Disulfide-Trapping Identifies a New, Effective Chemical Probe for Activating the Nuclear Receptor Human LRH-1 (NR5A2)

Abstract

Conventional efforts relying on high-throughput physical and virtual screening of large compound libraries have failed to yield high-efficiency chemical probes for many of the 48 human nuclear receptors. Here, we investigated whether disulfide-trapping, an approach new to nuclear receptors, would provide effective lead compounds targeting human liver receptor homolog 1 (hLRH-1, NR5A2). Despite the fact that hLRH-1 contains a large ligand binding pocket and binds phospholipids with high affinity, existing synthetic hLRH-1 ligands are of limited utility due to poor solubility, low efficacy or significant off-target effects. Using disulfide-trapping, we identified a lead compound that conjugates with remarkably high-efficiency to a native cysteine residue (Cys346) lining the hydrophobic cavity in the ligand binding domain of hLRH-1. Guided by computational modeling and cellular assays, the lead compound was elaborated into ligands PME8 and PME9 that bind hLRH-1 reversibly (no cysteine reactivity) and increase hLRH-1 activity in cells. When compared with the existing hLRH-1 synthetic agonist RJW100, both PME8 and PME9 showed comparable induction of the LRH-1 dependent target gene CYP24A1 in human HepG2 cells, beginning as early as 3 h after drug treatment. The induction is specific as siRNA-mediated knock-down of hLRH-1 renders both PME8 and PME9 ineffective. These data show that PME8 and PME9 are potent activators of hLRH-1 and suggest that with further development this lead series may yield useful chemical probes for manipulating LRH-1 activity in vivo.

LRH-1 mitigates intestinal inflammatory disease by maintaining epithelial homeostasis and cell survival

“…To ascertain if ligand binding is necessary for hLRH-1-mediated rescue, we next attempted to salvage organoid viability with the well-characterized ligand-binding-defective variant of hLRH-1 (PM; Fig. 3a ) 26 , 27 . Bulky hydrophobic residues were modeled in the binding pocket to impede ligand uptake without effecting protein integrity, as previously demonstrated in cultured cell lines 26 .…”

“…Indeed, the human PM variant is stably expressed in intestinal organoids (Fig. 3c , right), and despite the fact that the hPM retains modest transcriptional activity 26 , 27 , it failed to rescue TNFα-induced cell death in Lrh1 IEC-KO intestinal organoids (Fig. 3f , light blue bar).…”

NR5A2 discovering compounds that block tumor growth in PDAC

“…Computational modeling suggested a structure of the small molecule complex with NR5A2 and cellular assays were used to find an analog with no sulfur atom. This analog is termed PME8 and is a good activator, with A dissociation constant of 5 μM …”

“…This analog is termed PME8 and is a good activator, with A dissociation constant of 5 μM. 10 This technical approach could be used with protein complexes of NR5A2 such as with DAX1 peptide or β-catenin and suggest that with further development this chemical probes that are specific antagonists.…”

Development of a new class of liver receptor homolog-1 (LRH-1) agonists by photoredox conjugate addition

“…Towards this end, several laboratories have madesignificant advances in developing potent LRH-1 modulators. [13][14][15] Despite these advances, rational design has been difficult, in partbecause of the large, highly hydrophobic LRH-1 ligand binding pocket. Due to this lipophilicity and a scarcity of sites for anchoring polar interactions, even highly similar compounds can bind unpredictably, 15,16 further complicating systematic agonist development.…”