Toward flexibility-activity relationships by NMR spectroscopy: dynamics of Pin1 ligands.

Drug design involves iterative ligand modifications. For flexible ligands, these modifications often entail restricting conformational flexibility. However, defining optimal restriction strategies can be challenging if the relationship between ligand flexibility and biological activity is unclear. Here, we describe an approach for ligand flexibility-activity studies using Nuclear Magnetic Resonance (NMR) spin relaxation. Specifically, we use (13)C relaxation dispersion measurements to compare site-specific changes in ligand flexibility for a series of related ligands that bind a common macromolecular receptor. The flexibility changes reflect conformational reorganization resulting from formation of the receptor-ligand complex. We demonstrate this approach on three structurally similar but flexibly differentiated ligands of human Pin1, a peptidyl-prolyl isomerase. The approach is able to map the ligand dynamics relevant for activity and expose changes in those dynamics caused by conformational locking. Thus, NMR flexibility-activity studies can provide information to guide strategic ligand rigidification. As such, they help establish an experimental basis for developing flexibility-activity relationships (FAR) to complement traditional structure-activity relationships (SAR) in molecular design.