Development of drug design methodologies

INPHARMA

The new NMR-based methodology INPHARMA, developed by our group, provides access to the relative binding mode of low-affinity ligands to a common target. The method requires two competitively binding ligands and a model of the structure of the apo-receptor. INPHARMA is based on the observation of interligand, spin-diffusion mediated, transferred-NOE (Nuclear Overhauser Effect) data, between the two ligands L1 and L2. As the ligands are competitive binders, such NOEs do not originate from direct transfer of magnetization between L1 and L2, but rather from a spin-diffusion process mediated by the protons of the receptor binding pocket and are, therefore, dependent on the specific interactions of each of the two ligands with the protein (Fig. 1). Thus, a number of such intermolecular NOE peaks describe the relative orientation of the two ligands in the receptor-binding pocket. In accordance with existing SBDD workflows, the experimental information derived from the INPHARMA NOEs is used to select the correct binding mode among many possible binding orientations obtained by molecular docking.

Figure 1. Schematic representation of the principle of the INPHARMA NOEs.

 

INPHARMA has been validated on a system consisting of multiple ligands binding the catalytic subunit of the Protein Kinase A (PKA) and of Cyclin-dependent Kinase 2 (CDK2) in collaboration with Sanofi-Aventis (1-3) The availability of crystal structures for this system has allowed verifying the efficiency and the accuracy of INPHARMA. In addition, we have used INPHARMA to determine the binding mode of epothilone A, discodermolide and tubulysin to tubulin (4-6).

This methodology has raised a considerable interest in the pharmaceutical industry. The daily work of pharmaceutical discovery is often limited by the inability to obtain high-resolution crystal structures of the target receptor in complex with the lower affinity ligands (lead structures) that are commonly identified by high-throughput screening or by fragment-based lead discovery. Accordingly, SBDD benefits from methods, such as INPHARMA, which provide receptor-ligand structures of novel ligands or fragments in relation to the known co-crystal structure of a reference ligand, thus allowing the structural alignment of different chemical series.

The experimental information derived from the INPHARMA NOEs is currently used to select the correct binding mode among binding orientations generated by molecular docking. These binding poses are ranked by evaluating the agreement between the theoretical INPHARMA NOEs calculated for each pair of TL1 and TL2 complex models with the experimental values. Such a procedure has the advantage of being well integrated in typical SBDD workflows; however, the results of the INPHARMA analysis are dependent on the availability of a structural model for the apo-receptor and on the performance of the docking program, which in turn depends on the quality of the receptor structure. To overcome these limitations, we are currently developing the methodology in two directions: 1. Use INPHARMA data to directly calculate the structure of the protein-ligand complex starting from a low-resolution model of the apo-receptor; 2. Use INPHARMA data to perform ligand superposition without a receptor structure.

 

References

 

1.        Orts, J. et al. Crystallography-independent determination of ligand binding modes. Angew. Chemie, Int. Ed. 47, 7736–40 (2008).

2.        Skjærven, L. et al. Accounting for conformational variability in protein-ligand docking with NMR-guided rescoring. J. Am. Chem. Soc. 135, 5819–27 (2013).

3.        Carlomagno, T. et al. Identification of new hit scaffolds by INPHARMA-guided virtual screening. Med. Chem. Commun. 6, 1501–1507 (2015).

4.        Reese, M. et al. Structural Basis of the Activity of the Microtubule-Stabilizing Agent Epothilone A Studied by NMR Spectroscopy in Solution. Angew. Chemie, Int. Ed. 119, 1896–1900 (2007).

5.        Sánchez-Pedregal, V. M. et al. The tubulin-bound conformation of discodermolide derived by NMR studies in solution supports a common pharmacophore model for epothilone and discodermolide. Angew. Chem. Int. Ed. Engl. 45, 7388–94 (2006).

6.        Kubicek, K., Grimm, S. K., Orts, J., Sasse, F. & Carlomagno, T. The tubulin-bound structure of the antimitotic drug tubulysin. Angew. Chem. Int. Ed. Engl. 49, 4809–12 (2010).