ABSTRACT
ERG2, emopamil binding protein (EBP), and sigma-1 receptor (sigma(1)) are enzymes of sterol metabolism and an enzyme-related protein, respectively, that share high affinity for various structurally diverse compounds. To discover novel high-affinity ligands, pharmacophore models were built with Catalyst based upon a series of 23 structurally diverse chemicals exhibiting K(i) values from 10 pM to 100 microM for all three proteins. In virtual screening experiments, we retrieved drugs that were previously reported to bind to one or several of these proteins and also tested 11 new hits experimentally, of which three, among them raloxifene, had affinities for sigma(1) or EBP of <60 nM. When used to search a database of 3525 biochemicals of intermediary metabolism, a slightly modified ERG2 pharmacophore model successfully retrieved 10 substrate candidates among the top 28 hits. Our results indicate that inhibitor-based pharmacophore models for sigma(1), ERG2, and EBP can be used to screen drug and metabolite databases for chemically diverse compounds and putative endogenous ligands.
Subject(s)
Carrier Proteins/chemistry , Receptors, sigma/chemistry , Steroid Isomerases/chemistry , Animals , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Databases, Factual , Guinea Pigs , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Isomerism , Ligands , Models, Molecular , Quantitative Structure-Activity Relationship , Radioligand Assay , Receptors, sigma/antagonists & inhibitors , Receptors, sigma/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Steroid Isomerases/antagonists & inhibitors , Steroid Isomerases/metabolism , Sigma-1 ReceptorABSTRACT
EBP (emopamil-binding protein) is a high-affinity binding protein for [3H]emopamil and belongs to the family of so-called sigma receptors. Mutations that disrupt EBP's 3beta-hydroxysteroid sterol delta8-delta7 isomerase activity (EC 5.3.3.5) impair cholesterol biosynthesis and cause X-chromosomal dominant chondrodysplasia punctata. We identified a human cDNA for a novel EBPL (EBP-like protein) with a calculated mass of 23.2 kDa. Amino acid sequence alignments and phylogenetic analysis revealed that EBPL is distantly related to EBP (31% identity and 52% similarity) and found in animals but not in plants. EBPL is encoded by four exons on human chromosome 13q14.2 covering 30.7 kb, and a partially processed EBPL pseudogene was found on 16q21. The EBPL mRNA was expressed ubiquitously and most abundant in liver, lung and kidney. Upon heterologous expression in yeast EBPL had no detectable 3beta-hydroxysteroid sterol delta8-delta7 isomerase and sigma-ligand-binding activity. Nine out of ten amino acid residues essential for catalytic activity of EBP were conserved in EBPL. Replacement of the only differing residue (EBP-Y111W) reduced catalytic activity of EBP. Transfer of the divergent residue from EBP to EBPL (EBPL-W91Y) and chimaerization of EBP and EBPL at various positions failed to restore catalytic activity of EBPL. Chemical cross-linking induced homodimerization of EBPL and EBP. Whereas mevinolin increased the mRNA for EBP and DHCR7 (delta7-sterol reductase) in HepG2 cells, it had no effect on mRNAs for EBPL and sigma1 receptor, indicating that EBP and EBPL expression are not co-ordinated. We propose that EBPL has a yet-to-be-discovered function other than cholesterol biosynthesis.
