Binding thermodynamics at the human cannabinoid CB1 and CB2 receptors

Abstract

The thermodynamic parameters G°, H° and S° of the binding equilibrium of agonists and antagonists at cannabinoid CB 1 and CB 2 receptors were determined by means of affinity measurements at different temperatures and van't Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB 1 antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB 1 and CB 2 receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB 1 and CB 2 binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.Keywords: Binding mechanisms; Binding thermodynamics; drug development; enthalpy-entropy compensation; Pharmacokinetics. Page 4 of 27A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 INTRODUCTIONThe thermodynamic analysis of the binding equilibrium of a drug to its receptor allows us to evaluate the two components, standard enthalpy ( H°) and standard entropy ( S°), of the standard free energy ( G°) of the binding equilibrium [1]. It is often assumed that H° and S° terms represent the two classes of factors responsible for the drug-receptor recognition phenomenon: nonbonded interactions, as hydrogen bonding and multipolar or dispersive interactions (which are mainly to be related to the enthalpic term), and solvent reorganization (which is most properly associated with the entropic one) [2]. There are two main strategies for the evaluation of G°, H° A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Milano, Italy). All other reagents were purchased from Sigma-Aldrich (Milano, Italy). Cell Culture.hCHO-CB 1 and hCHO-CB 2 were grown adherently and mantained in Ham's Medium with nutrient mixture F12 (Ham's/F12), containing 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 g/ml), L-glutamine (2 mM) and Geneticin (G418, 0,4 mg/ml) at 37°C in 5% CO 2 /95% air.Cells were split 2 or 3 times weekly at a ratio between 1:5 and 1:20. Membrane Preparation.For membrane preparation the culture medium was removed. The cells were washed with PBS and scraped off T75 flasks in ice-cold hypotonic buffer (5 mM Tris HCl, 2 mM EDTA, pH 7.4). The ce...

Thermodynamics and Docking of Agonists to the β2-Adrenoceptor Determined Using [3H](R,R′)-4-Methoxyfenoterol as the Marker Ligand

“…This observation was confirmed using the approach developed by Borea et al (2000) (Merighi et al, 2010), in which the data obtained in this study and the data previously obtained using [ 3 H]CGP-12177 as the radioligand (Jozwiak et al, 2010a) were placed in a scatter plot of ϪT⌬S°versus ⌬H°. As seen in Fig.…”

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the β2-adrenoceptor

“…(R,R')- and (S,S')-fenoterol and (R,S')- and (S,R')-fenoterol, reflect the specific binding interactions with the β 2 -AR. The designation of the binding process as enthalpy-driven or entropy-driven is the sum of the enthalpic (ΔH°) and entropic (ΔS°) contributions of these interactions; in which ΔH° is associated with hydrogen bond formation, polar interactions and van der Waals interactions and ΔS° reflects hydrophobic interactions produced by an increase in solvent entropy arising from the conformational changes in the receptor involving hydrophobic groups and the release of water upon binding [3,5,10]. …”

“…[4]. This principle has been recently reviewed and appears to hold for the β-AR, adenosine A 1 and A 2A , A 2B and A 3 , cannabinoid CB 1 and CB 2 , histamine H 3 , glycine, GABA A , serotonin 5-HT 3 , nicotinic acetylcholine and purinergic P2X 3 receptors [4,5]. However, “thermodynamic agonist-antagonist discrimination” does not appear to apply to cholecystokinin CCK2, dopamine D 2 , histamine H 1 , δ- and μ-opiod, purinergic P2X 1 and serotonin 5-HT 1A receptors [4,5].…”

Is there a link between selectivity and binding thermodynamics profiles?

“…Binding thermodynamics of agonists and antagonists of cannabinoid CB1 and CB2 receptors were determined by van't Hoff analysis [19] a methodology that is generally considered to be less reliable than ITC measurements, and highly influenced by the heat capacity change of the system. In this case, similarly to many GPCR targets, binding thermodynamics separates the ligands by their functional activity.…”