No abstract

Abstract. Mutations producing constitutively active G-protein coupled receptors have been found in the pathophysiology of several diseases, implying that inverse agonists at the constitutively active receptors may have preferred therapeutic applications. Because of the involvement of 5-HT 2A receptors in mediating many cardiovascular diseases, constitutively active mutants of the 5-HT 2A receptor may be responsible for the disease states. Thus, the purpose of the present study was to investigate the inverse agonist activity of sarpogrelate, a selective 5-HT 2A -receptor antagonist, and its active metabolite, M-1; and we compared their activities with those of other 5-HT 2A -receptor antagonists such as ritanserin, ketanserin, and cyproheptadine. Using a constitutively active mutant (C322K) of the human 5-HT 2A receptor, we demonstrated that like other 5-HT 2A -receptor antagonists, sarpogrelate acts as a potent inverse agonist by significantly reducing basal inositol phosphate levels. However, there were no significant differences between sarpogrelate and other 5-HT 2A -receptor antagonists for their inverse agonist activity. Compared with the wild type receptor, mutant receptor displayed significantly higher affinity for 5-HT and lower affinity for sarpogrelate. These results indicate that stabilization of the inactive conformation of the 5-HT 2A receptor may be a key component of the mechanism of action of sarpogrelate.

Site-directed mutagenesis was used to investigate the molecular interactions involved in sarpogrelate binding

Abstract. The purpose of the present study was to examine 5-hydroxytryptamine (5-HT) 2A 3 H]ketanserin (K d 2.0 nM vs 0.8 nM for the wild-type receptor) and had reduced affinity for sarpogrelate (pK i value of 5.71 vs 9.06 for the wild-type receptor). The Trp6.48Ala mutant also showed the greatest decrease in sensitivity to sarpogrelate (pK b value 8.81 for wild-type and 5.11 for mutant) in inhibiting agonist-stimulated IP formation. These results provide direct evidence that Asp3.32, Trp3.28, and less importantly, Trp6.48 are responsible for the interaction between the 5-HT 2A receptor and sarpogrelate. In addition, these results support the findings obtained from molecular modeling studies.

Based on radio-ligand binding and molecular modeling studies, sarpogrelate shows a moderate selectivity for 5-HT(2B) versus 5-HT(2A) receptors. To confirm the modeling data of sarpogrelate to 5-HT(2B) receptors predicting interaction of sarpogrelate towards Asp135 in helix 3 of 5-HT(2B) receptors, we constructed and characterized the mutation of this residue by site-directed mutagenesis. The Asp135Ala mutant did not exhibit any affinity for [(3)H]rauwolscine. Therefore, it was not possible to find sarpogrelate affinity to the mutant using [(3)H]rauwolscine. The mutation also abolished agonist-stimulated inositol phosphates formation. These results provide evidence that Asp135 is important for the interaction between 5-HT(2B) receptors and sarpogrelate.