Discovery Of Chalkophomycin,a Natural Copper(Ⅱ) Metallophore With Antitumor Activity

Metal ions play essential roles in life by coordination with small molecules,proteins,and nucleic acids.Although the coordination of copper ions in many proteins and methanobactins is known,the coordination chemistry of Cu（II）in natural products and their biological functions remain under-explored.Small molecules capable of binding copper ion are generally referred as chalkophores,or copper-binding metallophores.Several chalkophores have recently been discovered,including methanobactins,yersiniabactin,as well as diisonitrile natural products SF2768 and xanthocillins.However,to our knowledge,there are few single-crystal X-ray structure of natural chalkophores binding to copper（II）,such as the 1.6?X-ray structure of Cu（II）-bleomycin in complex with the bleomycin binding protein.In this Thesis,we first screened the production of bioactive natural products of our actinomycete strain collection by varying the fermentation conditions.Six natural products（1–6）were isolated ad structurally characterized from Streptomyces sp.CB00271,including a new Cu（II）-binding natural product,chalkphomycin（CHM,1）.We next evaluated the antitumor activity of CHM（1）and potential mechanism of action.The main research contents are as follows:Discovery of chalkophomycin,a Copper（II）metallophore from Streptomyces sp.CB00271.We obtained six natural products（1–6）by fermentation of S.sp.CB00271,isolated from Yunnan province from our actinomycetes strain collection.Compound 1 was a new type of chalkophore,which we named chalkphomycin（CHM,1）.The X-ray crystal structure indicated that CHM（1）containing a tetracoordinated square-coordination system coordinated to Cu（II）,which was different from many synthesized Cu（II）complexes.The coordination system of CHM（1）included a rare bidentate diazeniumdiolate and a heterocyclic1H-pyrrole 1-oxide-oxazoline.Due to the paramagnetic effect of Cu（II）in CHM（1）,we treated CHM（1）with HCl to obtain its copper-free ligand,apo-chalkophomycin（apo-CHM,1’）,and its hydroxylated product.Their structures were subsequently determined by HR-ESI-MS and NMR techniques.Apo-CHM not only chelates Cu²⁺rapidly to form a stable Cu（II）complex,but also is capable to chelate Fe²⁺and Fe³⁺.The minimum inhibitory concentrations of CHM（1）and apo-CHM（1’）against a variety of gram-positive bacteria were 4–8μg/m L.The half-life of CHM（1）and apo-CHM（1’）in 10%serum were about 6 and 12 h,respectively.The antitumor activity and mode of mechanism study of chalkophomycin.First,we tested the cytotoxicity of CHM（1）and apo-CHM（1’）on selected tumor cells by CCK-8,exhibiting IC₅₀values of 0.18±0.05 to 0.98±0.01μM and 0.49±0.03 to 2.43±0.10μM,respectively.CHM（1）showed potent copper-dependent toxicity to these cancer cells,which is more than ten-fold potent than cisplatin.The ROS level and NO level of melanoma B16-F10 cells treated by CHM（1）and apo-CHM（1’）were analyzed using DCFH-DA ROS fluorescent probe,fluorescent inverted microscope,and NO detection kits.Both CHM（1）and apo-CHM（1’）can significantly increase the ROS and NO levels in the treated cells.In addition,both CHM（1）and apo-CHM（1’）blocked the treated cells in their G1-phase;CHM（1）can induce obviously late apoptosis of treated cells,while apo-CHM（1’）cannot induce cell apoptosis.Finally,CHM（1）exhibited comparable antitumor activity with cisplatin in B16-F10 melanoma mouse xenografts,while with reduced toxicity;In contrast,apo-CHM（1’）exhibited no significant antitumor activity.We further explored the antitumor mechanism of CHM（1）.We found that CHM（1）and apo-CHM（1’）can interact with DNA by molecular docking,UV-vis spectroscopy,fluorescence spectroscopy,and isothermal titration calorimetry.CHM（1）showed copper-dependent DNA cleavage activity under reducing environments,and the DNA-damage mode of action of CHM（1）was different from bleomycin Z.CHM（1）can also cause significant DNA damage in neutral comet assays,while apo-CHM（1’）treatment did not result in obvious DNA damage.Therefore,we reasoned that one of the main targets of CHM（1）is nucleic acids,likely DNA.In summary,a novel natural chalkophore,CHM（1）was discovered from S.sp.CB00271,and its structure and antitumor effect were also studied.This study not only provides a new molecular scaffold for the development of novel copper-containing antitumor drugs,but also is instrumental for continuing search of new natural products from soil Streptomyces strains.Due to the important role of copper homeostasis in both human physiology and diseases,chalkophore CHM（1）and its ligand may also become excellent tool molecules to study the biological functions of copper ions.There are 78 figures,24 tables,and 295 references included in this thesis.