Conformational constraint in protein ligand design and the inconsistency of binding entropy

Abstract

It is an accepted practice in ligand design to introduce conformational constraint with the expectation of improving affinity, justified by the theoretical possibility that an unfavorable change in binding entropy will be reduced. This rationale of minimizing the entropic penalty through imposing structural constraints upon a ligand, however, has been voiced more often than verified. Here we examine three modified cyclic peptides, along with multiple versions of their linear control analogs, and determine their thermodynamic parameters when binding the same host, the third PDZ domain (PDZ3) of the mammalian postsynaptic density-95 (PSD-95) protein. To begin a two-stage investigation, the initial evaluation involved solution binding studies with isothermal titration calorimetry (ITC), which provided the changes in Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) upon formation of the protein-ligand complex. In the second stage, a selected macrocycle along with two matched linear controls were subjected to more rigorous analysis by ITC, which included (1) change in heat of buffer ionization (DeltaH(ion)) titrations, to examine the role of proton transfer events; (2) change in heat capacity (DeltaC(p)) determinations, to indirectly probe the nature of the binding surface; and (3) osmotic stress experiments, to evaluate desolvation effects and quantitate water release. Together, these demonstrate that the entropic relationship between a macrocyclic ligand and a linear counterpart can be a complex one that is difficult to rationalize. Further, the addition of constraint can, counterintuitively, lead to a less favorable change in binding entropy. This underscores the need to use matched linear control ligands to assure that comparisons are made in a meaningful manner.

Bridging Calorimetry and Simulation through Precise Calculations of Cucurbituril–Guest Binding Enthalpies

“…8−13 Similarly, the efforts of medicinal chemists to enhance binding by making ligands more rigid, and thereby presumably reducing the entropy penalty, sometimes enhances the binding enthalpy instead of the entropy. 14,15,23 The ability to informatively decompose binding enthalpies, as done here, should help resolve such calorimetric paradoxes. Moreover, by combining an enthalpy calculation with a precise calculation of the binding free energy, 63−67 one can immediately obtain the binding entropy, a quantity which has been notoriously difficult to obtain at high precision from simulations.…”

Geometrically Constrained Derivatives of Indolylglyoxylamides as Ligands Binding the GABAA/BzR Complex

“…As the Gibbs energy comprises both enthalpic and entropic contributions, since G° = H° -T S°, optimization of the ligand-protein interaction should necessarily be obtained if the structure of the compound is modified so as to obtain both a more negative binding enthalpy and a more positive binding entropy. As a consequence, it may happen that constrained ligands, compared with their flexible parent compounds, bind target proteins with more favourable free energies, and accordingly a higher binding affinity, because they benefit from more favourable binding enthalpies, although displaying unfavourable entropies of binding [32][33][34][35][36].…”

“…Insertion of a carbonyl group within the side chain, to obtain arylalkylaminocarbonyl derivatives, led to similar results. Indeed, in both the (29-31) and the (32)(33)(34) series, an appreciable binding affinity was displayed only by compounds (29) (K i : 1.7 M) and (32) (K i : 1.6 M), unsubstituted on the side phenyl ring. Insertion of both an electronwithdrawing atom, like chloro, and an electron-releasing group, like OCH 3 , in the para position of their distal phenyl ring significantly reduced the activity.…”

Synthesis of a macrocycle based on Linked Amino Acid Mimetics (LAAM)

“…11 When applied to peptides, macrocyclization can enhance the resistance to protease degradation and improve the affinity by restricting conformational flexibility. 12,13 Here, we have developed the concept of Linked Amino Acids Mimetic (LAAM) subunits for the formation of a library of macrocyclic peptide structures and we envision their use in primary screening or for lead optimization, especially for protein-protein interactions.…”