Structural Optimization And Anti-hepatocellular Carcinoma Activity Evaluation Of An Aldolase B Activator

Most of primary liver cancer is hepatocellular carcinoma（HCC）,which is one of the most common clinical malignancies,with high incidence and mortality.At present,many multi-target anti-HCC drugs have been used in clinical treatment,but they are accompanied by great toxic and side effects.Fructose-biphosphatase aldolase B is a key enzyme in glycolysis,and studies show that ALDOB plays an important role in the development and development of HCC,making it an important target for therapeutic research of HCC in recent years.However,no selective small molecule regulators for ALDOB has been reported yet.According to the results found in the previous study of our group,compound YH67507can directly act on ALDOB and inhibit the proliferation of HCC cell to some extent.Based on the previous study,ALDOB was targeted for the lead compound YH67507 for structural optimization and virtual screening by computer-aided drug design.The synthesis and structural analysis of top hit compounds yielded a batch of chalcone derivatives.Then the synthesized compounds were evaluated in vitro and in vivo.In the end,compound MD33was selected as the preferred compound,which could lay the foundation for the research on related new drugs.Methods:1.Structural optimization of the lead compound and calculation of the lowest energy conformation were performed in Sybyl X 2.0 resulted in a molecular database containing over 500 chalcone derivatives;then the Surflex Dock function was used to perform the virtual docking of the small molecules with the human ALDOB protein.2.The potential-active compounds were synthesized using Clayson-Schmidt condensation and Baker-Venkararman rearrangement,and were structure-resolved by nuclear magnetic resonance spectra（NMR）.3.The affinity of the compounds to ALDOB were determined using Microscale thermophoresis（MST）;the effect of the compounds on the proliferation capacity of HCC cells was determined using Cell Counting Kit-8（CCK-8）.In the subcutaneous transplantation tumor model of nude mice,the tumor suppression rate and related biochemical indexes were determined to evaluate the in vivo anti-HCC activity of compound MD33.Results:1.By screening the docking sites,Total Score and Consistency Score（C Score）,29 potentially active compounds（C Score≧4 and Total Score>7.00）were selected,and it was also found that top hit compounds usually produced hydrogen bonding interactions with specific amino acids（Arg 42,Lys146,Arg303）in human ALDOB.2.¹H and ¹³C NMR results showed that the structure of the 29 synthesized compounds were correct.3.MST experiments showed that compounds 1-（2,6-dihydroxy-4-methoxyphenyl）-3-（3,4-dimethoxyphenyl）propan-1-one（MB17）,1-（2,6-dihydroxy-4-methoxyphenyl）-3-（2-hydroxyphenyl）propan-1-one（MB20）,（E）-3-（4-（tert-butyl）phenyl）-1-（2-hydroxy-4-methoxy-6-（methoxymethoxy）phenyl）prop-2-en-1-one（MB26）,（E）-3-（3,4-dimethoxyphenyl）-1-（2-hydroxy-4-methoxy-6-（methoxymethoxy）phenyl）prop-2-en-1-o ne（MB30）,（E）-1-（2-hydroxy-4,6-bis（methoxymethoxy）phenyl）-3-（2,4,5-trimethoxyphenyl）prop-2-en-1-one（MD28）,and 1-（3,4-dimethoxyphenyl）-3-（2,4,6-trihydroxyphenyl）propane-1,3-dione（MD33）can bind directly to the ALDOB protein in vitro;CCK-8 results showed that compounds mentioned above could dose and time dependently inhibit proliferation of HCC cells HepG2.Among them,compound MD33 had the strongest effect(K_Dis 5.13μM,and half inhibitory concentration(IC₅₀)was 0.744±0.12μM).Compound MD33 was determined as the preferred compound based on the results above.4.Compound MD33 showed a significant dose-dependent in vivo anti-HCC effect,with inhibitory rates of 52.87%and 73.43%（with concentrations of 20 and 40 mg/kg,respectively）against subcutaneous tumor in nude mice.The serum levels of glucose and glycated serum protein in the treated nude mice were not significantly different from the model group,suggesting that MD33 has no effect on the level of glucose metabolism.The results of the aspartate transaminase and alanine amino transferase have no significantly different from the model group,suggesting that MD33 did not have hepatotoxicity.In conclusion,compound MD33 can bind efficiently to ALDOB,time-and dose-dependently inhibit the proliferation of HCC cells HepG2,and can significantly inhibit the growth of subcutaneous transplanted tumors.MD33 has a simple structure,short synthetic route and high yield,which shows the potential to further development of novel anti-HCC drugs targeted for ALDOB.