Discovery And Mechanism Of Action Study Of Acetyltransferases And Deubiquitinating Enzymes Inhibitors

Proteins are the most important components for living system and the major executor responsible for living activity.Post-translational modification,one of the most fundamental reactions in eukaryotes,is the necessary process for protein to mature and the molecular basis for the diversity of function and structure in proteins.There are many kinds of post-translational modification,including phosphorylation,ubiquitination,methylation,acetylation,glycosylation and fatty acylation.The variety of post-translational modification of proteins regulates different biological network,increasing the complexity of living activities and the ability for organisms to adapt to the rapidly changing external environment.The disorder of post-translational modification is implicated in many diseases,including cancer,Alzheimer’s disease and diabetes.Therefore,post-translational-modification-related proteins have become the hottest targets for drug development.Acetylation and ubiquitination,as the earliest modification identified,are widely distributed and occur through the whole lifetime in cells.They regulate various biological process including gene transcription and expression,location and stabilization of protein and DNA repairment and have become the popular field for biological and clinical research.Acetylation refers to adding the acetyl group to the lysin of substrate protein by covalent linkage.It is regulated by three kinds of proteins including “writer” acetyltransferases,“eraser” deacetylases and “reader”,mainly bromodomains.Nowadays,the marketed drugs targeting acetylation focus on deacetylases,for example panobinostat.The drugs targeting “reader” are mainly in clinical research,for example OTX-015.In contrast,the development of drugs targeting acetyltransferases still lags behind,just staying in pre-clinical research.Only two kinds of inhibitors,A-485 for p300 and WM-1119 for MOZ,have potential for further clinical research.Ubiquitination refers to covalently attaching mono-or poly-ubiquitin to substrate proteins.Protein ubiquitylation is achieved on the sequential cascade reactions by three classes of enzymes including ubiquitin-activating(E1),ubiquitin-conjugating(E2),and ubiquitin-ligating enzymes(E3).The removal of ubiquitination is accomplished by deubiquitinating enzymes.Currently,the marketed drugs targeting ubiquitination are a class of immunomodulatory drugs focusing on ubiquitin-ligating enzyme CRBN,for example thalidomide,lenalidomide.The drugs targeting ubiquitin-activating enzymes,ubiquitin-conjugating enzymes and deubiquitinating enzymes are all in clinical or pre-clinical research.Especially for deubiquitinating enzymes,there is only one drug coming into clinical research,VLX1570 for USP14.In general,the drug development for acetyltransferases and deubiquitinating enzymes is relatively backward.Therefore,it is urgent to exploit novel drug discovery platforms,which will help us break through adversity in pharmaceutical research for these targets.In this paper,we discovered novel inhibitors against histone acetyltransferase GCN5 and deubiquitinating enzyme USP8 and investigated their mechanism of action.For GCN5,we first developed a high throughput screening platform based on Alphascreen technology.We screened in house compound library containing approximate 20,000 compounds and discovered a hit compound DC_G16.By radioactive acetylation assay,nuclear magnetic resonance(NMR)assay and surface plasmon resonance(SPR)assay,we validated the inhibition and binding activity of DC_G16 against GCN5 catalytic domain.Further structure optimization led to the discovery of DC_G16-11 with 5-fold improved potency.Radioactive acetylation assay and SPR assay gave convincing evidence for the higher inhibitory activity and binding affinity of DC_G16-11 than DC_G16 and presented good selectivity against other epigenetic targets.Molecular docking proposed that DC_G16-11 occupied the pocket of H3 substrate in a competitive manner and pointed out the importance of halogen atom in this binding mode.Cellular assays proved DC_G16-11 could inhibit the cell viability for MV4-11 cell lines with minimal effects on normal cells.Further study in biological mechanism showed DC_G16-11 dose-dependently reduced the acetylation level of H3K14 in MV4-11 cell lines and induced cell apoptosis and cell cycle arrest.For USP8,we established a high throughput screening platform based on Ubiquitin-Rho-110-based fluorometric assay and discovered a novel inhibitor DC-U43 by screening in house compound library.SPR assay proved DC-U43 directly bound to USP8 catalytic domain protein.Based on DC-U43,we performed chemical optimization and got DC-U43-10.Gel-based di-ubiquitin cleavage assay and SPR assay were carried out to validate the inhibitory and binding activity for DC-U43-10.Molecular docking predicted the putative binding mode of DC-U43-10.Then,we evaluated the activity of DC-U43-10 in cellular level in H1975 cell lines.Colony formation assay showed that DC-U43-10 inhibited the proliferation of H1975,presenting its potential in drug exploitation.In summary,our study set up high throughput screening platforms and discovered new small molecular inhibitors against GCN5 and USP8,respectively.These inhibitors built the foundation for the future development of chemical probes for acetylation and ubiquitination and provided new ideals for the biological and pharmaceutical research for post-translational modification.