Fragment deconstruction of small, potent factor Xa inhibitors: exploring the superadditivity energetics of fragment linking in protein-ligand complexes.

Predictable thermodynamic additivity is one of the cornerstones of classical covalent chemistry, allowing accurate calculation of energy terms for complete processes by addition of terms for individual components. However this principle breaks down in complex noncovalent systems, such as biological systems, in which the energetics of individual components are not truly independent of each other. This complicates predicting protein structure and folding and, the focus of this work, the prediction of ligand binding to proteins. Molecular recognition in protein–ligand complexes predominantly occurs through multiple noncovalent interactions, whereas their contribution to the total free-energy of binding (DG) is often unevenly distributed over the contact interface. The identification of ligands as “molecular anchors” for high affinity regions in proteins (“hot spots”) is fundamental for fragment-based drug discovery, 3] indicating the similarity of ligandand protein-centric concepts. Often highaffinity ligands encompass more than one fragment in proximal protein sites; in a few cases, individual fragments in two neighboring sites could be linked to result in high binding affinity. Ideally, the DG of linked fragments should be significantly greater than the sum of DG increments from each fragment. This overproportional increase (“superadditivity”) is attributed to the fact that each fragment loses a significant part of its rigid body rotational and translational entropy upon complex formation. Thus, the sum of DG for two fragments includes two unfavorable rigid body entropy barrier terms, whereas the joined molecule is only affected by one of these terms. Any ligand has to overcome this barrier because of entropy loss upon association to its site. The nonadditivity for DG contributions is defined as linker coefficient E corresponding to the difference between the sums of fragment affinity and the final ligand [Eq. (1)]. DGfinal 1⁄4 DGfrag1 þ DGfrag2 þ DGlink with DGlink 1⁄4 R T ln E ð1Þ