Modification And Enzymatic Activities Of Nitrovinyl Benzoate And Other Two Types Covalent Inhibitors Against Hu-FBPase

Diabetes,as one of the world’s largest non-communicable diseases,has been plaguing people all over the world.The incidence of type 2 diabetes(T2DM)is also increasing rapidly.The development of corresponding therapeutic drugs has always been an important issue for researchers.Human liver fructose-1,6-bisphosphatase(Fructose-1,6-bisphosphatase,referred to as Hu-FBPase)is an important rate-limiting enzyme in the gluconeogenesis pathway.Studies have found that the inhibition of Hu-FBPase activity can achieve the effect of lowering blood glucose,so Hu-FBPase is one of the important potential targets for the treatment of T2DM.At present,there are two types of inhibitors against Hu-FBPase.One type is designed for AMP allosteric site,but such inhibitors have poor selectivity and relatively large side effects.Another is substrate site,which is rarely reported.In recent years,there have been more and more reports of covalent inhibitors targeting specific amino acids and covalent drugs have been successfully marketed.Because covalent inhibitors have the advantages of strong selectivity and long action time,they have received more and more attention and research in the field of drug discoveryThe work of this thesis is mainly to modify the structure of the Hu-FBPase covalent inhibitors compounds.In the previous work,our lab discovered a new covalent binding regulation site(C128 site),located near the Hu-FBPase substrate site.For the initial screening study at C128 site,our lab obtained two hit compounds of nitrostyrene(HS)and benzyl isothiocyanate(SF2).In this thesis,the non-covalent parts of the above two compounds were further modified,and nitrovinyl benzoate(SHZ series)and benzyl isothiocyanate series(SF series)were designed and synthesized respectively.In addition to covalent drug design method based on covalent warhead,it can also be covalently designed based on reported crystal structures of non-covalent compounds.A class of noncovalent sulfonylurea inhibitors targeting allosteric site of AMP has been reported in the literature.First,we obtained their analogues through framework modification,and then tried to introduce suitable covalent warheads to obtain new covalent inhibitory compounds.The specific research work of this thesis is as follows:(1)Early research by our lab found that nitrostyrene(HS,IC50=3.50±0.30 μM)can bind to the C128 site on Hu-FBPase,thereby inhibiting its activity.In order to further improve inhibitory activity,the framework of the HS was modified.On the basis of retaining the original nitrostyrene structure,the framework of the compound was extended by introducing a linker ester bond on the benzene ring.Therefore,nitrovinyl benzoate compounds(SHZ series)were designed,synthesized and tested for their inhibitory activity.There are 9 compounds below 1 μM,the best is SHZ13(IC50=0.20±0.03 μM),which is increased by 15 folds compared with that of HS.(2)Thioisocyanate compounds contain R-N=C=S unsaturated double bond structure,which is easy to undergo nucleophilic addition reaction with sulfhydryl groups on cysteine and form a covalent bond group.When screening compounds,we found that hit compound benzyl isothiocyanate(SF2)had a moderate inhibitory activity(IC50=112.31±7.94 μM).In order to further improve its activity,the structure of hit compound was modified,and a series of compounds with different substituents were obtained.A total of 22 compounds of benzyl isothiocyanate(SF series)were synthesized,A total of 22 benzyl isothiocyanate(SF series)compounds were synthesized,and the inhibitory activity of compound SF25(IC50=7.59±1.88 μM)increased by an order of magnitude compared with that of hit compound SF2.Mutant experiments showed that the C128 site had a significant effect on the inhibitory activity of SF compounds.(3)A class of sulfonylurea compounds have been reported in the design of noncovalent inhibitors for AMP allosteric site.Based on the structure of its typical compound R03 crystal complex(PDB:2WBB),we found that cysteine C179 exists near the AMP allosteric site,and the distance between R03 and C179 is only 7.5 人.Inspired by the previous research on the mechanism of Hu-FBPase covalent allosteric inhibition,we first modified the framework of R03 to obtain 27 analogues of WB series compounds(noncovalent inhibitors).On this basis,we initially tried to introduce different covalent warheads and linker group to discover novel covalent inhibitory compounds that target C179.Among the WB series of non-covalent inhibitors,WB31 compound(IC50=1.05±0.08 μM)has the best inhibitory activity.The inhibitory activity of the SWB1 compound with the nitroethylene covalent warhead is IC50=0.222±0.01 μM.Mutation experiment data preliminarily support that it is a covalent inhibitor targeting C179,and the specific mechanism of action still needs to be further studied.