The Study On Virtual Screening And Molecular Modelling Of Tubulin Inhibitors

Microtubule（MT）,as an important component of the cytoskeleton,is the basis of cell growth and proliferation.MT involved in cell mitosis regulation and tumorigenesis,which has been considered as an attract target for cancer treatment.It was reported that microtubule-targeting agents（MTAs）can disrupt the dynamic equilibrium of microtubules,induces cell cycle arrest in the G2/M phase,and eventually leads to cell apoptosis.MTAs exert anti-cancer effects by disrupting the dynamic equilibrium state of assembled and disassembled at a reversible manner.MTAs have become important anti-cancer drugs in the clinical tumor treatment.Therefore,discovery of novel MTAs lead compounds has become a research hotspot in the area of anti-cancer drugs development.The aim of this study is that identification of some potential MTAs targeting Taxane and Colchicine sites of tubulin by using computer-aided virtual screening methods,and molecular dynamics simulations.The main contents are as follows:Chapter 1 Research review.This chapter introduces the research significance,the structure and function of microtubules,the research progress on active sites of MT and MTAs,and the current progress of computer-aided drug design（CADD）methods.Chapter 2 Virtual screening and molecular simulation studies of microtubule-stabilizing agents targeting Taxane site.Agents interacting with Taxane site can promote microtubule polymerization and inhibit microtubule depolymerization,which are known as microtubule-stabilizing agents（MSAs）.In this study,the structure-based methods（pharmacophore model,molecular docking and interaction molecular fingerprinting similarity）and property-based methods（ADME/T assessment,toxicity prediction,false positive compound（PAINS）filter and drug-likeness evaluation）were applied to discover novel microtubule-stabilizing agents.17 hit compounds（P1-P17）were identified by virtual screening.The anti-proliferative activity of P1-P17 against HeLa cancer cells was measured by MTT assays.P2 exhibited the best inhibition at a concentration of 10μM.The anti-proliferative activities(IC₅₀)of P2 against four cancer cells HeLa,HepG2,MCF-7,and A549 were 16.30±0.30,17.30±0.57,13.79±0.07,and 9.21±0.42μM,respectively.In addition,the in vitro tubulin polymerization assay revealed P2 promoted tubulin polymerization by 104.08%（15μM）and 117.66%（45μM）,respectively.The results of molecular dynamics simulations showed that P2 could stably bind to the Taxane site,and form stable hydrogen bonding interactions with PRO274 and ARG284 in the M-loop,and hydrophobic interactions with VAL23,ALA233,PHE272,PRO274,ARG369 and LEU371 in the hydrophobic pocket.TheΔG_bind of Tubulin-P2 was-68.25±12.98 kJ/mol.The energy decomposition results also revealed the binding pattern of P2 was similar to paclitaxel.As a consequence,P2 could promote microtubule polymerization,which considered as a novel microtubule-stabilizing agent.Chapter 3 Virtual screening and molecular simulation studies of microtubule-destabilizing agents targeting Colchicine site.Agents interacting with Colchicine site can inhibit microtubule polymerization and promote microtubule depolymerization,which are known as microtubule-destabilizing agents（MDAs）.In this study,the shape similarity,machine learning classification model LGBMClassifier,molecular docking,IFP similarity,property-based ADME/T assessment,toxicity prediction,PAINS filter,and drug-likeness evaluation methods were used to identify microtubule-destabilizing agents.Finally,21 hit compounds C1-C21 were selected,which were clustered into four classes（Cluster1-Cluster4）according to molecule scaffold.Four representatives C5,C12,C14 and C20 were selected.The results of molecular dynamics simulations revealed that four compounds could stably bind to Colchicine sites.The conformational changes of tubulin induced by the C5 and C20 were similar to reference compound colchicine.Binding mode analysis revealed that the four compounds formed interactions with residues ofβCYS241,βLEU248,βALA250,βLEU255,βMET259,βALA316,βILE318,βARG352,andβALA354 onβS8,βS9,βH7,βH7,βH8,andβT7,and formed hydrogen bonds withαSER178,αTHR179,andαVAL181 onαT5.TheΔG_bind values were-108.8±13.6,-77.7±13.5,-80.3±10.8,and-90.6±10.3 kJ/mol for C5,C12,C14 and C20,respectively.The energy decomposition results showed that interactions of the four compounds and colchicine were similar.The C5 and C12displayed better characteristics for Colchicine site targeting,which considered as novel microtubule-destabilizing agents.Chapter 4 Conclusion and outlook.This research identified potential MTAs by computer-aided virtual screening and pharmacological experiments targeting Taxane and Colchicine sites.Binding modes of these compounds binding to tubulin were elucidated using molecular dynamics simulations.Comparative analysis with reference inhibitors concluded that these compounds have potential as MTAs,but more pharmacological validation and structure optimization are needed.In conclusion,the virtual screening and molecular modelling approach were used to discover novel microtubule inhibitors.These screened candidates could provide more novel lead compounds for antitumor drug development targeting microtubules.