Polyphenols Substances Flynn Protease (furin) Activity Inhibition

furin is the first identified mammalian PC and the most extensively studied member of the known PCs.On one hand,furin plays important roles in various biological processes such as the activation and secretion of peptides and peptide hormones,but on another hand,furin, because it processes growth factors,viral glycoproteins and bacterial toxins,is also involved in many diseases such as cancer as well as viral and bacterial diseases.Searching for small molecule inhibitors to furin is not only helpful for understanding the function of furin in vivo but also for the development of potential anti-cancer,anti-viral and anti-bacterial drugs.Tea polyphenols are small molecules.Compared to large molecules like for example peptides they can have,if selected carefully,several advantages such as better cell permeability,higher specificity,higher stability and less side effects.In this study,we focus on three candidate inhibitors to furin which are the components of green tea.They are (-)-epigallocatechingallate,(-)-EGCG,(-)-epigallocatechin,(-)-EGC and Quercetin.The inhibition to furin by these three inhibitors was evaluated in vitro and "ex vivo".(1) The "in vitro" furin cleavage system was optimized.The optimal pH value and the optimal calcium ion concentration were evaluated.The Km and Vmax values were determined in the optimized system.The results show that,the optimal pH is 6.0 and the optimal calcium concentration is 1 mM.The Km and Vmax value of the furin chromogenic substrate(Pyr-Arg-Thr-Lys-Arg-AMC) was 76.38μmol/L and 2.254 RFU/s/ng furin.(2) Based on the measurement of fluorescent signal caused by the cleavage of a chromogenic substrate by furin,the inhibition of furin by the three candidate inhibitors was evaluated.Ki values and inhibition types were determined using the Dixon plot method.The results show that the three candidate inhibitor all show inhibition towards furin.Dixon plot results show that the inhibition types are all non-competitive.And the Ki value is 2.09 for EGC,8.14 for EGCG and 27.5 for Quercetin.(3) At micro molar concentration,some small molecules can self-associate into colloidal aggregates that non-specifically bind to protein and therefore can inhibit enzymes in a non specific way.These pseudo-inhibitors are called promiscuous inhibitors.In this study, we performed three experiments to verify whether the three candidate inhibitors tested are promiscuous inhibitors.The experiments performed are:addition of a low concentration of detergent,addition of BSA and centrifuge the inhibitor solutions in order to eliminate possible aggregates.The results show that 0.01%concentration of the detergent does not diminish the inhibition.Centrifugation of the inhibitor solutions also does not reduce the inhibition. Therefore the three candidate inhibitors are most likely not promiscuous inhibitors.Incubation of furin in the presence of BSA increases the furin activity,probably because the enzyme is stabilised by this protein.As a consequence addition of BSA cannot in this specific case be used as a discriminating factor for verifying the promiscuous character of the candidate inhibitors.(4)In the ex vivo test,A furin substrate plasmid was transfected into cells which will be supplied with radioactive-labelled cysteine and methionine.In this way the cells will produce the substrate precursor-protein which will be cleaved by furin carboxy-terminally of RHRR into a mature substrate.The secreted proteins are then radioactively marked and can be detected autoradiographically.The result shows that in case of EGCG and Quercetin,when the inhibitors was added.There is a gradual decrease of the amount of mature BAFF in the medium.While no signifficant effect was seen on the amount of precursor BAFF in the cell lysate.To address whether the demonstrated effect of some of these tested polyphenols is limited to BAFF or whether there is also an effect on the amount of mature product of other furin substrates.The processing of TGF-beta and IGF-1R were also evaluated.The result shows that when EGCG and Quercetin was added,there is a gradual decrease of the mature TGF-beta and IGF 1R,While the amount of precursor TGF-beta is increased.No significant effect was seen on the amount of precursor IGF-1R.These candidate inhibitors is probably inhibit the furin substrate thought its effect to furin.EGC does not show any inhibition ex vivo,probably because it could not go through the cell membrane.