LSD1 Inhibitor Screening And The Biological Acitivity Study Of The Candidate Compounds

Epigenetics is the study of heritable phenotype changes without involving changes in DNA sequences,such as DNA modifications,RNA modifications and histone modifications.Histone modification is an important part for transcriptional regulation.This work is carried out with LSD1（Histone Lysine Specific Demethylase 1）,which is the first identified histone demethylase that specifically removes the methyl groups from histones H3K4 and H3K9.LSD1 is reported to be overexpressed in a variety of cancers,such as gastric cancer and leukemia.Lack of LSD1 functionality can inhibit the proliferation and metastasis ability of cancer cells.So,the obtaining of LSD1-targeting inhibitors can be a new strategy for cancer treatment.Nowadays,the screening platforms for LSD1 inhibitors are limited.Most of the platforms are focusing on the inhibition rate but insufficient in detecting the binding between inhibitors and LSD1.Some assays,such as the assay based on H₂O₂quantification cannot evaluate the compounds which react with H₂O₂,so the establishment of multiple screening platforms can make up for the limitations of screening.After getting good inhibitors,a series of experiments were performed to get the inhibitory activities in cancer cells.Therefore,the results can provide new ideas for the optimization of LSD1 inhibitors and that is why this topic is carried out around these issues.The detailed work was listed as below:（1）Expression and purification of LSD1 recombinant proteinIn this study,affinity chromatography and ion exchange chromatography were used to purify the LSD1 recombinant protein with purity>95%,which could meet the purity needs of later inhibitor screening and enzyme kinetics research.（2）Establishment of LSD1 inhibitor screening platformIn this work,the experimental design of LSD1 inhibitor screening platform was constructed from three aspects:inhibition activity,compounds-LSD1 binding and compounds-LSD1 kinetics.Firstly,FDH（Formaldehyde dehydrogenase）was expressed and purified then used for quantification of the demethylation activity of LSD1;After that,SYPRO Orange was used to monitor the fluorescence value in real time to show the folding or unfolding of LSD1,which can get the information whether the inhibitor can bind to LSD;Finally,the application of SPR（Surface plasmon resonance technology）can obtain the enzyme kinetics and affinity data to make up for the insufficient in detecting the binding behavior between inhibitors and LSD1 of existing LSD1 inhibitor screening platforms.（3）Study on the anti-tumor activities of inhibitorsFrom the screening and optimization of the compound library in our group,we obtained a total of four high-efficiency LSD1 targeting inhibitors,one form stilbene derivatives,one form indole derivates and another two form triazolopyrimidines derivates.Then the inhibitors are evaluated in both molecular and cellular level.DYC-8c is a promising inhibitor from stilbene derivatives which can reversibly inhibits LSD1 activity at the molecular level by FAD competition,resulting in IC₅₀=0.28μM with fast binding（Kon=1.82×10³ 1/（M?s））and slow dissociation(Koff=1.00×10^-2 1/s)acts on LSD1.At the cellular level,DYC-8c can effectively promote the expression of CD86（Cluster of Differentiation 86）in leukemia THP-1 cells and promote its myeloid differentiation,also,can inhibit its proliferation and clonal formation ability with IC₅₀ of 5.76μM.In addition,it can gradually increased the accumulation of the LSD1 substrate H3K4me2 in THP-1 cells,indicating that it inhibits LSD1 activity in cells.XHW-9e is a promising inhibitor from indole derivates which can irreversibly inhibits LSD1 activity at the molecular level with IC₅₀ of 1.23μM This is the first irreversible LSD1 inhibitor that is not derived from monoamine oxidase inhibitors and does not contain cyclopropylamine.The LSD1 recombinant protein was targeted by XHW-9e and with fast binding（Kon=2.98×10³ 1/（M?s））and slow dissociation(Koff=6.05×10^-2 1/s).At the cellular level,XHW-9e can effectively inhibit the proliferation and clonality of THP-1.The IC₅₀ of proliferation inhibition is 1.20μM and then promotes the expression of CD86 and promotes myeloid differentiation.WS-996 and WS-812 are both the novel LSD1-targeting inhibitor that are from triazolopyrimidines derivates.WS-996 inhibits LSD1 activity by competing with FAD in a reversible manner with IC₅₀=34.45 nM.In THP-1,the proliferation inhibition effect of IC₅₀=1.65μM is achieved,the differentiation of THP-1 can be promoted.The thermal stability of LSD1 in cells can be effectively enhanced,indicating that it targets intracellular LSD1 with tight bingding.Compared with WS-996,WS-812 has similar inhibition ability and behavior with IC₅₀=38.15 nM and K_D=6.23μM.In addition,WS-812 not only inhibits cell proliferation and clonal formation,promotes differentiation of leukemia THP-1 cells,but also inhibits cell proliferation and wound healing of gastric cancer cell MGC-803.In summary,this study used the two-step method to obtain high-purity LSD1recombinant protein for the first time.Three LSD1 inhibitor screening platforms were established based on inhibition activity,compounds-LSD1 binding and compounds-LSD1 kinetics,four new LSD1 inhibitors were found and be evaluated.Among them,three of them are reversible inhibitors,the last one is the first irreversible LSD1inhibitor that is not derived from monoamine oxidase inhibitors and does not contain cyclopropylamine.All of the results both from the establishment of LSD1 inhibitor screening platform and study on the anti-tumor activities of inhibitors give a strong support for the discovery of anti-tumor drugs targeting LSD1.