New High-Throughput Screen Discovers Novel Ligands of Full-Length Nuclear Receptor LRH-1

Abstract

Nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) is a lipid-regulated transcription factor and an important drug target for several liver diseases. Advances toward LRH-1 therapeutics have been driven recently by structural biology, with fewer contributions from compound screening. Standard LRH-1 screens detect compound-induced interaction between LRH-1 and a transcriptional coregulator peptide, an approach that excludes compounds that regulate LRH-1 through alternative mechanisms. Here, we developed a FRET-based LRH-1 screen that simply detects compound binding to LRH-1, applying it to discover 58 new compounds that bind the canonical ligand-binding site in LRH-1 (2.5% hit rate), also supported by computational docking. Four independent functional screens identified 15 of these 58 compounds to also regulate LRH-1 function in vitro or in living cells. Although one of these 15 compounds, abamectin, directly binds LRH-1 and regulates full-length LRH-1 in cells, abamectin failed to regulate the isolated ligand-binding domain in standard coregulator peptide recruitment assays using PGC1α, DAX-1, or SHP. Abamectin treatment of human liver HepG2 cells selectively regulated endogenous LRH-1 ChIP-seq target genes and pathways associated with known LRH-1 functions in bile acid and cholesterol metabolism. Thus, the screen reported here can discover compounds not likely to have been identified in standard LRH-1 compound screens but which bind and regulate full-length LRH-1 in cells.

A novel heuristic of rigid docking scores positively correlates with full-length nuclear receptor LRH-1 regulation.

“…Prioritizing hit compounds identified from a primary compound screen is important for drug development[1–6] as follow-up secondary assays are resource-intensive, particularly cell-based assays of target protein function[7,8]. Cell-based assays monitor compound activity in a physiologically relevant environment, increasing confidence in results[7,9–11] while retaining compatibility with higher-throughput screening formats[12].…”

“…Thus, there is a need for docking approaches that have been validated with wet-lab data, correlating docking scores with compound activity in cell-based assays[2–4,23,24] particularly for allosterically-regulated targets[5,20,22]. Here, we discovered and validated one such approach, retrospectively correlating published cell-based functional data[1] with new rigid-body docking of compounds to Liver Receptor Homolog-1 (LRH-1, NR5A2 ), an allosterically regulated nuclear receptor and drug target for several human diseases.…”

“…To address this, we executed an LRH-1 compound screen that identified 58 new compounds which simply bind directly to LRH-1 from the 2322 compound Spectrum Discovery library[1]. This FRET-based screen measured the ability of each compound to compete with a probe installed in the canonical ligand binding site of LRH-1, suggesting these compounds also bind LRH-1 at the canonical ligand binding site.…”

“…Surprisingly and against the standard dogma of nuclear receptor regulation, there was almost no overlap in these compounds. The compounds either regulated coregulator binding to the LBD, or the compounds regulated full-length LRH-1 assays in cells, with no overlap, even though these compounds were all identified based on their ability to directly bind pure, recombinant LRH-1[1].…”

“…Here, we examined the interaction of these compounds with LRH-1 by applying PyRx computational rigid docking[72] to predict relative docked binding energies to a total of 19 structural models of LRH-1 (18 crystal structures of the isolated LBD and 1 integrative model of the full-length LRH-1[73]). We then correlated those docking results retrospectively with published cell-based functional data from our previous study (439 total functional assay measurements)[1]. We found that docking to the full-length model of LRH-1[73] predicts different binding positions for compounds active on full-length LRH-1, which were closer to the entrance of the ligand binding pocket and Helix 6, an important helix that helps mediate ligand activation of LRH-1 [37,64,70,71,74–76].…”