Thermodynamics and docking of agonists to the beta2-adrenoceptor determined using [(3)H](R,R')-4-methoxyfenoterol as the marker ligand
Mol Pharmacol. 2012 Jun;81(6):846-54. doi: 10.1124/mol.111.077347. Epub 2012 Mar 20
Toll L, Pajak K, Plazinska A, Jozwiak K, Jimenez L, Kozocas JA, Tanga MJ, Bupp JE, Wainer IW
G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the β(2)-adrenergic receptor, we developed a new agonist radioligand for binding assays. [(3)H](R,R')-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total β(2)-adrenoceptor (AR) population as determined with the antagonist [(3)H]CGP-12177. The β(2)-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and K(i) values determined for fenoterol analogs model much better the cAMP activity of the β(2)-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [(3)H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β(2)-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β(2)-adrenoceptor.
The stereoselective sulfate conjugation of 4'-methoxyfenoterol stereoisomers by sulfotransferase enzymes
Chirality. 2012 Oct;24(10):796-803. doi: 10.1002/chir.22072. Epub 2012 Jun 29
Iyer LV, Ramamoorthy A, Rutkowska E, Furimsky AM, Tang L, Catz P, Green CE, Jozwiak K, Wainer IW
The presystemic sulfate conjugation of the stereoisomers of 4'-methoxyfenoterol, (R,R')-MF, (S,S')-MF, (R,S')-MF, and (S,R')-MF, was investigated using commercially available human intestinal S9 fractions, a mixture of sulfotransferase (SULT) enzymes. The results indicate that the sulfation was stereospecific and that an S-configuration at the β-OH carbon of the MF molecule enhanced the maximal formation rates with (S,R')-MF (S,S')-MF (R,S')-MF ≈ (R,R')-MF, and competition studies demonstrated that (S,R')-MF is an effective inhibitor of (R,R')-MF sulfation (IC(50) = 60 μM). In addition, the results from a cDNA-expressed human SULT isoform screen indicated that SULT1A1, SULT1A3, and SULT1E1 can mediate the sulfation of all four MF stereoisomers. Previously published molecular models of SULT1A3 and SULT1A1 were used in docking simulations of the MF stereoisomers using Molegro Virtual Docker. The models of the MF-SULT1A3 and MF-SULT1A1 complexes indicate that each of the two chiral centers of MF molecule plays a role in the observed relative stabilities. The observed stereoselectivity is the result of multiple hydrogen bonding interactions and induced conformational changes within the substrate-enzyme complex. In conclusion, the results suggest that a formulation developed from a mixture of (R,R')-MF and (S,R')-MF may increase the oral bioavailability of (R,R')-MF.
Structural and functional interaction of (±)-2-(N-tert-butylamino)-3'-iodo-4'-azidopropiophenone, a photoreactive bupropion derivative, with nicotinic acetylcholine receptors
Neurochem Int. 2012 Dec;61(8):1433-41. doi: 10.1016/j.neuint.2012.10.011. Epub 2012 Oct 26
Arias HR, Feuerbach D, Targowska-Duda KM, Aggarwal S, Lapinsky DJ, Jozwiak K
The pharmacological properties of (±)-2-(N-tert-butylamino)-3'-iodo-4'-azidopropiophenone [(±)-SADU-3-72], a photoreactive analog of bupropion (BP), were characterized at different muscle nicotinic acetylcholine receptors (AChRs) by functional and structural approaches. Ca²⁺ influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1β1γδ) and adult (hα1β1εδ) muscle AChRs, respectively. (±)-SADU-3-72 binds with high affinity to the [³H]TCP site within the resting or desensitized Torpedo AChR ion channel, whereas BP has higher affinity for desensitized AChRs. Molecular docking results indicate that both SADU-3-72 enantiomers interact with the valine (position 13') and serine (position 6') rings. However, an additional domain, between the outer (position 20') and valine rings, is observed in Torpedo AChR ion channels. Our results indicate that the azido group of (±)-SADU-3-72 may enhance its interaction with polar groups and the formation of hydrogen bonds at AChRs, thus supporting the observed higher potency and affinity of (±)-SADU-3-72 compared to BP. Collectively our results are consistent with a model where BP/SADU-3-72 and TCP bind to overlapping sites within the lumen of muscle AChR ion channels. Based on these results, we believe that (±)-SADU-3-72 is a promising photoprobe for mapping the BP binding site, especially within the resting AChR ion channel.