The Development & Application Of Affinity Selection-Mass Spectrometry Screening Methodologies For Drug Discovery

In the primary stage of drug discovery,developments of novel screening technologies play an important role in rapidly identifying active compounds for biological targets from either synthesized or herbal libraries.Mass spectrometry based screening techniques are such developments that have received much attention.Among them,affinity selection mass spectrometry(ASMS)techniques,which do not depend on drug target function for their readout,outperform traditional drug discovery techniques in discovering and characterizing new lead compounds.Particularly,the ASMS techniques combined with highly effective separation methods,such as ultrafiltration,size-exclusion chromatography,and frontal affinity chromatography,have been successfully used for screening active ligands over a broad range of drug targets.Presently,however,there is no one-size-fits-all ASMS method,as each has its positive aspects and drawbacks.This dissertation is specifically focused on the development,optimization and application of ultrafiltration(UF)and ultracentrifugation(UC)-based ASMS methods,thereby expanding the impact and use of ASMS techniques in drug discovery.The first part of this work is the demonstration and application of a newly developed UF-ASMS method.We showed that UF-ASMS is a reliable method to evaluate ligand binding to a target protein without prior knowledge of the protein or compounds and is especially helpful in primary screening due to its rapidity.In the following,we screened 2000 compounds and found that 0.25% of them are potential PTP1 B inhibitors.Moreover,we used the crude extract of Chinese red yeast rice(RYR),a traditional medicine with a known anti-diabetic effect,as a representative tool material to investigate active ingredients with specific PTP1 B inhibitory activity.This investigation demonstrated that the most attractive advantage of the UF-ASMS method is the rapid detection and precise identification of bioactive compounds from natural products via a single assay without extra isolation and purification steps.As a result,we identified at least one compound in the RYR extract as a potential PTP1 B inhibitor by the ESI ionization mode and confirmed its structure as monascorubramine by mass spectrometryElevated Energy(MSE).The specific binding behavior of monascorubramine to PTP1 B is verified by comparing the binding behavior of monascorubramine to 6 randomly chosen target proteins and a homolog of PTP1 B named T cell protein tyrosine phosphatase(TCPTP).This complete strategy is especially helpful for the discovery of unknown bioactive compounds from natural products,which would be easily missed through compound isolation or when present at low levels.The next part presents a work of validating the UC-ASMS approach that can overcome the membrane effect caused by the filter involved in the UF-ASMS method.Moreover,it dramatically reduces the consumption of proteins and compounds compared to those for the UF-ASMS approach,although the screening speed is lower than the UF-ASMS.We utilized the UC-ASMS platform to screen potential PTP1 B ligands from the 2000 compounds,which were screened by UF-ASMS.It is found that the results from the two methods were positively correlated and highly reliable by comparing their in vitro PTP1 B inhibitory activities.Therefore,it is considered that the ultrafiltration and ultracentrifugation are exchangeable and complementary techniques for separating protein-ligand complexes prior to the ligand identification by MS depending on screening prerequisites,such as throughput,protein expression levels,and required additives.In addition,we developed a simultaneous multi-target selection approach based on the strategy of rate-zonal density gradient centrifugation.In this method,an applied centrifugal driving force is used to separate the mixed proteins and their respective ligands in the centrifugal medium based on the different molecular weights of proteins.The ligand binding ability is assessed by comparing the molecular distribution of compounds with the respective protein distribution after centrifugation once the solution is fractionated.Theoretically,a specific ligand will have a distribution that is essentially identical to that of its target protein.This method would expand the applications of ASMS techniques from the single target screening to the simultaneous screening of multiple targets.It would be particularly useful for screening multi-target directed ligands(MTDLs)that can interact with multiple targets for the specific pathogenic events.