GPCRs are a group of membrane receptors with seven transmembrane helices. They sense various signals from outside of the cells, transfer the signals to intracellular pathways and modulate cellular responses to environment. The ligands of GPCRs include odorants, hormones, neurotransmitters and chemokines. GPCRs are targets of approximately 30% commercially available medicines.
Drugs that target the orthosteric pockets of GPCRs (where native hormones or neurotransmitters bind) often have problems with selectivity, especially for GPCRs within a single subfamily. For example, there are nine adrenergic receptors in human; all of them can be activated by adrenaline. The orthosteric pockets between these adrenergic receptors are highly conserved. Drugs that target the orthosteric pocket of one receptor may also interact with other receptors, which may cause side effects. Unlike orthosteric ligands, allosteric modulators may bind to less conserved regions and are more likely to be selective. The majority of GPCR structures reported to date were obtained in complex with orthosteric antagonists; only a few structures were obtained with allosteric ligands. The Kobilka Lab at Tsinghua University has established collaborations with other groups to develop allosteric modulators for GPCRs, and to use structural biology to understand how allosteric modulators perform their functions.
Compound 15 (Cmpd-15) is an allosteric modulator of β2AR that was recently isolated from a DNA-encoded small-molecule library by Professor Robert Lefkowitz’s group (Ahn, S. et al. 2017). Biochemical and pharmacologic studies showed that Cmpd-15 crosses the plasma membrane and binds to the intracellular side of the β2AR. Orthosteric β-adrenergic receptor antagonists, known as beta-blockers, are amongst the most prescribed drugs in the world and Cmpd-15 is the first allosteric beta-blocker.
In this work, Brian Kobilka’s group used a β2AR – T4 lysozyme fusion protein (β2AR–T4L) for crystallographic studies. Initially they obtained a 2.5Å crystal structure of β2AR–T4L in complex with carazolol and Cmpd-15. Cmpd-15 could only be added to 200 uM in crystal conditions due to solubility limit. The structure revealed weak positive electron density near the cytoplasmic ends of transmembrane (TM) segments 1,2,6 and 7. But it was impossible to unambiguously dock Cmpd-15 into the density. To improve the solubility of Cmpd-15, a carboxylic acid functionalized polyethelyene glycol group was added to the position used for coupling the compound to its DNA tag. The modified compound is named Cmpd-15PA. A 2.7Å crystal structure was obtained for β2AR–T4L bound to both carazolol and Cmpd-15PA. The crystal structure clearly revealed how Cmpd-15PA interacts with the β2AR. It binds to a pocket formed by TMs 1, 2, 6, 7, helix 8 and intracellular loop 1 (ICL1). Structure comparison, molecular dynamic simulations and biochemical characterizations suggest Cmpd-15PA functions in two different ways. One is stabilizing the receptor in the inactive conformation by restricting TM6 in the inactive state. The other is directly competing with transducers like G protein or arrestins to interfere with signal transduction.
In December 2016, crystal structures of two chemokine receptors (CCR2 and CCR9) bound to intracellular negative allosteric modulators were reported in Nature (Zheng, Y. et al. 2016. Oswald, C. et al. 2016). While the modulators are chemically distinct and the interacting residues are not conserved, the binding locations are similar between allosteric pockets on the two chemokine receptors and the Cmpd-15PA binding pocket on the β2AR. These results suggest that the location of this intracellular allosteric pocket may be conserved amongst diverse GPCRs, and there may be potential therapeutic value in targeting this surface for drug discovery.
This work is the result of a collaboration between Professor Brian Kobilka’s lab at Tsinghua University, Professor Robert Lefkowitz’s lab at Duke University, Professor Chen Xin’s lab at Changzhou University, and Professor Ron Dror’s lab at Stanford University. Professor Brian Kobilka and Professor Robert Lefkowitz are corresponding authors of the paper. Dr. Xiangyu Liu from School of Medicine, Tsinghua University and Dr. Seungkirl Ahn from Duke University Medical Center are co-first authors of the paper. The work is supported by grants from Beijing Advanced Innovation Center for Structural Biology. Crystal diffraction and data collection was supported by Spring-8 synchrotron radiation facility, Japan.
