Study On The Interactions Of Protein With Small Drug Molecules By Ultrafiltration Mass Spectrometry

Biochemical applications of mass spectrometry (MS) are important in thepharmaceutical industry. They comprise compositional analyses of biomolecules,especially proteins, and methods that measure molecular functions such as ligandbinding. In early state of drug discovery, MS is used to characterize essential reagentsand to structural biology. A number of MS-based methods have been proposed for usein high-throughput screening (HTS).Ultrafiltration is mainly used for solute concentration, desalting, and bufferexchange. The technique, however, has been also successfully adapted to a varietyof biological samples for the purification and concentration of a wide range ofbiomolecules including therapeutic proteins, industrial enzymes, natural proteinproducts, and diagnostic antibodies. Ultrafiltration-mass spectrometry (UF-MS) is amethod with a variety of uses for the discovery and development of biologicallyactive small molecules, including the screening of combinatorial libraries and naturalproduct extracts for biologically active compounds, investigation of thermodynamicand kinetic ligand-receptor binding parameters, high-throughput metabolic screening.Solution phase ligand-screening assays that use ultrafiltration mass spectrometry areuseful for "reverse pharmacology" studies in which a macromolecular receptor ofinterest has been isolated, but ligands for the receptor are needed.In this study, three rhubarb anthraquinone analogues (chrysophanol、rhein、aloe-emodin), five compounds from licorice extract (liquiritin、glycyrrhizic acid、liquiritigenin、licorice furfuran ketone、licoricone), four FAK inhibitors (Y11、PF-562271、 PF-573228、 PF-03814735) were identified by liquidchromatography-mass spectrometry (LC-MS). The ultrafiltration LC-MS method usedto investigate the binding activities those small molecular drugs with protein receptorswas reliable and reproducible. A soluble HSA was incubated with each sample, andthen an aliquot of each mixture was injected into the Millipore fitted with a10000MW cutoff ultrafiltration membrane. After the unbound and weakly boundcompounds were washed away, the released ligands were identified using LC-MS.The results showed that these small molecular drugs had strong protein receptorsbinding activities.The combination of ultrafiltration extraction and mass spectrometric detectionprovides a sensitive and selective method to screen and character small drugmolecules bound to their target proteins, and is applicable to the analysis ofbiologically active compounds from combinatorial libraries and botanical extracts.