A Phase Separation Based High Throughput Screening(HTS) Method For Methyltransferase Inhibitors

Biomacromolecule liquid-liquid phase separation(LLPS)is often driven by multivalent interactions to form biomacromolecular condensates separated from the surrounding liquid environment.Due to surface tension,biomacromolecular condensates exhibit spherical morphologies like oil droplets in water.LLPS prevalently happens in all cell types,and plays key roles in various biological activities.The droplet formation is a direct manifestation of interactions.As a molecular switch,methylation changes the intermolecular interaction,so the methylation can be indicated by the LLPS droplet formation.Labelling the LLPS system with fluoresence protein allows the detection of methylation by the protein fluorescence in the droplet,by which we can design a LLPS based high throughput screening(HTS)method for methyltransferase inhibitors.We successfully constructed assay systems for protein lysine methylation,DNA Cp G methylation and RNA adenosine N6 methylation.The system of protein lysine methylation is further characterized and utilized in the thesis.This system is composed of a 14-meric SH3 complex and a 14-meric SH3 ligand,polyproline motif(PRM),complex.These two complexes undergo LLPS.One of the complex is fused with the chromo domain(CD)of human HP1 which can bind to methylated Histone 3 lysine 9(H3K9).To overcome the weak binding affinity between CD and H3K9 me,we implement a substrate peptide composed of Histone 3 N-terminal tail and a KKETPV sequence flanking a flexible linker.We obtained a hexameric PDZ domain complex,which can multimerize H3 tail by interacting with the KKETPV motif.This multimerized H3K9 tail has tighter binding to puncta enriched with CD.We labeled the LLPS system with GFP and the hexameric system with m Cherry.The m Cherry signal in puncta is the readout for methylation.Z factor of the protein methylation assay system is about 0.8.This method was successfully used to screen inhibitors of the methyltransferase SUV39H1.