Conformational Preference for the Binding of Biaryl Substrates and Inhibitors to the Active Site of Phenylethanolamine N-Methyltransferase

Article abstract of 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 α-methylbenzylamine inhibitors.For bound substrates, this region is located in the vicinity of the para position of the aromatic ring, while for bound α-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-α-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 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 (K_m = 26 μM) that is approximately 10 times lower than that for 9 ("m-bipenyl", K_m = 297 μM); in the α-methylbenzylamine inhibitors, fluorenyl derivative 11 ("m-biphenyl", K_i = 4.14 μM) is approximately 40 times better than 12 ("p-biphenyl", K_i =185 μM) 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 thr PNMT active site.Thus, conformational restriction of ethanolamine 5 (K_m = 82 μM) and α-methylbenzylamine 7 (K_i = 89 μM) as in 11 (K_i = 4.14 μM) 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 α-methylbenzylamine inhibitors is essentially a flat, hydrophobic pocket.