Discovery And Biological Activity Evaluation Of Small Molecules Inhibiting The Prion Protein Aggregation

Prion diseases were caused by misfolding and aggregation of related proteins,and these diseases related with protein misfolding and aggregation were also defined as conformational diseases.At present,there were few specific therapy medicines towards prion diseases and discovery of inhibitors which could inhibit protein from misfolding and aggregation has been widely accepted as an efficient strategy to fight against prion diseases.Currently,the reported inhibitors can be classified into two categories:low molecular weight chemicals and peptides,among them low molecular weight chemicals have attracted much attention.It is a conventional strategy in drug discovery with the help of virtual screening to screen potential small molecules binding to specific targets from large chemical libraries and then evaluating their biological activities using experimental assays.Virtual screening can greatly reduce the experimental cost and shorten the time of experimental cycle.In order to screen new chemicals which could bind and interact with prion protein,a variety of biological assays combining with molecular dynamics simulation were applied to discover potential prion inhibitors.In the first work,we performed virtual screening to screen small molecules that can stabilize the normal conformation of prion.First of all,121 compounds having favorable interactions with prion were screened from the Chemdiv and Specs database against¹.6 million compounds using virtual screening method.Then based on the steady-state fluorescence quenching approach,14compounds were tested as fluorescence quenchers at different extent towards prion.At the same time,among the 14 compounds,5 were proved to quench endogenous prion fluorescence at a concentration dependent manner.Subsequently,binding affinities between the 5 compounds and prion were monitored in real-time using SPR assay and the results indicated that the 5 compounds could bind to prion protein with different affinities.Compound 3253-0207 had the strongest binding affinity with the binding constant of²6?M.After that,the SDS-PAGE and thioflavin T fluorescence assays qualitatively and quantitatively confirmed that compound 3253-0207 could strongly block prion protein from aggregation at 20?M.At last,all atom molecular dynamics simulation was carried out to evaluate the interaction between compound 3253-0207 and prion.The results suggested that compound 3253-0207 bound to prion protein could stabilize the overall structure of prion and then interrupt the conversion from PrP^C to PrP^Sc.In this work,a new inhibitor with novel scaffold that could stabilize prion protein was discovered using multiple biological methods combining with molecular dynamics simulation.These methods used here could provide new guidance strategy for future drug discovery against prion protein.In the second work,we investigated the molecular mechanism between prion and resveratrol and its deriviates extracting from natural plants.First,the interaction between prion and resveratrol derivatives were determined using steady-state fluorescence quenching assay and finally three positives,named as JZ-I,JZ-II and JZ-XIII,were identified,,where JZ-II is resveratrol.Then the fibrillation inhibition abilities of the three compounds were tested using the dye thioflavin T which could specifically bind to?-sheet rich structures and the results demonstrated that the compounds could obviously inhibit prion aggregation at 5?M and the fibrillation process was totally interrupted at 100?M.The following western blotting further confirmed the fibrillation inhibition abilities of these compounds:quantities of prion monomer were more in the compounds containing samples than the compounds absent samples.Finally,the results of molecular dynamics simulation indicated that the inhibition mechanism of resveratrol was not to stabilize the natural monomer of PrP^C but to stabilize the peptide PrP_127-147 through hydrogen bonds and?-?stacking interactions which could further reduce the flexibility and the propensity of aggregation of this peptide.The third work of this paper is to screen inhibitors of prion protein from the EGCG analogs library.21 compounds were first obtained using single concentration screening based on the ThT fluorescence assay from the EGCG analogs library which contains⁹0 compounds.Among the 21hits,three compounds?compound codes:0074-0036,0325-0409 and D715-2679?were identified to inhibit prion fibrillation with different concentrations using concentration dependent prion aggregation inhibition assay.Molecular dynamics simulation results suggested that the three compounds could bind and interact with prion at absolutly different binding sites:compound0325-0409 at a pocket consisted of?1,?2 and?2;compound 0074-0036 at the pocket surrounded by?1-?1 loop and?1;compound D715-2679 at the pocket consisted of residues from?1 and C-terminal of?3.Interactions between the three compounds and residues in the binding pocket of prion stabilized the complexes and reduced structural flexibility of?1,C-terminal of?2 and N-terminal of?3 and further decreased propensity of prion conversion to misfolded conformation.Here,using multiple biological approaches together with molecular dynamics simulation,new structural diversity prion inhibitors were discovered and the molecular mechanisms of prion fibrillation inhibition of these compounds were also investigated,the results obtained here could help to further screenning new inhibitors of prion protein.