Target Identification And Antitumor Mechanism Evaluation Of Barbigerone,T007-1,Withaferin A As Small Molecular Inhibitors Targeting Tubulin

Microtubules are hollow cylindrical tubes composed of 13 arranged protofilaments,which are formed by the head-to-tail connection of α-and β-tubulin heterodimers.Microtubules show an inherent behavior of dynamic instability,which changes frequently between the growth stage and the shortening stage.Microtubules play many roles in a variety of cellular functions,including cell division,intracellular transport and localization of organelles in cells.In the process of normal mitosis,microtubules separate sister chromosomes into daughter cells through the mechanism of time and space synchronization mediated by cell cycle checkpoints,thus completing cell proliferation.Inhibition of microtubule assembly during mitosis leads to cell cycle arrest in G2/M phase,and eventually leads to apoptosis and death.Therefore,microtubules have become an important target for anti-tumor research.Such as clinical chemotherapy drugs: paclitaxel,vinorelbine and eribulin.Microtubule targeting agents(MTAs)are composed of many kinds of compounds.At present,there are six kinds of tubulin binding sites,which are paclitaxel site,colchicine site,vinblastine site,maytansine site,Laulimalide/Peloruside A site and Pironetin site.Plants are one of the main sources of MTAs which have been found to occupy these sites and have significant anti-tumor activity.There are at least 500,000 species in the plant kingdom,and only less than 10 percent of them have been phytochemically investigated for pharmacological applications.For example,isoflavonoid compounds is one of the largest and most widespread class of natural products with various biological activities including anti-cancer.Microtubule is proved to be one of the cellular targets of some anti-cancer isoflavones,such as genistein and glaziovianin A.However,due to lack of crystal structure of tubulin with any isoflavones,details of the interaction of isoflavones with tubulin remains elusive and the development of isoflavone-based compounds as novel anticancer agents has stalled.Barbigerone is natural products which was firstly separated from seed of a leguminous plant Tephrosia barbigera.We proved that it has tubulin inhibitory activity and anti-tumor activity,and we solved the crystal structure of tubulin-Barbigerone complex for the first time.It laid the foundation for the further research of isoflavones targeting tubulin.At present,microtubule targeting agents is a kind of anticancer drugs widely used in clinic,such as paclitaxel and Eribulin.Tubulin inhibitors can be divided into two categories according to their functions: tubulin depolymerizing agents(destabilizing agents)and tubulin polymerizing agents(stabilizing agents).Tubulin depolymerizing agents inhibits microtubule polymerization.Tubulin polymerizing agents enhances tubulin polymerization and microtubule stabilization.Both tubulin depolymerizing agents and polymerizing agents have been shown to generally interfere with microtubule dynamics,but does not change the total microtubule mass at a wide range of concentrations.A number of small molecules have been identified that promote degradation of tubulin,such as Withaferin A,T0070907,isothiocyanates.The action mechanism of these compounds is different from that of conventional microtubule polymerizers and these small-molecule tubulin degradation agents may represent a third novel class of tubulin inhibitors,but the related molecular mechanism is not clear.Therefore,we made a detailed study on the binding sites and the mechanism of tubulin degradation induced by Withaferin A,T0070907 and their derivatives.It reveals the in-depth mechanism of these compounds promoting tubulin degradation,which is helpful for the subsequent discovery and development of tubulin degradation agents.Chapter 1: Target identification of natural isoflavone Barbigerone and its anti-tumor mechanism with its derivatives.Here,we found Barbigerone,an isoflavone with anticancer activity,inhibit tubulin polymerization by binding to the colchicine site of β-tubulin.Firstly,we confirmed that Barbigerone can directly bind to purified tubulin by SPR assay,and then we solved the crystal structure of tubulin-Barbigerone complex at 2.33 (?)resolution,which unambiguously determine the orientation and position of Barbigerone in the colchicine-binding site.Illuminated by the detailed interactions between Barbigerone and tubulin,we synthetized a serial of Barbigerone derivatives,and obtained compound 0412.Both Barbigerone and 0412 could inhibit cancer cell proliferation,inhibit tubulin polymerization,induce G2/M phase cell cycle arrest and apoptosis,and has anti-vascular activity,manifested by inhibiting migration and capillary-like tubes formation of HUVEC.Importantly,in an H460 xenograft model,both Barbigerone and 0412 showed anticancer activity.In all these activities tested,0412 is more effective than Barbigerone.These results indicate that we have synthesized more effective isoflavone derivatives under the guidance of the crystal structure of tubulin-Barbigerone complex.In conclusion,our research provides an important research basis for the development of flavonoids.Chapter 2: The action site and anti-tumor mechanism of tubulin degrading agents T007-1.It has been reported that T0070907 can promote the degradation of tubulin.In this chapter,we synthesized the derivative T007-1 of T0070907.We deeply studied the action site of T007-1 and its mechanism of promoting tubulin degradation.The anti-tumor activity in vitro showed that both T007-1 and T0070907 had antiproliferative activity,and T007-1 had the better effect.Label-free quantitative proteomic analysis revealed that T007-1 promotes tubulin degradation with high selectivity.Microscale thermophoresis assay showed that T007-1 could directly interact with tubulin.In the study of tubulin degradation activity(and depolymerization activity)of T007-1,T007-1 down-regulated tubulin in a posttranscriptional way and induced tubulin degradation through proteasome pathway.Immunofluorescence and microtubule kinetics experiments showed that T007-1 not only had degradation activity,but also inhibited tubulin polymerization.In addition,T007-1 also has the characteristics of conventional tubulin inhibitors,which can induce G2/M phase cycle arrest and apoptosis.Competitive binding experiments of colchicine site inhibitors(colchicine and plinabulin)showed that T007-1 interacted with colchicine site of tubulin to promote tubulin degradation.Mass spectrometry findings showed covalent binding of both T0070907 and T007-1 to Cys239 of β-tubulin.In order to prove that the covalent binding of T007-1with Cys239 is the reason for its tubulin degradation activity,we studied T007-1 by immunoblotting and point mutation assay.The results show that T007-1 exerted a degradative effect on tubulin isoforms possessing Cys239(β2,β4,and β5(β))but not those containing Ser239(β3,β6)or mutant β-tubulin with a C239 S substitution.In addition,180422(derivative of T007-1)was synthesized to verify the active group(Chlorine atom on nitrobenzene)of T007-1.The results showed that the antiproliferative activity and tubulin degradation activity of 180422 were lost,and it was confirmed that the Chlorine atom on nitrobenzene of T007-1 was the key functional group for covalent bonding with Cys239.In summary,the covalent interaction between T007-1 and the Cys239 of β-tubulin promoted the degradation of tubulin.We believe that these small molecules represent the third class of new tubulin inhibitors,which play an anti-tumor effect by promoting tubulin degradation.Chapter 3: The action site and anti-tumor mechanism of tubulin degrading agent Withaferin A.Withaferin A(WIT)is a natural product possessing a wide range of pharmacologic activities.Previous studies have reported that WIT covalently binds to tubulin Cys303 and down-regulate the level of tubulin although the underlying mechanisms remain to be established.In the current investigation,we detected the protein levels of α-tubulin and β-tubulin in cancer cells after drug treatment by western blotting,and detected the m RNA levels of α-tubulin and β-tubulin in cancer cells after drug treatment by PCR.The results show that WIT induces down-regulation of tubulin in a post-transcriptional manner.The EBI assay and competitive binding experiments with four colchicine site targeted tubulin inhibitors(Colchicine,Nocodazole,Plinabulin and Combretastatin A-4)further revealed that WIT interacts with the colchicine site of tubulin to promote degradation.Furthermore,cancer cells pretreated with proteasome inhibitor MG132 can inhibit the degradation of tubulin by WIT,indicating that WIT induces tubulin degradation through proteasome pathway.Immunofluorescence and Western blotting experiments showed that WIT irreversibly inhibited tubulin polymerization,and mass spectrometry results disclosed binding to Cys239 and Cys303 sites of β-tubulin.Interestingly,WIT promoted degradation of the β-tubulin isoforms containing Cys239 [β2,β4,and β5(β)] but had no effect on those containing Ser239(β3 and β6).Moreover,a C239 S but not C303 S mutation in β-tubulin completely abolished the degradation effect of WIT,suggesting that the Cys239-WIT covalent bond accounts for this activity.Finally,DWIT(structural analogue of WIT)was selected to verify the unsaturated double bonds in the A ring of WIT.The results showed that DWIT did not promote the degradation of tubulin in He La cells and did not inhibit the viability of cells.It is confirmed that the A ring containing unsaturated double bonds is its pharmacophore.Our collective findings demonstrate that the natural compound WIT forms a covalent bond with Cys239 of β-tubulin to promote tubulin degradation,providing another example of a covalent modifier of Cys239 of β-tubulin as an effective tubulin degradation agent with therapeutic potential.Finally,we investigated whether tubulin degradation is a common biochemical consequence of covalent modification of Cys239 in β-tubulin by small molecules.To this end,small molecules reported to form covalent interactions with Cys239 of β-tubulin,such as T138067 and EBI,were selected for analysis.The results show that all of them could induce tubulin degradation,but had no effect on mutant C239 S β-tubulin.Overall,our findings suggest that covalent modification of Cys239 of β-tubulin by small molecules presents an effective strategy to promote tubulin heterodimer degradation.