Grunewald G L, Carter A E, Sall D J, Monn J A
Department of Medicinal Chemistry, University of Kansas, Lawrence 66045.
J Med Chem. 1988 Jan;31(1):60-5. doi: 10.1021/jm00396a010.
We have previously described regions of steric bulk tolerance in the aromatic-ring binding site of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) for phenylethanolamine substrates and alpha-methylbenzylamine inhibitors. For bound substrates, this region is located in the vicinity of the para position of the aromatic ring, while for bound alpha-methylbenzylamine inhibitors, it is located in the region complementary to the meta position. In the present study, we sought to determine the preferred conformation of the biaryl portion of (m-phenylphenyl)- and (p-phenylphenyl)ethanolamine (4 and 5, respectively) as well as for m-phenyl- and p-phenyl-alpha-methylbenzylamine (7 and 8, respectively) for PNMT active site interactions. Planar derivatives of 4, 5, 7, and 8 were obtained through the synthesis of 2-(1-fluorenyl)-2-hydroxyethylamine (9), 2-(2-fluorenyl)-2-hydroxyethylamine (10), 1-(1-fluorenyl)ethylamine (11), and 1-(2-fluorenyl)ethylamine (12). The four fluorene derivatives were examined for in vitro activity as substrates and inhibitors of the PNMT-catalyzed reaction. As in the case of 4, 5, 7, and 8, we have observed a positional preference for the alkylamine side chain with respect to the biphenyl skeleton present in 9-12. Thus, fluorenylethanolamine 10 ("p-biphenyl") displays a Michaelis constant (Km = 26 microM) that is approximately 10 times lower than that for 9 ("m-biphenyl", Km = 297 microM); in the alpha-methylbenzylamine inhibitors, fluorenyl derivative 11 ("m-biphenyl", Ki = 4.14 microM) is approximately 40 times better than 12 ("p-biphenyl", Ki = 185 microM) for in vitro inhibition of PNMT. In each case, conformational restriction of the biaryl system present in 4, 5, 7, and 8, such that the aromatic rings are coplanar, resulted in enhanced affinity for the PNMT active site. Thus, conformational restriction of ethanolamine 5 (Km = 82 microM) as in 10 (Km = 26 microM) and alpha-methylbenzylamine 7 (Ki = 89 microM) as in 11 (Ki = 4.14 microM) leads, in each case, to a stronger enzyme-ligand dissociable complex. These results, in conjunction with others from these laboratories, indicate that the PNMT active site beyond the zone that interacts with the central aromatic ring portion of phenylethanolamine substrates and alpha-methylbenzylamine inhibitors is essentially a flat, hydrophobic pocket.
我们之前已经描述过苯乙醇胺N-甲基转移酶（PNMT，EC 2.1.1.28）的芳香环结合位点中对于苯乙醇胺底物和α-甲基苄胺抑制剂的空间位阻耐受区域。对于结合的底物，该区域位于芳香环对位附近，而对于结合的α-甲基苄胺抑制剂，它位于与间位互补的区域。在本研究中，我们试图确定（间苯基苯基）-和（对苯基苯基）乙醇胺（分别为4和5）以及间苯基-和对苯基-α-甲基苄胺（分别为7和8）的联芳基部分对于PNMT活性位点相互作用的优选构象。通过合成2-（1-芴基）-2-羟乙胺（9）、2-（2-芴基）-2-羟乙胺（10）、1-（1-芴基）乙胺（11）和1-（2-芴基）乙胺（12）获得了4、5、7和8的平面衍生物。对这四种芴衍生物作为PNMT催化反应的底物和抑制剂的体外活性进行了检测。与4、5、7和8的情况一样，我们观察到烷基胺侧链相对于9-12中存在的联苯骨架有位置偏好。因此，芴基乙醇胺10（“对联苯”）的米氏常数（Km = 26 μM）比9（“间联苯”，Km = 297 μM）低约10倍；在α-甲基苄胺抑制剂中，芴基衍生物11（“间联苯”，Ki = 4.14 μM）在体外抑制PNMT方面比12（“对联苯”，Ki = 185 μM）好约40倍。在每种情况下，4、5、7和8中存在的联芳基系统的构象限制，使得芳香环共面，导致对PNMT活性位点的亲和力增强。因此，乙醇胺5（Km = 82 μM）如10（Km = 26 μM）那样的构象限制以及α-甲基苄胺7（Ki = 89 μM）如11（Ki = 4.14 μM）那样的构象限制，在每种情况下都导致形成更强的酶-配体可解离复合物。这些结果，与这些实验室的其他结果一起，表明PNMT活性位点在与苯乙醇胺底物和α-甲基苄胺抑制剂的中心芳香环部分相互作用的区域之外基本上是一个扁平的疏水口袋。
