Design,Synthesis,and Evaluation Of Galactosed Derivatives As Targeting Molecules To Liver Cancer Cells

Liver cancer is the fifth most common tumor and the third most deadly disease in the world,causing about 400,000 deaths in China per year and accounting for 51%of the total number of deaths in the world.Hepatocellular carcinoma(HCC)is the most common and malignant tumor with high mortality of all the types of liver cancers,accounting for 75-90%cases of liver cancer.In addition,the specific biomarkers for HCC have not been identified yet.There is an urgent need in developing the methods for detection and treatment of HCC through improving the targeting efficiency to enrich drugs at the focus site.The asialoglycoprotein receptor(ASGP-R)is an endocytic C-type lectin receptor mainly expressed on the surface of mammalian liver parenchymal cells and was demonstrated by numerious studies to be an effective target for many diagnostic and therapeutic drugs to bind and enter hepatocytes.Although the research and development of liver targeting drugs with ASGP-R as receptor is a hot spot in the international pharmaceutical industry,the research of sugar targeting molecular systems specifically targeting ASGP-R oligomers of liver cancer cells is insufficient.ASGP-R is a heterooligomer composed of two homologous subunits ASGP-RH1(46 kDa)and ASGP-RH2(50 kDa)in a ratio of 2-5:1,among which the trimer consisting of two ASGP-RH1 and one ASGP-RH2 is the most abundant.The ASGP-R recognizes ligands with terminal,asialylated galactose(Gal)or N-acetylgalactosamine(GalNAc)residues and functions via clathrin-dependent,receptor-mediated endocytosis.It has been reported that the transcriptional levels of ASGP-RH1 and ASGP-RH2 in HCC cells are significantly different from those in normal liver cells.Currently,the reported HCC targeting drugs and materials usually use the monovalent GalNAC and Gal or polymer-carried GalNAC and Gal as the targeting functional groups,and there is a lack of systematic studies on the differences in the selection ability of targeting molecules to the HCC cells and normal liver cells.The structure of the multivalent sugar ligands,including the type of sugar,the length of linker,and the spatial distance between sugar moieties,could significantly inlufence their binding efficience to ASGP-R.In order to design and construct the multivalent sugar ligands,we first performed the simulations of ASGP-R heterotrimer structure,and used this structure to perform the computational ligand-protein docking.The fitness degree and binding efficiency of 14 of reported sugar ligands was then compared.The △G values of the trivalent ligands were between-11.07 kcal/mol and-16.13 kcal/mol,the △G values of the bivalent were between-11.11 kcal/mol and-13.28 kcal/mol,and the △G values of the monovalent were between-7.06 kcal/mol and-9.18 kcal/mol,which reflected that the trivalent,bivalent and monovalent ligands diaplayed the successive decrease of the affinity to ASGP-R.Based on this result,we designed 11 kinds of galactose ligands with 1-3 antenna numbers,4-12 carbon atom numbers of linkers,and the gallic acid or amino-isophthalic as the acid scaffolds,respectively.These ligands were respectively docked with simulated ASGP-R heterotrimer.The results showed that the △G value of the trivalent ligand N3C12 was-14.47 kcal/mol,which was 6.69 kcal/mol lower than that of the monovalent ligand N1C4(-7.78 kcal/mol).These results above led to a conclusion that the trivalent ligand has the higher affinity in binding with the ASGP-R heterotrimer.Moreover,the△G value of N3C12 was even lower than those of the reported two ligands TG6(-13.55 kcal/mol)and TG8(-14.14 kcal/mol)which display strong binding affinity to ASGP-R proved by the experiments.We than speculated that N3C12 might have a better binding ability with ASGP-R then the reported ligands.Base on the results of the rational design,we synthesized the single-tentacle ligand N1C4(Gal-DEG-NH2)through 7 steps of chemical synthesis reactions and the three-tentacle galactose-targeting molecule N3C12(TGal-PEG-NH2)through 12 steps of chemical synthesis reactions with galactic acid as the scaffold.The structures of the compounds were determined by NMR and MS.The total yield of Gal-DEG-NH2 and TGal-PEG-NH2 was 22.4%and 16.7%,respectively.We also used Quantitative Real-time PCR(qPCR)to detect the transcriptional differences of the two ASGP-R subunits in various tumor and normal cells including HCC hepatic tumor cells(HepG2)and normal hepatic cells(L-02).The results showed that the transcription level of the ASGP-RH1 and ASGP-RH2 in liver tumor cells were about 3300 times and 2300 times higher than those in normal liver cells,respectively.This result also provides the theoretical basis for further studies on the targeting abilities of sugar ligands.The two subunits of ASGP-R showed nonsimultaneous transcription in other tumor cells,including human breast tumor cell MCF-7,human oral tumor cell KB,human prostate tumor cell PC3,and the transcription levels of the single subunit of ASGP-R in these cells was also significantly lower than that of human hepatic tumor cell HepG2.We then studied the targeting efficiency of galactose ligands to tumor cells at the cellular level.We first labeled three ligands TGal-PEG-NH2,Gal-DEG-NH2 and galactose with the fluorescent molecule Rhodamine B(RhB)through covalent modification.The RhB labeled ligands were incubated with various cells.Among the three ligands,the TGal-PEG-NH2 showed the highest targeting efficiency to HepG2 cells followed by the Gal-DEG-NH2 and the Gal.The inhibition assay showed that the fluorescence intensities of Gal-DEG-RhB were reduced in HepG2 by the prior addition of excess galactose,suggesting that the Gal-DEG-NH2 was introduced into the cells through the specific binding of ASGP-R.However,the prior addition of excess galactose could not significantly inhibit the accumulation of TGal-PEG-RhB in both HepG2 and L-02 cells,which could due to the much stronger affinity of TGal-PEG-NH2 to ASGP-R than that of Gal.The above results indicate that TGal-PEG-NH2,the three-tentacled galactose ligand,has strong targeting ability to hepatocyte.Morevoer,TGal-PEG-NH2 displayed the significant selectivity to liver tumor cells versus normal liver cells.Therefore,TGal-PEG-NH2 is an ideal targeting molecule for liver tumor cells.