A NOVEL-APPROACH FOR COMPARING LIGAND-BINDING RESULTS FROM TITRATION AND COMPETITION ANALYSES TO STUDY HORMONE MIMICS

Assessment of a compound's ability to mimic or disrupt a hormone's act ivity in a target cell requires evaluation of a complex cascade diffic ult to reproduce in vitro. Inhibition of radiolabeled hormone binding by an unlabeled compound has been used as a preliminary estimate of a compound's ability to impersonate a hormone. We describe an efficient and reproducible approach for simultaneous determinations of the compe titive behavior of a candidate hormone mimic in which an array of liga nd binding isotherms is generated in the presence of various concentra tions of the test compound. Influence of a candidate hormone mimic on radioligand binding affinity (apparent Kd or Ka values) of the recepto r protein is determined directly from Scatchard plots using Lundon One -Site(R) Software. Competition curves generated either from ligand tit ration data or from analysis of binding inhibition of a candidate mimi c at a single ligand concentration using Lundon Compete(R) Software yi elded apparent Kd values of the hormone competitor. Wild-type estrogen receptors from calf uterus and estrone as the candidate estrogen mimi c were used to demonstrate the utility of the approach, which may be a pplied to a broad range of hormone receptors and their cognate ligands .