title: Novel yeast-based strategy unveils antagonist binding regions on the nuclear xenobiotic receptor PXR.
abstract: BACKGROUND
  Ketoconazole binds to and antagonizes pregnane X receptor (PXR) activation.
  
  
  RESULTS
  Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding.
  
  
  CONCLUSIONS
  Ketoconazole genetically interacts with specific PXR surface residues.
  
  
  CONCLUSIONS
  A yeast-based genetic method to discover novel nuclear receptor interactions with ligands that associate with surface binding sites is suggested. The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple "gain-of-function" mutants that were "resistant" to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications.
authors.affiliation: Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Li
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Redinbo
authors.forename: Madhukumar
authors.initials: M
authors.lastname: Venkatesh
authors.forename: Sean
authors.initials: S
authors.lastname: Ekins
authors.forename: Anik
authors.initials: A
authors.lastname: Chaudhry
authors.forename: Nicolin
authors.initials: N
authors.lastname: Bloch
authors.forename: Abdissa
authors.initials: A
authors.lastname: Negassa
authors.forename: Paromita
authors.initials: P
authors.lastname: Mukherjee
authors.forename: Ganjam
authors.initials: G
authors.lastname: Kalpana
authors.forename: Sridhar
authors.initials: S
authors.lastname: Mani
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D025141:Drug Resistance, Fungal; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D062105:Molecular Docking Simulation; D016296:Mutagenesis; D015688:Oncogene Protein pp60(v-src) / Q000096:biosynthesis / Q000235:genetics; D000077297:Pregnane X Receptor; D011485:Protein Binding; D011987:Receptors, Steroid / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D012293:Rifampin / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000378:metabolism; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques; D015262:Xenobiotics
chemical_list: D000935:Antifungal Agents; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D015262:Xenobiotics; D015688:Oncogene Protein pp60(v-src); D007654:Ketoconazole; D012293:Rifampin
keywords: Antagonist; Drug Development; Drug Metabolism; Ketoconazole; Mutagenesis; Nuclear Receptors; Pregnane X Receptor; Protein Drug Interactions; Proteomics; Yeast Two-hybrid