The thyroid hormone analogue SKF L-94901: nuclear occupancy and serum binding studies

Abstract

1. We studied a brominated thyroid hormone analogue, SKF L-94901, which has the potential to lower serum cholesterol without adverse cardiovascular effects. This compound is about 50% as active as tri-iodothyronine (T3) in liver nuclear receptor binding in vivo but only 1% as active in vitro and has nearly 200 times more enzyme-inducing activity in liver than in heart. Our aim was to examine the interaction of SKF L-94901 with [125I]T3 binding to the intact nuclei in whole cells, isolated nuclei and nuclear extracts of human HeLa cells and to investigate the binding of this compound to human serum. 2. Relative to thyroxine (T4), the affinity of this compound for T4-binding globulin was 0.0035%, for transthyretin 1.66% and for albumin 1.26%. Low affinity for serum proteins, with a relatively high circulating free fraction, could explain why SKF L-94901 is more potent in vivo than in vitro. 3. Human HeLa cell nuclei, isolated after whole-cell incubations, bound [125I]T3 with high affinity (Kd = 78 ± 8 pmol/l, mean ± sem), which was displaceable by T3 analogues in the order Triac {[4-(4-hydroxy-3-iodophenoxy)-3,5-di-iodophenyl]acetic acid} > T3 > T4 ≫ reverse T3. Similar high-affinity (Kd = 58 ± 6 pmol/l, mean ± sem) and identical specificity was observed in high-salt (0.4 mol/l KCl) nuclear extracts. In nuclei of whole cells incubated with [125I]T3 and SKF L-94901, the analogue was 0.8% as potent as T3, whereas in experiments with nuclear extract, the analogue was 7.7% as potent as T3. Results from incubation of T3 with isolated nuclei were virtually identical to those obtained with nuclear extracts. 4. These results suggest an extranuclear component may be involved in restricting access of SKF L-94901 to the nucleus. Whether such mechanisms account for observed differences in its effects on different tissues with reduced influence of SKF L-94901 on cardiac tissue remains to be established. 5. We conclude that SKF L-94901 is weakly bound in serum and shows less potent competition for T3 nuclear binding after incubation of whole cells than after incubation with nuclear extracts or isolated nuclei. This compound may allow further analysis of intracellular mechanisms of thyroid hormone transport and action.