HDAC6 Selective Antitumor Lead Compound Discovery Based On DNA-encoded Chemical Libraries Technology

Traditional drug discovery and development（R&D）is a long,expensive,and risky systematic project.Medicinal chemists have been striving to discover lead compounds cost-effectively to accelerate drug discovery.In recent years,DNA-encoded chemical library technology（DELT）has become a popular technology in drug discovery and development due to its powerful technology advantages,bringing disruptive changes to traditional drug discovery and development.The mastery and application of disruptive and cutting-edge technologies for new drug development is the driving force for the transformation of China’s pharmaceutical R&D from generic to creation and pharmaceutical industry from a large country to a strong one.The purpose of this study is to establish a technology platform for DNA-encoded compound libraries and use the technology for drug discovery research.The incidence of cancer has been increasing due to various objective factors such as changes in living environment and lifestyle and increasing survival pressure.Current antitumor drugs are not yet able to meet the demand for clinical use,thus there is an urgent need for antitumor drug development against confirmed cancer targets.Histone deacetylase 6（HDAC6）,plays an important role in the regulation of epigenetic reversibility and cellular life processes.HDAC6 has been found to be involved in the oncogenesis and development of tumor diseases through multiple pathways and is considered a very promising target for antitumor drug research.Due to the large number of HDAC family members,the study of selective HDAC6 inhibitors is extremely challenging.At the same time,hydroxamic acid inhibitors have many defects,which affect the wide application of the inhibitors in clinical practice.The research of non-hydroxamate HDAC6 inhibitors requires a new approach.In order to discover novel selective HDAC6 inhibitors,this study conducted an exploratory study of selective HDAC6 antitumor inhibitors using DNA-encoded chemical library technology,expecting to discover novel selective HDAC6 inhibitors and make some contribution to antitumor drug development.Technical platform for building DNA-encoded compound librariesThe starting material AOPHPP was prepared by amidation,Fmoc removal and T4DNA ligase ligation reaction using DNA primer Head piece（HP）as the starting material,and purified by preparative liquid chromatography with a purity of 90%.Different structural types of chemical small molecule blocks are required for compound synthesis in DNA-encoded chemical libraries.Based on the designed DNA-encoded chemical library,we selected and purchased five types of small molecule blocks as building blocks for the construction of the DNA-encoded chemical library,including 70 Fmoc-amino acids,40 amines,40 carboxylic acids,40 haloaromatics and60 boronic acids or boron esters.To increase the structural diversity of the DNA-encoded chemical library,we designed and synthesized more than 50 scaffolds with vertical structures.Suitable single crystals were obtained for some of the scaffolds,and the crystal structures of nearly 20 scaffolds were confirmed by X-ray single crystal diffraction.DNA-encoded chemical libraries are constructed by using DNA-compatible reactions to convert small molecule blocks into final small molecule compounds.Therefore,we explored DNA-compatible reactions such as amidation,nitroreduction,sulfonation reaction,nucleophilic substitution,nucleophilic addition,imine reduction,and Suzuki using HP as a substrate and obtained suitable reaction conditions and screened some of the building blocks to eliminate the poorly reacting ones.The HPLC analysis method,HPLC-MS method and agarose gel electrophoresis method were established for quality control of the construction of DEL libraries.Construction of DEL libraries of Panobinostat derivatives and DEL libraries of new structural typesThree scaffords were synthesized from ethyl 2-methylindole-5-carboxylate,methyl indole-2-acetate,and 2-methyl-5-bromoindole,and a DEL library of Panobinostat derivatives with 15,300 molecules was constructed for activity screening.A three-dimensional DEL library（120,000 molecules）and a four-dimensional DEL library（6 million molecules）were constructed using the synthesized 50 scaffolds.HDAC6 protein was recombinantly expressed,and the constructed DNA-encoded chemical libraries were validated and screened.Six hit were synthesized and tested for activity.The results showed that these six Panobinostat derivatives exhibits excellent inhibition against HDAC1,HDAC2,HDAC 3,HDAC 6,HDAC 8 with IC₅₀ in the nanomolar range,while the inhibition of HDAC4,HDAC5,HDAC7 and HDAC9subtypes was significantly diminished.Compound 6(IC₅₀=6 n M)inhibits HDAC6 at the same level as Panobinostat(IC₅₀=7.2 n M).The in vitro antitumor activity of six targets were evaluated against three tumor cell lines,HL60,Hela and RPMI8226.In vitro antitumor assays showed that compound 7 showed the best inhibitory effect against HL60 with IC₅₀=0.9μM,while 6 showed IC₅₀=5.5μM against HL60.Compounds inhibited RPMI8226 tumor cells with IC₅₀ ranging from 1.4μM to 7.6μM,with 6 showing IC₅₀=1.4μM against RPMI8226.Compounds showed poor inhibitory effect against Hela tumor cells.ConclusionIn this study,we established a DNA-encoded chemical library technology platform.Based on DNA-encoded chemical library technology,we constructed a DEL library of Panobinostat derivatives and a non-hydroxamate DEL library and screened the HDAC6protein to obtain 6,which was extremely active in enzyme level experiments.Compound 6 can be used as a lead compound for further structure optimization studies.The screening of non-hydroxamate DEL library yielded hits with novel scaffords and novel zine binding group.This study laid some foundation for the development of HDAC6 inhibitors.