Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β2-adrenergic receptor (β2AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β2AR. We show that the select ligands bind preferentially to different predicted conformers of β2AR, and identify a role of β2AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as "computational probes" to systematically identify protein conformers with likely biological significance. © 2012 Isin et al

Receptor-Mediated Enhancement of Beta Adrenergic Drug Activity by Ascorbate In Vitro and In Vivo

RATIONALE: Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors. OBJECTIVES: Extending this work to beta 2 adrenergic systems in vitro and in vivo. METHODS: Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction. MEASUREMENTS: Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep. MAIN RESULTS: Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3-10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol's effect on heaves and produces a 10-fold enhancement of albuterol activity in "asthmatic" sheep. CONCLUSIONS: Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are probably caused by a novel mechanism involving phosphorylation of aminergic receptors and have clinical and drug-development applications

Host-Guest Interactions between Candesartan and Its Prodrug Candesartan Cilexetil in Complex with 2-Hydroxypropyl-β-cyclodextrin: On the Biological Potency for Angiotensin II Antagonism

Renin−angiotensin aldosterone system inhibitors are for 25 a long time extensively used for the treatment of cardiovascular and renal 26 diseases. AT1 receptor blockers (ARBs or sartans) act as antihypertensive 27 drugs by blocking the octapeptide hormone Angiotensin II to stimulate 28 AT1 receptors. The antihypertensive drug candesartan (CAN) is the active 29 metabolite of candesartan cilexetil (Atacand, CC). Complexes of 30 candesartan and candesartan cilexetil with 2-hydroxylpropyl-β-cyclodextrin 31 (2-HP-β-CD) were characterized using high-resolution electrospray 32 ionization mass spectrometry and solid state 13C cross-polarization/ 33 magic angle spinning nuclear magnetic resonance (CP/MAS NMR) 34 spectroscopy. The 13C CP/MAS results showed broad peaks especially in 35 the aromatic region, thus confirming the strong interactions between 36 cyclodextrin and drugs. This experimental evidence was in accordance with molecular dynamics simulations and quantum mechanical calculations. The 37 synthesized and characterized complexes were evaluated biologically in vitro. It was shown that as a result of CAN’s complexation, CAN exerts higher 38 antagonistic activity than CC. Therefore, a formulation of CC with 2-HP-β-CD is not indicated, while the formulation with CAN is promising and needs 39 further investigation. This intriguing result is justified by the binding free energy calculations, which predicted efficient CC binding to 2-HP-β-CD, and 40 thus, the molecule’s availability for release and action on the target is diminished. In contrast, CAN binding was not favored, and this may allow easy 41 release for the drug to exert its bioactivity

Discovery of a stable tripeptide targeting the N‑domain of CRF1 receptor

The corticotropin-releasing factor (CRF) and its CRF1 receptor (CRF1R) play a central role in the maintenance of homeostasis. Malfunctioning of the CRF/CRF1R unit is associated with several disorders, such as anxiety and depression. Non-peptide CRF1R-selective antagonists have been shown to exert anxiolytic and antidepressant efects on experimental animals. However, none of them is in clinical use today because of several side efects, thus demonstrating the need for the development of other more suitable CRF1R antagonists. In an efort to develop novel CRF1R antagonists we designed, synthesized and chemically characterized two tripeptide analogues of CRF, namely (R)-LMI and (S)-LMI, having their Leu either in R (or D) or in S (or L) confguration, respectively. Their design was based on the crystal structure of the N-extracellular domain (N-domain) of CRF1R/CRF complex, using a relevant array of computational methods. Experimental evaluation of the stability of synthetic peptides in human plasma has revealed that (R)-LMI is proteolytically more stable than (S)-LMI. Based on this fnding, (R)-LMI was selected for pharmacological characterization. We have found that (R)-LMI is a CRF antagonist, inhibiting (1) the CRF-stimulated accumulation of cAMP in HEK 293 cells expressing the CRF1R, (2) the production of interleukins by adipocytes and (3) the proliferation rate of RAW 264.7 cells. (R)-LMI likely blocked agonist actions by interacting with the N-domain of CRF1R as suggested by data using a constitutively active chimera of CRF1R. We propose that (R)-LMI can be used as an optimal lead compound in the rational design of novel CRF antagonists

Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody

G protein-coupled receptors (GPCRs) are integral membrane proteins that play an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. Thus far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists(1,2) or receptor stabilization by mutagenesis(3-5). Many natural agonists such as adrenaline, which activates the β(2)-adrenoceptor (β(2)AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β(2)AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here, we present structures of active-state β(2)AR bound to three chemically distinct agonists: the ultra high-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ~80 pM. The adrenaline-bound receptor structure is overall similar to the others, but shows substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β(2)AR and related GPCRs