Fluorescence Visualization Analysis For The Activity Of Anti-tuberculosis Target Enzymes And Traditional Chinese Medicine Intervention

Background:Tuberculosis（TB）,an infectious disease caused by Mycobacterium tuberculosis（Mtb）,is the second leading cause of death from a single infectious agent.In recent years,the emergence and spread of drug-resistant Mycobacterium tuberculosis has further increased the difficulty of TB control.In addition,the long treatment cycle and poor compliance of patients aggravate the occurrence of drug-resistant TB.Therefore,the development of new anti-TB targets,new anti-TB drugs and novel anti-TB strategies are crucial for TB treatment.China has rich resources and a long culture of traditional Chinese medicine（TCM）,accumulated a wealth of drug knowledge,with the deepening of TCM research,the action mechanism of TCM becoming clearer,and the clinical use of TCMs is more and more common.Natural products come from a wide range of sources,including Chinese herbs,animal and microbial chemical components or their metabolites.They are rich in structure,pharmacological effects and play an important role in clinical drug development.In this study,three anti-tuberculosis target enzymes were used to construct a high-throughput and visual fluorescence screening system for enzyme active inhibitors,hoping to discover new anti-TB drugs from natural products and optimize the treatment plans.N-acetyltransferase 2（NAT2）can metabolize the first-line anti-tuberculosis drug Isoniazid（INH）to acetylisoniazid（Ac INH）and participates in the synthesis of mycolic acid and the metabolic inactivation of INH in Mtb.Taking NAT2 as an anti-tuberculosis target,a high-throughput and visual detection method for NAT2 activity was established to screen inhibitors derived from natural products.It can not only inhibit the synthesis of mycolic acid,but also reduce the metabolic inactivation of INH,and enhance the anti-tuberculosis effect of combination drugs.β-lactamase（Blac）is the fundamental cause of Mtb resistance toβ-lactam antibiotics,which can destroy theβ-lactam ring of antibiotics and make them lose their antibacterial effect.The natural inhibitors of Blac were evaluated by the fluorescence visualization enzyme activity screening platform,and the natural resistance of Mtb toβ-lactam antibiotics was reversed,to explore a new anti-tuberculosis treatment scheme.Thioredoxin system is composed of thioredoxin,thioredoxin reductase（TrxR）,and reduced niacinamide adenine dinucleotide phosphate,which is an important antioxidant system in Mtb and plays a key role in maintaining the redox balance,DNA synthesis,oxidation defense and other physiological processes.TrxR is a credible anti-tuberculosis target,thus fluorescent probe was used to rapidly screen TrxR inhibitors in natural products and explored potential anti-tuberculosis compounds.In summary,a high-throughput and visual fluorescence detection method for the activity of tuberculosis target enzymes was developed,an evaluation system for the interaction between natural products and target enzymes was constructed,potential anti-tuberculosis active ingredients were explored,and new anti-tuberculosis strategies were developed,BDPN could be used as a high-connotation molecular tool for the treatment of tuberculosis.Part 1 The potential applications of fluorescence visualization analysis for N-acetyltransferase 2 and Traditional Chinese Medicine inhibitor screening in the anti-tuberculosisObjectives:An off-on fluorescent probe for detection of NAT2 activity was designed and developed,the fluorescent properties of the probe were characterized,the fluorescence imaging function of the probe was evaluated,and the interaction between TCM and NAT2 was explored,the antibacterial effect of NAT2 natural inhibitor and the anti-tuberculosis effect of combined with anti-tuberculosis drug INH was investigated.Methods:（1）A fluorescent probe（BDPN）toward NAT2 based on4,4-difluoro-boradiazaindacene（BODIPY）was developed using aniline as the recognition group of NAT2 and fluorescence quenching group.（2）The NAT2 activity evaluation system was established to characterize the fluorescence properties of BDPN probe.（3）The imaging function of BDPN was used to visualize the endogenous NAT2of Mtb.（4）The TCM inhibitory effect toward NAT2 was high-throughput screened by BDPN,and the active ingredients were separated by activity tracing.The effect of active ingredients on Mtb cell wall was observed by scanning electron microscopy and transmission electron microscopy,and the antibacterial activity in vitro was investigated.（5）Investigate the effect of active ingredients combined with anti-TB drug INH on the metabolic inactivation process of INH,and the effect of the combined drug on the anti-TB activity.Results:（1）The fluorescent probe BDPN has an obvious absorption peak at 498 nm with weak fluorescence intensity,by catalysis of NAT2,BDPN produced an obvious fluorescence signal at 512 nm,indicating that BDPN could be used as an off-on fluorescent probe for NAT2 activity detection;（2）The molecular orbitals and electron distribution under light excitation were investigated by chemical calculation,and the fluorescence mechanism of BDPN as an off-on fluorescent probe for NAT2 activity was further elucidated;（3）BDPN displayed the good stability and specificity in a variety of biological mediators.Among various chemical inhibitors,only quercetin,a selective inhibitor of NAT2,can block acetylation.Molecular docking results further verified the specificity of BDPN to NAT2,indicating that BDPN can be used to selectively detect NAT2 activity in complex biological samples;（4）The confocal imaging was performed on the endogenous NAT2 of Mtb H37Ra and M.smegmatis mc²155 by using BDPN,and BDPN produced obvious fluorescence signal under the catalysis of NAT2;（5）By virtue of the superior properties of BDPN,Psoraleae Fructus was selected from 168kinds of TCM with the strongest inhibition on NAT2,and active monomers with strong inhibitory effect were isolated,among which Isobavachalcone had the strong antibacterial effect that could significantly inhibit the fluorescence signal of BDPN in Mtb H37Ra and M.smegmatis mc²155;（6）The scanning electron microscopy and transmission electron microscopy showed that the morphology of Mtb H37Ra treated by Isobavachalcone was significantly changed,the integrity of the cell wall was destroyed,and the outline was not clear;（7）Isobavachalcone can inhibit the growth rate of Mtb H37Ra and slow down the metabolic inactivation rate of INH,thereby increasing the drug concentration and improving the anti-TB effect of INH in Mtb H37Ra.Conclusions:（1）In vitro metabolic evaluation confirmed that BDPN could be used as a highly selective off-on fluorescent probe for NAT2 detection in complex biological systems;（2）Visual detection of endogenous NAT2 in Mtb H37Ra and M.smegmatis mc²155;（3）Through high-throughput,visual screening and active tracer isolation,the potent inhibitor Isobavachalcone was obtained,which could affect the formation of cell wall of Mtb H37Ra,change bacterial morphology and inhibit the growth of Mtb;（4）Isobavachalcone can reduced the metabolic inactivation of INH and enhance the anti-tuberculosis effect,BDPN provides a fluorescent molecular tool for the discovery of potential anti-tuberculosis drugs and the implementation of new anti-tuberculosis strategies.Part 2 Fluorescence high-throughput screening ofβ-lactamase inhibitors to improve the antibiotic treatment strategies for tuberculosisObjectives:A near-infrared（NIR）fluorescent probe for real-time detecting the activity ofβ-lactamase（Blac）was designed and developed,the fluorescent properties of the probe were characterized,the fluorescence imaging function of the probe was evaluated,the interaction between TCM and Blac was explored,and use it in combination withβ-lactam antibiotics to alleviate the natural resistance of Mycobacterium tuberculosis and achieve the purpose of anti-tuberculosis.Methods:（1）The specific fluorescent probe LXMB of Blac was developed by introducingβ-lactam ring as the recognition site,using the hemi-cyanine as the fluorophore;（2）The fluorescence mechanism was explained by chemical calculation;（3）The fluorescence properties of the probe LXMB was characterized and the Blac activity evaluation system was established;（4）Elucidation of the interaction between LXMB and Blac by molecular docking;（5）High-throughput screening of TCM inhibitory effect on Blac,the active ingredients were isolated under the instructions of fluorescence tracing,and the interaction between active ingredients and Blac was explained by molecular docking;（6）The endogenous Blac of Mtb H37Ra and M.smegmatis mc²155 were visualized using the imaging function of LXMB;（7）The antibacterial effect of Blac inhibitor combined withβ-lactam antibiotics against Mtb H37Ra was investigated.Results:（1）The fluorescent probe LXMB had an obvious absorption peak at 588 nm with weak fluorescence intensity,a new absorption peak was generated at 700 nm and an obvious fluorescence signal was observed at 724 nm after catalyzed by Blac,indicating that LXMB could be used as an off-on fluorescent probe for detecting Blac.（2）The fluorescence mechanism of LXMB was elucidated by chemical calculation,which further indicated that LXMB was an off-on NIR fluorescent probe for detecting Blac activity.（3）LXMB showed excellent stability in a variety of endogenous substances and showed good photostability within 120 min.The detection of Blac had a wide linear range and LXMB showed excellent selectivity for Blac in a variety of biological enzymes.Among a variety of chemical inhibitors,only sulbactam sodium,a Blac specific inhibitor,can inhibit the catalytic process of Blac.The reasons for the high selectivity and good affinity of LXMB for Blac were explained in molecular docking experiments.These results indicated that LXMB could be used to detect Blac activity in complex biological samples;（4）The Galla Chinensis with strong inhibitory effect on Blac was screened out from 260 TCM,and the active component tannic acid was isolated by activity tracing;（5）The IC₅₀value of tannic acid was determined,and the interaction between tannic acid and Blac was analyzed by molecular docking;（6）In confocal imaging experiments,LXMB produced obvious fluorescence signal catalyzed by endogenous Blac,which was attenuated by tannic acid;phagocytosis of LXMB labeled Mtb H37Ra in macrophages was observed in fluorescence imaging,indicating that LXMB has excellent imaging performance;（7）Tannic acid could enhance the anti-tuberculosis effects of manyβ-lactam antibiotics.Conclusions:（1）Metabolic phenotype,computational chemistry and molecular docking experiments indicated that LXMB could be used as a specific off-on NIR fluorescent probe for Blac detection in complex biological systems;（2）LXMB could realize the visual detection of the endogenous Blac of Mtb H37Ra and M.smegmatis mc²155,and fluorescence imaging of the labeled Mtb H37Ra in macrophages.（3）LXMB was used for high-throughput screening of Blac inhibitors,Tannic acid could be combined withβ-lactam antibiotics to significantly enhanced the anti-tuberculosis effect.Therefore,LXMB could be used to analyze the resistance mechanism of Mtb toβ-lactam antibiotics and provide a high-content fluorescent molecular tool for the development of Blac inhibitors,to improve the anti-tuberculosis effect ofβ-lactam antibiotics.Part 3 Development of fluorescent probe for thioredoxin reductase and screening of potential antituberculosis drugs in natural productsObjectives:A NIR fluorescent probe for detecting the activity of thioredoxin reductase（TrxR）was designed and developed,the fluorescence properties of the probe were characterized,the fluorescence imaging function of the probe was evaluated,the interaction between TCM and natural products with TrxR was explored,and the anti-tuberculosis effect of the active ingredients was evaluated.Methods:（1）Using 1,3-dichloro-7-amino-9,9-dimethyl-2（9H）-acridone（DDAN）as fluorophore,disulfide bonds were introduced as the recognition site to develop the specific fluorescent probe DDAT for TrxR;（2）Fluorescence properties of DDAT were characterized,TrxR activity evaluation system was established,and the interaction between DDAT and TrxR was analyzed by molecular docking;（3）The inhibitory effect of Chinese herbal medicines and natural products toward TrxR was further screened by high-throughput screening system.The active ingredients were isolated and identified using the activity tracing approach,and the interaction between the active ingredients and TrxR was analyzed by molecular docking;（4）The imaging function of DDAT was used to visualize the endogenous TrxR of Mtb H37Ra;（5）The minimum inhibitory concentration（MIC）of Brazilin and Gliotoxin to Mtb H37Ra was determined by resazurin assay,and the effect of Gliotoxin on the growth rate of Mtb H37Ra was detected.The effect of active ingredients on the morphological structure of Mtb H37Ra was observed by propidium iodide penetration test and scanning electron microscope.Results:（1）DDAT generated DDAN under the catalysis of TrxR,the absorption peak was redshifted,and a new absorption peak was generated at 610 nm,and the fluorescence signal was significantly enhanced at 666 nm,indicating that DDAT could be used as an off-on NIR fluorescent probe for detecting TrxR;（2）DDAT had a stable fluorescence intensity in the p H range of 3.0-9.0,showed excellent stability in a variety of endogenous substances,and showed a high selectivity toward TrxR in a variety of enzymes.The fluorescence intensity and TrxR concentration show a good linear relationship,and the specificity of DDAT for TrxR is further proved by chemical inhibition experiments.The interaction between DDAT and TrxR was analyzed by molecular docking experiments,indicating that DDAT has excellent stability and selectivity,and could be used to detect TrxR activity in complex biological samples;（3）Sappan Lignum and Gliotoxin with strong inhibitory effects on TrxR were screened from 260 Chinese herbal medicines and 180 natural products.And the active component of Brazilin was isolated under the activity tracing,and their interaction with TrxR was analyzed by molecular docking;（4）The endogenous TrxR of Mtb H37Ra was detected by DDAT and produced a significant fluorescence signal catalyzed by TrxR,which could be attenuated by the inhibitors Ebselen and Gliotoxin;（5）Brazilin and Gliotoxin had significant inhibitory effects on Mtb H37Ra,and the integrity of the cell wall of Mtb H37Ra was destroyed and the morphology of Mtb H37Ra was changed significantly after Brazilin and Gliotoxin treatment.Conclusions:（1）Metabolic phenotype and molecular docking experiments indicated that DDAT could be used as a highly selective NIR fluorescent probe for the detection of TrxR in complex biological systems;（2）DDAT could be used to visualize the endogenous TrxR in Mtb H37Ra;（3）DDAT was used for high-throughput screening of TrxR inhibitors to exert anti-TB effect.DDAT can be used to analyze the activity of TrxR in Mtb and provide a useful fluorescent molecular tool for the screening of TrxR specific inhibitors and the development of new anti-tuberculosis drugs.