The INPHARMA method: protein-mediated interligand NOEs for pharmacophore mapping.

In structure-based drug design, the relative orientation of two competitive ligands A and B in the receptor binding pocket plays a central role in the design of high-affinity drug candidates from weakly bound lead compounds. In this communication we report on the observation of interligand spin-diffusion-mediated transferred-NOE data, which have the potential to be used for the determination of the relative orientation of two competitive ligands in the receptor binding pocket. In many cases, the orientation of one ligand in the binding pocket is known, for example, from crystallography or fluorescence resonance energy transfer (FRET) data. In these cases it is desirable to develop a methodology that allows the determination of a unique binding mode and pharmacophore model for a second competitive ligand. We anticipate that the interligand transferred NOEs described here could provide the experimental basis for a methodology that is applicable to any combination of ligands weakly bound to a common receptor. The conformation of a ligand in the binding pocket of a macromolecular receptor can be accessed by NMR spectroscopy in solution by two approaches. If the molecular weight of the complex is of the order of 50 kDa or smaller and C/Nlabeled receptor can be produced in vitro, a detailed threedimensional picture of the ligand and of its interactions with the receptor binding pocket can be obtained with standard NMR spectroscopy methods. However, in other cases the macromolecular receptor is too large for NMR studies in solution or it cannot be synthesized in vitro to introduce NMR-active isotopes. In these instances, NMR spectroscopy can be applied to determine the receptor-bound conformation of the ligand, provided the rate constant for the dissociation of the complex (koff) is much larger than the relaxation constants of the ligand in the complex and the difference between the resonance frequencies of the free and bound ligand forms (a fast-exchange regime). Under this condition, which implies a dissociation constant (Kd) value in the low micromolar to millimolar range, information on the ligand structure is derived from transferred NOEs (tr-NOE) and transferred cross-correlated relaxation rates (tr-CCRR). 4] The determination of the receptor-bound conformation of the ligand leaves open the question of its orientation in the receptor binding pocket. The relative orientation of two competitive ligands weakly bound to a common receptor could be determined by the simple and novel approach that we propose here. A NOESY spectrum of a mixture of the two ligands in the presence of the common receptor is recorded. Under the fast-exchange condition described above, and provided that the two ligands A and B bind competitively to the macromolecular receptor T, intermolecular tr-NOE peaks between the two ligands A and B can be observed in the NOESY spectrum. A NOE peak between a proton HA of ligand A and a proton HB of ligand B in the presence of the receptor T originates from spin diffusion involving a proton HT of the receptor. During the NOESYmixing time, ligand A binds to receptor Tand HA transfers its magnetization to HT (Figure 1). The complex AT