Screening Proteases Inhibitors From The Compound And Traditional Chinese Medicinal Library

Protease inhibitors refers to a class of compounds which can combine with some groups of the active sites or allosteric sites of proteases to inhibit the activities of proteases and can be widely found in animals, plants, microbes and viruses. Protease inhibitors have wide biological effects, such as anti-tumor, anti-virus, anti-fungal, anti-inflammatory and anti-infective. Protease inhibitors have been clinically used to treat high blood pressure, periodontitis, AIDS, blood clots and other diseases. However, when chemical synthetic protease inhibitor drugs are used in the clinical application, there are many problems, such as low bioavailability, side effects and long-term use will cause drug resistance. But protease inhibitors from natural plants have many advantages, such as good environmental compatibility, safety and low cost. So it will be the future trend of drug development to screen inhibitors from natural plants, and it will also be very promising for the development and application. Therefore, recently it is a very hot point in medicine and biology to screen effective protease inhibitors from Chinese medicines.Cathepsins are important members of papain-like cysteine protease family and exist mainly in lysosome. They can easily be activated in weak acid environments and are not stable in neutral and alkaline solution. In human, cathepsins mainly participate in protein degradation, also they are involved in activation of certain protein precursors or protease systems which play a variety of physiological functions, such as osteolysis and immune function. They are closely connected with arthritis, osteoporosis, tumor invasion and metastasis and other diseases. As such, in recent years, they become a class of target proteases which are received extensive attention. MMPs are a family of zinc-dependent endopeptidases which are secreted to extracellular matrax, They are the most important proteases which are involved in degradation of extracellular matrix, and can degrade almost all components of extracellular matrix. MMPs are closely related with tumor, cardiovascular diseases and inflammation. Therefore, it will be very promising to develop inhibitors against MMPs. uPA is an important activator for exogenous plasminogen, and uPA which is secreted by tissues can cleave plasminogen to produce active plasmin. Plasmin can not only degrade ECM and basement membrane, but also can activate a variety of protein precursors and growth factors such as SF/HGF and TGF. uPA plays an important role in a variety of physiological and pathological processes, such as rheumatoid arthritis, inflammation and tumor migration and invasion. Therefore, uPA is also a promising protease target.In this experiment, matrix metalloproteinases, cathepsins and uPA are chosen as the targets to screen inhibitors from 300 kinds of Chinese medicines and more than 100 kinds of compounds at the concentrations of 100μg/ml. It was found that five kinds of Chinese medicines including Flos caryophyli, Fructus canari, Sanguisorba officinalis, Folium Pyrrosiae could inhibit the activity of MMP-14, with inhibitory rates of 98.8%, 95.5%, 82%, 78%, 65.6%, respectively; eight kinds of Chinese medicines including Tuckahoe, puffball, gallnut, rhizoma cimcifugae, Polygonum multiflorum, Sterculia lychnophora, catechu, white front could inhibit the activity of Cat L with inhibitory rates of 88%, 87.6%, 86.5%, 85.5%, 83.2%, 83.6%, 76.5%, 75%, respectively; four kinds of Chinese medicines including Sanguisorba officinalis, black snake, gallnut and Sterculia lychnophora could inhibit the activity of uPA with inhibitory rates of 90.5%, 91.5%, 79.7%, 65.26%. Then we further measured the dose-dependent inhibitory activities of Flos caryophyli and Fructus canari against MMP-14, it was found that Flos caryophyli and Fructus canari could inhibit MMP-14 activity with IC50 of 0.5μg/ml, 0.25μg/ml. Meanwhile, we also measured the dose-dependent inhibitory activities of against uPA, it was found that Sanguisorba officinalis and black snake could inhibit MMP-14 activity with IC50 of 0.5μg/ml, 30.5μg/ml. Discovery of Chinese medicines which can inhibit the activities of these proteases plays an important role in development of medicine, and also lays the foundation for making full use of traditional Chinese medicine resources to search for new protease inhibitor.When screening from compound library, it was found that tanshinone IIA sulfonate (STS) could effectively inhibit the activity of Cat L in a time-dependent manner, with increasing preincubation time, STS displayed increasing inhibitory potency towards Cat L, with IC50 values falling from 10μM at 0 h to 0.8μM at 5 h. Further studies showed that STS could also effectively inhibit the activities of Cat B, C and K at 20μM with inhibitory rates of 55%, 66%, 47%, respectively. However, cryptotanshinone and tanshinone IIA, which have similar structures to STS, could hardly inhibit the activities of Cat L, B, C and K at 20μM. It was indicated that the inhibitory effect of STS on cathepsins might be structural specificity.Tumor invasion and metastasis is a multi-step, complex process. It is essential for tumor cells to permeate through extracellular matrix barriers to form invasion and metastasis. According to the reports, cathepsin L locates in lysosomes to degrade proteins in normal cells. However, in pathological conditions, cathepsin L can secrete to extracellular matrix which can not only degrade extracellular matrix, but also cleave a variety of protein precursors and other enzymes to promote tumor invasion and metastasis. As such, we want to know whether STS can inhibit the migration and invasion of tumor cells through inhibiting the activities of cathepsin L. Recently Yoko Hashimoto et al. reported that a kind of 32-kDa activated Cat L could be secreted into extracellular matrix by HT1080 cells to facilitate tumor development. So HT1080 cells were chosen to investigate whether STS could inhibit cell migration and invasion. It was found the cell viability was not affected by STS below 90μM. Through wound healing assay and Transwell chamber assay, it was found that STS could effectively inhibit HT1080 cell migration and invasion at 50μM. However, it was believed MMP especially MMP-2 and MMP-9 and uPA play an important role in cell migration and invasion. To exclude the possibility that STS inhibit HT1080 cell migration and invasion is due to the inhibitory activity of STS against MMP-2, MMP-9 and uPA, the inhibitory effect of STS towards MMP-2, MMP-9 and uPA was detected in vitro. It was shown that STS did not inhibit the activities of MMP-2 and MMP-9 at the concentration of 50μM. To further demonstrate STS inhibit HT1080 cell migration and invasion is due to the inhibitory activity of STS against cathepsins, a cathepsin broad-spectrum inhibitor E-64 was chosen as the positive control, it was found that E-64 could inhibit migration and invasion at 100μM.In short, we screened protease inhibitors from 300 kinds of Chinese medicines and compounds using three proteases as the target, and eventually we got several Chinese medicines and compounds which could effectively inhibit the activities of these proteases, then the compound was chosen to study the anti-tumor activity. The study will not only play a guiding role in the treatment of diseases with Chinese medicine in the clinical application, but also provides a theoretical basis for treatment of related diseases and accumulates data for making full use of our traditional Chinese medicine resources and discovery and development of new protease inhibitors. Also we have found several derivatives from Salvia miltiorrhiza BUNGE that could inhibit the activities of several cysteine cathepsins. It will be very promising to use these compounds as the lead compounds, develop more specific cathepsins inhibitors for cancer therapy.