Competition of milrinone, a non-iodinated cardiac inotropic agent, with thyroid hormone for binding sites on human serum prealbumin (TBPA).

Milrinone [2-methyl-5-cyano-(3,4'-bipyridin)-6(1H)-one] is a positive cardiac inotropic agent recently shown to have thyromimetic activity in vitro in a rabbit myocardial membrane Ca2+-ATPase system [K. M. Mylotte et al., Proc. natn. Acad. Sci. U.S.A. 82, 7974 (1985)]. In the present studies, milrinone was examined for activity as an inhibitor of iodothyronine binding by human serum thyroid hormone transport proteins, thyroxine-binding globulin (TBG), prealbumin (TBPA) and albumin. Polyacrylamide gel electrophoresis at pH 9.0 of sera equilibrated with [125I]thyroxine showed that milrinone competed with L-thyroxine (T4) for binding sites on TBPA (10 and 100 microM milrinone caused 61 and 73% reductions, respectively, in T4 binding to TBPA, P less than 0.01); T4 displaced from TBPA was bound by TBG and albumin. Comparable reductions in T4 binding to TBPA were observed in electrophoretic studies conducted at pH 7.4. Binding of triiodo-L-thyronine (T3) to TBPA was electrophoretically confirmed and shown to be decreased in the presence of milrinone. Electrophoresis of purified TBPA also demonstrated that [14C]milrinone co-migrated with this transport protein and that milrinone displaced tracer T4 from TBPA. Amrinone, the 2-H-5-NH2 analog of milrinone, had less than 5% of the activity of milrinone as an inhibitor of T4 binding in electrophoretic studies. Scatchard analysis of T4 and milrinone binding to purified TBPA, measured by equilibrium dialysis, showed two classes of binding sites, with association constants, respectively, of 6.1 X 10(7) M-1 and 1.6 X 10(6) M-1 for T4, and 1.7 X 10(6) M-1 and 8.9 X 10(2) M-1 for milrinone. Computer graphic modeling of the binding of milrinone to the T4 site in the crystal structure of TBPA showed that milrinone best occupied this site when the substituted bipyridine ring overlapped the phenolic ring of T4. In this orientation the 5-cyano group, which has an electronegativity similar to that of iodine, occupied the same volume as the 5'-iodine of T4. The 5-amino group of amrinone lacks these characteristics. In this orientation, the keto function of milrinone overlapped the T4 4'-hydroxyl and could participate in similar intermolecular interactions. Thus, milrinone, a non-iodinated bipyridine, and thyroid hormone share structural and biochemical homologies and compete for the same binding site on TBPA.