Establish A Mass Spectrometry-based Approach For Inhibitors Screening Of Targeted Proteins

The molecular basis of protein-ligand interactions is significantly valuable for the early stage of drug discovery. A variety of analytical tools including nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) have been employed to discover specific ligands targeted therapeutic proteins. Recently, mass spectrometry has been increasingly developed for using in drug discovery by its high sensitivity, selectivity and rapid simultaneous measurement of multiple ligands. Two major mass spectrometry based approaches have been developed for examination of protein-ligand interactions, one is direct ESI-MS assay based on direction detection of protein-ligand complexes which can be used to evaluate the binding stoichiometry and affinity; the other one is affinity liquid chromatography-mass spectrometry (LC/MS) assay based on detection the ligands released from protein-ligand complexes to evaluate the binding specificity.We established both a ESI-MS and ultrafiltration-LC/MS based assay to identify New Delhi metallo-β-lactamase1(NDM-1) which can help bacteria resist almost all the antibiotics. Side by side comparison of the two approaches shows that the indirect approach has better reproducibility and tolerance of interference than the direct approach. The combination of the results of direct and indirect MS based approaches, competition assays, structural simulation and enzymatic activity approach; we found a new compound that can be developed as a drug candidate inhibited NDM-1.We succeed in screening the NDM-1’s potent ligand out of a mixture of small fragments, which demonstrates that we can use this approach for more complex screening system. We then established the ultrafiltration-LC/MS assay for fragment screening targeted hepatitis C virus NS5B RNA polymerase. After a primary screening and validation screening of a384-fragment library against NS5B, we discovered12fragment hits and estimated their binding affinity by SPR, we also got7NS5B-fragment complexes; all these data can be used for further fragments optimization and get promising drug candidates.