Thermodynamics and mechanism of the interaction of willardiine partial agonists with a glutamate receptor: implications for drug development

Abstract

Understanding the thermodynamics of binding of a lead compound to a receptor can provide valuable information for drug design. The binding of compounds, particularly partial agonists, to subtypes of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor is, in some cases, driven by increases in entropy. Using a series of partial agonists based on the structure of the natural product, willardiine, we show that the charged state of the ligand determines the enthalpic contribution to binding. Willardiines have uracil rings with pKa values ranging from 5.5 to 10. The binding of the charged form is largely driven by enthalpy, while that of the uncharged form is largely driven by entropy. This is due at least in part to changes in the hydrogen bonding network within the binding site involving one water molecule. This work illustrates the importance of charge to the thermodynamics of binding of agonists and antagonists to AMPA receptors and provides clues for further drug discovery.

Figure 1

Thermograms showing raw (top) and integrated (bottom) data for NW (A) and IW (B) displacement of glutamate from the GluA2 LBD at 20 °C. The molar ratio is the ratio of ligand to protein. Fits were performed as described by Sigurskjold et al. (C) Calculated ΔΔH, −TΔΔS°, and ΔΔG values for displacement of glutamate by the five willardiine derivatives. (D) Structures of the willardiine derivatives.

Figure 2

(A) Ionization states of the uracil ring of NW. Similar ionization states are possible for the other willardiine derivatives. (B) Effect of a change in pH on the thermodynamic parameters for NW (left) and HW (right) at 10 °C. For both compounds, the pH at which the ionized state is favored is shown on the left and that for which the un-ionized state is favored is shown on the right. (C) Structure of the binding site, showing the direct interactions between NW and the GluA2 LBD [Protein Data Bank (PDB) entry 3RTW(32)]. (D) Structure of the binding site for NW showing the hydrogen bonding network associated with the nitrogen at position 3 of the willardiine ring in the charged form (pH 6.5, PDB entry 3RTW(32)). On the left is the structure and on the right a schematic. In the schematic, NW is colored blue, the protein black, and water green and the H-bonds are colored red. (E) Structure of NW bound to the GluA2 LBD obtained at pH 3.5 (PDB entry 4Q30). At this pH, the ring is largely uncharged. The formatting of this panel is similar to that of panel D. Note the change in the H-bonding network. The potential H-bond between the hydroxyl of S654 and the uracil carbonyl is shown with a question mark because the distance between the two oxygens is relatively long, 3.5 Å, and the angle between this H-bond and the H-bond with the water is not favorable (80°).

Figure 3

Dependence of ΔΔH on temperature.