Construction Of Near-Infrared Fluorescence Detection And Imaging Method For Dipeptidyl Peptidase Family And Its Application In Screening Of Active Compounds Of Chinese Herbal Medicine

Serine Hdrolase is a collective name for a series of hydrolase enzymes with serine as the catalytic active site.It has become a therapeutic target for diseases such as the nervous system,immune system,endocrine metabolism and so on.As the only peptidase family of serine hydrolases,the Dipeptidyl Peptidase(DPP)family has many subtypes with different organs,tissues,cell distribution and functions.Among them,Dpeptidyl Pptidase4(DPP4)and Proly Edopeptidase(PREP)are currently recognized as the two most important peptidases.DPP4 is an FDA-approved target for the treatment of diabetes.In addition,because DPP4 is widely distributed in lymphocytes and kidney tissues,studies have shown that DPP4 is also a marker of immune function and kidney damage disease;PREP interacts with the nervous system in brain tissue Many neurodegenerative diseases are related.At present,studies have shown that PREP combines with many protein molecules in the nervous system and aggregates to form a copolymer.The increase of such copolymers may be one of the factors leading to the progression of neurodegenerative diseases,but Its mechanism of action is still unclear.In order to clarify the role of DPP family proteins in the physiological role and pathological environment in the body,the distribution of DPP family in cells or tissues and the detection of functional effects are particularly important.However,there are currently many fixed methods based on gene level,transcription level,and protein level detection.The detection of a protein in complex biological samples requires a separate system.As the DPP family with enzyme activity,the activity-based detection method can better detect the function of the protein,but there are still many technical challenges,such as lack of selectivity and sensitivity for protein detection,and even interference from the matrix.Therefore,the establishment of an effective DPP family of small molecule detection methods and the provision of effective molecular tools for the disconnection between basic biological research and clinical drug research based on physiological and pathological changes are currently urgent problems to be solved.In this thesis,using the near-infrared fluorescent core structure as the starting template compound,two "off-on" probes with high signal-to-noise ratio,strong tissue penetration,and small damage to the body are developed with high selectivity for DPP family peptidases.,Respectively named GP-ACM and Z-GP-ACM.On the basis of these two probe substrates,a microplate reader was used to establish and optimize a specific test method for the detection of DPP4 and PREP in vitro,and qualitative and quantitative determinations were performed on recombinant protein,cell homogenate,and tissue homogenate,respectively.Then it was evaluated in cell and tissue imaging detection research;a high-throughput detection method was built to screen and evaluate the DPP inhibitory components in traditional Chinese medicine.The main results of the research are as follows:1.The selectivity,sensitivity and anti-interference ability of GP-ACM to DPP4 were synthesized and evaluated,and the commercial probe GP-AMC was compared with the fluorescent probe GP-BAN and DPP4 probe GP-ACM developed by the research group GP.It has greater advantages in selectivity and anti-interference ability;comprehensively,GP-BAN has better sensitivity in the detection of biological samples in vitro,and its selectivity and anti-interference ability are also stronger.Combined with the above probe evaluation,the detection of DPP4 in different cells S9 by GP-ACM has a strong correlation with other specific detection results.The results show that GP-ACM can selectively detect the activity of DPP4 in a complex system.Combined with the advantages of near-infrared small molecule probes in tissue penetration and imaging sensitivity,it can realize fluorescence imaging of DPP4 activity in cells in vitro.Detection.In addition,the probe GP-ACM further performed fluorescence imaging of DPP4 in different tissues.The results show that the probe GP-ACM can perform fluorescence imaging of DPP4 in different regions of kidney,intestine,and liver tissues.2.The selectivity,sensitivity and anti-interference ability of the Z-GP-ACM probe for the detection of PREP activity were synthesized and evaluated,and the commercial fluorescent probe Z-GP-AMC was compared with that of the PREP probe Z-GP-ACM.There are obvious advantages in selectivity,sensitivity and anti-interference ability.Combined with the above probe evaluation,Z-GP-ACM also has a strong correlation between the detection results of PREP in different mouse brain homogenates and other specific detection results.The results show that Z-GP-ACM can selectively detect the activity of PREP in a complex system,combined with the advantages of near-infrared small molecule probes in tissue penetration and imaging sensitivity,it can achieve the activity of PREP in living cells in vitro Fluorescence imaging detection.In addition,the probe Z-GP-ACM can perform fluorescence imaging of PREP in different areas of three different anatomical surfaces in brain tissue.And through the immunofluorescence imaging detection,the feasibility of the probe Z-GP-ACM to image PREP in the brain tissue was verified.3.By evaluating the effects of six Chinese medicinal solutions in Liuwei Dihuang Pills on the activity of DPP4,it was screened out that Cortex Moutan had a certain inhibitory effect on DPP4,and the main components in Cortex Moutan were further screened for inhibitory activity.It was found that quercetin and kaempferol have a certain inhibitory effect on DPP4,with IC50 of 74.90 ± 6.17 μM;9.16 ± 0.69,respectively.Kaempferol inhibited the hydrolysis of GP-BAN by DPP4 through non-competitive inhibition with a Ki of 16.70 μM..4.Aimed at the main components of Dungflower,Dinggong Teng,Maqianzi,Huashan Ginseng,Stephanotis and Ginkgo which have nervous system effects,evaluate their inhibitory effects on PREP activity,and find that biflavonoids have strong PREP inhibitory ability,And through the structure-activity relationship analysis of bisflavonoids,the results show that the inhibitory activity is strongest when the 7,4’,4’’’ positions in the bisflavonoids are methoxy groups.Sciadopitysin inhibits PREP most strongly,with IC50 of 0.31 ± 0.06 μM,and inhibited the hydrolysis of Z-GP-ACM by PREP through noncompetitive inhibition,with Ki of 0.28 μM.In this study,two DPP4 and PREP activity detection probes with advantages of nearinfrared fluorescence were developed and evaluated.It has not only constructed a detection method for DPP4 and PREP in complex biological samples,but also greatly improved the efficiency and selectivity of DPP4 and PREP in cell and tissue imaging.At the same time,the PREP active fluorescent probe was used for fluorescence imaging of animal tissues for the first time.It provides a new detection method for the physiological or pathological changes of DPP4 and PREP in biological tissues.In addition,by screening the main substances that affect the activity of the DPP family in Chinese medicinal materials and their components,the application of fluorescent probes based on functional evaluation in the modernization of Chinese medicine has been explored.