org-41841 and thienopyrimidine

A thienopyr(im)idine (Org41841) activates the luteinizing hormone (LH) receptor but does not compete with the natural ligand binding site and does not show agonistic action on the follicle-stimulating hormone receptor (hFSHR) at sub-millimolar concentrations. When this drug is preincubated at sub-micromolar concentrations with host cells expressing the hFSHR, and then washed out, binding analysis and assessment of receptor-effector coupling show that it increases plasma membrane expression of the hFSHR. Real-time PCR shows that this effect did not result from increased hFSHR mRNA accumulation. It is possible that Org41841 behaves as a pharmacoperone, a drug which increases the percentage of newly synthesized receptor routing to the membrane. Like pharmacoperones for other receptors, this drug was able to rescue a particular mutant hFSHR (A(189)V) associated with misrouting and endoplasmic reticulum retention, although other mutants could not be rescued. This is potentially the first member of the pharmacoperone drug class which binds at a site that is distinctive from the ligand binding site.

Topics: Animals; Cell Membrane; Chlorocebus aethiops; COS Cells; Dose-Response Relationship, Drug; Humans; Models, Biological; Protein Binding; Protein Transport; Pyridines; Pyrimidines; Receptors, FSH; Receptors, LH; Thiophenes; Transfection

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.

Topics: Amino Acid Sequence; Cell Line; Cyclic AMP; Genetic Vectors; Humans; Hydrogen Bonding; Ligands; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Polymerase Chain Reaction; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Pyrimidines; Receptors, LH; Receptors, Thyrotropin; Recombinant Fusion Proteins; Signal Transduction; Thiophenes; Transfection