Discovery Of Novel Cdc25 Inhibitors Via Miniaturized Synthesis And Virtual Screening

Cancer is one of the most lethal diseases in the world today,and with the emergence of tumor resistance,cancer becomes more and more difficult to be effectively shut down via the prevalent clinical drugs.Therefore,the research and development of anti-tumor drugs based on new mechanisms and new targets is of great significance.It is well known that one of the most prominent features of tumor cells is uncontrolled cell proliferation caused by an aberrant cell cycle.Cdc25 phosphatases(cell division cyclin 25 phosphatase),a kind of cell cycle regulators,are overexpressed in a variety of tumor cells which facilitate the tumor cell proliferation.Therefore,effectively inhibiting the activity of Cdc25 phosphatase can thus effectively inhibit the division and proliferation of tumor cells.Currently,there are no approved drugs targeting Cdc25 phosphatases in the market,so the research and development of new anti-tumor drugs targeting Cdc25 phosphatase is of great research value.However,due to the flat and positively charged active site of Cdc25 phosphatase,the discovery of chemical entities with desired bio-activity is difficult.Meanwhile,the catalytic domain between three isoforms of Cdc25 phosphatases is highly conserved which makes it complicated to identify selective inhibitors for certain isoforms.Therefore,Cdc25 phosphatase is classified as "undruggable target".In view of these situations,the fragment-based drug design strategy,miniaturized synthesis technology and computer-aided drug design methods were employed in this thesis to discover the potential hit or lead compounds.This thesis is mainly divided into the following three parts:(Ⅰ)Discovery of novel Cdc25 phosphatase inhibitors via miniaturized synthesisMost drugs are developed through iterative rounds of chemical synthesis and biochemical testing to optimize the affinity of a particular compound for a protein target of therapeutic interest which waste a lot of time.Therefore,how to rapidly synthesize diverse small molecules from privileged fragments has become a bottleneck in the development of new drugs.In recent years,various new synthetic methodologies and concepts have been proposed for the assembly of compound collections.Among them,miniaturized synthesis integrated with parallel synthesis methods,diversity-oriented synthesis concepts and rapid biological screening technology is generally regarded as a robust,sensitive and economically friendly method in modern drug discovery.The previous research of our group has utilized CuAAC click reaction to introduce diverse substituent fragments to the privileged naphthoquinone scaffold of NSC663284 which resulted in the identification of M2N12,a Cdc25C selective inhibitor.(Cdc25A:IC50 =0.53±0.03 μM;Cdc25B:IC50=1.39±0.95 μM;Cdc25C:IC50=0.09±0.01 μM).Consequently,with an aim of discovering novel selective Cdc25 inhibitors,the Chapter 2 continued to the structural modification of this privileged naphthoquinone scaffold.It is reported that the possible binding site of naphthoquinone compounds is the active site,which contains the catalytic Cys473.Since the active site is rich in phenylalanine and arginine,we have designed several aromatic or heterocyclic fragments,bearing the azide or amine groups,with structural and electrical diversity as the privileged fragment libraries for the subsequent substitution decoration of naphthoquinone scaffold.And then,the naphthoquinone fragment was linked with the privileged azide or amine fragment via the CuAAC and amide coupling reaction based miniaturized synthesis which is monitored by TLC and LC-MS.Finally,this chapter quickly constructed four focused libraries based on the privileged naphthoquinone scaffold which totally contains 138 compounds.Afterwards,rapid biological screening,scale-up synthesis and biological evaluation have disclosed several compounds with desired bio-activity and isoform selectivity.For example,compound M5N36(Cdc25A:IC50=0.15 ±0.05 μM;Cdc25B:IC50=0.19 ± 0.06 μM;IC50=0.06± 0.04 μM)exhibited a superior inhibitory activity against Cdc25 phosphatase,in comparison of positive control NSC663284(Cdc25A:IC50=0.27± 0.02 μM;Cdc25B:IC50=0.42±0.01 μm;Cdc25C:IC50 =0.23± 0.01 μM).Notably,comparing the inhibitory activity of M5N36 against three Cdc25 phosphatase isoforms,we found that M5N36 have also showed a potential selectivity toward Cdc25C.Next,the M5N36 was evaluated for its in vitro cytotoxic activity against several tumor cells lines with over-expression of the Cdc25 phosphatases.The results showed that compound M5N36 exhibited the best anti-tumor activities towards MDA-MB-231 and Raji,with IC50 values of 2.00 ± 0.81 μM and 1.20 ±0.47 μM,respectively,which are equipotent to that of positive control NSC663284(MDA-MB-231:IC50=2.04 ±0.21 μM;Raji:IC50=1.00±0.66 μM).Western-blot assay have demonstrated that compound M5N36 promoted apoptosis of tumor cells by inhibiting Cdc25 phosphatase in MDA-MB-231 cells.Then,the binding mode and physicochemical properties of all three target compounds in this chapter were predicted,utilizing molecular simulation.The three target compounds were all accurately accord with the "Lipinski’s rule of five".(Ⅱ)Discovery of novel bivalent Cdc25 phosphatase inhibitors based on naphthoquinone as the privileged scaffoldAs mentioned before,the active site of Cdc25 phosphatase is surprisingly flat which leads to difficulties in combination of small molecule inhibitors,but the adjacent "swimming pool" site and the substrate recognition site(20 (?) from the active site)is large and deep,and is consequently favorable for compounds to combine with.These two sites are rich in arginine and tyrosine which is similar to the active site.Thus,these two sites are also favorable to accommodate naphthoquinone fragment.Based on the above analysis of protein structure,the idea of designing Cdc25 phosphatase dual-site inhibitors was proposed,in order to further improve the binding affinity of inhibitors toward Cdc25 phosphatase.Thus,in Chapter 3,two privileged naphthoquinone fragments were combined by linkers with different lengths and flexibility,in order to realize the dual-site inhibition.And all these compounds were synthesized by CuAAC click reaction based miniaturized synthesis.Next,rapid biological screening technology,scale-up synthesis and biological evaluation have demonstrated that compound M5N43(Cdc25A:IC50=0.14 ± 0.01 μm;Cdc25B:IC50=0.37± 0.06 μM;Cdc25C:IC50=0.67± 0.04 μM)was the most potential compound which showed a higher inhibitory activity against Cdc25 phosphatase than that of NSC663284(Cdc25A:IC50=0.82± 0.07μM;Cdc25B:IC50=1.31±0.14 μM;IC50=1.44± 0.37μM).Particularly,M5N43 showed the best inhibitory activity against Cdc25A which was nearly 6 times more than that of NSC663284.However,the antitumor activity of the compound in liver cancer cells(A549,IC50=6.29± 0.69 μM),triple negative breast cancer cells(MDA-MB-231,IC50=13.05±0.38 μM)and breast cancer cells(McF-7,IC50=6.72±0.21μM)was lower than that of NSC663284(A549:IC50=2.14±0.32 μM;MDA-MB-231:IC50=5.06±0.43 μM;McF-7,IC50=1.07 ± 0.21 μM),which may be caused by the poor physicochemical properties of the compound,and makes it difficult to penetrate the cell membrane.Next,we have conducted the molecular simulation to predict the binding modes and physicochemical properties of the lead compounds reported in Chapters 3.Subsequently,we employed a series of biological mechanism studies to further confirm that M5N42 and M5N43 have inhibited Cdc25 phosphatase in cellular level,which provided ideas and research bases for subsequent compound design and structure modification.(Ⅲ)Discovery of novel Cdc25 phosphatase inhibitors with non-quinone structure via virtual screeningUp to now,Cdc25 phosphatase inhibitors have been extensively studied for more than 20 years.Quinones are still one of the most representative Cdc25 phosphatase inhibitors,which may produce excessive reactive oxygen species(ROS)while exerting anti-tumor activity in cells.ROS is able to oxidize cysteine from normal proteins and is potentially toxic for the normal cells.Therefore,it is of great significance to develop novel Cdc25 phosphatase inhibitors without quinone motifs.Herein,in Chapter 4,a docking-based virtual screening(VS)of a large commercial library was used to identify non-quinone structure-based Cdc25B inhibitors.Prior to virtual screening,the Cdc25B substrate:phosphorylated threonine/tyrosine was docked to the Cdc25B active site separately to evaluate Cdc25B as a structural model for virtual screening as well as to test the performance of the docking and scoring.The calculated structural determinants of the phosphate group of these compounds matched well with the location of a free sulfate molecule observed in the Cdc25B crystal structure that should mimic the incoming phosphate group of the substrate(RMSD=0.18)which finally demonstrated the reliability of such constructed screening model.Following the success of the control dockings,virtual screening calculations were performed with the Cdc25B crystal structure model,using a database of 3.84 million compound structures.As a result,we have shortlisted 19 compounds for experimental validation as inhibitors of Cdc25B.The VS was successful discovering 2 compounds,Y11 and Y19,with moderate bio-activities and totally new skeletons.The two compounds showed moderate inhibitory activity and selectivity against Cdc25B(Y1:Cdc25A:IC50>500 μM,Cdc25B:IC50=156.30⒈7.19 μM,Cdc25C:IC50>500 pM;Y19:Cdc25A:IC50>500 μM,Cdc25B:IC50=118.85⒈12.07 μM,Cdc25C:IC50>500 μM).However,only compound Y19 showed weak antitumor activity against A549 cell line,and its IC50 value was 193.60⒈ 4.27 μM.In summary,with the aim of discovering novel anti-cancer agents,we have chosen Cdc25 phosphatase,an important regulatory protein in the process of tumor proliferation,as our target.And found a promising Cdc25C selective inhibitor M5N36 which possessed nearly 4 times higher bio-activity than that of the positive control NSC663284 by utilizing miniaturized synthesis and rapid biological screening methods.Subsequently,we have used the same method to find a Cdc25A selective inhibitor M5N43 which possessed nearly 6 times higher bio-activity against Cdc25A than that of NSC663284.Besides,we have successfully established a virtual screening method towards Cdc25B phosphatase,and used this method to discover two hit compounds with new structures,which provided a new scaffold for the future discovery of Cdc25B phosphatase selective inhibitors,and further enriched the scaffold diversity of Cdc25 phosphatase inhibitors.