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

Abstract

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...

The (R)-enantiomer of the 6-chromanol derivate SUL-121 improves renal graft perfusion via antagonism of the α1-adrenoceptor

“…Interestingly, prazosin and SUL-150 (Fig. 5C) formed contacts with PHE312 and ASP106 confirmed in prazosin binding by mutagenesis 11,15 , whereas SUL-151 (Fig. 5D) did not show interactions with these residues.…”

“…5B). The quinazoline scaffold of non-protonated prazosin was docked close to TM5 to form a hydrogen bond between the 6,7-methoxy groups and SER188 11,12 , a hydrogen bond between the furan oxygen and SER83, and between the prazosin carboxamide and GLN177 side chain; van der Waals interactions with side chains of PHE86, VAL107, ILE178, SER192 11 , PHE289 13 , MET292 and PHE312 12,14 ; π-π interactions with PHE288 and PHE312 12 ; and a π-hydrogen bond interaction with the side chain carboxyl group and backbone peptide carboxamide of ASP106 12,15 . Thus, the proposed binding mode of non-protonated prazosin utilized interactions that were in accord with those described in previous literature.…”

Orthosteric Binding of ρ-Da1a, a Natural Peptide of Snake Venom Interacting Selectively with the α1A-Adrenoceptor

“…We investigated the involvement of residues within the orthosteric pocket of α 1 -ARs that are known to interact with agonists and/or antagonists: F86 2.64 [33], D106 3.32 (D125 in α 1B -AR) [34]–[36], F187 5.41 [37], S188 5.42 and S192 5.46 [38], F288 6.51 (F310 on α 1B -AR) [18], [39], M292 6.55 [40] and F308 7.35 and F312 7.39 [18], [41] (superscripts refer to the Ballesteros-Weinstein numbering system for residues in 7TM helices). In addition, we tested the positions F193 5.47 and F281 6.44 , predicted to play a role in the stabilization of the active state of α 1A -AR [42] (Table 1).…”

“…The negative charge of D106 3.32 is expected to interact with the positive charge of biogenic amines [51], and no binding of 3 H-prazosin to the α 1A -AR variants D106 3.32 A or D106 3.32 A/N167F is observed [36]. The homologous D125 3.32 A variant of the α 1B -AR has been expressed but showed no change in affinity for HEAT [52], although another study has shown a total loss of affinity for HEAT [34].…”

Kinetic Characterization of an Intestinal Trefoil Factor Receptor

“…This technique has been used to determine receptor density, affinity, and dissociation constants. Reaction time, reaction temperature, the number of cells, and relative concentrations of labeled and unlabeled ligands impact results [17], [18]. In general, the number of cells should be more than 10 5 /mL, the time for reaction equilibrium should be 30-60 min, and the relative concentrations of unlabeled to labeled ligand should be over 4000∶1.…”