Biophysical studies of protein-ligand interactions and the discovery of FKBP12 inhibitors

Abstract

The principal aim of this study was to discover, through virtual screening, new nonimmunosuppressive inhibitors for the human immunophilin FKBP12, a target of the immunosuppressant drugs rapamycin and FK506. The enzyme acts as peptidyl-prolyl isomerase catalysing protein folding in the cell. Structurally similar isomerase domains are important for molecular recognition in multi-domain chaperone proteins. FKBP inhibitors have been shown to have protective effects against nerve damage and are therefore interesting targets for the treatment of neurodegenerative diseases. Virtual screening has been used to discover novel inhibitors for protein drug targets. Recent advances in computational power and the availability of large virtual libraries, such as the EDULISS database at Edinburgh University, have enhanced the appeal of this approach. X-ray structures of known protein-ligand complexes were examined to obtain an understanding of the key non-covalent interactions in the FKBP12 binding pocket. Virtual screening hits were selected using macromolecular docking and programs that employed a ligand-based approach. The bulk of the virtual screening in this study used Edinburgh University’s in-house program LIDAEUS. In the course of this study nearly three hundred compounds were screened in the laboratory using biophysical and biochemical binding assays. Thirty four compounds were found to have an affinity for FKBP12 of less than one hundred micromolar. To test virtual hits, it was necessary to select the most appropriate medium-throughput biophysical assay. The aim was to employ methods with sufficient sensitivity to detect compounds with affinity in the order of one hundred micromolar, coupled with the capacity to screen hundreds of compounds in a week. This study used a wide variety of biophysical techniques, these including: electrospray ionisation mass spectrometry, surface plasmon resonance and isothermal titration calorimetry. There was a particular emphasis on the quality of data from electrospray ionisation mass spectrometry. A correlation was found between the cone voltages that gave 50 % dissociation of the complex with the enthalpic contribution to the free energy of binding. From the careful examination of the differences in charge-state distributions between a pure protein and a protein-ligand mixture, it was possible to determine if a protein-ligand complex had been present in solution prior to dissociation during the electrospray process. This observation provides the basis for an assay that could be of general utility in detecting very weak inhibitors.