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.
Highlights
Liver Receptor Homolog 1 (LRH-1, NR5A2) is among several nuclear receptors (NRs) that still lack a high affinity, selective chemical probe [1]
Receptor-ligand interactions can greatly change the size of the ligand binding pocket, as evidenced by the contracted binding pocket observed when hLRH-1 ligand binding domains (LBDs) is bound to either the shorter-chain phospholipid ligand DLPC [5] or the synthetic ligand GSK8470 [7], compared to the higher-affinity phosphoinositide ligands PIP2 and PIP3 [8]
Covalent docking was used to visualize conjugated compounds bound within the hLRH-1 ligand binding domain (PDB:3PLZ).The ligand binding pocket is an hourglass shaped structure with the bottom portion nearest to the solvent
Summary
Liver Receptor Homolog 1 (LRH-1, NR5A2) is among several nuclear receptors (NRs) that still lack a high affinity, selective chemical probe [1]. Human LRH-1 LBD structures bound to either endogenous or exogenous phospholipid ligands reveal the two lipid tails buried within and occupying the entire length of the hydrophobic pocket, and the headgroup positioned at the mouth of the pocket [2, 3, 5]. Mouse LRH-1 contains a salt-bridge at the mouth of the pocket that greatly diminishes the binding of phospholipid ligands [2, 6]. Receptor-ligand interactions can greatly change the size of the ligand binding pocket, as evidenced by the contracted binding pocket observed when hLRH-1 LBD is bound to either the shorter-chain phospholipid ligand DLPC [5] or the synthetic ligand GSK8470 [7], compared to the higher-affinity phosphoinositide ligands PIP2 and PIP3 [8]. For hLRH-1, standard virtual screening methods that survey a static structure might fail to capture the structural dynamics of the hydrophobic ligand binding pocket
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