Bioluminescent Probes Based On Firefly Luciferase For Detection Of Disease Biomarkers And Its Applications In Related Animal Models

Bioluminescence is a common phenomenon in nature.Luciferases catalyze the oxidation reactions of luciferins,and bioluminescence was released in the meanwhile.Bioluminescence is different from fluorescence because it does not require external excitation light,so that it cannot cause photobleaching as well as autofluorescence.Among various bioluminescent systems,firefly luciferase-luciferin is the most well-developed and widely-used one.It has been extensively applied in high-throughput screening(HTS)of compounds,detection of enzyme activity and cell tracking.Biomarkers are indicators that can be objectively assessed and measured in normal physiological processes,pathological processes,or pharmacological responses to therapeutic interventions.Protein is a common disease marker and the abnormal activity of some enzymes is associated with many pathological processes.Bioluminescence imaging technology(BLI)can be applied to the detection of enzyme activity,therefore,the development of bioluminescent probes for biomarkers is of great significance.By modification of the substrate of luciferase,this study has expanded the scope of bioluminescent imaging for different enzymes recognized as the biomarkers of several diseases.Several bioluminescent probes were well developed for the detection of pantetheinase,fibroblast activation protein,and ?-glutamine transpeptidase based on the "cage" strategy.Moreover,bioluminescent probes for tyrosinase was also developed based on the Bioluminescent Enzyme-Induced Electron Transfer(BioLeT)mechanism.In the second chapter,a bioluminescent probe for pantetheinase was developed.Pantetheinase,a glycosylphosphatidylinositol(GPI)anchored enzyme,overexpresses in intestine,liver,and kidney with various biological functions such as its linkage to the inflammation and some metabolic diseases.It can hydrolyze pantetheine to cysteamine,an antioxidant,and pantothenic acid(Vitamin B5)that is an essential component of coenzyme A(CoA).Until now,very few analytic methods were developed for this enzyme,thus hampering the further investigation of its biological functions.This part describes the design,synthesis,and biological examination of a highly sensitive bioluminogenic probe for pantetheinase with a limit of detection of 1.14 ng/mL.Furthermore,animal experiments validated that our probe can be applied to detecting the endogenous pantetheinase activity in vivo.To the best of our knowledge,this is the very first bioluminogenic probe achieving the detection of pantetheinase level in vivo.In the third chapter,two bioluminescent probes for fibroblast activation protein(FAP)were developed.FAP,as a crucial member of cell surface glycoprotein,highly expresses in reactive fibroblasts of tumors and several fibrosis diseases.It is a potential target for drug design and also reported as a prodrug strategy to increase the therapeutic window of some anticancer agents.Thereafter,it is urgently needed to improve the efficiency of detection method toward FAP.In this work,we developed the first bioluminogenic probe for FAP with a limit of detection(LOD)of 0.254 ng/mL,which could be applied to evaluating the efficiency of FAP inhibitors in vitro.The experiments of transgenic mice and tumor-bearing nude mice validated our probe could reflect the endogenous FAP level in vivo.Furthermore,this probe was used to reflect FAP up-regulation in the lung homogenates of the bleomycin-induced idiopathic pulmonary fibrosis(IPF)mice.In the fourth chapter,we introduce a bioluminescent probe for y-glutamyl transpeptidase(GGT).Increased GGT level was regarded as a biomarker of diseases such as liver sickness and stroke.Moreover,plasma GGT is associated with an elevated danger of developing cancer in epidemiologic studies.This bioluminescent probe processed high sensitivity and specificity toward GGT so that was utilized for detection of enzyme in vitro and in vivo.This probe can be recognized by GGT and released strong bioluminescence when it reacted with luciferase.After confirming the performance of probe in vitro and in cells,we applied it for the detection of endogenous GGT activity in a xenograft mice model.In the fifth chapter,we present bioluminogenic probes for tyrosinase.Tyrosinase,a copper-containing enzyme that catalyzes the oxidation of both phenols and catechols,is a key enzyme for eumelanin biosynthesis.It has been reported to be associated with melanoma and Parkinson's disease.Therefore,a convenient tool for the detection of tyrosinase activity in different biochemical and pathological manners is urgently desirable.Herein,based on bio luminescent enzyme-induced electron transfer(BioLeT)mechanism,a new class of bioluminogenic probes for tyrosinase was developed.We prepared two aminoluciferin derivatives with 4-hydroxyl phenylethyl group and 3,4-dihydroxylphenylethyl group,which have different HOMO energy levels.The aminoluciferin bearing 4-hydroxylphenylethy group(HP-AL)processed lower HOMO energy level and stronger luminescence than the one with 3,4-dihydroxylphenylethyl group(DHP-AL).Moreover,the bioluminescence of DHP-AL disclosed apparently bioluminescent augment when incubated with tyrosinase.LC-MS and UV-Vis absorption spectra analysis indicated that the reactions between DHP-AL and tyrosinase were different from the other fluorescent probes.Then,it was employed for the bioluminescent imaging of tyrosinase in living cells and in tumor-bearing mice model.In summary,we have developed bioluminescence probes for the detection of the activity of four enzymes based on different strategies.The abnormal expression of these enzymes is related to different diseases,and our probes could reflect the abnormal level of these enzymes in the related animal models.We believed that our probes would play an important role in studying the biological functions of these biomarkers.