A multi-step NMR based screening assay has been developed for identifying and evaluating chemical leads for their ability to bind a target protein. The multi-step NMR assay provides structure-related information while being an integral part of a structure based drug discovery and design program. The fundamental principle of the multi-step NMR assay is to combine distinct 1D and 2D NMR techniques in such a manner that the inherent strengths and weakness associated with each technique is complementary to each other in the screen. By taking advantage of the combined strengths of a variety of 1D and 2D NMR experiments, it is possible to minimize protein requirements and experiment time while being able to verify ligand binding, identify the ligand binding site and differentiate between non-specific and stoichiometric binders. Furthermore, the quality and physical behavior of the ligand is readily evaluated to determine its appropriateness as a chemical lead. An important strength of the multi-step NMR screen is its inherent flexibility, permitting the details of the assay to be fined-tuned to the particulars of the protein target being screened.

The multi-step NMR assay provides a flexible alternative to standard HTS screening protocols. Additionally, the multi-step NMR assay may be used in combination with HTS screens to expedite the analysis of results and prioritize lead compounds. The unique aspect of the NMR methodology provides information that is critical to a structure-based drug design program that is absent in traditional HTS assays. NMR provides direct evidence that the compound of interest binds to the desired protein target. Additionally, information on the stoichiometry of binding, the identification of the ligand-binding site and the conformation of the bound ligand are readily achievable. Furthermore, NMR easily provides information on the physical behavior of the ligand itself. NMR rapidly identifies such properties as relative solubility, purity and stability that are fundamental for a good lead compound. Verification that the compound binds stoichiometricly to a functional part of the protein while exhibiting good physical behavior suggests a high likelihood that a co-structure will be achieved. Collecting this information is an intrinsic function of the multi-step NMR assay which provides a powerful mechanism to prioritize potential lead candidates, especially since obtaining a co-structure of the ligand and the protein target is an essential component of a structure-based drug design program.