Glennon R A, Dukat M, Westkaemper R B, Ismaiel A M, Izzarelli D G, Parker E M
Department of Medicinal Chemistry, School of Pharmacy, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0540, USA.
Mol Pharmacol. 1996 Jan;49(1):198-206.
Although the beta-adrenergic receptor antagonist (-)-propranolol binds with relatively low affinity at human 5-hydroxytryptamine1D beta receptors (Ki = 10,200 nM), it displays significantly higher affinity (Ki = 17 nM) at its species homolog, 5-HT1B receptors, and at a mutant 5-HT1D beta receptor (Ki = 16 nM), where the threonine residue at position 355 (T355) is replaced with an asparagine residue (i.e., a T355N mutant). Propranolol contains two oxygen atoms, an ether oxygen atom and a hydroxyl oxygen atom, and it has been speculated that the enhanced affinity of propranolol for the T355N mutant receptor is related to the ability of the asparagine residue to hydrogen bond with the ether oxygen atom. However, the specific involvement of the propranolol oxygen atoms in binding to the wild-type and T355N mutant 5-HT1D beta receptors has never been addressed experimentally. A modification of a previously described 5-HT1D beta receptor graphic model was mutated by replacement of T355 with asparagine. Propranolol was docked with the wild-type and T355N mutant 5-HT1D beta receptor models in an attempt to understand the difference in affinity of the ligand for the receptors. The binding models suggest that the asparagine residue of the mutant receptor can form hydrogen bonds with both oxygen atoms of propranolol, whereas the threonine moiety of the wild-type receptor can hydrogen-bond only to one oxygen atom. To test this hypothesis, we prepared and examined several analogues of propranolol that lacked either one or both oxygen atoms. The results of radioligand binding experiments are consistent with the hypothesis that both oxygen atoms of propranolol could participate in binding to the mutant receptor, whereas only the ether oxygen atom participates in binding to the wild-type receptor. As such, this is the first investigation of serotonin receptors that combines the use of molecular modeling, mutant receptors generated by site-directed mutagenesis, and synthesis to investigate structure/affinity relationships.
尽管β-肾上腺素能受体拮抗剂（-）-普萘洛尔与人5-羟色胺1Dβ受体的结合亲和力相对较低（Ki = 10200 nM），但它在其物种同源物5-HT1B受体以及突变型5-HT1Dβ受体（Ki = 16 nM）上显示出显著更高的亲和力，在突变型5-HT1Dβ受体中，第355位的苏氨酸残基（T355）被天冬酰胺残基取代（即T355N突变体）。普萘洛尔含有两个氧原子，一个醚氧原子和一个羟基氧原子，据推测，普萘洛尔对T355N突变体受体亲和力的增强与天冬酰胺残基与醚氧原子形成氢键的能力有关。然而，普萘洛尔的氧原子在与野生型和T355N突变体5-HT1Dβ受体结合中的具体作用从未通过实验得到证实。通过将T355替换为天冬酰胺，对先前描述的5-HT1Dβ受体图形模型进行了突变。将普萘洛尔与野生型和T355N突变体5-HT1Dβ受体模型对接，以试图了解配体对受体亲和力的差异。结合模型表明，突变体受体的天冬酰胺残基可以与普萘洛尔的两个氧原子形成氢键，而野生型受体的苏氨酸部分只能与一个氧原子形成氢键。为了验证这一假设，我们制备并检测了几种缺少一个或两个氧原子的普萘洛尔类似物。放射性配体结合实验的结果与以下假设一致：普萘洛尔的两个氧原子都可以参与与突变体受体的结合，而只有醚氧原子参与与野生型受体的结合。因此，这是首次将分子建模、定点诱变产生的突变体受体以及合成相结合来研究5-羟色胺受体结构/亲和力关系的研究。
