the nearby positively charged Lys236 or with other amino acid residues on different transmembrane portions of the enzyme. As a result, the increased pK i values of prazosin and tamsulosin might be the result of the breakdown of this "receptor constrain" by the chemically unreactive, hydrophobic alanine mutation.…”

Section: Agonist-stimulated Ip Accumulationmentioning

confidence: 80%

“…As for the binding sites for prazosin and tamsulosin in α 1d -AR, it is evident that amino acids on the 3rd, 4th, and 5th TMDs of α 1d -AR are important according to a study that analyzed the binding sites with the receptor by computer-based homology modeling (17). Especially, Asp176, Glu238, and Ser258 on α 1d -AR are supposed to interact with α 1 -AR antagonists.…”

Section: Agonist-stimulated Ip Accumulationmentioning

confidence: 99%

“…Previous molecular modeling studies of α 1d -AR in our laboratory (17) predicted that the amino group of prazosin and tamsulosin make electrostatic interactions with the carboxylate side chain of Asp176 in TMD III, which is highly conserved in all GPCR-binding amine ligands. This aspartate is involved in high-affinity binding of agonists as well as antagonists at both the β 2 -(18) and α 2A -AR (19).…”

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Amino Acids of the Human α1d-Adrenergic Receptor Involved in Antagonist Binding

et al. 2008

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Abstract. Computer simulations of the human α 1d -adrenergic receptor (α 1d -AR) based on the crystal structure of rhodopsin have been combined with experimental site-directed mutagenesis to investigate the role of residues in the transmembrane domains in antagonist binding. Our results indicate that the amino acids Asp176 in the third transmembrane domain (TMD), Glu237 in TMD IV, and Ser258 in TMD V of α 1d -AR were directly involved in prazosin and tamsulosin binding. The Asp176Ala mutant did not exhibit any affinity for [ Competition binding experiments showed that prazosin affinity had increased to 5-fold and 3-fold in the Glu237Ala and Ser258Ala mutants, respectively, versus wild-type; and tamsulosin affinity only increased in the Ser258Ala mutant (2-fold vs wild-type). It seems that these two residues constrain the receptor by interaction with other residues and this disruption of the interaction increased the receptor's binding affinity towards antagonists. However, the Glu237Ala and Ser258Ala mutant receptors retained the ability to stimulate the formation of myo-[ 3 H]inositol but had activities lower than that of the wild-type receptor. The present results provide direct evidence that these amino acid residues are responsible for the interactions between α 1d -AR and the radioligand [

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“…8) Previous molecular modeling studies of a 1a -AR in our laboratory predicted that the protonated amino group of prazosin, tamsulosin and KMD-3213 make ionic interactions with the carboxylate side chain of Asp106 in TMD III, which is highly conserved in all GPCR-binding amine ligands. 9) Docking study and subsequent molecular simulation studies with a 1a -AR antagonist/receptor complex by other investigators have also revealed the same aspartate (Asp106) to be interacting with doxazosin (a quinazoline based antagonist, with a similar basic structure of prazosin) and tamsulosin. 10) Pedretti and colleagues published a homology model of a 1a -AR where both the agonist and antagonist interacted with Asp106.…”

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confidence: 92%

Mutational Analysis of the .ALPHA.1a-Adrenergic Receptor Binding Pocket of Antagonists by Radioligand Binding Assay

Ahmed

Hossain

Bhuiyan

et al. 2008

Biological & Pharmaceutical Bulletin

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The a 1 -adrenergic receptors (a 1 -AR) belong to Class A of the super family of G protein coupled receptors (GPCRs). They comprise at least three cloned subtypes, a 1a -, a 1b -and a 1d -ARs.1,2) a 1 -ARs are of particular therapeutic interest due to their important roles in the control of blood pressure, in the contraction and growth of smooth muscle and in renal and hepatic metabolism. The a 1a -AR located in large abundance in the prostate is thought to be influential in the condition of benign prostatic hyperplasia (BPH), a considerable health problem for aging man.3) The development of high affinity a 1a -AR antagonists is of paramount importance for the treatment of BPH.To rationally design selective drugs, an understanding of subtype differences in the ligand-binding pockets would be invaluable. In this paper the attention is focused on a 1a -AR due to its role in neurotransmission processes, in pharmacological treatment of BPH. 4,5) Indeed, many researchers are involved in mutagenesis studies on the a 1a -AR, which revealed the key residues of both agonist and antagonist binding sites. 6) In contrast to the wealth of information about the agonist binding pocket of adrenergic receptors, the knowledge about the binding mode of antagonists is much more limited. Three consecutive residues set on second extracellular loop (Gln177, Ile178 and Asn179) are involved in interaction with some a 1a -AR selective antagonists. 7) Two conserved phenylalanine residues on transmembrane domain (TMD) VII (Phe308 and Phe312) are involved in p-p stacking with almost all a 1a -AR antagonists. 8)Previous molecular modeling studies of a 1a -AR in our laboratory predicted that the protonated amino group of prazosin, tamsulosin and KMD-3213 make ionic interactions with the carboxylate side chain of Asp106 in TMD III, which is highly conserved in all GPCR-binding amine ligands. 9)Docking study and subsequent molecular simulation studies with a 1a -AR antagonist/receptor complex by other investigators have also revealed the same aspartate (Asp106) to be interacting with doxazosin (a quinazoline based antagonist, with a similar basic structure of prazosin) and tamsulosin. 10)Pedretti and colleagues published a homology model of a 1a -AR where both the agonist and antagonist interacted with Asp106. 11)KMD-3213 is a selective a 1a -AR antagonist.12,13) a 1a -AR selective antagonist would be very useful for the treatment of BPH without any untoward effect on the blood pressure. By homology modeling of a 1a -AR with KMD-3213, we identified a key amino acid residue responsible for higher binding affinity to a 1a -AR. The study revealed that carboxamide nitrogen of KMD-3213 forms an ionic bond with the carboxylate side chain of Gln167 in TMD IV.9) Prazosin and tamsulosin are proposed to interact with the side chain of Asp106 in TMD III.The objectives of this study are both the construction of mutant a 1a -ARs such as Asp106Ala, double mutants Asp106Ala-Gln167Phe and Gln167Phe by site-directed mutagenesis and the analysis of their interac...

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Identification of binding sites of prazosin, tamsulosin and KMD-3213 with α1-adrenergic receptor subtypes by molecular modeling

Cited by 35 publications

References 12 publications

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of .ALPHA.1b-Adrenergic Receptor Antagonist Binding

Amino Acids of the Human α1d-Adrenergic Receptor Involved in Antagonist Binding

Mutational Analysis of the .ALPHA.1a-Adrenergic Receptor Binding Pocket of Antagonists by Radioligand Binding Assay

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