Compound Profiling Using a Panel of Steroid Hormone Receptor Cell-Based Assays

Abstract

A major focus in the current discovery of drugs targeting nuclear receptors (NRs) is identifying drugs with reduced side effects by improving selectivity, not only from other receptors but also by selective modulation of the NR of interest. Cellular assays not only provide valuable information on functional activity, potency, and selectivity but also are ideally suited for differentiating partial agonists and antagonists. The ability to partially activate a receptor is believed to be closely tied to the ability to selectively modulate the NR, resulting in expression of a subset of the normally regulated genes. To this end, the authors have built a complete panel of cell-based steroid hormone receptor assays for the androgen receptor, estrogen receptor alpha, estrogen receptor beta, glucocorticoid receptor, mineralocorticoid receptor, and progesterone receptor by stably engineering a Gal4 DNA-binding domain/nuclear receptor ligand-binding domain fusion protein into an upstream activation sequence beta-lactamase reporter cell line. Each assay was validated with known agonists and antagonists for correct pharmacology and high-throughput compatibility. To demonstrate the utility of these assays, the authors profiled 35 pharmacologically relevant compounds in a dose-response format against the panel in both agonist and antagonist modes. The results demonstrated that selective estrogen receptor modulators can be identified and differentiated, as well as mixed and partial agonists and antagonists easily detected in the appropriate assays. Importantly, a comparison of the chimeric assays with fulllength reporter gene assay data from the literature shows a good degree of correlation in terms of selectivity and pharmacology of important ligands. Taken together, these steroid hormone receptor assays provide good selectivity, sensitivity, and appropriate pharmacology for high-throughput screening and selectivity profiling of modulators of steroid hormone receptors. (Journal of Biomolecular Screening 2008:755-765)

Two Panels of Steroid Receptor Luciferase Reporter Cell Lines for Compound Profiling

“…Similarly both AR reporter cell lines respond to dihydrotestosterone in the picomolar range: EC 50 (U2OS-AR/3xGRE) = 4 pM and EC 50 (U2OS-AR/MMTV) = 3 pM (Table 1c ) and MR to aldosterone in the subnanomolar range: EC50(U2OS-MR/MMTV) = 88 pM (Table 1g ). We also compared data generated with our cell lines to values in the literature and found our data comparable to literature reports for all receptor/promoter combinations [11, 12, 14, 19-21]. …”

Two Panels of Steroid Receptor Luciferase Reporter Cell Lines for Compound Profiling

“…Similarly both AR reporter cell lines respond to dihydrotestosterone in the picomolar range: EC 50 (U2OS-AR/3xGRE) = 4 pM and EC 50 (U2OS-AR/MMTV) = 3 pM (Table 1c) and MR to aldosterone in the subnanomolar range: EC50(U2OS-MR/MMTV) = 88 pM (Table 1g). We also compared data generated with our cell lines to values in the literature and found our data comparable to literature reports for all receptor/promoter combinations [11,12,14,[19][20][21]. ER (1a, 1b), AR (1c, 1d), GR (1e, 1f) …”

“…Several luciferase reporter systems have been developed recently using different cell lines [11][12][13][14]. Here we report on a development of two panels of U2OS-based luciferase steroid receptor reporter cell lines using two different reporter formats.…”

Mode of action prediction of ligands of steroid hormone receptors

“…Так, минералокортикоидные рецепторы могут связывать кортизол, а рецепторы прогестерона способны связывать помимо гестагенов также глюкокортикоиды, минералокортикоиды и в меньшей степени андрогены [2]. Некоторые стероиды, не являющиеся гормонами, могут взаимодействовать со всеми 5 типами, обуславливая развитие определенных эффектов [3,4]. Таким образом, при создании лекарственного прототипа возникает проблема селективности лиганда, когда желательно заранее определить спектр возможных взаимодействий конкретного вещества.…”