Takafumi Nagatomo

Identification of the binding sites and selectivity of sarpogrelate, a novel 5-HT2 antagonist, to human 5-HT2A, 5-HT2B and 5-HT2C receptor subtypes by molecular modeling

The aim of the present study was to investigate the binding sites interactions and the selectivity of sarpogrelate to human 5-HT(2) receptor family (5-HT(2A), 5-HT(2B) and 5-HT(2C) receptor subtypes) using molecular modeling. Rhodopsin (RH) crystal structures were used as template to build structural models of the human serotonin-2A and -2C receptors (5-HT(2A)R, 5-HT(2C)R), whereas for 5-HT(2B)R, we used our previously published three-dimensional (3D) models based on bacteriorhodopsin (BR). Sarpogrelate, a novel 5-HT(2)R antagonist, was docked to the receptors. Molecular dynamics (MD) simulations produced the strongest interaction for 5-HT(2A)R/sarpogrelate complex. Upon binding, sarpogrelate constraints aromatic residues network (Trp(3.28), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2A)R; Phe(3.35), Phe(6.51), Trp(7.40) in 5-HT(2B)R; Trp(3.28), Phe(3.35), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2C)R) in a stacked configuration, preventing activation of the receptor. The models suggest that the structural origin of the selectivity of sarpogrelate to 5-HT(2A)R vs both 5-HT(2B)R and 5-HT(2C)R comes from the following results: (1) The tight interaction between the antagonist and the transmembrane domain (TMD) 3. Asp(3.32) neutralizes the cationic head and interacts simultaneously with carboxylic group hydrogen of the antagonist molecule. (2) Due to steric hindrance, Ser(5.46) (vs Ala(5.46) in 5HT(2B) and 5HT(2C)) prevents sarpogrelate to enter deeply inside the hydrophobic core of the helix bundle and to interact with Pro(5.50). (3) The side chain of Ile(4.56) (vs Ile(4.56) in 5HT(2B)R and Val(4.56) in 5HT(2C)R) constraints sarpogrelate to adjust its position by translating toward the strongly attractive Asp(3.32). These results are in good agreement with binding affinities (pKi) of sarpogrelate for 5-HT(2) receptor family expressed in transfected cell.

Binding and functional affinity of sarpogrelate, its metabolite M-1 and ketanserin for human recombinant alpha-1-adrenoceptor subtypes

Serotonin (5-HT2) antagonists show high affinity for the α1-adrenoceptor (α1-AR) in addition to the 5-HT2 receptor. In the present study we compared the pharmacological characteristics of a new 5-HT2 antagonist sarpogrelate and its active metabolite M-1 with those of ketanserin on human recombinant α1-AR subtypes. In the binding study, sarpogrelate, M-1 and ketanserin produced concentration-dependent inhibition of 3H-prazosin binding to α1-ARs. Among the three drugs, ketanserin showed the highest affinity for α1a-, α1b- and α1d-ARs (pKi 8.0, 8.3 and 7.6, respectively). Sarpogrelate had a relatively low affinity for the three subtypes (6.3 , 6.4 and 6.3, respectively) and M-1 showed medium affinity (7.1, 7.1 and 6.1, respectively). Chinese hamster ovary (CHO) cells expressing each α1-AR subtype showed concentration-dependent inositol phosphate (IP) accumulation in response to phenylephrine. The concentration response curves were shifted to the right by three drugs, and the pKb values were close to the pKi values in the binding study. In addition to these effects, sarpogrelate and M-1, but not ketanserin produced an increase in the basal IP level of α1d-expressed CHO cells, although the increase was less than that of phenylephrine. The present results indicate that sarpogrelate and M-1 have antagonistic activity to the three α1-AR subtypes, but their affinities are significantly lower than those of ketanserin.

Binding sites of valsartan, candesartan and losartan with angiotensin II receptor 1 subtype by molecular modeling

AIMSnThis study was designed to examine the importance of interaction in the binding of selective angiotensin II receptor antagonists to angiotensin II type 1 receptor using molecular modeling. The AT(1) antagonists used in this study were valsartan, candesartan and losartan.nnnMAIN METHODSnAT(1) receptor structural model was constructed by homology modeling using structural models of rhodopsin photointermediates. Through molecular modeling, possible binding sites for these drugs were suggested to lie between transmembrane domains (TM) 3, 5, and 6 of AT(1) receptor.nnnKEY FINDINGSnThe carboxylic acid group and tetrazole ring of valsartan possibly interact with Lys199 of TM5 and Ser109 of TM3 and Asn295 of TM7 of AT(1) receptor, respectively. In candesartan, carboxylic group, tetrazole ring, and ethoxy group oxygen possibly interact with Lys199 of TM5, Ser109 of TM3 and Asn295 of TM7 and Gln257 of TM6, respectively. In losartan, tetrazole ring and hydroxymethyl group possibly interact with Asn295 of TM7 and Ser109 of TM3, respectively.nnnSIGNIFICANCEnThe results of the present study suggested that candesartan interacts at a higher number of binding sites compared to valsartan whereas losartan has a lower number of binding sites with the amino acid residues of the AT(1) receptor. These findings are consistent with the data of the radioligand-binding studies of the antagonists with the AT(1) receptor.

Binding and functional affinity of some newly synthesized phenethylamine and phenoxypropanolamine derivatives for their agonistic activity at recombinant human β3‐adrenoceptor

β3‐Adrenoceptor is the predominant β‐adrenoceptor in adipocytes and has drawn much attention during the investigation for anti‐obesity and antidiabetes therapeutics. Thirteen new compounds have been evaluated for their potencies and efficacies as β3‐adrenoceptor agonists on human β3‐ adrenoceptor expressed in COS‐7 and Chinese hamster ovary (CHO) cells using radio ligand binding assay and cyclic AMP (cAMP) accumulation assay. Phenoxypropanolamine derivatives, SWR‐0334NA (([E)‐[4‐[5‐[(3‐phenoxy‐2‐hydroxypropyl)amino]‐2‐pentene−3‐yl] phenoxy]acetic acid sodium salt), SWR‐0335SA ((E)‐[4‐[5‐[(3‐phenoxy‐2‐hydroxypropyl)amino]‐2‐pentene−3‐yl] phenoxy] acetic acid ethanedioic acid), SWR‐0342SA (S‐(Z)‐[4‐[[1‐[2‐[(2‐hydroxy−3‐phenoxypropyl)]amino]ethyl]‐1‐pro‐penyl]phenoxy] acetic acid ethanedioic acid), SWR‐0348SA‐SITA ((E)‐[4‐[5‐[(3‐phenoxy‐2‐hydroxy‐propyl)amino]‐2‐hexene−3‐yl] phenoxy]acetic acid ethanedioic acid) and SWR‐0361SA ((E)‐N‐methyl‐[4‐[5‐[(3‐phenoxy‐2‐hydroxypropyl)amino]‐2‐pentene−3‐yl]phenoxy]acetoamide ethanedioic acid) showed higher agonistic activity for the β3‐adrenoceptor. Among the compounds tested, SWR‐0334NA exhibited full agonist activity (%Emax = 100.26) despite its lower binding affinity (pK1 = 6.11). Compounds SWR‐0338SA((E)‐[4‐[5‐[(2‐phenyl‐2‐hydroxyethyl)amino]‐2‐pentene−3‐yl]phenoxy]acetic acid ethanedioic acid), SWR‐0339SA (S‐(E)‐[4‐[5‐[(3‐phenoxy‐2‐hydroxypropyl)amino]‐2‐pentene−3‐yl] phenoxy] acetic acid ethanedioic acid), SWR‐0345HA ((E)‐2‐methyl−3‐[4‐[2‐(2‐phenyl‐2‐hydroxyethyl‐amino)ethoxy] phenyl]‐2‐propenoic acid ethyl ester hydrochloride), SWR‐0358SA ((E)‐(2‐methoxy‐ethyl)‐[4‐[5‐[(3‐phenoxy‐2‐hydroxypropyl) amino]‐2‐pentene−3‐yl]phenoxy]acetoamide ethanedioic acid) and SWR‐0362SA ((E)‐1‐[[[4‐[5‐[(3‐phenoxy‐2‐hydroxypropyl)amino]‐2‐pentene −3‐yl]phenoxy]‐acetyl]carbonyl]piperidine ethanedioic acid) had moderate agonistic activity and were phenethylamine and phenoxypropanolamine derivatives. Compounds SWR‐0065HA ([4‐[2‐[3‐[[(3,4‐dihydro‐4‐oxo‐[1,2,4]‐triazino(4,5‐a)indol)‐lyl]oxy]‐2‐hydroxypropylamino]ethoxy]phenyl]acetic acid methyl ester hydrochloride), SWR‐0098NA ((E)‐[4‐[3‐[(2‐phenyl‐2‐hydroxyethyl)amino]‐1‐butenyl] phenoxy]‐acetic acid sodium salt) and SWR‐0302HA ([4‐[[4‐[2‐(3‐chlorophenoxy‐2‐hydroxypropyl)amino]‐E‐2‐butenyl]oxy]phenoxy]acetic acid hydrochloride) had very low binding affinity towards β3‐adreno‐ceptors and they did not induce cAMP accumulation. We concluded that compounds SWR‐0334NA, SWR‐0335SA, SWR‐0342SA, SWR‐0348SA‐SITA and SWR‐0361SA were potential agonists of human β3‐ adrenoceptor. Further investigation on their selectivity towards β3‐adrenoceptor could be useful for the exploration of the physiological properties of the β3‐adrenoceptor.

Practical access to four stereoisomers of naftidrofuryl and their binding affinity towards 5-hydroxytryptamine 2A receptor

Naftidrofuryl oxalate (Praxilene®, 1) has been used for the treatment of intermittent claudication for more than 30 years. It selectively blocks vascular and platelet 5-hydroxytryptamine 2 (5-HT(2)) receptors. This drug is marketed as a mixture of four stereoisomers, and so far there is no individual biological evaluation on the single isomers. The purpose of this study is to provide an improved method for the preparation of all four stereoisomers of naftidrofuryl, and more importantly, to distinguish them in terms of their binding affinity to 5-hydroxytryptamine 2A (5-HT(2A)) receptor. The bioassay results revealed that the C-2S configuration of naftidrofuryl was crucial for the binding affinity with 5-HT(2A) receptor, and the C-2 configuration was less important for binding. In conclusion, our study may pave the way to develop single naftidrofuryl isomers with C-2S configuration as inhibitors of 5-HT(2A) receptor that have clinical significance as vasodilators and CNS agents.

Insurmountable antagonism of AT‐1015, a 5‐HT2 antagonist, on serotonin‐induced endothelium‐dependent relaxation in porcine coronary artery

The purpose of this study was to examine the inhibitory effects of AT‐1015, a newly synthesized 5‐HT2 receptor antagonist, on serotonin‐induced endothelium‐dependent relaxation in U 46619 (5 times 10−9m)‐precontracted porcine coronary artery pre‐incubated with ketanserin (3 times 10−6m), and then compare its effects with another potent 5‐HT2 antagonist, ritanserin. The investigation showed that AT‐1015 (10−8−10−6m) caused rightward shift with significant inhibition of maximum relaxation response induced by serotonin in porcine coronary artery with endothelium. Ritanserin caused a rightward shift of serotonin‐induced relaxation without decreasing maximum response at 10−9 and 10−8m, but it inhibited the maximum relaxation response at 10−7m. The study showed that AT‐1015 and ritanserin had no inhibitory effect on bradykinin‐induced relaxation in porcine coronary artery with endothelium. Thus, these findings suggested that AT‐1015 at concentrations of 10−8−10−6m caused noncompetitive blockade of serotonin‐induced endothelium‐dependent relaxation in porcine coronary artery. The antagonistic effects of AT‐1015 on serotonin‐induced relaxation were different from that of ritanserin, except at 10−7m ritanserin. The variation of inhibitory effects between these two 5‐HT2 antagonists may be due to the different chemical structure and/or interaction sites at the receptor.

ASSESSMENT OF HOMOLOGOUS INTERNALIZATION OF CONSTITUTIVELY ACTIVE N111G MUTANT OF AT1 RECEPTOR

Constitutively active mutants (CAMs) of G-protein-coupled receptors mimic the active conformation of the receptor in their ability to activate second messenger systems in the absence of agonist. They have revealed novel properties of drugs that reverse the basal levels of constitutive activity, indicating that the drugs have the inverse agonist activity. Internalization plays an important role in receptor endocytosis and signal transduction. The present chapter provides the investigation of the internalization behavior of CAM N111G of Angiotensin II type 1 (AT(1)) receptor and correlates the result with the mechanism of constitutive activity of the mutant. Both wild-type (WT) and N111G mutant receptors were transiently expressed in COS-7 cells and total inositol phosphate production was measured in presence and absence of the angiotensin II receptor blockers (ARBs). The binding affinities toward agonist and ARBs were also determined. We found that the ARBs have the inverse agonist activity in CAM N111G of AT(1) receptor. The internalization of the mutant, which was much lower than WT receptor, was significantly increased in presence of the ARBs. The results indicate that internalization of CAM N111G of AT(1) receptor is induced by the ARBs, which may be an important characteristic of inverse agonist activities of the ARBs in N111G.

Mutagenesis of important amino acid reveals unconventional homologous internalization of β1-adrenergic receptor

AIMSnThe study was designed to examine the internalization of Asp104Lys mutant of beta(1)-adrenergic receptor (beta(1)-AR) and compared to other mutant (Asp104Ala) and wild type receptors. Moreover, this study needs to perform the role of GRK2 (betaARK1) and beta-arrestin1 on this internalization of Asp104Lys mutant of beta(1)-AR.nnnMAIN METHODSnBinding affinity, functional potency of agonist and agonist-induced internalization were determined for wild type and both mutants of beta(1)-ARs stably expressed in HEK 293 cells as assessed by [(3)H] CGP12177 radioligand. We have performed GRK2 and beta-arrestin1 expression levels by western blot analysis and also performed internalization of this mutant receptor after over expression and deletion of beta-arrestin1 gene.nnnKEY FINDINGSnIn the present study, the binding affinity of (-)-isoproterenol for both mutants were significantly decreased compared to wild type. Though the mutant Asp104Ala showed agonist-induced receptor activation, interestingly this mutant was not internalized. However, the mutant Asp104Lys, which showed uncoupling with G protein, was internalized 31.77+/-3.13% from cell surface. Asp104Lys mutant produced the same level of GRK2 expression in (-)-isoproterenol induced stimulation of wild type receptor and addition of (-)-isoproterenol further increased GRK2 expression in mutant receptors. In addition, overexpression of beta-arrestin1 in mutant Asp104Lys promoted (39.75+/-2.19%) and knockdown of beta-arrestin1 by siRNA decreased (3.55+/-1.75%) internalization compared to Asp104Lys mutant of beta(1)-ARs.nnnSIGNIFICANCEnThe present studies suggest that Asp104Lys mutant beta(1)-ARs triggers unconventional homologous internalization induced by G protein independent signals, where GRK2 and beta-arrestin1 play an important role for beta(1)-AR internalization.

Comparison of the binding affinity of some newly synthesized phenylethanolamine and phenoxypropanolamine compounds at recombinant human β‐ and α1‐adrenoceptor subtypes

We evaluated six new compounds, SWR‐0065HA ([4‐[2‐[3‐[[(3,4‐dihydro‐4‐oxo‐[1,2,4]‐triazino(4,5‐a)indol)‐lyl]oxy]‐2‐hydroxypropylamino]ethoxy]phenyl]acetic acid methyl ester hydrochloride), SWR‐0098NA ((R*R*‐UE)‐(E)‐[4‐[3‐[(2‐phenyl‐2‐hydroxyethyl)amino]‐1‐butenyl]phenoxy]acetic acid sodium salt), SWR‐0315NA ((E, Z)‐[4[[1‐[2‐[(3‐phenoxy‐2‐hydroxy propyl)]amino]ethyl]‐1‐propenyl]phenoxy]acetic acid sodium), SWR‐0338SA ((E)‐[4‐[5‐[(2‐phenyl‐2‐hydroxyethyl)amino]‐2‐pentene‐3‐yl]phenoxy] acetic acid ethanedioic acid), SWR‐0342SA ((S)‐(Z)‐[4‐[[1‐[2‐[(2‐hydroxy‐3‐phenoxypropyl)]amino] ethyl]‐1‐propenyl]phenoxy]acetic acid ethanedioic acid) and SWR‐0345HA ((E)‐2‐methyl‐3‐[4‐[2‐(2‐phenyl‐2‐hydroxyethylamino)ethoxy]phenyl]‐2‐propenoic acid ethyl ester hydrochloride) for their potencies as selective ligands at human β‐adrenoceptors expressed in COS‐7 cells and compared the binding affinities for human α1‐adrenoceptors expressed in Chinese hamster ovary (CHO) cells using a radioligand‐binding assay. Phenoxypropanolamine derivatives SWR‐0315NA and SWR‐0342SA showed higher binding affinities for β‐adrenoceptor subtypes; SWR‐0065HA, however, showed a higher affinity for only β2‐adrenoceptors, accounting for 3‐fold and 6‐fold selectivity against β1‐ and β3‐adrenoceptors. Compounds SWR‐0315NA and SWR‐0342SA did not show any binding selectivity for any of the subtypes. However, functionally these two compounds are selective for β3‐adrenoceptors. Among the phenylethanolamine derivatives, SWR‐0338SA and SWR‐0345HA showed 9‐fold and 16‐fold higher binding selectivity for β3‐adrenoceptors against β1‐adrenoceptors, respectively, whereas they both showed a 7‐fold higher binding selectivity for β3‐adrenoceptors against β2‐adrenoceptors. SWR‐0098NA did not show any significant binding affinity for any of the β‐adrenoceptor subtypes. These compounds, except for SWR‐0098NA, were not found to possess any significant binding affinity for α1‐adrenoceptor subtypes over that for β‐adrenoceptor subtypes. However, SWR‐0098NA has about a 3‐fold to 22‐fold higher binding selectivity for α1‐adrenoceptor subtypes against β‐adrenoceptor subtypes, making it difficult for use in a β‐adrenoceptor receptor study. Compounds SWR‐0315NA and SWR‐0342SA have similar binding potency for α1‐adrenoceptors as adrenaline (epinephrine), proving the finding of this manuscript that this phenoxypropanolamine group of β‐adrenoceptor ligands could also be used as α1‐adrenoceptor ligands. Functional assays have to be performed to confirm their agonistic activity.