title: Biased small-molecule ligands for selective inhibition of HIV-1 cell entry via CCR5.
abstract: Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small-molecule receptorantagonistsfor inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small-molecule CCR5agonistsas HIV-1 fusion inhibitors. A virus-free cell-based fusion reporter assay, based on mixing "effector cells" (expressing HIVEnvand luciferase activator) with "target cells" (expressing CD4, CCR5 wild type or a selection of well-described mutations, and luciferase reporter), was used as fusion readout. Receptor expression was evaluated by ELISA and fluorescence microscopy. On CCR5 WT, Maraviroc and Aplaviroc inhibited fusion with high potencies (EC50values of 91 and 501 nM, respectively), whereas removal of key residues for both antagonists (Glu283Ala) or Maraviroc alone (Tyr251Ala) prevented fusion inhibition, establishing this assay as suitable for screening of HIV entry inhibitors. Both ligands inhibited HIV fusion on signaling-deficient CCR5 mutations (Tyr244Ala and Trp248Ala). Moreover, the steric hindrance CCR5 mutation (Gly286Phe) impaired fusion, presumably by a direct hindrance of gp120 interaction. Finally, the efficacy switch mutation (Leu203Phe) - converting small-molecule antagonists/inverse agonists to full agonists biased toward G-protein activation - uncovered that also small-moleculeagonistscan function as direct HIV-1 cell entry inhibitors. Importantly, no agonist-induced receptor internalization was observed for this mutation. Our studies of the pharmacodynamic requirements for HIV-1 fusion inhibitors highlight the possibility of future development of biased ligands with selective targeting of the HIV-CCR5 interaction without interfering with the normal functionality of CCR5.
authors.affiliation: Department of Neuroscience and Pharmacology Faculty of Health and Medical Sciences The Panum Institute University of Copenhagen Copenhagen Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Berg
authors.affiliation: Department of Neuroscience and Pharmacology Faculty of Health and Medical Sciences The Panum Institute University of Copenhagen Copenhagen Denmark.
authors.forename: Katja
authors.initials: K
authors.lastname: Spiess
authors.affiliation: Department of Neuroscience and Pharmacology Faculty of Health and Medical Sciences The Panum Institute University of Copenhagen Copenhagen Denmark; Department of Medicine Infectious Disease Unit Herlev Hospital Copenhagen Denmark.
authors.forename: Hans R
authors.initials: HR
authors.lastname: Lüttichau
authors.affiliation: Department of Neuroscience and Pharmacology Faculty of Health and Medical Sciences The Panum Institute University of Copenhagen Copenhagen Denmark.
authors.forename: Mette M
authors.initials: MM
authors.lastname: Rosenkilde
keywords: CCR5; HIV‐1; drug development; efficacy switch; fusion; ligand bias; small‐molecule inhibitors