Thermodynamic analysis in fragment-based drug discovery

Thermodynamic analysis provides access to the determinants of binding affinity, enthalpy and entropy. In fragment-based drug discovery (FBDD), thermodynamic analysis provides a powerful tool to discriminate fragments based on their potential for successful optimization. The thermodynamic data generated by FBDD studies can in turn be used to better understand the forces that drive biomolecular interactions. In this review, the technologies that enable thermodynamic analysis of fragment-protein complexes are discussed. In addition, the available thermodynamic data on fragment-protein complexes is summarized and several key studies which highlight the role of thermodynamics in FBDD are discussed in more detail. Although, thermodynamic analysis is not yet applied widely within the FBDD field, the first success stories are starting to appear, exemplifying its value in the development of a more efficient fragment optimization process and a better understanding of ligand-protein interactions. Chapter 3 Thermodynamic analysis in fragment-based drug discovery 40 Introduction Fragment-based drug discovery (FBDD) is making an impact as an efficient and effective drug discovery method. 1 With small libraries of a few thousand structures and hit rates reaching 10%, FBDD provides a valuable alternative to HTS. 2 A crucial aspect of FBDD is the efficient optimization of the binding affinity of fragment hits towards high affinity clinical candidates. Ultimately, binding affinity is governed by the changes in enthalpy and entropy (see Glossary) that occur upon formation of a ligand-protein complex. The small size of fragments simplifies the process of relating the thermodynamic profile of fragments to the interactions made during binding. When combined with structural data, the stepwise fragment growing process provides an ideal dataset with which to improve our understanding of the thermodynamics of binding. In this manner both FBDD, and our understanding of the thermodynamics of binding, have much to gain from the application of thermodynamic analysis in FBDD. Thermodynamics in drug discovery Thermodynamic analysis provides access to the constituents of the Gibbs energy of binding (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) (see Glossary). Enthalpy, or heat energy, is associated with direct binding forces such as hydrogen bonding, van der Waals forces and π-π interactions. Entropy, a term determined by the number of accessible states of a system, is associated with conformational freedom and the hydrophobic effect (see Glossary). The thermodynamic changes that occur when a ligand binds to its respective protein binding site are schematically depicted in Figure 1. Improving the enthalpic contribution to binding generally involves optimizing the polar interactions made by a ligand. This may be achieved by strengthening already existing interactions within the binding site or by forming new interactions by adding additional polar groups to a ligand. However, a poorly optimized polar interaction may not simply provide a lower favorable contribution to the enthalpy change on binding, it may actually result in an unfavorable contribution to enthalpy. Desolvation of polar functionalities comes at a cost, which can only be overcome when the positioning of interacting groups obeys strict angle and distance requirements. 3 As a consequence, enthalpic optimization is a difficult route by which to improve affinity. However, due to the specificity of polar interactions, enthalpy driven ligand binding may provide a major selectivity advantage. 4 Interestingly, retrospective analysis of the thermodynamics of HIV-protease inhibitors and cholesterol lowering statins, shows that the later best in class compounds, which outperformed earlier first in class compounds, had been enthalpically optimized. 5 While enthalpic optimization can provide highly selective high affinity drugs, medicinal chemists tend to optimize entropy, as shown in a recent study comparing synthetic and natural drugs. 6 Unfortunately, too much focus on entropic optimization by constraining ligands in their bioactive conformation, and by the addition of hydrophobic groups, may in the end result in poorly soluble compounds, with reduced selectivity and higher chances of attrition. 7,8 A further complication to thermodynamic optimization results from enthalpy-entropy compensation. As a polar interaction becomes tighter (enthalpically favorable), the binding atoms Chapter 3 Thermodynamic analysis in fragment-based drug discovery 41 become locked into a tighter conformational geometry (entropically unfavorable). 9,10 This means that optimizing enthalpy necessarily adversely affects entropy and vice versa. Since we are looking at interactions occurring in water, desolvation and the ordering or disordering of water surrounding the binding partners may play an important role in enthalpy-entropy compensation. 10 As a result, large favorable changes in enthalpy or entropy often result in only minor gains in binding affinity. Figure 1: In this simplified schematic diagram, four stages of ligand binding are shown. The colors indicate the degree of conformational freedom of the ligand, protein and water as indicated on the key. 1) Prior to complex formation, the ligand and the protein binding site are solvated by water. 2) and 3) As the ligand approaches the binding pocket, the ligand and the protein’s conformational freedom become increasingly restricted, resulting in an unfavorable entropic contribution. In addition, the polar moieties become partially desolvated, resulting in an unfavorable change in enthalpy. At the same time, the formation of enthalpic van der Waals interactions, as well as the hydrophobic effect (see Glossary), ensure that this process is favorable. 4) Eventually, the ligand reaches a binding conformation and a significant favorable contribution to enthalpy is made as the ligand and protein become locked together, resulting in an unfavorable entropy contribution. At this crucial stage, the net effect of the polar interactions, desolvation, conformational constraint and the hydrophobic effect, will determine the final thermodynamic profile of the ligand. Chapter 3 Thermodynamic analysis in fragment-based drug discovery 42 For an extensive overview on the use of thermodynamics in drug discovery, the reader is referred to the book on drug-receptor thermodynamics edited by Raffa. 11 An overview of the studies in which isothermal titration calorimetry (ITC) (see below) has been applied to attain thermodynamic data is published annually, and represents a good starting point for those interested in an overview on more recent studies. 12-18 Finally, several online databases that contain data on the thermodynamics of ligand-protein interactions exist, providing access to relevant studies. 6,19,20